Stem Cell Treatment for Degenerative Disc Disease

Stem Cell Treatment for Degenerative Disc Disease

Degeneration of the intervertebral disc, often called "degenerative disc disease" (DDD) of the spine, is a condition that can be painful and can greatly affect the quality of one's life.

STEM CELL TREATMENT DEGENERATIVE DISC DISEASE

While disc degeneration is a normal part of aging and for most people is not a problem, for certain individuals a degenerated disc can cause severe constant chronic pain. Often, degenerative disc disease can be successfully treated without surgery. One or a combination of treatments such as physical therapy, chiropractic manipulative therapy (CMT) and other chiropractic treatments, osteopathic manipulation, anti-inflammatory medications such as nonsteroidal anti-inflammatory drugs, traction, or spinal injections often provide adequate relief of these troubling symptoms.

Degenerative discs typically show degenerative fibrocartilage and clusters of chondrocytes, suggestive of repair. Inflammation may or may not be present. Histologic examination of disc fragments resected for presumed DDD is routine to exclude malignancy.

Fibrocartilage replaces the gelatinous mucoid material of the nucleus pulposus as the disc changes with age. There may be splits in the annulus fibrosis, permitting herniation of elements of nucleus pulposus. There may also be shrinkage of the nucleus pulposus that produces prolapse of the annulus with secondary osteophyte formation at the margins of the adjacent vertebral body.

The pathologic findings in DDD include protrusion, spondylolysis, and/or subluxation of vertebrae (sponylolisthesis) and spinal stenosis.

STEM CELL TREATMENT FOR DEGENERATIVE DISC DISEASE

Stem Cell Treatment and Degenerative Disc Disease NIH Streaming Database

Related Articles Stem cell regeneration of the intervertebral disk. Orthop Clin North Am. 2011 Oct;42(4):555-62, viii-ix Authors: Sakai D Abstract The use of stem cell applications has been explored and aimed at regenerating the intervertebral disk. The microenvironment in which cells of the intervertebral disk reside is harsh; however, researchers have reported on many applications for stem cells, including research aimed at defining and stimulating endogenous stem cell populations, methods to induce stem cell differentiation toward intervertebral disk cell phenotype in vivo, and direct transplantation of stem cells into damaged intervertebral disk to promote transplanted site-dependant differentiation. Successful results have been reported, although limitations remain. This article reviews the current status of stem cell research as applied to the intervertebral disk. PMID: 21944591 [PubMed - indexed for MEDLINE]
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Related Articles Notochordal cells in the adult intervertebral disc: new perspective on an old question. Crit Rev Eukaryot Gene Expr. 2011;21(1):29-41 Authors: Risbud MV, Shapiro IM Abstract The intervertebral disc is a tissue positioned between each of the vertebrae that accommodates applied biomechanical forces to the spine. The central compartment of the disc contains the nucleus pulposus (NP) which is enclosed by the annulus fibrosus and the endplate cartilage.The NP is derived from the notochord, a rod-like structure of mesodermal origin. Development of the notochord is tightly regulated by interactive transcription factors and target genes. Since a number of these molecules are unique they have be used for cell lineage and fate mapping studies of tissues of the intervertebral disc. These studies have shown that in a number of species including human, NP tissue retains notochordal cells throughout life. In the adult NP, there are present both large and small notochordal cells, as well as a progenitor cell population which can differentiate along the mesengenic pathway. Since tissue renewal in the intervertebral disc is dependent on the ability of these cells to commit to the NP lineage and undergo terminal differentiation, studies have been performed to assess which signaling pathways may regulate these activities. The notch signaling pathway is active in the intervertebral disc and is responsive to hypoxia, probably through HIF-1a. From a disease viewpoint, it is hypothesized that an oxemic shift, possibly mediated by alterations in the vascular supply to the tissues of the disc would be expected to lead to a failure in notochordal progenitor cell activation and a decrease in the number of differentiated cells. In turn, this would lead to decrements in function and enhancement of the effect of agents that are known to promote disc degeneration. PMID: 21967331 [PubMed - indexed for MEDLINE]
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Related Articles Investigating the possibility of intervertebral disc regeneration induced by granulocyte colony stimulating factor-stimulated stem cells in rats. Adv Orthop. 2011;2011:602089 Authors: Tzaan WC, Chen HC Abstract Intervertebral disc (IVD) degeneration is a multifactorial process that is influenced by contributions from genetic predisposition, the aging phenomenon, lifestyle conditions, biomechanical loading and activities, and other health factors (such as diabetes). Attempts to decelerate disc degeneration using various techniques have been reported. However, to date, there has been no proven technique effective for broad clinical application. Granulocyte colony-stimulating factor (GCSF) is a growth factor cytokine that has been shown to enhance the availability of circulating hematopoietic stem cells to the brain and heart as well as their capacity for mobilization of mesenchymal bone marrow stem cells. GCSF also exerts significant increases in circulating neutrophils as well as potent anti-inflammatory effects. In our study, we hypothesize that GCSF can induce bone marrow stem cells differentiation and mobilization to regenerate the degenerated IVD. We found that GCSF had no contribution in disc regeneration or maintenance; however, there were cell proliferation within end plates. The effects of GCSF treatment on end plates might deserve further investigation. PMID: 21991419 [PubMed]
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Related Articles Structured bilaminar coculture outperforms stem cells and disc cells in a simulated degenerate disc environment. Spine (Phila Pa 1976). 2012 May 1;37(10):813-8 Authors: Allon AA, Butcher K, Schneider RA, Lotz JC Abstract STUDY DESIGN: This study explores the use of bilaminar coculture pellets of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. The pellets were tested under conditions that mimic the degenerative disc. OBJECTIVE: Our goal was to optimize our cell-based therapy in vitro under conditions representative of the eventual diseased tissue. SUMMARY OF BACKGROUND DATA: Harnessing the potential of stem cells is an important strategy for regenerative medicine. Our approach directed the behavior of stem cells by mimicking embryonic processes underlying cartilage and intervertebral disc development. Prior experiments have shown that bilaminar coculture can help differentiate MSC and substantially improve new matrix deposition. METHODS: We have designed a novel spherical bilaminar cell pellet (BCP) where MSCs are enclosed in a shell of NPC. There were 3 groups: MSC, NPC, and BCP. The pellets were tested under 3 different culture conditions: 1) in a bioreactor that provides pressure and hypoxia (mimicking normal disc conditions): 2) with inflammatory cytokines (IL-1b and TNF-a); and 3) a bioreactor with inflammation (mimicking painful disc conditions). RESULTS: When cultured in the bioreactor, the NPC pellets produced significantly more glycosaminoglycans (GAGs) per cell than the other groups: 70% to 80% more than the BCP and the MSC alone. When cultured in an inflammatory environment, the MSC and BCP groups produced 30% to 34% more GAGs per cell than NPC (P < 0.05). When the pellets were cultured in a bioreactor with inflammation, the BCP made 25% more GAGs per cell than the MSC and 57% more than the NPC (P < 0.05). CONCLUSION: This study shows that BCPs outperform controls in a simulated degenerated disc environment. Adapting inductive mechanisms from development to trigger differentiation and restore diseased tissue has many advantages. As opposed to strategies that require growth factor supplements or genetic manipulations, our method is self-sustaining, targeted, and minimally invasive injection. PMID: 22024902 [PubMed - indexed for MEDLINE]
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Related Articles Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate. PLoS One. 2011;6(10):e26285 Authors: Liu LT, Huang B, Li CQ, Zhuang Y, Wang J, Zhou Y Abstract Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering. PMID: 22028847 [PubMed - indexed for MEDLINE]
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Related Articles Study to determine the presence of progenitor cells in the degenerated human cartilage endplates. Eur Spine J. 2012 Apr;21(4):613-22 Authors: Huang B, Liu LT, Li CQ, Zhuang Y, Luo G, Hu SY, Zhou Y Abstract INTRODUCTION: Cartilage endplate (CEP) degeneration is usually accompanied by loss of cellularity, and this loss may be a crucial key factor in initiation and development of degenerative disc disease. The study of cell types in degenerated CEP could help in understanding CEP etiopathogenesis, and may help in devising new treatments, especially if the presence of progenitor cells could be demonstrated. The aim of this study was to determine if progenitor cells existed in degenerated human CEP. MATERIALS AND METHODS: Cells isolated from CEP were cultured in a three-dimensional agarose suspension to screen for proliferative cell clusters. Cell clusters were then expanded in vitro and the populations were analyzed for colony forming unit, immunophenotype, multilineage induction, and expression of stem cell-related genes. RESULTS: The presence of progenitor cells in degenerated human CEP is indicated by the results of CFU, immunophenotype, multilineage induction, and expression of stem cell-related genes. CONCLUSIONS: We believe that this is the first study which has conclusively shown the presence of progenitor cells in degenerated CEP. The finding of this study may influence the clinical management of degenerative disc disorder. PMID: 22033570 [PubMed - indexed for MEDLINE]
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Related Articles Evaluation of platelet-rich plasma and hydrostatic pressure regarding cell differentiation in nucleus pulposus tissue engineering. J Tissue Eng Regen Med. 2013 Mar;7(3):244-52 Authors: Mietsch A, Neidlinger-Wilke C, Schrezenmeier H, Mauer UM, Friemert B, Wilke HJ, Ignatius A Abstract Generation of a biological nucleus pulposus (NP) replacement by tissue engineering appears to be a promising approach for the therapy of early stages of intervertebral disc degeneration. Thereby, autologous mesenchymal stem cells (MSCs) represent an attractive cell source compared to cells of the NP that are already altered in their phenotype due to degenerative processes. This study compares the influence of 3D pellet culture and alginate beads, as well as that of different media compositions, by the addition of human platelet-rich plasma (PRP) or transforming growth factor (TGF-β1 ) in interaction with hydrostatic pressure on chondrogenic differentiation of human MSCs compared to NP cells. We found that gene expression of the chondrogenic markers aggrecan, collagen type 2 and collagen type 1 and Sox9 was considerably lower in cells cultivated with PRP compared to TGF-β1 . Immunohistology confirmed this result at protein level in pellet culture. Additionally, the pellet culture system was found to be more suitable than alginate beads. A positive influence of hydrostatic pressure could only be shown for individual donors. In summary, in comparison to TGF-β1 , human PRP did not induce adequate chondrogenic differentiation for both culture systems and cell types used. The mixture of growth factors in PRP promoted proliferation rather than chondrogenic differentiation. Based on these results, an application of PRP in human NP tissue-engineering approaches cannot be recommended. PMID: 22162329 [PubMed - indexed for MEDLINE]
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Related Articles Growth factors and anticatabolic substances for prevention and management of intervertebral disc degeneration. Stem Cells Int. 2012;2012:897183 Authors: Longo UG, Petrillo S, Franceschetti E, Maffulli N, Denaro V Abstract Intervertebral disc (IVD) degeneration is frequent, appearing from the second decade of life and progressing with age. Conservative management often fails, and patients with IVD degeneration may need surgical intervention. Several treatment strategies have been proposed, although only surgical discectomy and arthrodesis have been proved to be predictably effective. Biological strategies aim to prevent and manage IVD degeneration, improving the function and anabolic and reparative capabilities of the nucleus pulposus and annulus fibrosus cells and inhibiting matrix degradation. At present, clinical applications are still in their infancy. Further studies are required to clarify the role of growth factors and anticatabolic substances for prevention and management of intervertebral disc degeneration. PMID: 25098367 [PubMed]
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Related Articles [An innovative disc repair for the degeneration]. Nihon Rinsho. 2011 Dec;69(12):2220-4 Authors: Mochida J Abstract Experimental studies in various animals showed that using a coculture system with direct cell-to-cell contact with mesenchymal stem cells (MSCs) significantly upregulated the biological activity of nucleus pulposus (NP) cells. Activated NP cells can be reinserted into the disc to inhibit intervertebral disc degeneration. In human cells, the positive effects of the coculture system with direct cell-to-cell contact seen in animal studies were also evaluated and cell proliferation, DNA synthesis, and PG synthesis were significantly upregulated. Chromosome abnormalities and tumorigenesis were not observed in the activated NP cells. Then the authors have started the specially designed clinical trial under the control of Ministry of Health, Labour and Welfare and now post operative follow-up has been continued with expected good outcome. PMID: 22242323 [PubMed - indexed for MEDLINE]
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Related Articles Overenthusiastic interpretations of a nonetheless promising study. Transplantation. 2012 Feb 15;93(3):e6-7; author reply e7-9 Authors: Kovacs FM, Abraira V, Gérvas J, Arana E, Peul WC, Schoene ML, Corbin TP PMID: 22277960 [PubMed - indexed for MEDLINE]
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Related Articles Quantitative analysis of exogenous IGF-1 administration of intervertebral disc through intradiscal injection. J Biomech. 2012 Apr 30;45(7):1149-55 Authors: Huang CY, Travascio F, Gu WY Abstract Exogenous administration of IGF-1 has been proposed as a therapy for disc degeneration. The objectives of this study were to develop a numerical model for quantitatively analysing exogenous administration of IGF-1 into the intervertebral disc (IVD) via intradiscal injection and to investigate the effects of IGF-1 administration on distribution of glucose and oxygen in the IVD. In this study, the reversible binding reaction between IGF-1 and IGF binding proteins was incorporated into the mechano-electrochemical mixture model. The model was used to numerically analyse transport of IGF-1, glucose, oxygen and lactate in the IVD after IGF-1 administration. The enhancement of IGF-1 on lactate production was also taken into account in the theoretical model. The numerical analyses using finite element method demonstrated that the binding reactions significantly affect the time-dependent distribution of IGF-1 in the IVD. It was found that the region affected by IGF-1 was smaller and the duration of the therapeutic IGF-1 level was longer in the degenerated disc with a higher concentration of IGF binding proteins. It was also found that the IGF-1 injection can reduce glucose concentration and increase lactate accumulation (i.e., lower pH) in the IVD and these influences were regulated by the IGF-1 binding reactions. This study indicated the complexity of intradiscal administration of growth factors, which needs to be fully analysed in order to achieve a successful outcome. The new theoretical model developed in this study can serve as a powerful tool in analysing and designing the optimal treatments of growth factors for disc degeneration. PMID: 22365501 [PubMed - indexed for MEDLINE]
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Related Articles My aching back. World Neurosurg. 2012 Sep-Oct;78(3-4):248-51 Authors: Shamji MF, Hurlbert RJ PMID: 22366745 [PubMed - indexed for MEDLINE]
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Related Articles Transplantation of human adipose-derived stem cells in a rabbit model of traumatic degeneration of lumbar discs. World Neurosurg. 2012 Sep-Oct;78(3-4):364-71 Authors: Chun HJ, Kim YS, Kim BK, Kim EH, Kim JH, Do BR, Hwang SJ, Hwang JY, Lee YK Abstract OBJECTIVE: The purpose of the present study is to assess the possibility of disc regeneration by treatment with adipose-derived stem cells (ADSCs) in a rabbit model of degenerative disc disease, and to evaluate the efficacy of a percutaneous technique for constructing a model of degenerative disc disease in rabbits. METHODS: The study sample consisted of 20 mature male New Zealand white rabbits. Intervertebral discs were injured in each rabbit by a percutaneous technique at L2-3, L3-4, and L4-5 under C-arm guidance with a 19-gauge spinal needle. Magnetic resonance images (MRI) were checked at 6, 9, 12, and 15 weeks after injury to evaluate disc degeneration. Nineteen weeks after injury, ADSCs were injected into the L4-5 disc space, with saline injected into the L3-4 disc as a control, using a 21-gauge spinal needle. Histologic confirmations of degenerated discs were performed at 10 and 18 weeks after injury with safranin O and trichrome stains. RESULTS: MRI revealed intervertebral disc degeneration from 9 weeks after injury, and full degeneration at 15 weeks after injury, when compared with uninjured control discs. We confirmed the proliferation of ADSCs at the L4-5 level in 10-week rabbits after cell injection. Histologically, the ADSC-injected discs exhibited elevated extracellular matrix secretion and little ossification of damaged cartilage in the nucleus pulposus compared with degenerative control discs. CONCLUSIONS: These results suggest that the injection of ADSCs into injured lumbar discs could be an effective treatment for degenerative disc disease by promoting the cartilage regeneration. PMID: 22381275 [PubMed - indexed for MEDLINE]
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Related Articles Immunoselected STRO-3+ mesenchymal precursor cells and restoration of the extracellular matrix of degenerate intervertebral discs. J Neurosurg Spine. 2012 May;16(5):479-88 Authors: Ghosh P, Moore R, Vernon-Roberts B, Goldschlager T, Pascoe D, Zannettino A, Gronthos S, Itescu S Abstract OBJECT: Chronic low-back pain of discal origin is linked strongly to disc degeneration. Current nonsurgical treatments are palliative and fail to restore the disc extracellular matrix. In this study the authors examined the capacity of ovine mesenchymal precursor cells (MPCs) to restore the extracellular matrix of degenerate discs in an ovine model. METHODS: Three adjacent lumbar discs of 24 adult male sheep were injected intradiscally with chondroitinase-ABC (cABC) to initiate disc degeneration. The remaining lumbar discs were used as normal controls. Three months after cABC injection, the L3-4 discs of all animals were injected with either a high dose (4 × 10(6) cells, in 12 sheep) or low dose (0.5 × 10(6) cells, in 12 sheep) of MPCs suspended in hyaluronic acid (HA). The adjacent L4-5 degenerate discs remained untreated; the L5-6 discs were injected with HA only. The animals were euthanized at 3 or 6 months after MPC injections (6 sheep from each group at each time point), and histological sections of the lumbar discs were prepared. Radiographs and MR images were obtained prior to cABC injection (baseline), 3 months after cABC injection (pretreatment), and just prior to necropsy (posttreatment). RESULTS: Injection of cABC decreased the disc height index (DHI) of target discs by 45%-50%, confirming degeneration. Some recovery in DHI was observed 6 months after treatment in all cABC-injected discs, but the DHI increased to within baseline control values only in the MPC-injected discs. This improvement was accompanied by a reduction in MRI degeneration scores. The histopathology scores observed at 3 months posttreatment for the high-dose MPC-injected discs and at 6 months posttreatment for the low-dose MPC-injected discs were significantly different from those of the noninjected and HA-injected discs (p <0.001) but not from the control disc scores. CONCLUSIONS: On the basis of the findings of this study, the authors conclude that the injection of MPCs into degenerate intervertebral discs can contribute to the regeneration of a new extracellular matrix. PMID: 22404141 [PubMed - indexed for MEDLINE]
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Related Articles Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc. J Transl Med. 2012;10:49 Authors: Liang C, Li H, Tao Y, Zhou X, Li F, Chen G, Chen Q Abstract BACKGROUND: Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions. METHODS: ADMSCs were harvested from young (aged 8-12 years, n = 6) and mature (aged 33-42 years, n = 6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for 2 weeks. Cell viability was measured by annexin V-FITC and PI staining and cell proliferation was measured by MTT assay. The expression of aggrecan and collagen-I was detected by real-time quantitative polymerase chain reaction and Western blot analysis. RESULTS: IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-I. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD. CONCLUSION: IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain. PMID: 22424131 [PubMed - indexed for MEDLINE]
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Related Articles Homing of mesenchymal stem cells in induced degenerative intervertebral discs in a whole organ culture system. Spine (Phila Pa 1976). 2012 Oct 15;37(22):1865-73 Authors: Illien-Jünger S, Pattappa G, Peroglio M, Benneker LM, Stoddart MJ, Sakai D, Mochida J, Grad S, Alini M Abstract STUDY DESIGN: Homing of human bone marrow-derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs). OBJECTIVE: To investigate in a whole organ culture whether metabolic and mechanical challenges can induce BMSC recruitment into the IVD. SUMMARY OF BACKGROUND DATA: Cells from injured tissues release cytokines and mediators that enable the recruitment of progenitor cells. BMSCs have the ability to survive within the IVD. METHODS: Bovine IVDs with or without endplates were cultured for 1 week under simulated physiological or degenerative conditions; disc cells were analyzed for cell viability and gene expression, whereas media was analyzed for nitric oxide production and chemotaxis. Homing of BMSCs was investigated by supplying PKH-labeled human BMSCs onto cultured IVDs (1 × 10(6) cells/disc on d 8, 10, and 12 of culture); on day 14, the number of homed BMSCs was microscopically assessed. Moreover, a comparative study was performed between transduced BMSCs (transduced with an adenovirus encoding for insulin-like growth factor 1 [IGF-1]) and nontransduced BMSCs. Disc proteoglycan synthesis rate was quantified via (35)S incorporation. The secretion of IGF-1 was evaluated by enzyme-linked immunosorbent assay on both simulated physiological and degenerative discs. RESULTS: Discs cultured under degenerative conditions showed reduced cell viability, upregulation of matrix degrading enzymes, and increased nitric oxide production compared with simulated physiological discs. Greater homing occurred under degenerative compared with physiological conditions with or without endplate. Media of degenerative discs demonstrated a chemoattractive activity toward BMSCs. Finally, discs homed with IGF-1-transduced BMSCs showed increased IGF-1 secretion and significantly higher proteoglycan synthesis rate than discs supplied with nontransduced BMSCs. CONCLUSION: We have demonstrated for the first time that degenerative conditions induce the release of factors promoting BMSC recruitment in an ex vivo organ culture. Moreover, IGF-1 transduction of BMSCs strongly increases the rate of proteoglycan synthesis within degenerative discs. This finding offers a new delivery system for BMSCs and treatment strategy for IVD regeneration. PMID: 22433498 [PubMed - indexed for MEDLINE]
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Related Articles Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells. PLoS One. 2012;7(3):e33739 Authors: Strassburg S, Hodson NW, Hill PI, Richardson SM, Hoyland JA Abstract Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells. PMID: 22438989 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell-mediated osteogenesis: therapeutic potential for bone tissue engineering. Biologics. 2012;6:47-57 Authors: Neman J, Hambrecht A, Cadry C, Jandial R Abstract Intervertebral disc degeneration often requires bony spinal fusion for long-term relief. Current arthrodesis procedures use bone grafts from autogenous bone, allogenic backed bone, or synthetic materials. Autogenous bone grafts can result in donor site morbidity and pain at the donor site, while allogenic backed bone and synthetic materials have variable effectiveness. Given these limitations, researchers have focused on new treatments that will allow for safe and successful bone repair and regeneration. Mesenchymal stem cells have received attention for their ability to differentiate into osteoblasts, cells that synthesize new bone. With the recent advances in scaffold and biomaterial technology as well as stem cell manipulation and transplantation, stem cells and their scaffolds are uniquely positioned to bring about significant improvements in the treatment and outcomes of spinal fusion and other injuries. PMID: 22500114 [PubMed]
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Related Articles Adrenal insufficiency following spinal cord injury: an underrecognized cause of hemodynamic instability? World Neurosurg. 2012 Sep-Oct;78(3-4):252 Authors: Wang MY PMID: 22548876 [PubMed - indexed for MEDLINE]
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Related Articles Mesenchymal stem cell for prevention and management of intervertebral disc degeneration. Stem Cells Int. 2012;2012:921053 Authors: Longo UG, Papapietro N, Petrillo S, Franceschetti E, Maffulli N, Denaro V Abstract Intervertebral disc degeneration (IVD) is a frequent pathological condition. Conservative management often fails, and patients with IVD degeneration may require surgical intervention. Several treatment strategies have been proposed, although only surgical discectomy and arthrodesis have been proved to be predictably effective. The aim of biological strategies is to prevent and manage IVD degeneration, improve the function, the anabolic and reparative capabilities of the nucleus pulposus and annulus fibrosus cells, and inhibit matrix degradation. At present, clinical applications are still in their infancy. Further studies are required to clarify the role of mesenchymal stem cells and gene therapy for the prevention and treatment of IVD degeneration. PMID: 22550520 [PubMed]
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Related Articles Lumbar degenerative disc disease: current and future concepts of diagnosis and management. Adv Orthop. 2012;2012:970752 Authors: Taher F, Essig D, Lebl DR, Hughes AP, Sama AA, Cammisa FP, Girardi FP Abstract Low back pain as a result of degenerative disc disease imparts a large socioeconomic impact on the health care system. Traditional concepts for treatment of lumbar disc degeneration have aimed at symptomatic relief by limiting motion in the lumbar spine, but novel treatment strategies involving stem cells, growth factors, and gene therapy have the theoretical potential to prevent, slow, or even reverse disc degeneration. Understanding the pathophysiological basis of disc degeneration is essential for the development of treatment strategies that target the underlying mechanisms of disc degeneration rather than the downstream symptom of pain. Such strategies ideally aim to induce disc regeneration or to replace the degenerated disc. However, at present, treatment options for degenerative disc disease remain suboptimal, and development and outcomes of novel treatment options currently have to be considered unpredictable. PMID: 22567411 [PubMed]
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Related Articles Stem cell therapy for degenerative disc disease. Adv Orthop. 2012;2012:961052 Authors: Drazin D, Rosner J, Avalos P, Acosta F Abstract Low back pain is widely recognized as one of the most prevalent pathologies in the developed world. In the United States, low back pain is the most common health problem for adults under the age of 50, resulting in significant societal and personal costs. While the causes of low back pain are myriad, it has been significantly associated with intervertebral disc (IVD) degeneration. Current first-line therapies for IVD degeneration such as physical therapy and spinal fusion address symptoms, but do not treat the underlying degeneration. The use of tissue engineering to treat IVD degeneration provides an opportunity to correct the pathological process. Novel techniques are currently being investigated and have shown mixed results. One major avenue of investigation has been stem cell injections. Mesenchymal stem cells (MSCs) have shown promise in small animal models, but results in larger vertebrates have been mixed. PMID: 22593830 [PubMed]
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Related Articles Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction? Swiss Med Wkly. 2012;142:w13598 Authors: Chan SC, Gantenbein-Ritter B Abstract The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline. PMID: 22653467 [PubMed - indexed for MEDLINE]
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Related Articles Diversity of intervertebral disc cells: phenotype and function. J Anat. 2012 Dec;221(6):480-96 Authors: Pattappa G, Li Z, Peroglio M, Wismer N, Alini M, Grad S Abstract The intervertebral disc (IVD) is a moderately moving joint that is located between the bony vertebrae and provides flexibility and load transmission throughout the spinal column. The disc is composed of different but interrelated tissues, including the central highly hydrated nucleus pulposus (NP), the surrounding elastic and fibrous annulus fibrosus (AF), and the cartilaginous endplate (CEP), which provides the connection to the vertebral bodies. Each of these tissues has a different function and consists of a specific matrix structure that is maintained by a cell population with distinct phenotype. Although the healthy IVD is able to balance the slow matrix turnover of synthesis and degradation, this balance is often disturbed, leading to degenerative disorders. Successful therapeutic management of IVD degeneration requires a profound understanding of the cellular and molecular characteristics of the functional IVD. Hence, the phenotype of IVD cells has been of significant interest from multiple perspectives, including development, growth, remodelling, degeneration and repair. One major challenge that complicates our understanding of the disc cells is that both the cellular phenotype and the extracellular matrix strongly depend on disc maturity and health and as a consequence are continuously evolving. This review delineates the diversity of the cell types found in the intervertebral disc, with emphasis on human, but with reference to other species. The cells of the NP appear rounded and express a proteoglycan-rich matrix, whereas the more elongated AF cells are embedded in a collagen fibre matrix and the CEPs represent a layer of cartilage. Even though all disc cells have often been referred to as 'intervertebral disc chondrocytes', distinct phenotypical differences in comparison with articular chondrocytes exist and have been reported recently. The availability of more specific markers has also improved our understanding of progenitor cell differentiation towards an IVD cell phenotype. Ultimately, new cell- and tissue-engineering approaches to regenerative therapies will only be successful if the specific characteristics of the individual tissues and their context in the function of the whole organ, are taken into consideration. PMID: 22686699 [PubMed - indexed for MEDLINE]
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Related Articles [Promotion effect of notochordal cells conditioned medium on proliferation and differentiation of bone marrow mesenchymal stem cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 May;26(5):601-6 Authors: Ma K, Shao Z, Wang B, Zhang Y, Yang S, Liu T Abstract OBJECTIVE: To investigate the effect of notochordal cells (NCs) conditioned medium (NCCM) on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: NCs and BMSCs were isolated from the thoracolumbar intervertebral disc and the femurs of 4-week-old Japanese white rabbits, respectively. NCs were cultured with DMEM/F12 medium containing 15% FBS for 5 days to prepare NCCM. The experiment consisted of 2 groups: BMSCs were cultured with NCCM in experimental group and with DMEM/F12 medium containing 15% FBS in control group. The proliferation of BMSCs was assessed by cell counting kit-8 at 1, 3, 5, 7, 9, and 14 days after culture; the expression of proteoglycan and collagen type II were determined by immunofluorescence and real-time fluorescent quantitative PCR at 7 and 14 days after culture. RESULTS: NCs and BMSCs were successfully isloated. At 5, 7, 9, and 14 days, the number of BMSCs in the experimental group was significantly more than those in the control group (P < 0.05). At 7 and 14 days, there was no expression or less expression of proteoglycan and collagen type II in the control group; however, there was a lot of expression of proteoglycan and collagen type II in the experimental group, and the expressions were higher at 14 days than at 7 days. At 7 and 14 days after culture, the mRNA expressions of proteoglycan and collagen type II were significantly higher in the experimental group than in the control group (P < 0.05), and at 14 days than at 7 days in the experimental group (P < 0.05). CONCLUSION: NCCM can promote the proliferation and the differentiation of BMSCs into chondroyte-like cells, which provides the basis for NCs and BMSCs as seed cells in the treatment of degenerative disc disease. PMID: 22702058 [PubMed - indexed for MEDLINE]
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Related Articles [Cell therapy in treating age-related intervertebral disc pathology]. Adv Gerontol. 2012;25(1):105-11 Authors: Anisimov SV Abstract Pathology of the spine significantly affects the quality of life and is associated with a high rate of disability. Prevalence of the pathology of the spine increases with advanced age. It may have various causes and is associated with a wide spectrum of significant and variable symptoms. Low back pain (back pain) is the most common syndrome. It is mostly caused by the degeneration of the intervertebral discs, associated with unspecific and irreversible structural alterations in the latter. Surgical treatment of degenerative disc disease (DDD) is symptomatic, thus requiring a development of alternative approaches. In the current review, the principles of regenerative cellular therapy and cell replacement therapy of DDD are analyzed. Various cell types utilized as cellular therapy substrates are compared. PMID: 22708454 [PubMed - indexed for MEDLINE]
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Related Articles New treatments and imaging strategies in degenerative disease of the intervertebral disks. Radiology. 2012 Jul;264(1):6-19 Authors: Lotz JC, Haughton V, Boden SD, An HS, Kang JD, Masuda K, Freemont A, Berven S, Sengupta DK, Tanenbaum L, Maurer P, Ranganathan A, Alavi A, Marinelli NL Abstract Magnetic resonance (MR) imaging in patients with persistent low back pain and sciatica effectively demonstrates spine anatomy and the relationship of nerve roots and intervertebral disks. Except in cases with nerve root compression, disk extrusion, or central stenosis, conventional anatomic MR images do not help distinguish effectively between painful and nonpainful degenerating disks. Hypoxia, inflammation, innervation, accelerated catabolism, and reduced water and glycosaminoglycan content characterize degenerated disks, the extent of which may distinguish nonpainful from painful ones. Applied to the spine, "functional" imaging techniques such as MR spectroscopy, T1ρ calculation, T2 relaxation time measurement, diffusion quantitative imaging, and radio nucleotide imaging provide measurements of some of these degenerative features. Novel minimally invasive therapies, with injected growth factors or genetic materials, target these processes in the disk and effectively reverse degeneration in controlled laboratory conditions. Functional imaging has applications in clinical trials to evaluate the efficacy of these therapies and eventually to select patients for treatment. This report summarizes the biochemical processes in disk degeneration, the application of advanced disk imaging techniques, and the novel biologic therapies that presently have the most clinical promise. PMID: 22723559 [PubMed - indexed for MEDLINE]
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Related Articles Percutaneous image-guided delivery for the transplantation of mesenchymal stem cells in the setting of degenerated intervertebral discs. J Vasc Interv Radiol. 2012 Aug;23(8):1084-1088.e6 Authors: Prologo JD, Pirasteh A, Tenley N, Yuan L, Corn D, Hart D, Love Z, Lazarus HM, Lee Z Abstract PURPOSE: The delivery of mesenchymal stem cells (MSCs) to their site of action has remained a technical hurdle for clinical researchers in the expanding field of stem cell-based therapy. The purpose of this study was to test the feasibility of percutaneous image-guided needle delivery of bone marrow-derived human MSCs (hMSCs) to degenerated intervertebral discs (IVDs) in a preclinical model and to assess the containment of these cells within the IVDs. MATERIALS AND METHODS: Degeneration was induced in the lumbar IVDs of four 28-35-kg female pigs. Approximately 100,000 iodine-124 2'-fluoro-2'-deoxy-1β-D-arabinofuranosyl-5-iodouracil-labeled hMSCs were delivered under fluoroscopic guidance to one of the affected discs in each of the animals. The remaining levels served as internal controls. The animals were imaged by computed tomography (CT) and positron emission tomography (PET) immediately after delivery and 3 days after the procedure. Fifteen days after transplantation, immunohistochemical staining was performed on harvested discs to confirm the presence of delivered hMSCs. RESULTS: After refinement of the technique, PET-CT images on the day of cell transplantation showed initial deposition of the delivered radiolabeled MSCs to the IVD. An additional PET-CT study obtained 3 days later confirmed persistence and containment of activity in the IVD. Findings of histologic evaluation for the presence of human Alu sequences were positive in the treated discs and negative in the controls. CONCLUSIONS: Image-guided needle delivery of MSCs for treatment of degenerated IVDs is feasible as demonstrated in this preclinical model. Trials of this minimally invasive technique in humans are warranted. PMID: 22739647 [PubMed - indexed for MEDLINE]
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Related Articles The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus. Biomaterials. 2012 Nov;33(33):8256-64 Authors: Chen WH, Zeng R, Lo WC, Tina Chen SY, Lai TY, Williams DF, Deng WP Abstract Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations. PMID: 22938762 [PubMed - indexed for MEDLINE]
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Related Articles The presence of local mesenchymal progenitor cells in human degenerated intervertebral discs and possibilities to influence these in vitro: a descriptive study in humans. Stem Cells Dev. 2013 Mar 1;22(5):804-14 Authors: Brisby H, Papadimitriou N, Brantsing C, Bergh P, Lindahl A, Barreto Henriksson H Abstract Low back pain is common and degenerated discs (DDs) are believed to be a major cause. In non-degenerated intervertebral discs (IVDs) presence of stem/progenitor cells was recently reported in different mammals (rabbit, rat, pig). Understanding processes of disc degeneration and regenerative mechanisms within DDs is important. The aim of the study was to examine the presence of local stem/progenitor cells in human DDs and if these cell populations could respond to paracrine stimulation in vitro. Tissue biopsies from the IVD region (L3-S1) were collected from 15 patients, age 34-69 years, undergoing surgery (spinal fusion) and mesenchymal stem cells (MSCs) (iliac crest) from 2 donors. Non-DD cells were collected from 1 donor (scoliosis) and chordoma tissue was obtained from (positive control, stem cell markers) 2 donors. The IVD biopsies were investigated for gene and protein expression of: OCT3/4, CD105, CD90, STRO-1, and NOTCH1. DD cell cultures (pellet mass) were performed with conditioned media from MSCs and non-degenerated IVD cells. Pellets were investigated after 7, 14, 28 days for the same stem cell markers as above. Gene expression of OCT3/4 and STRO-1 was detected in 13/15 patient samples, CD105 in 14/15 samples, and CD90 and NOTCH1 were detected 15/15 samples. Immunohistochemistry analysis supported findings on the protein level, in cells sparsely distributed in DDs tissues. DDs cell cultures displayed more undifferentiated appearance with increased expression of CD105, CD90, STRO-1, OCT3/4, NOTCH1, and JAGGED1, which was observed when cultured in conditioned cell culture media from MSCs compared to cell cultures cultured with conditioned media from non-DD cells. Expression of OCT3/4 (multipotency marker) and NOTCH1 (regulator of cell fate), MSC-markers, CD105, CD90, and STRO-1, indicate that primitive cell populations are present within DDs. Furthermore, the possibility to influence cells from DDs by paracrine signaling /soluble factors from MSCs and from nondegenerated IVD cells was observed in vitro indicating that repair processes within human DDs may be stimulated. PMID: 23025667 [PubMed - indexed for MEDLINE]
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Related Articles Epidural abscess and cauda equina syndrome after percutaneous intradiscal therapy in degenerative lumbar disc disease. Spine J. 2012 Nov;12(11):e1-4 Authors: Subach BR, Copay AG, Martin MM, Schuler TC, DeWolfe DS Abstract BACKGROUND CONTEXT: Percutaneous intradiscal therapies are gaining popularity as a regenerative treatment option for spinal disc degeneration. The risks, benefits, and possible complications associated with such procedures have been poorly defined. As these procedures are performed with increasing frequency, the likelihood that clinicians will be faced with significant complications also increases. PURPOSE: The purpose of this study is to describe a significant complication of a percutaneous intradiscal bone marrow and adipose tissue transplantation for symptomatic lumbar disc degeneration. STUDY DESIGN: The study design is a case report. METHODS: Two weeks after an injection of adipose cells, bone marrow aspirate and plasma into his L3-L4 and L5-S1 lumbar discs, a 64-year-old patient presented to the emergency room with cauda equina syndrome, fever, and back pain. Magnetic resonance imaging diagnosed L3-L4 disc extrusion, discitis with osteomyelitis, and epidural abscess, resulting in emergency decompressive surgery. An epidural abscess was drained, extruded disc material was removed, and cultures obtained. Five days later, once afebrile on antibiotics, he underwent a definitive interbody arthrodesis and stabilization. RESULTS: Cauda equina syndrome resolved, osteomyelitis (methicillin-resistant Staphylococcus epidermidis) was treated, and instrumented arthrodesis stabilized the involved segment. CONCLUSIONS: Complications associated with the intradiscal injection of agents, such as stem cells, fibrin glue, adipose tissue, or bone marrow, have been poorly defined. Given the nature of the degenerating disc, serious adverse events, including discitis, osteomyelitis, and extrusion of disc contents, may occur. PMID: 23131581 [PubMed - indexed for MEDLINE]
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Related Articles Regenerative potential of decellularized porcine nucleus pulposus hydrogel scaffolds: stem cell differentiation, matrix remodeling, and biocompatibility studies. Tissue Eng Part A. 2013 Apr;19(7-8):952-66 Authors: Mercuri JJ, Patnaik S, Dion G, Gill SS, Liao J, Simionescu DT Abstract Nucleus pulposus (NP) tissue regeneration has been proposed as an early stage interventional therapy to combat intervertebral disc degeneration. We have previously reported on the development and characterization of a novel biomimetic acellular porcine NP (APNP) hydrogel. Herein, we aimed to evaluate this material for use as a suitable scaffold for NP tissue regeneration. Human-adipose-derived stem cells (hADSCs) were cultured for 14 days on APNP hydrogels in chemically defined differentiation media and were analyzed for an NP-cell-like mRNA expression profile, evidence of hydrogel remodeling including hydrogel contraction measurements, extracellular matrix production, and compressive dynamic mechanical properties. The innate capacity of the hydrogel itself to induce stem cell differentiation was also examined via culture in media lacking soluble differentiation factors. Additionally, the in vivo biocompatibility of non-crosslinked and ethyldimethylaminopropyl carbodiimide/N-hydroxysuccinimide and pentagalloyl glucose crosslinked hydrogels was evaluated in a rat subdermal model. Results indicated that hADSCs expressed putative NP-cell-positive gene transcript markers when cultured on APNP hydrogels. Additionally, glycosaminoglycan and collagen content of hADSC-seeded hydrogels was significantly greater than nonseeded controls and cell-seeded hydrogels exhibited evidence of contraction and tissue inhibitors of metalloproteinase-1 production. The dynamic mechanical properties of the hADSC-seeded hydrogels increased with time in culture in comparison to noncell-seeded controls and approached values reported for native NP tissue. Immunohistochemical analysis of explants illustrated the presence of mononuclear cells, including macrophages and fibroblasts, as well as blood vessel infiltration and collagen deposition within the implant interstices after 4 weeks of implantation. Taken together, these results suggest that APNP hydrogels, in concert with autologous ADSCs, may serve as a suitable scaffold for NP tissue regeneration. PMID: 23140227 [PubMed - indexed for MEDLINE]
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Related Articles The role of Osteocel Plus as a fusion substrate in minimally invasive instrumented transforaminal lumbar interbody fusion. Clin Neurol Neurosurg. 2013 Jul;115(7):991-4 Authors: Ammerman JM, Libricz J, Ammerman MD Abstract BACKGROUND: Instrumented lumbar fusion has become an accepted and effective surgical technique used to address a wide variety of conditions of the lumbar spine. Iliac crest autograft remains the gold standard with regards to bony fusion substrate. Unfortunately there are significant potential disadvantages associated with autograft harvest, including pain, infection, iatrogenic fracture and bleeding. Osteocel Plus (OC+) is an allograft cellular bone matrix containing mesenchymal stem cells (MSCs) and osteoprogenitor cells combined with DBM and cancellous bone. OC+ is designed to mimic the osteobiologic profile of human autograft bone, thereby eliminating the risks of autograft harvest. METHODS: A retrospective chart review was conducted to identify all patients who had undergone a MITLIF with OC+ for degenerative lumbar conditions. Patient demographics including age, sex, history of risk factors for nonunion including: osteoporosis documented on DEXA scanning, diabetes mellitus, smoking or steroid use were examined and recorded. Successful arthrodesis was judged based on post-operative X-ray imaging. RESULTS: 23 patients at 26 spinal levels underwent a MITLIF with OC+. Twenty-one patients (91.3%) and 24 levels (92.3%) went on to achieve radiographic evidence of solid bony arthrodesis by 12 months post-op. Six patients (26%) demonstrated clear evidence of early interbody bone growth within 6 months of surgery. CONCLUSION: OC+ results in robust and reproducible lumbar interbody fusion, in both young and older patients. PMID: 23182179 [PubMed - indexed for MEDLINE]
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Related Articles [Time differences of bone marrow mesenchymal stem cells and nucleus pulposus-like cells in a non contact co-culture system]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Nov;26(11):1369-74 Authors: Chen L, Hu X, Fu L, Ye J Abstract OBJECTIVE: To investigate time differences in directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells (NPCs) in a non contact co-culture system so as to search for the best time for transplantation in vivo. METHODS: Six New Zealand white rabbits (aged 6 weeks, weighing 1.5-2.0 kg) were selected. BMSCs were collected and cultured for immunocytochemistry identification of CD34, CD44, CD45, and CD90; NPCs were isolated and identified immunocytochemically by RT-PCR. The 2nd passage BMSCs and the primary NPCs were co-cultured in a non contact co-culture system. The cell morphological changes were observed and the cell growth curves were made at 1, 3, and 5 passages after co-culture. The expressions of the aggrecan and collagen type II genes were detected by RT-PCR in BMSCs at 5, 10, and 15 days after co-culture; the expressions of the aggrecan and collagen type II proteins were detected by Western blot at 5, 10, 15, 20, 25, and 30 days after co-culture. RESULTS: The expressions of CD44 and CD90 were positive, CD34 and CD45 were negative in BMSCs. The expressions of the collagen type II and aggrecan were positive in NPCs. At 2 weeks after co-culture, the morphology of BMSCs changed obviously, the cells were polygonal and irregular shape. The cell growth rate showed no difference within 3 passages, but decreased obviously after 3 passages. RT-PCR showed that the expressions of collagen type II and aggrecan genes at 10 and 15 days were significantly higher than those at 5 days (P < 0.05), no significant difference was found between at 10 days and at 15 days (P > 0.05). Western blot showed that the expressions of collagen type II and aggrecan proteins gradually increased with time, and there was significant difference within 5, 10, and 15 days (P < 0.05), but no significant difference was found after 15 days of co-culture (P > 0.05). CONCLUSION: In a non contact co-culture system, BMSCs can differentiate into the NPCs. The expression of collagen type II and aggrecan can reach a stable level at 15 days after co-culture, and it is the suitable time for transplantation in vivo. PMID: 23230675 [PubMed - indexed for MEDLINE]
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Related Articles Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun. 2012;3:1264 Authors: Sakai D, Nakamura Y, Nakai T, Mishima T, Kato S, Grad S, Alini M, Risbud MV, Chan D, Cheah KS, Yamamura K, Masuda K, Okano H, Ando K, Mochida J Abstract Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard. PMID: 23232394 [PubMed - indexed for MEDLINE]
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Related Articles Molecular basis of intervertebral disc degeneration. Adv Exp Med Biol. 2012;760:114-33 Authors: Gopal D, Ho AL, Shah A, Chi JH Abstract Intervertebral disc (IVD) degeneration is a disease of the discs connecting adjoining vertebrae in which structural damage leads to degeneration of the disc and surrounding area. Degeneration of the disc is considered to be a normal process of aging, but can accelerate faster than expected or be precipitated by other factors. The scientific community has come a long way in understanding the biological basis and interpreting the lifestyle implications of IVD degeneration. Of all the diseases of the intervertebral disc, degeneration is the most common and has earned much attention due to its diversity in presentation and potential multiorgan involvement. We will provide a brief overview of the anatomic, cellular, and molecular structure of the IVD, and delve into the cellular and molecular pathophysiology surrounding IVD degeneration. We will then highlight some of the newest developments in stem cell, protein, and genetic therapy for IVD degeneration. PMID: 23281517 [PubMed - indexed for MEDLINE]
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Related Articles Clinical efficacy of stem cell mediated osteogenesis and bioceramics for bone tissue engineering. Adv Exp Med Biol. 2012;760:174-87 Authors: Neman J, Hambrecht A, Cadry C, Goodarzi A, Youssefzadeh J, Chen MY, Jandial R Abstract Lower back pain is a common disorder that often requires bony spinal fusion for long-term relief. Current arthrodesis procedures use bone grafts from autogenous bone, allogenic backed bone or synthetic materials. Autogenous bone grafts can result in donor site morbidity and pain at the donor site, while allogenic backed bone and synthetic materials have variable effectiveness. Given these limitations, researchers have focused on new treatments that will allow for safe and successful bone repair and regeneration. Mesenchymal stem cells (MSCs) have received attention for their ability to differentiate into osteoblasts, cells that synthesize the extracellular matrix and regulate matrix mineralization. Successful bone regeneration requires three elements: MSCs that serve as osteoblastic progenitors, osteoinductive growth factors and their pathways that promote development and differentiation of the cells as well as an osteoconductive scaffold that allows for the formation of a vascular network. Future treatments should strive to combine mesenchymal stem cells, cell-seeded scaffolds and gene therapy to optimize the efficiency and safety of tissue repair and bone regeneration. PMID: 23281520 [PubMed - indexed for MEDLINE]
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Related Articles Clinico-biomechanical issues and dynamic stabilization when considering stem cell treatment for degenerative disc disease. Transplantation. 2013 Jan 27;95(2):e1 Authors: Clemente JM, Clemente JV PMID: 23325008 [PubMed - indexed for MEDLINE]
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Related Articles The effects of microenvironment in mesenchymal stem cell-based regeneration of intervertebral disc. Spine J. 2013 Mar;13(3):352-62 Authors: Huang YC, Leung VY, Lu WW, Luk KD Abstract BACKGROUND CONTEXT: Recent studies have demonstrated new therapeutic strategy using transplantation of mesenchymal stem cells (MSCs), especially bone marrow-derived MSCs (BM-MSCs), to preserve intervertebral disc (IVD) structure and functions. It is important to understand whether and how the MSCs survive and thrive in the hostile microenvironment of the degenerated IVD. Therefore, this review majorly examines how resident disc cells, hypoxia, low nutrition, acidic pH, mechanical loading, endogenous proteinases, and cytokines regulate the behavior of the exogenous MSCs. PURPOSE: To review and summarize the effect of the microenvironment in biological characteristics of BM-MSCs for IVD regeneration; the presence of endogenous stem cells and the state of the art in the use of BM-MSCs to regenerate the IVD in vivo were also discussed. STUDY DESIGN: Literature review. METHODS: MEDLINE electronic database was used to search for articles concerning stem/progenitor cell isolation from the IVD, regulation of the components of microenvironment for MSCs, and MSC-based therapy for IVD degeneration. The search was limited to English language. RESULTS: Stem cells are probably resident in the disc, but exogenous stem cells, especially BM-MSCs, are currently the most popular graft cells for IVD regeneration. The endogenous disc cells and the biochemical and biophysical components in the degenerating disc present a complicated microenvironment to regulate the transplanted BM-MSCs. Although MSCs regenerate the mildly degenerative disc effectively in the experimental and clinical trials, many underlying questions are in need of further investigation. CONCLUSIONS: There has been a dramatic improvement in the understanding of potential MSC-based therapy for IVD regeneration. The use of MSCs for IVD degeneration is still at the stage of preclinical and Phase 1 studies. The effects of the disc microenvironment in MSCs survival and function should be closely studied for transferring MSC transplantation from bench to bedside successfully. PMID: 23340343 [PubMed - indexed for MEDLINE]
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Related Articles Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy. Spine J. 2013 Mar;13(3):273-83 Authors: Chan SC, Bürki A, Bonél HM, Benneker LM, Gantenbein-Ritter B Abstract BACKGROUND CONTEXT: Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE: To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN: Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS: Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS: A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS: By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols. PMID: 23353003 [PubMed - indexed for MEDLINE]
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Related Articles Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro. Biochem Biophys Res Commun. 2013 Mar 15;432(3):444-50 Authors: Fang Z, Yang Q, Luo W, Li GH, Xiao J, Li F, Xiong W Abstract Differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype under hypoxia has been proposed as a potential therapeutic approach for intervertebral disc degeneration. However, limited cell viability under hypoxic conditions has restricted MSC differentiation capacity and thus restricted its clinical application. In this study, we genetically modified MSCs with an anti-apoptotic GFP-Bcl-2 gene and evaluated cell survival and functional improvement under hypoxia in vitro. Rat bone marrow MSCs were transfected by lentiviral vectors with the GFP-Bcl-2 gene (GFP-Bcl-2-MSCs). Cell proliferation and apoptosis were assessed, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities. In addition, Alcian blue staining was used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. We found that the Bcl-2 gene protected MSCs against apoptosis. We also observed that Bcl-2 over-expression reduced apoptosis by 40.61% in non-transfected MSCs and 38.43% in vector-MSCs to 18.33% in Bcl-2-MSCs. At 3days, the number of viable Bcl-2-MSCs was approximately two times higher than the number of MSCs or vector-MSCs under hypoxic conditions. RT-PCR showed higher expression of chondrocyte-related genes (Sox-9, aggrecan and type II collagen) in GFP-Bcl-2-MSCs cultured under hypoxia. The accumulation of proteoglycans in the pellet was 86% higher in GFP-Bcl-2-MSCs than in the control groups. Furthermore, the ratio of proteoglycans/collagen II in GFP-Bcl-2-MSCs was 6.2-fold higher compared to the MSC and vector-MSC groups, which denoted a nucleus pulposus-like differentiation phenotype. Our findings support the hypothesis that anti-apoptotic gene-modified MSCs can differentiate into cells with a nucleus pulposus-like phenotype in vitro, which may have value for the regeneration of intervertebral discs using cell transplantation therapy. PMID: 23416353 [PubMed - indexed for MEDLINE]
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Related Articles Novel cartilage-derived biomimetic scaffold for human nucleus pulposus regeneration: a promising therapeutic strategy for symptomatic degenerative disc diseases. Orthop Surg. 2013 Feb;5(1):60-3 Authors: Yang Q, Zhao YH, Xia Q, Xu BS, Ma XL, Liu Y, Hu YC, Li HF, Miao J, Wang T, Ma JX, Sun XL Abstract Because current therapies have not always been successful and effective, the possibility of regenerating the nucleus pulposus (NP) through a tissue-engineered construct offers a novel therapeutic possibility for symptomatic degenerative disc diseases (DDDs). However, more research is necessary to identify the optimal scaffold, cell type and mixture of signal factors needed for NP regeneration. Numerous possible scaffolds for NP regeneration have been investigated; they have many shortcomings in common. Various biological scaffolds derived from decellularized tissue and organs have been successfully used in tissue engineering and received approval for use in humans. Regretfully, harvesting of human NP is difficult and only small amounts can be obtained. The macromolecules of cartilage, which include collagen and proteoglycan aggrecan, are similar to those of the extracellular matrix of immature NP. Recent studies have shown that adipose-derived stem cells (ADSC) can be induced to develop NP-like phenotypes when stimulated by appropriate signals. We thus reasonably postulated that an ideal NP scaffold for tissue engineering could be fabricated from decellularized cartilage matrix (DCM). Furthermore, a combination of ADSCs and DCM-derived biomimetic scaffolds would be advantageous in NP tissue engineering and, in the long run, could become an effective treatment option for symptomatic DDD. PMID: 23420750 [PubMed - indexed for MEDLINE]
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Related Articles An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints. J Orthop Res. 2013 Jun;31(6):831-7 Authors: Vo N, Niedernhofer LJ, Nasto LA, Jacobs L, Robbins PD, Kang J, Evans CH Abstract As human lifespan increases so does the incidence of age-associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint-related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age-related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time-dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age-dependent degenerative joint disease, and highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases. PMID: 23483579 [PubMed - indexed for MEDLINE]
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Related Articles Intervertebral disc regeneration: from the degenerative cascade to molecular therapy and tissue engineering. J Tissue Eng Regen Med. 2013 Mar 20; Authors: Vadalà G, Russo F, Di Martino A, Denaro V Abstract Low back pain is one of the major health problems in industrialized countries, as a leading source of disability in the working population. Intervertebral disc degeneration has been identified as its main cause, being a progressive process mainly characterized by alteration of extracellular matrix composition and water content. Many factors are involved in the degenerative cascade, such as anabolism/catabolism imbalance, reduction of nutrition supply and progressive cell loss. Currently available treatments are symptomatic, and surgical procedures consisting of disc removal are often necessary. Recent advances in our understanding of intervertebral disc biology led to an increased interest in the development of novel biological treatments aimed at disc regeneration. Growth factors, gene therapy, stem cell transplantation and biomaterials-based tissue engineering might support intervertebral disc regeneration by overcoming the limitation of the self-renewal mechanism. The aim of this paper is to overview the literature discussing the current status of our knowledge from the degenerative cascade of the intervertebral disc to the latest molecular, cell-based therapies and tissue-engineering strategies for disc regeneration. Copyright © 2013 John Wiley & Sons, Ltd. PMID: 23512973 [PubMed - as supplied by publisher]
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Related Articles TGF-β1 induces a nucleus pulposus-like phenotype in Notch 1 knockdown rabbit bone marrow mesenchymal stem cells. Cell Biol Int. 2013 Aug;37(8):820-5 Authors: Morigele M, Shao Z, Zhang Z, Kaige M, Zhang Y, Qiang W, Yang S Abstract We have investigated the effects of Notch1 knockdown and treatment with TGF-β1 on the regulation of the directional differentiation of mesenchymal stem cells (MSCs). MSCs were isolated from the femur bone of New Zealand rabbits and purified by using discontinuous gradient density centrifugation. Notch1 siRNAs were designed, synthesised and transiently transfected into these MSCs, and treated with TGF-β1. The MSCs were examined for morphology, stained with toluidine blue for proteoglycan analysis and gene and protein expression were measured with qRT-PCR and Western blotting respectively. They had an ellipse or fusiform shape and gathered in nests or swirls after being cultured for 7 days. Notch1 expression was knocked down with Notch1 siRNA (the silence rate was 47%; P < 0.001). After knockdown and TGF-β1 treatment, MSCs expressed more proteoglycan (P < 0.01), and higher levels of collagen II mRNA and protein than control cells (P < 0.001). Thus knockdown of Notch1 expression in MSCs may be useful in the treatment of intervertebral disc degeneration. PMID: 23554118 [PubMed - indexed for MEDLINE]
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Related Articles Potential of co-culture of nucleus pulposus mesenchymal stem cells and nucleus pulposus cells in hyperosmotic microenvironment for intervertebral disc regeneration. Cell Biol Int. 2013 Aug;37(8):826-34 Authors: Tao YQ, Liang CZ, Li H, Zhang YJ, Li FC, Chen G, Chen QX Abstract Nucleus pulposus mesenchymal stem cells (NPMSCs) are a potential cell source for intervertebral disc (IVD) regeneration, but little is known about their response to IVD-like high osmolarity (400 mOsm). This study was to investigate the viability, proliferation and protein biosynthesis of nucleus pulposus cells (NPCs), NPMSCs and co-cultured NPMSCs-NPCs under IVD-like high osmolarity conditions. NPCs and NPMSCs were isolated and cultured under standard and IVD-like high osmolarity conditions for 1 or 2 weeks. Cell viability was measured by annexin V-FITC and PI staining, and cell proliferation measured by MTT assay. The expression of SOX-9, aggrecan and collagen-II was measured by RT-PCR and Western blot analyses. IVD-like high osmolarity condition slightly inhibited cell viability and decreased the expression of SOX-9, aggrecan and collagen-II at the mRNA and protein levels in all groups compared with standard condition. NPMSCs could tolerate IVD-like high osmolarity, and NPCs-NPMSCs co-culture increased cell proliferation and the expression of SOX-9, aggrecan and collagen-II under both culture conditions, suggesting that co-culture of NPMSCs-NPCs has potential application for IVD regeneration. PMID: 23554141 [PubMed - indexed for MEDLINE]
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Related Articles [Regeneration strategies of intervertebral disc]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Feb;27(2):227-32 Authors: Fu W, Xiang Z Abstract OBJECTIVE: To review the research progress of the seed cells, scaffolds, growth factors, and the prospects for clinical application of the intervertebral disc regeneration. METHODS: The recent literature concerning the regeneration strategies and tissue engineering for treatment of degenerative intervertebral disc disease was extensively reviewed and summarized. RESULTS: Seed cells based on mesenchymal stem cells (MSCs) and multiple-designed biomimetic scaffolds are the hot topic in the field of intervertebral disc regeneration. It needs to be further investigated how to effectively combine the interactions of seed cells, scaffolds, and growth factors and to play their regulation function. CONCLUSION: The biological regeneration of intervertebral disc would have a very broad prospects for clinical application in future. PMID: 23596694 [PubMed - indexed for MEDLINE]
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Related Articles Human umbilical cord blood-derived mesenchymal stem cells in the cultured rabbit intervertebral disc: a novel cell source for disc repair. Am J Phys Med Rehabil. 2013 May;92(5):420-9 Authors: Anderson DG, Markova D, An HS, Chee A, Enomoto-Iwamoto M, Markov V, Saitta B, Shi P, Gupta C, Zhang Y Abstract OBJECTIVE: Back pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture. DESIGN: This study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant tracer specific for live cells. Human type II collagen gene expression (from the graft) was assessed by polymerase chain reaction. RESULTS: We have shown that hUCB-MSCs cultured in micromass are stained blue with Alcian blue dye, which suggests that proteoglycan-rich extracellular matrix is produced. In the cultured rabbit IVD explants, hUCB-MSCs survived for at least 4 wks and expressed the human type II collagen gene, suggesting that the injected hUCB-MSCs are differentiating into a chondrocyte-like lineage. CONCLUSIONS: This study demonstrates the abiity of hUBC-MSCs to survive and assume a chondrocyte-like phenotype when injected into the rabbit IVD. These data support the potential for hUBC-MSCs as a cell source for disc repair. Further measures of the host response to the injection and studies in animal models are needed before trials in humans. PMID: 23598901 [PubMed - indexed for MEDLINE]
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Related Articles Molecular and genetic advances in the regeneration of the intervertebral disc. Surg Neurol Int. 2013;4(Suppl 2):S94-S105 Authors: Maerz T, Herkowitz H, Baker K Abstract BACKGROUND: Owing to the debilitating nature of degenerative disc disease (DDD) and other spine pathologies, significant research has been performed with the goal of healing or regenerating the intervertebral disc (IVD). Structural complexity, coupled with low vascularity and cellularity, make IVD regeneration an extremely challenging task. METHODS: Tissue engineering-based strategies utilize three components to enhance tissue regeneration; scaffold materials to guide cell growth, biomolecules to enhance cell migration and differentiation, and cells (autologous, or allogeneic) to initiate the process of tissue formation. Significant advances in IVD regeneration have been made utilizing these tissue engineering strategies. RESULTS: The current literature demonstrates that members of the transforming growth factor beta (TGF-β) superfamily are efficacious in the regeneration of an anabolic response in the IVD and to facilitate chondrogenic differentiation. Gene therapy, though thwarted by safety concerns and the risk of ectopic transfection, has significant potential for a targeted and sustained regenerative response. Stem cells in combination with injectable, biocompatible, and biodegradable scaffolds in the form of hydrogels can differentiate into de novo IVD tissue and facilitate regeneration of the existing matrix. Therapies that address both anabolism and the inherent catabolic state of the IVD using either direct inhibitors or broad-spectrum inhibitors show extensive promise. CONCLUSION: This review article summarizes the genetic and molecular advances that promise to play an integral role in the development of new strategies to combat DDD and promote healing of injured discs. PMID: 23646279 [PubMed]
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Related Articles Fullerol nanoparticles suppress inflammatory response and adipogenesis of vertebral bone marrow stromal cells--a potential novel treatment for intervertebral disc degeneration. Spine J. 2013 Nov;13(11):1571-80 Authors: Liu Q, Jin L, Shen FH, Balian G, Li XJ Abstract BACKGROUND CONTEXT: Intervertebral disc degeneration, leading to chronic back pain, is a major health problem in western societies. Vertebral bone marrow has been considered to play an important role in nutrition supply and metabolic exchange for discs. Vertebral bone marrow lesions, including fatty marrow replacement and inflammatory edema, noted on magnetic resonance imaging were first described in 1988. PURPOSE: To investigate the potential of a free radical scavenger, fullerol nanoparticles, to prevent vertebral bone marrow lesion and prevent disc degeneration by inhibiting inflammation and adipogenic differentiation of vertebral bone marrow stromal cells (vBMSCs). STUDY DESIGN/SETTING: Fullerol nanoparticle solutions were prepared to test their in vitro suppression effects on mouse vBMSC inflammation and adipogenic differentiation compared with non-fullerol-treated groups. METHODS: With or without fullerol treatment, vBMSCs from Swiss Webster mice were incubated with 10 ng/mL interleukin-1 β (IL-1 β). The intracellular reactive oxygen species (ROS) were measured with fluorescence staining and flow cytometry. In addition, vBMSCs were cultured with adipogenic medium (AM) with or without fullerol. Gene and protein expressions were evaluated by real-time polymerase chain reaction and histologic methods. RESULTS: Fluorescence staining and flow cytometry results showed that IL-1 β markedly increased intracellular ROS level, which could be prevented by fullerol administration. Fullerol also decreased the basal ROS level to 77%. Cellular production of matrix metalloproteinase (MMP)-1, 3, and 13 and tumor necrosis factor alpha (TNF-α) induced by IL-1 β was suppressed by fullerol treatment. Furthermore, adipogenic differentiation of the vBMSCs was retarded markedly by fullerol as revealed by less lipid droplets in the fullerol treatment group compared with the adipogenic group. The expression of adipogenic genes PPARγ and aP2 was highly elevated with AM but decreased on fullerol administration. CONCLUSIONS: These results suggest that fullerol prevents the catabolic activity of vBMSCs under inflammatory stimulus by decreasing the level of ROS, MMPs, and TNF-α. Also, fat formation in vBMSCs is prevented by fullerol nanoparticles, and, therefore, fullerol may warrant further in vivo investigation as an effective biological therapy for disc degeneration. PMID: 23669123 [PubMed - indexed for MEDLINE]
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Related Articles Photochemically crosslinked collagen annulus plug: a potential solution solving the leakage problem of cell-based therapies for disc degeneration. Acta Biomater. 2013 Sep;9(9):8128-39 Authors: Chik TK, Ma XY, Choy TH, Li YY, Diao HJ, Teng WK, Han SJ, Cheung KM, Chan BP Abstract Intra-disc injection of mesenchymal stem cells (MSCs) to treat disc degeneration may lead to unfavorable complications, particularly osteophyte formation. Development of an effective method to block the injection portal, prevent the leakage of injected cells and materials and, hence, prevent osteophyte formation is of the utmost importance before MSC-based therapies can be applied in a clinical setting. Here we seek to alleviate the cell leakage problem and the associated complication osteophyte formation by developing an injectable annulus plug to block the injection portal during intra-disc delivery. Specifically, we fabricated a needle-shaped collagen plug by photochemical crosslinking and successfully delivered it intra-discally, in association with MSCs in collagen microsphere carriers, using a custom-made delivery device. The mechanical performance of the plug and its effectiveness in reducing cell leakage were evaluated ex vivo under compression and in torsion push-out tests. The results demonstrate that the plug survived physiologically relevant loadings and significantly reduced leakage and enhanced retention of the injected materials. Finally, a pilot in vivo study in rabbits was conducted to evaluate the performance of the plug. Microcomputed tomography imaging and histology revealed that the plug significantly reduced osteophyte formation. This work suggests the potential of the annulus plug as an adjunct or annulus closure device for intra-disc delivery of cells and materials. PMID: 23751592 [PubMed - indexed for MEDLINE]
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Related Articles [Fabrication and analysis of a novel tissue engineered composite biphasic scaffold for annulus fibrosus and nucleus pulposus]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Apr;27(4):475-80 Authors: Xu H, Xu B, Yang Q, Li X, Ma X, Xia Q, Zhang C, Wu Y Abstract OBJECTIVE: To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. METHODS: Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. RESULTS: The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 +/- 88.25) microm pore diameter of the annulus fibrosus phase, 82.98% +/- 7.02% porosity and 621.53% +/- 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 +/- 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. CONCLUSION: Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering. PMID: 23757878 [PubMed - indexed for MEDLINE]
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Related Articles Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model. Spine (Phila Pa 1976). 2013 Oct 15;38(22):E1377-87 Authors: Furtwängler T, Chan SC, Bahrenberg G, Richards PJ, Gantenbein-Ritter B Abstract STUDY DESIGN: In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1. OBJECTIVE: This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options. SUMMARY OF BACKGROUND DATA: Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an intervertebral disc degeneration model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant. METHODS: Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4, and HTRA1 were injected at a dose of 10 μg/mL each. Phosphate-buffered saline was injected as a control. Discs were cultured for 8 days and loaded diurnally (days 1-4 with ≈0.4 MPa for 16 hr) and left under free swelling condition from days 4 to 8 to avoid expected artifacts because of dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, GAG content, total collagen content, relative gene expression, and histological investigation. RESULTS: The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 than the day 0 control discs. Disc height was decreased after injection with HTRA1 and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3. CONCLUSION: MMP-3, ADAMTS-4, and HTRA1 provoked neither visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height that positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may, therefore, be necessary to induce disc degeneration. PMID: 23778376 [PubMed - indexed for MEDLINE]
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Related Articles Analysis of molecular expression in adipose tissue-derived mesenchymal stem cells : prospects for use in the treatment of intervertebral disc degeneration. J Korean Neurosurg Soc. 2013 Apr;53(4):207-12 Authors: Jin ES, Min J, Jeon SR, Choi KH, Jeong JH Abstract OBJECTIVE: Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor β3 (TGF-β3) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-β3, and co-cultured ADMSCs treated with TGF-β3. METHODS: ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-β3, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitativeanalysis of gene expression. RESULTS: Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-β3-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-β3 showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-β3 alone. CONCLUSION: ADMSC treated with TGF-β3 may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo. PMID: 23826475 [PubMed]
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Related Articles Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells. Spine J. 2013 Nov;13(11):1627-39 Authors: Peroglio M, Eglin D, Benneker LM, Alini M, Grad S Abstract BACKGROUND CONTEXT: The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE: To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN: In vitro and ex vivo whole-organ culture of hMSCs. METHODS: In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS: The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS: Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration. PMID: 23830827 [PubMed - indexed for MEDLINE]
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Related Articles [Research situation of stem cells transplantation for intervertebral disc degeneration]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 May;27(5):575-9 Authors: Wang F, Wu X, Wang Y, Bao J Abstract OBJECTIVE: To summarize the research situation of stem cells transplantation for intervertebral disc (IVD) degeneration.degeneration. METHODS: The original articles about stem cells transplantation for repair of IVD degeneration were extensively reviewed; the clinical applications, the mechanisms, and related factors to influence repair effect were analyzed; and obstaclesreviewed; the clinical applications, the mechanisms, and related factors to influence repair effect were analyzed; and obstacles RESULTS: Autogenic stem cells transplantation can repair IVD degeneration and effectively relieve the symptoms of low back and leg pain. Stem cells can differentiate into disc chondrocytes in the disc microenvironment, increase the production of various growth factors, and exert a trophic effect on disc cells. It is also evident that the transplanted stem cells can potentially protect disc cells from apoptosis and maintain an immune-privileged state in the IVD. Multiple factors such as tissue origin of stem cells, methods to pre-modulate the seeds, choice of injectable scaffolds, and even the severity of degeneration are closely related to the repair effects. To get a more efficient stem cell therapy, future researches are challenged to modulate the migration and distribution of stem cells in the IVD, avoid flow back, and better understand their ability to restore stemnness properties within the degenerative disc niche. CONCLUSION: Stem cells transplantation is proven to be a promising biological approach for repair of IVD degeneration. PMID: 23879095 [PubMed - indexed for MEDLINE]
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Related Articles Impact of direct cell co-cultures on human adipose-derived stromal cells and nucleus pulposus cells. J Orthop Res. 2013 Nov;31(11):1804-13 Authors: Sun Z, Liu ZH, Zhao XH, Sun L, Chen YF, Zhang WL, Gao Y, Zhang YZ, Wan ZY, Samartzis D, Wang HQ, Luo ZJ Abstract Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose-derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co-cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co-cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co-culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up-regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP-1, TGF-β1, IGF-1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up-regulated. Collectively, these results suggest that direct co-cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD. PMID: 23913869 [PubMed - indexed for MEDLINE]
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Related Articles Stemming the Degeneration: IVD Stem Cells and Stem Cell Regenerative Therapy for Degenerative Disc Disease. Adv Stem Cells. 2013;2013 Authors: Sivakamasundari V, Lufkin T Abstract The intervertebral disc (IVD) is immensely important for the integrity of vertebral column function. The highly specialized IVD functions to confer flexibility and tensile strength to the spine and endures various types of biomechanical force. Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain and includes the more severe degenerative lumbar scoliosis, disc herniation and spinal stenosis. DDD is a multifactorial disorder whereby an imbalance of anabolic and catabolic factors, or alterations to cellular composition, or biophysical stimuli and genetic background can all play a role in its genesis. However, our comprehension of IVD formation and theetiology of disc degeneration (DD) are far from being complete, hampering efforts to formulate appropriate therapies to tackle DD. Knowledge of the stem cells and various techniques to manipulate and direct them to particular fates have been promising in adopting a stem-cell based regenerative approach to DD. Moreover, new evidence on the residence of stem/progenitor cells within particular IVD niches has emerged holding promise for future therapeutic applications. Existing issues pertaining to current therapeutic approaches are also covered in this review. PMID: 23951558 [PubMed - as supplied by publisher]
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Related Articles Identification of novel nucleus pulposus markers: Interspecies variations and implications for cell-based therapiesfor intervertebral disc degeneration. Bone Joint Res. 2013;2(8):169-78 Authors: Rodrigues-Pinto R, Richardson SM, Hoyland JA Abstract Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc. Cite this article: Bone Joint Res 2013;2:169-78. PMID: 23958792 [PubMed]
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Related Articles Dual release of dexamethasone and TGF-β3 from polymeric microspheres for stem cell matrix accumulation in a rat disc degeneration model. Acta Biomater. 2013 Dec;9(12):9423-33 Authors: Liang CZ, Li H, Tao YQ, Peng LH, Gao JQ, Wu JJ, Li FC, Hua JM, Chen QX Abstract Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported that a new nanostructured three-dimensional poly(lactide-co-glycolide) (PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(l-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSC)-seeded PLGA microspheres into the rat intervertebral disc could regenerate the degenerated disc. Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix-associated gene expression were evaluated in normal controls (NCs) (without operations), a degeneration control (DC) group (with needle puncture, injected only with Dulbecco's modified Eagle's medium), a PLGA microspheres (PMs) treatment group (with needle puncture, PLGA microspheres only injection), and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSC injection) for a 24-week period. The results showed that at 24 weeks post-transplantation, the PM and PMA groups regained disc height values of ∼63% and 76% and MRI signal intensities of ∼47% and 76%, respectively, compared to the NC group. Biochemistry, immunohistochemistry and gene expression analysis also indicated the restoration of proteoglycan accumulation in the discs of the PM and PMA groups. However, there was almost no restoration of proteoglycan accumulation in the discs of the DC group compared with the PM and PMA groups. Taken together, these data suggest that ADSC-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat degenerative intervertebral disc. PMID: 23973308 [PubMed - indexed for MEDLINE]
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Related Articles The use of stem cells for the treatment of spinal surgical conditions. Curr Stem Cell Res Ther. 2013 Nov;8(6):456-63 Authors: Aftab S, Chimutengwende-Gordon M, Malik A, Lee R Abstract Spinal pathologies are a major burden on society and individuals. Recent years have seen a large number of studies dedicated to the use of stem cells in spinal surgery. This review focuses on recent advances and controversies regarding the applications of stem cells in spinal fusion surgery, spinal cord injury and intervertebral disc degeneration. There are significant concerns regarding the ethics and risks of stem cell use. Animal models do not always accurately depict the human condition. While a great deal has been achieved, successful translation into clinical practice is needed. However there is no doubt that stem cells have a major role to play in the future management of spinal conditions. PMID: 24016321 [PubMed - indexed for MEDLINE]
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Related Articles An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration. Biomaterials. 2013 Dec;34(37):9430-40 Authors: Frith JE, Cameron AR, Menzies DJ, Ghosh P, Whitehead DL, Gronthos S, Zannettino AC, Cooper-White JJ Abstract Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications. PMID: 24050877 [PubMed - indexed for MEDLINE]
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Related Articles MR monitoring of minimally invasive delivery of mesenchymal stem cells into the porcine intervertebral disc. PLoS One. 2013;8(9):e74658 Authors: Barczewska M, Wojtkiewicz J, Habich A, Janowski M, Adamiak Z, Holak P, Matyjasik H, Bulte JW, Maksymowicz W, Walczak P Abstract PURPOSE: Bone marrow stem cell therapy is a new, attractive therapeutic approach for treatment of intervertebral disc (IVD) degeneration; however, leakage and backflow of transplanted cells into the structures surrounding the disc may lead to the formation of undesirable osteophytes. The purpose of this study was to develop a technique for minimally invasive and accurate delivery of stem cells. METHODS: Porcine mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIO, Molday ION rhodamine) and first injected into the explanted swine lumbar IVD, followed by ex vivo 3T MRI. After having determined sufficient sensitivity, IVD degeneration was then induced in swine (n=3) by laser-evaporation. 3 x 10(6) SPIO-labeled cells embedded within hydrogel were injected in 2 doses using a transcutaneous cannula and an epidural anesthesia catheter. T2-weighted MR images were obtained at 3T before and immediately after cell infusion. Two weeks after injection, histological examination was performed for detection of transplanted cells. RESULTS: MSCs were efficiently labeled with Molday ION rhodamine. Cells could be readily detected in the injected vertebral tissue explants as distinct hypointensities with sufficient sensitivity. MR monitoring indicated that the MSCs were successfully delivered into the IVD in vivo, which was confirmed by iron-positive Prussian Blue staining of the tissue within the IVD. CONCLUSION: We have developed a technique for non-invasive monitoring of minimally invasive stem delivery into the IVD at 3T. By using a large animal model mimicking the anatomy of IVD in humans, the present results indicate that this procedure may be clinically feasible. PMID: 24058619 [PubMed - indexed for MEDLINE]
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Related Articles Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro. PLoS One. 2013;8(9):e75548 Authors: Chen J, Lee EJ, Jing L, Christoforou N, Leong KW, Setton LA Abstract A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD. PMID: 24086564 [PubMed - indexed for MEDLINE]
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Related Articles Utilization of stem cells in alginate for nucleus pulposus tissue engineering. Tissue Eng Part A. 2014 Mar;20(5-6):908-20 Authors: Wang H, Zhou Y, Huang B, Liu LT, Liu MH, Wang J, Li CQ, Zhang ZF, Chu TW, Xiong CJ Abstract In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, etc. evaluations, we found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs (p<0.05); additionally, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray (p>0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. All in all, our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases. PMID: 24102374 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell regeneration of degenerated intervertebral discs: current status (update). Curr Pain Headache Rep. 2013 Dec;17(12):377 Authors: Gilbert HT, Hoyland JA, Richardson SM Abstract Low back pain, strongly associated with intervertebral disc (IVD) degeneration, affects a large proportion of the population and has major social and economic costs. Current treatments remain inadequate, targeting the symptoms without addressing the underlying cause. As such, efforts are being directed towards development of therapies aimed at alleviating pain through the restoration of IVD function. The potential of cell-based therapies for the treatment of IVD degeneration are being actively explored, with an emphasis on cell/biomaterial tissue engineering. Adult mesenchymal stem cells, capable of differentiating down the discogenic lineage, have shown promise as a suitable cell source for IVD tissue engineering. However, a number of factors, (discussed in this review), remain to be addressed, including development of a differentiation protocol to produce the correct cell phenotype, identification of suitable biomaterials for cell delivery/implantation, and ensuring cell survival and correct function upon implantation into the degenerate IVD. PMID: 24234817 [PubMed - indexed for MEDLINE]
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Related Articles Stem cells in preclinical spine studies. Spine J. 2014 Mar 1;14(3):542-51 Authors: Werner BC, Li X, Shen FH Abstract BACKGROUND CONTEXT: The recent identification and characterization of mesenchymal stem cells have introduced a shift in the research focus for future technologies in spinal surgery to achieve spinal fusion and treat degenerative disc disease. Current and past techniques use allograft to replace diseased tissue or rely on host responses to recruit necessary cellular progenitors. Adult stem cells display long-term proliferation, efficient self-renewal, and multipotent differentiation. PURPOSE: This review will focus on two important applications of stem cells in spinal surgery: spine fusion and the management of degenerative disc disease. STUDY DESIGN: Review of the literature. METHODS: Relevant preclinical literature regarding stem cell sources, growth factors, scaffolds, and animal models for both osteogenesis and chondrogenesis will be reviewed, with an emphasis on those studies that focus on spine applications of these technologies. RESULTS: In both osteogenesis and chondrogenesis, adult stem cells derived from bone marrow or adipose show promise in preclinical studies. Various growth factors and scaffolds have also been shown to enhance the properties and eventual clinical potential of these cells. Although its utility in clinical applications has yet to be proven, gene therapy has also been shown to hold promise in preclinical studies. CONCLUSIONS: The future of spine surgery is constantly evolving, and the recent advancements in stem cell-based technologies for both spine fusion and the treatment of degenerative disc disease is promising and indicative that stem cells will undoubtedly play a major role clinically. It is likely that these stem cells, growth factors, and scaffolds will play a critical role in the future for replacing diseased tissue in disease processes such as degenerative disc disease and in enhancing host tissue to achieve more reliable spine fusion. PMID: 24246748 [PubMed - in process]
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Related Articles Cell sources for nucleus pulposus regeneration. Eur Spine J. 2014 Jun;23 Suppl 3:S364-74 Authors: Kregar Velikonja N, Urban J, Fröhlich M, Neidlinger-Wilke C, Kletsas D, Potocar U, Turner S, Roberts S Abstract PURPOSE: There is increasing interest in the development of cell therapy as a possible approach for the treatment of degenerative disc disease. To regenerate nucleus pulposus tissue, the cells must produce an appropriate proteoglycan-rich matrix, as this is essential for the functioning of the intervertebral disc. The natural environment within the disc is very challenging to implanted cells, particularly if they have been subcultured in normal laboratory conditions. The purpose of this work is to discuss parameters relevant to translating different proposed cell therapies of IVD into clinical use. RESULTS: Several sources of cells have been proposed, including nucleus pulposus cells, chondrocytes and mesenchymal stem cells derived from bone marrow or adipose tissue. There are some clinical trials and reports of attempts to regenerate nucleus pulposus utilising either autologous or allogenic cells. While the published results of clinical applications of these cell therapies do not indicate any safety issues, additional evidence will be needed to prove their long-term efficacy. CONCLUSION: This article discusses parameters relevant for successful translation of research on different cell sources into clinically applicable cell therapies: the influence of the intervertebral disc microenvironment on the cell phenotype, issues associated with cell culture and technical preparation of cell products, as well as discussing current regulatory requirements. There are advantages and disadvantages of each proposed cell type, but no strong evidence to favour any one particular cell source at the moment. PMID: 24297331 [PubMed - indexed for MEDLINE]
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Related Articles Delivering mesenchymal stem cells in collagen microsphere carriers to rabbit degenerative disc: reduced risk of osteophyte formation. Tissue Eng Part A. 2014 May;20(9-10):1379-91 Authors: Li YY, Diao HJ, Chik TK, Chow CT, An XM, Leung V, Cheung KM, Chan BP Abstract Mesenchymal stem cells (MSCs) have the potential to treat early intervertebral disc (IVD) degeneration. However, during intradiscal injection, the vast majority of cells leaked out even in the presence of hydrogel carrier. Recent evidence suggests that annulus puncture is associated with cell leakage and contributes to osteophyte formation, an undesirable side effect. This suggests the significance of developing appropriate carriers for intradiscal delivery of MSCs. We previously developed a collagen microencapsulation platform, which entraps MSCs in a solid microsphere consisting of collagen nanofiber meshwork. These solid yet porous microspheres support MSC attachment, survival, proliferation, migration, differentiation, and matrix remodeling. Here we hypothesize that intradiscal injection of MSCs in collagen microspheres will outperform that of MSCs in saline in terms of better functional outcomes and reduced side effects. Specifically, we induced disc degeneration in rabbits and then intradiscally injected autologous MSCs, either packaged within collagen microspheres or directly suspended in saline, into different disc levels. Functional outcomes including hydration index and disc height were monitored regularly until 6 months. Upon sacrifice, the involved discs were harvested for histological, biochemical, and biomechanical evaluations. MSCs in collagen microspheres showed advantage over MSCs in saline in better maintaining the dynamic mechanical behavior but similar performance in hydration and disc height maintenance and matrix composition. More importantly, upon examination of gross appearance, radiograph, and histology of IVD, delivering MSCs in collagen microspheres significantly reduced the risk of osteophyte formation as compared to that in saline. This work demonstrates the significance of using cell carriers during intradiscal injection of MSCs in treating disc degeneration. PMID: 24372278 [PubMed - indexed for MEDLINE]
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Related Articles Clinical challenges and opportunities of mesenchymal stem cells in musculoskeletal medicine. PM R. 2014 Jan;6(1):70-7 Authors: Centeno CJ Abstract The use of stem cells in orthopedics has been researched for many years, with robust animal data that show efficacy in cartilage healing, tendon repair, and intervertebral disk treatment. Early clinical data are also just starting to be published, and these results are encouraging. Safety data in large case series, some that lasted for many years, have also been published. The field of tissue engineering with stem cells in musculoskeletal impairments has the potential to reduce morbidity and improve clinical outcomes. The regulatory environment for this area of medicine is still developing. PMID: 24439149 [PubMed - indexed for MEDLINE]
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Related Articles Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System. Stem Cells Int. 2013;2013:326828 Authors: Bucher C, Gazdhar A, Benneker LM, Geiser T, Gantenbein-Ritter B Abstract Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration. PMID: 24454406 [PubMed]
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Related Articles Differentiation of menstrual blood-derived stem cells toward nucleus pulposus-like cells in a coculture system with nucleus pulposus cells. Spine (Phila Pa 1976). 2014 Apr 20;39(9):754-60 Authors: Hu X, Zhou Y, Zheng X, Tian N, Xu C, Wu W, Li F, Zhu S, Zheng Y, Xue E, Yu Y, Zhang X, Xu H Abstract STUDY DESIGN: Human stromal stem cells derived from menstrual blood (MenSCs) and nucleus pulposus (NP) cells were cocultured under normal or low oxygen (O2) condition. OBJECTIVE: To assess the differentiation capability of MenSCs toward nucleus pulposus cells under normal or low oxygen condition. SUMMARY OF BACKGROUND DATA: Given the proliferative capacity and pluripotentiality of mesenchymal stem cells, mesenchymal stem cells transplantation is thought to be a promising approach to managing intervertebral disc degeneration. METHODS: Using coculture plates with 0.4-μm pore size polyethylene terephthalate track-etched inserts, MenSCs and NP cells (1:1 ratio) were cocultured with cell-to-cell contact for 2 weeks in normal (20% O2) or low oxygen tension (2% O2), respectively. Extracellular matrix accumulation was quantified by dimethylmethylene blue assay, histological staining, and quantitative reverse-transcription polymerase chain reaction. Novel characteristic human NP markers cytokeratin-19 (KRT19), carbonic anhydrase XII (CA12), and forkhead box F1 (FoxF1) were also detected by quantitative reverse-transcription polymerase chain reaction. RESULTS: The result of quantitative reverse-transcription polymerase chain reaction showed that aggrecan and COL2A1 genes expression was significantly increased in differentiated MenSCs (P < 0.05). There was significantly more COL2A1 gene expression in normoxic group than that in low O2 group (P < 0.05). But no significant difference was observed in aggrecan gene expression between normoxic group and low O2 group. These aforementioned results were also confirmed by histological analysis. We also found that the characteristic NP markers (KRT19, CA12, FoxF1) were significantly upregulated in differentiated MenSCs. Moreover, low O2 tension (2%) further enhanced these genes expression (P < 0.05). CONCLUSION: In our study, MenSCs were successfully differentiated into NP-like cells and may become a new source of seed cells for the treatment of intervertebral disc degeneration in the future. LEVEL OF EVIDENCE: N/A. PMID: 24503685 [PubMed - in process]
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Related Articles A comparison of intravenous and intradiscal delivery of multipotential stem cells on the healing of injured intervertebral disk. J Orthop Res. 2014 Jun;32(6):819-25 Authors: Tam V, Rogers I, Chan D, Leung VY, Cheung KM Abstract A major hurdle of cellular therapy for biological treatment of intervertebral disk (IVD) degeneration is the delivery method where current delivery methods are limited to intradiscal injection which can potentially cause further degeneration. Recent studies indicated that multipotential stem cells (MPSCs) from human umbilical cord blood home to injured sites and induce local therapeutic changes, thereby potentially addressing the drawbacks of direct delivery. We tested the effects of these cells on injured IVD using a mouse model of puncture-induced degeneration via two delivery methods. Caudal IVD underwent needle puncture, and MPSCs were injected indirectly (intravenously), or directly (intradiscally) into the nucleus pulposus. IVD were harvested for histological, gene and protein analysis after 14 weeks. Our finding showed limited homing ability of the MPSCs. However, regardless of delivery method, no engraftment or expansion of MPSCs was observed at the injured site. Contrasting to direct injection, intravenous injection neither improved the degeneration status, nor preserve disk height, however, both delivery methods increased glycosaminoglycan (GAG) protein and Acan gene expression relative to controls, suggesting possible paracrine effects. Identifying the mechanisms by which MPSCs act on endogenous IVD cells would provide insights into the potential of these cells to treat IVD injuries and degeneration. PMID: 24578095 [PubMed - indexed for MEDLINE]
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Related Articles Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther. 2014;16(2):R67 Authors: Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA Abstract INTRODUCTION: Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs. METHODS: Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-β3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM). RESULTS: GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-β or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs. CONCLUSIONS: The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies. PMID: 24618041 [PubMed - in process]
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Related Articles Effects of hypoxia on differentiation from human placenta-derived mesenchymal stem cells to nucleus pulposus-like cells. Spine J. 2014 Oct 1;14(10):2451-8 Authors: Ni L, Liu X, Sochacki KR, Ebraheim M, Fahrenkopf M, Shi Q, Liu J, Yang H Abstract BACKGROUND CONTEXT: Low back pain is a frequently occurring disease caused by intervertebral disc degeneration. Mesenchymal stem cells (MSCs) are a possible treatment modality. Studies have shown MSCs can be transformed into nucleus pulposus-like cells under normoxic conditions. However, this is not a true representation of the hypoxic environment nucleus pulposus cells experience during in vivo growth and differentiation. PURPOSE: To determine the effects of a hypoxic environment on the differentiation of human placenta-derived mesenchymal stem cells (PMSCs) to nucleus pulposus-like cells. STUDY DESIGN: An experimental study. METHODS: Placenta-derived mesenchymal stem cells were cultured and the mesenchymal lineage was confirmed by flow cytometry. Two groups of PMSCs were then cultured under different oxygen concentrations creating a hypoxic group and normoxic group. The proliferation of cells in each group was compared by cell counting kit-8 on Day 1, 3, 5, and 7. Real-time polymerase chain reaction on Days 3 and 7 compared the expressions of Sox-9, Type II collagen, aggrecan, and hypoxia inducible factor-1α (HIF-1α) between the two groups. Immunofluorescence was used to compare the expression of Type II collagen between the two groups after 14 days. RESULTS: Placenta-derived mesenchymal stem cells were successfully isolated and cultured. Mesenchymal markers were positive. On Days 3 and 5, the hypoxic group had a significantly higher proliferation rate than the normoxic group (p<.05). The expression of Sox-9 and HIF-1α was significantly higher (p<.05) in the hypoxic group at Days 3 and 7. Type II collagen and aggrecan expressions were significantly higher (p<.05) in the hypoxic group at Day 7. The hypoxic group stained more positive for Type II collagen at Day 14. CONCLUSIONS: Hypoxic conditions lead to an increased differentiation and proliferation of nucleus pulposus-like cells. Placenta-derived mesenchymal stem cells cultured in nucleus pulposus inducing media and a hypoxic environment show enhanced expression of the nucleus pulposus-like cell markers, Sox-9, Type II collagen, aggrecan, and HIF-1α. PMID: 24662208 [PubMed - in process]
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Related Articles Update on canine MSC markers. Cytometry A. 2014 May;85(5):379-81 Authors: Tryfonidou MA, Schumann S, Armeanu S, Harichandan A, Sivasubramaniyan K, Mollenhauer J, Bühring HJ PMID: 24677755 [PubMed - indexed for MEDLINE]
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Related Articles Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model. PLoS One. 2014;9(4):e93570 Authors: Sun W, Zhang K, Liu G, Ding W, Zhao C, Xie Y, Yuan J, Sun X, Li H, Liu C, Tang T, Zhao J Abstract OBJECTIVE: The effect of Sox9 on the differentiation of bone marrow mesenchymal stem cells (BMSCs) to nucleus pulposus (NP)-like (chondrocyte-like) cells in vitro has been demonstrated. The objective of this study is to investigate the efficacy and feasibility of Sox9-transduced BMSCs to repair the degenerated intervertebral disc in a rabbit model. MATERIALS AND METHODS: Fifty skeletally mature New Zealand white rabbits were used. In the treatment groups, NP tissue was aspirated from the L2-L3, L3-L4, and L4-L5 discs in accordance with a previously validated rabbit model of intervertebral disc degeneration and then treated with thermogelling chitosan (C/Gp), GFP-transduced autologous BMSCs with C/Gp or Sox9-transduced autologous BMSCs with C/Gp. The role of Sox9 in the chondrogenic differentiation of BMSCs embedded in C/Gp gels in vitro and the repair effect of Sox9-transduced BMSCs on degenerated discs were evaluated by real-time PCR, conventional and quantitative MRI, macroscopic appearance, histology and immunohistochemistry. RESULTS: Sox9 could induce the chondrogenic differentiation of BMSCs in C/Gp gels and BMSCs could survive in vivo for at least 12 weeks. A higher T2-weighted signal intensity and T2 value, better preserved NP structure and greater amount of extracellular matrix were observed in discs treated with Sox9-transduced BMSCs compared with those without transduction. CONCLUSION: Sox9 gene transfer could significantly enhance the repair effect of BMSCs on the degenerated discs. PMID: 24691466 [PubMed - in process]
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Related Articles Mesenchymal progenitor cells combined with pentosan polysulfate mediating disc regeneration at the time of microdiscectomy: a preliminary study in an ovine model. J Neurosurg Spine. 2014 Jun;20(6):657-69 Authors: Oehme D, Ghosh P, Shimmon S, Wu J, McDonald C, Troupis JM, Goldschlager T, Rosenfeld JV, Jenkin G Abstract OBJECT: Following microdiscectomy, discs generally fail to undergo spontaneous regeneration and patients may experience chronic low-back pain and recurrent disc prolapse. In published studies, formulations of mesenchymal progenitor cells combined with pentosan polysulfate (MPCs+PPS) have been shown to regenerate disc tissue in animal models, suggesting that this approach may provide a useful adjunct to microdiscectomy. The goal of this preclinical laboratory study was to determine if the transplantation of MPCs+PPS, embedded in a gelatin/fibrin scaffold (SCAF), and transplanted into a defect created by microdiscectomy, could promote disc regeneration. METHODS: A standardized microdiscectomy procedure was performed in 18 ovine lumbar discs. The subsequent disc defects were randomized to receive either no treatment (NIL), SCAF only, or the MPC+PPS formulation added to SCAF (MPCs+PPS+SCAF). Necropsies were undertaken 6 months postoperatively and the spines analyzed radiologically (radiography and MRI), biochemically, and histologically. RESULTS: No adverse events occurred throughout the duration of the study. The MPC+PPS+SCAF group had significantly less reduction in disc height compared with SCAF-only and NIL groups (p < 0.05 and p < 0.01, respectively). Magnetic resonance imaging Pfirrmann scores in the MPC+PPS+SCAF group were significantly lower than those in the SCAF group (p = 0.0213). The chaotropic solvent extractability of proteoglycans from the nucleus pulposus of MPC+PPS+SCAF-treated discs was significantly higher than that from the SCAF-only discs (p = 0.0312), and using gel exclusion chromatography, extracts from MPC+PPS+SCAF-treated discs also contained a higher percentage of proteoglycan aggregates than the extracts from both other groups. Analysis of the histological sections showed that 66% (p > 0.05) of the MPC+PPS+SCAF-treated discs exhibited less degeneration than the NIL or SCAF discs. CONCLUSIONS: These findings demonstrate the capacity of MPCs in combination with PPS, when embedded in a gelatin sponge and sealed with fibrin glue in a microdiscectomy defect, to restore disc height, disc morphology, and nucleus pulposus proteoglycan content. PMID: 24702507 [PubMed - indexed for MEDLINE]
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Related Articles An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration. Eur Spine J. 2014 Sep;23(9):1803-14 Authors: Rodrigues-Pinto R, Richardson SM, Hoyland JA Abstract Cell-based regenerative medicine therapies have been proposed for repairing the degenerated intervertebral disc (a major cause of back pain). However, for this approach to be successful, it is essential to characterise the phenotype of its native cells to guarantee that implanted cells differentiate and maintain the correct phenotype to ensure appropriate cell and tissue function. While recent studies have increased our knowledge of the human nucleus pulposus (NP) cell phenotype, their ontogeny is still unclear. The expression of notochordal markers by a subpopulation of adult NP cells suggests that, contrary to previous reports, notochord-derived cells are retained in the adult NP, possibly coexisting with a second population of cells originating from the annulus fibrosus or endplate. It is not known, however, how these two cell populations interact and their specific role(s) in disc homeostasis and disease. In particular, notochordal cells are proposed to display both anabolic and protective roles; therefore, they may be the ideal cells to repair the degenerate disc. Thus, understanding the ontogeny of the adult NP cells is paramount, as it will inform the medical and scientific communities as to the ideal phenotype to implant into the degenerate disc and the specific pathways involved in stem cell differentiation towards such a phenotype. PMID: 24777668 [PubMed - indexed for MEDLINE]
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Related Articles Link N and mesenchymal stem cells can induce regeneration of the early degenerate intervertebral disc. Tissue Eng Part A. 2014 Nov;20(21-22):2942-9 Authors: Mwale F, Wang HT, Roughley P, Antoniou J, Haglund L Abstract Link N is a naturally occurring peptide that can stimulate proteoglycan synthesis in intervertebral disc (IVD) cells. IVD repair can also potentially be enhanced by mesenchymal stem cell (MSC) supplementation to maximize extracellular matrix (ECM) production. In a previous study, we have shown that Link N can inhibit osteogenesis and increase the chondrogenesis of MSCs in vitro. The aim of the present study was to determine the potential of MSCs and Link N alone or in combination with regard to tissue repair in the degenerate disc. Bovine IVDs with trypsin-induced degeneration were treated with MSCs, Link N, or a combination of MSCs and Link N. Trypsin-treated discs were also injected with phosphate-buffered saline to serve as a degeneration control. The ECM proteins and proteoglycans were extracted from the inner nucleus pulposus (NP) of the discs, and sulfated glycosaminoglycans (GAGs) were analyzed by the dimethyl methylene blue dye-binding assay. The expression of type II collagen was analyzed by western blot. To track the MSCs after injection, MSCs were labeled with PKH67 and observed under confocal microscopy after the 2 week culture period. The GAG content significantly increased compared with the degeneration control when degenerate discs were treated with MSCs, Link N, or a combination of both Link N and MSCs. Histological analysis revealed that the newly synthesized proteoglycan was able to diffuse throughout the ECM and restore tissue content even in areas remote from the cells. The quantity of extractable type II collagen was also increased when the degenerate discs were treated with MSCs and Link N, either alone or together. MSCs survived, integrated in the tissue, and were found distributed throughout the NP after the 2 week culture period. MSCs and Link N can restore GAG content in degenerate discs, when administered separately or together. Treatment with MSCs and Link N can also increase the expression of type II collagen. The results support the concept that biological repair of disc degeneration is feasible, and that the administration of either MSCs or Link N has therapeutic potential in early stages of the disease. PMID: 24786145 [PubMed - in process]
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Related Articles Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin-hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution. Eur Spine J. 2014 Sep;23(9):1837-47 Authors: Omlor GW, Fischer J, Kleinschmitt K, Benz K, Holschbach J, Brohm K, Anton M, Guehring T, Richter W Abstract PURPOSE: Cell therapy would be favorably performed immediately after nucleotomy, to restore intervertebral disc functionality and to slow down disc degeneration. Promising results were reported from small animal models but remaining problems, especially in larger animals, include loss of vital cells due to annular damage at the injection site and detrimental intradiscal conditions. The aim of the present study was to optimize cell-based disc therapy using a new albumin-hyaluronan hydrogel together with bone marrow-derived mesenchymal stem cells in a large porcine disc model. METHODS: Luciferase cell labeling was evaluated to follow-up stem cells metabolically up to 7 days in 3D cell cultures mimicking the harsh disc environment with low oxygen and glucose concentrations. As a pilot in vivo study, the implant was injected into porcine discs after removal of ~10% of nucleus volume and animals were killed immediately after surgery (n = 6) and 3 days later (n = 6). 24 discs were analyzed. Implant persistence and cell activity (luciferase + WST assay) were observed simultaneously. RESULTS: In vitro cell culture with reduction of glucose (20, 5, 0.5, 0 mM) and oxygen (21, 5, 2%) significantly decreased metabolic cell activity and luciferase activity after 3 days, with no recovery and a further decrease after 7 days, establishing luciferase activity as a metabolic sensor. During 3 days of 3D culture with disc-like conditions, luciferase activity decreased to 8%. In vivo, initial implant volume shrank to 61% at day 3 with evidence for hydrogel compression. Luciferase activity in vivo at day 3 was 2% without referencing but 23% after referencing to in vitro cell adaptation, and 38% after additional consideration of detected implant volume loss. CONCLUSION: In vitro analysis up to 7 days established for the first time luciferase activity as a metabolic sensor for mesenchymal stem cells used in regenerative disc therapy. Under the present protocol, short-term in vivo analysis after 3 days suggests improved implant retainment inside the disc and persistence of metabolically active cells; however, further studies will have to prove long-term in vivo outcome. PMID: 24801573 [PubMed - indexed for MEDLINE]
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Related Articles Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics. Eur Cell Mater. 2014;27:5-11 Authors: Benneker LM, Andersson G, Iatridis JC, Sakai D, Härtl R, Ito K, Grad S Abstract Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects. PMID: 24802611 [PubMed - indexed for MEDLINE]
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Related Articles Intervertebral disc and stem cells cocultured in biomimetic extracellular matrix stimulated by cyclic compression in perfusion bioreactor. Spine J. 2014 Sep 1;14(9):2127-40 Authors: Tsai TL, Nelson BC, Anderson PA, Zdeblick TA, Li WJ Abstract BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration. PURPOSE: To tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD. STUDY DESIGN: An extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD. METHODS: An optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production. RESULTS: Among all cocultured groups, hAF cells and hMSCs in the ratio of 2:1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers. CONCLUSIONS: We demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The results show that our approach provides both favorable physical and chemical cues through cell-matrix and cell-cell interactions and mechanobiological induction to enhance IVD generation ex vivo. Our findings may lead to viable tissue engineering applications of generating a functional biological IVD for the treatment of disc degeneration. PMID: 24882152 [PubMed - in process]
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Related Articles Native nucleus pulposus tissue matrix promotes notochordal differentiation of human induced pluripotent stem cells with potential for treating intervertebral disc degeneration. J Biomed Mater Res A. 2014 Jun 2; Authors: Liu Y, Fu S, Rahaman MN, Mao JJ, Bal BS Abstract Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T), cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18), and they displayed the ability to generate NP-like tissue in vitro, which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs, and eventually towards regenerative therapies for disc degeneration. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014. PMID: 24889905 [PubMed - as supplied by publisher]
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Related Articles Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification. Bone. 2014 Sep;66:232-9 Authors: Xu HG, Yu YF, Zheng Q, Zhang W, Wang CD, Zhao XY, Tong WX, Wang H, Liu P, Zhang XL Abstract Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification. PMID: 24970040 [PubMed - in process]
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Related Articles Fluoroscopy assisted minimally invasive transplantation of allogenic mesenchymal stromal cells embedded in HyStem reduces the progression of nucleus pulposus degeneration in the damaged interverbal disc: a preliminary study in rabbits. ScientificWorldJournal. 2014;2014:818502 Authors: Subhan RA, Puvanan K, Murali MR, Raghavendran HR, Shani S, Abdullah BJ, Abbas AA, Mohamed JA, Kamarul T Abstract This study was conducted to develop a technique for minimally invasive and accurate delivery of stem cells to augment nucleus pulposus (NP) in damaged intervertebral discs (IVD). IVD damage was created in noncontiguous discs at L4-L5 level; rabbits (N = 12) were randomly divided into three groups: group I treated with MSCs in HyStem hydrogel, group II treated with HyStem alone, and group III received no intervention. MSCs and hydrogel were administered to the damaged disc under guidance of fluoroscopy. Augmentation of NP was assessed through histological and MRI T2 mapping of the NP after eight weeks of transplantation. T2 weighted signal intensity was higher in group I than in groups II and III (P < 0.05). Disc height index showed maximum disc height in group I compared to groups II and III. Histological score of the degenerative index was significantly (P < 0.05) lower in group I (8.6 ± 1.8) than that in groups II (11.6 ± 2.3) and III (18.0 ± 5.7). Immunohistochemistry staining for collagen type II and aggrecan staining were higher in group I as compared to other groups. Our results demonstrate that the minimally invasive administration of MSCs in hyaluronan hydrogel (HyStem) augments the repair of NP in damaged IVD. PMID: 24983002 [PubMed - indexed for MEDLINE]
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Related Articles Molecular Basis of Intervertebral Disc Degeneration and Herniations: What Are the Important Translational Questions? Clin Orthop Relat Res. 2014 Jul 15; Authors: Kadow T, Sowa G, Vo N, Kang JD Abstract BACKGROUND: Intervertebral disc degeneration is a common condition with few inexpensive and effective modes of treatment, but current investigations seek to clarify the underlying process and offer new treatment options. It will be important for physicians to understand the molecular basis for the pathology and how it translates to developing clinical treatments for disc degeneration. In this review, we sought to summarize for clinicians what is known about the molecular processes that causes disc degeneration. RESULTS: A healthy disc requires maintenance of a homeostatic environment, and when disrupted, a catabolic cascade of events occurs on a molecular level resulting in upregulation of proinflammatory cytokines, increased degradative enzymes, and a loss of matrix proteins. This promotes degenerative changes and occasional neurovascular ingrowth potentially contributing to the development of pain. Research demonstrates the molecular changes underlying the harmful effects of aging, smoking, and obesity seen clinically while demonstrating the variable influence of exercise. Finally, oral medications, supplements, biologic treatments, gene therapy, and stem cells hold great promise but require cautious application until their safety profiles are better outlined. CONCLUSIONS: Intervertebral disc degeneration occurs where there is a loss of homeostatic balance with a predominantly catabolic metabolic profile. A basic understanding of the molecular changes occurring in the degenerating disc is important for practicing clinicians because it may help them to inform patients to alter lifestyle choices, identify beneficial or harmful supplements, or offer new biologic, genetic, or stem cell therapies. PMID: 25024024 [PubMed - as supplied by publisher]
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Related Articles A systematic review of the safety and efficacy of mesenchymal stem cells for disc degeneration: insights and future directions for regenerative therapeutics. Stem Cells Dev. 2014 Nov 1;23(21):2553-67 Authors: Yim RL, Lee JT, Bow CH, Meij B, Leung V, Cheung KM, Vavken P, Samartzis D Abstract Intervertebral disc degeneration is associated with low-back pain. Mesenchymal stem cells (MSCs) have been used to "regenerate" the disc. The aim of this study was to perform a systematic review of comparative controlled studies that have assessed the safety and efficacy of using MSCs for disc regeneration. Literature databases were extensively searched. Trial design, subject-type, MSC sources, injection method, disc assessment, outcome intervals, and complication events were assessed. Validity of each study was performed. Twenty-four animal studies were included with 20.8% of the studies reporting randomization of groups. Trials in humans fulfilling inclusion criteria were not noted. The studies represented 862 discs that were injected with MSCs and 1,603 discs as controls. All three types of MSCs (ie, bone marrow, synovial, and adipose tissues) showed successful inhibition of disc degeneration. Bone-marrow-derived MSCs demonstrated superior quality of repair compared with other non-MSC treatments. A 2.7% overall complication rate was noted, whereby complications were noted only in rabbits. Overall, evidence suggested that MSCs increased disc space height in the majority of animal models. This is the first systematic review to assess the safety and efficacy of MSCs for the treatment of disc degeneration. Short-term MSC transplantation is safe and effective; however, additional, larger, and higher-quality studies are needed to assess the long-term safety and efficacy. Inconsistencies in methodological design and outcome parameters prevent any robust conclusions. Human-based clinical trials are needed. Recommendations are further made to improve efficacy, reduce potential complications, and standardize techniques for future studies. PMID: 25050446 [PubMed - in process]
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Related Articles Human umbilical cord mesenchymal stem cell transplantation for the treatment of chronic discogenic low back pain. Pain Physician. 2014 Jul-Aug;17(4):E525-30 Authors: Pang X, Yang H, Peng B Abstract BACKGROUND: Chronic low back pain is one of the major causes of disability and thus has a major socioeconomic impact. Intervertebral disc degeneration is the main cause of chronic low back pain. Treatment for chronic discogenic low back pain has traditionally been limited to either conservative management or surgical fusion. If conservative treatment fails, then surgical fusion is commonly considered. Current treatments are limited to treat the symptoms and not the underlying biologic alterations of the disc. OBJECTIVE: Human umbilical cord tissue-derived mesenchymal stem cells (HUC-MSCs) contain stem cells and possess the ability to regenerate degenerative discs. Based on the results of previous in vitro and animal experiments, we conducted a preliminary study to test the feasibility and safety and to obtain an early indication for the therapeutic value of HUC-MSC transplantation in patients with chronic discogenic low back pain. STUDY DESIGN: This is the first study involving treatment of chronic low back pain using HUC-MSC transplantation. SETTING: The study was performed at a spine center in China. METHODS: Two patients with chronic discogenic low back pain were treated with HUC-MSC transplantation. An 11-point visual analog scale (VAS, 0-10) and Oswestry Disability Index (ODI, 0-100) were used to assess the back pain symptoms and the lumbar function, respectively. RESULTS: After transplantation, the pain and function improved immediately in the 2 patients. The VAS and ODI scores decreased obviously during a 2-year follow-up period. LIMITATIONS: The shortcoming of this study is that it is a preliminary study with only 2 patients. CONCLUSION: The clinical outcomes indicated that HUC-MSC transplantation is a favorable alternative method for the treatment of chronic discogenic low back pain. PMID: 25054402 [PubMed - indexed for MEDLINE]
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Related Articles Differential Response of Encapsulated Nucleus Pulposus and Bone Marrow Stem Cells in Isolation and Coculture in Alginate and Chitosan Hydrogels. Tissue Eng Part A. 2014 Sep 8; Authors: Naqvi SM, Buckley CT Abstract Cell-based therapies may hold significant promise for the treatment of early stage degeneration of the intervertebral disc (IVD). Given their propensity to proliferate and ability to form multiple tissue types, mesenchymal stem cells (MSCs) have been proposed as a potential cell source to promote repair of the nucleus pulposus (NP). However, for any successful cell-based therapy, a carrier biomaterial may be essential for targeted delivery providing key biophysical and biochemical cues to facilitate differentiation of MSCs. Two widely used biomaterials for NP regeneration are chitosan and alginate. The primary objective of this study was to assess the influence of alginate and chitosan hydrogels on bone marrow stem cells (BM) and NP cells in isolation or in coculture. A secondary objective of this study was to investigate coculture seeding density effects of BM and NP cells and simultaneously explore which cell type is responsible for matrix formation in a cocultured environment. Porcine NP and BM cells were encapsulated in alginate and chitosan hydrogels separately at two seeding densities (4×10(6) and 8×10(6) cells/mL) or in coculture (1:1, 8×10(6) cells/mL). Constructs (diameter=5 mm, height=3 mm) were maintained under IVD-like conditions [low-glucose, low (5%) oxygen] with or without transforming growth factor-β3 (TGF-β3) supplementation for 21 days. Results demonstrated differential viability depending on hydrogel type. NP cells remained viable in both biomaterial types whereas BM viability was diminished in chitosan. Further, hydrogel type was found to regulate sulfated glycosaminoglycan (sGAG) and collagen accumulation. Specifically, alginate better supports sGAG accumulation and collagen type II deposition for both NP and BM cell types compared with chitosan. Having identified that alginate more readily supports cell viability and matrix accumulation, we further explored additional effects of seeding density ratios (NP:BM-1:1, 1:2) for coculture studies. Interestingly, in coculture conditions, the BM cell population declined in number while NP cells increased, indicating that MSCs may in fact be signaling NP cells to proliferate rather than contributing to matrix formation. These findings provide exciting new insights on the potential of MSCs for NP tissue regeneration strategies. PMID: 25060596 [PubMed - as supplied by publisher]
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Related Articles Cell and molecular biology of intervertebral disc degeneration: current understanding and implications for potential therapeutic strategies. Cell Prolif. 2014 Oct;47(5):381-90 Authors: Wang SZ, Rui YF, Lu J, Wang C Abstract Intervertebral disc degeneration (IDD) is a chronic, complex process associated with low back pain; mechanisms of its occurrence have not yet been fully elucidated. Its process is not only accompanied by morphological changes, but also by systematic changes in its histological and biochemical properties. Many cellular and molecular mechanisms have been reported to be related with IDD and to reverse degenerative trends, abnormal conditions of the living cells and altered cell phenotypes would need to be restored. Promising biological therapeutic strategies still rely on injection of active substances, gene therapy and cell transplantation. With advanced study of tissue engineering protocols based on cell therapy, combined use of seeding cells, bio-active substances and bio-compatible materials, are promising for IDD regeneration. Recently reported progenitor cells within discs themselves also hold prospects for future IDD studies. This article describes the background of IDD, current understanding and implications of potential therapeutic strategies. PMID: 25112472 [PubMed - indexed for MEDLINE]
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Related Articles Stem cell therapy for intervertebral disk regeneration. Am J Phys Med Rehabil. 2014 Nov;93(11 Suppl 3):S122-31 Authors: Gou S, Oxentenko SC, Eldrige JS, Xiao L, Pingree MJ, Wang Z, Perez-Terzic C, Qu W Abstract Intervertebral disk degeneration has been considered an irreversible process characterized by a decrease in cell viability, attenuation of proteoglycan and type II collagen synthesis, and dehydration of nucleus pulposus. Stem cell therapy specifically addresses the degenerative process and offers a potentially effective treatment modality. Current preclinical studies show that mesenchymal stem cells have the capacity to repair degenerative disks by differentiation toward chondrocyte-like cells, which produce proteoglycans and type II collagen. There has been evidence that mesenchymal stem cell transplantation into the intervertebral disk increases the intradiskal magnetic resonance imaging T2 signal intensity, increases the disk height, and decreases the degenerative grade in animal models. Appropriate selection of cell carriers/matrix is important because it may prevent cell leakage into the spinal canal and provide an environment that facilitates cell proliferation and differentiation. Although human cell therapy trials for degenerative disk disease are on the horizon, potential issues might arise. The authors hereby review the current state of regenerative cell therapy in degenerative disk disease, with emphasis in cell source, techniques for cellular expansion, induction, transplantation, potential benefit, and risks of the use of this novel medical armamentarium in the treatment of degenerative disk disease. PMID: 25122106 [PubMed - indexed for MEDLINE]
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Related Articles Percutaneous injection of autologous bone marrow concentrate cells significantly reduces lumbar discogenic pain through 12 months. Stem Cells. 2014 Sep 3; Authors: Pettine KA, Murphy MB, Suzuki RK, Sand TT Abstract Degenerative disc disease (DDD) induces chronic back pain with limited non-surgical options. In this open label pilot study, twenty-six patients (median age 40 years; range 18-61) received autologous bone marrow concentrate (BMC) disc injections (13 one level, 13 two levels). Pre-treatment Oswestry Disability Index (ODI) and Visual Analogue Scale (VAS) were performed to establish baseline pain scores (average 56.5 and 79.3 respectively), while MRI were independently scored according to the modified Pfirrmann scale. Approximately 1 mL of BMC was analyzed for total nucleated cell (TNC) content, colony forming unit-fibroblast (CFU-F) frequency, differentiation potential, and phenotype characterization. The average ODI and VAS scores were reduced to 22.8 and 29.2 at 3 months, 24.4 and 26.3 at 6 months, and 25.0 and 33.2 at 12 months, respectively (p≤0.0001). Eight of 20 patients improved by one modified Pfirrmann grade at one year. The average BMC contained 121x10(6) TNC/mL with 2,713 CFU-F/mL (synonymous with mesenchymal stem cells). Although all subjects presented a substantial reduction in pain, patients receiving greater than 2,000 CFU-F/mL experienced a significantly faster and greater reduction in ODI and VAS. Subjects older than 40 years who received fewer than 2,000 CFU-F/mL experienced an average pain reduction of 33.7% (ODI) and 29.1% (VAS) at 12 months, while all other patients' average reduction was 69.5% (ODI, p=0.03) and 70.6% (VAS, p=0.01). This study provides evidence of safety and feasibility in the non-surgical treatment of DDD with autologous BMC and indicates an effect of mesenchymal cell concentration on discogenic pain reduction. Stem Cells 2014. PMID: 25187512 [PubMed - as supplied by publisher]
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Related Articles Gene Expression Profile Analysis of Human Mesenchymal Stem Cells from Herniated and Degenerated Intervertebral Discs Reveals Different Expression of Osteopontin. Stem Cells Dev. 2014 Oct 29; Authors: Marfia G, Navone SE, Di Vito C, Tabano S, Giammattei L, Di Cristofori A, Gualtierotti R, Tremolada C, Zavanone M, Caroli M, Torchia F, Miozzo M, Rampini P, Riboni L, Campanella R Abstract Gene expression analysis provides an effective methodology to identify clinically relevant genes implicated in intervertebral disc (IVD) pathology. The analysis of gene profile in mesenchymal stem cells (MSCs) from human herniated IVD (H-IVD) and degenerated IVD (D-IVD) has not yet been investigated. We present in this study a characterization of MSCs isolated from clinically categorized H-IVD and D-IVD disc samples. H-IVD-MSCs and D-IVD-MSCs showed multipotent mesenchymal differentiation ability, expressing positivity for adipogenic, osteogenic, and chondrogenic markers with an immunophenotypical profile representative of MSCs. FACS analyses revealed a higher expression of CD44 in D-IVD-MSCs compared to H-IVD-MSCs. Gene expression profile revealed that most genes under investigation displayed large variations and were not significantly different in the two types of analyzed IVD-MSCs. Conversely, the gene expression of osteopontin (OPN), a protein involved in bone matrix mineralization and extracellular matrix destruction, was found markedly increased (more than 400-fold) in D-IVD-MSCs compared to H-IVD-MSCs. Moreover, the OPN protein expression was detectable only in D-IVD-MSCs, and its levels were directly related with D-IVD severity. These findings suggest that an abnormal expression of OPN in D-IVD-MSCs occurs and plays a pivotal role in the pathophysiological process of human disc degeneration. We speculate that the regulation of the OPN pathway might be a therapeutic target to counteract disc degeneration. PMID: 25203751 [PubMed - as supplied by publisher]
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Related Articles Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus. Bioconjug Chem. 2014 Oct 20; Authors: Fontana G, Srivastava A, Thomas D, Lalor P, Dockery P, Pandit A Abstract Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair. PMID: 25290910 [PubMed - as supplied by publisher]
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Related Articles Biological performance of cell-encapsulated methacrylated gellan gum-based hydrogels for nucleus pulposus regeneration. J Tissue Eng Regen Med. 2014 Nov 5; Authors: Tsaryk R, Silva-Correia J, Oliveira JM, Unger RE, Landes C, Brochhausen C, Ghanaati S, Reis RL, Kirkpatrick CJ Abstract Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue-engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte-like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow-derived MSCs and nasal chondrocytes (NCs) to modified gellan gum-based hydrogels was investigated. Both ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels show no cytotoxicity in extraction assays with MSCs and NCs. Furthermore, the materials do not induce pro-inflammatory responses in endothelial cells. Moreover, MSCs and NCs can be encapsulated into the hydrogels and remain viable for at least 2 weeks, although apoptosis is observed in phGG-MA. Importantly, encapsulated MSCs and NCs show signs of in vivo chondrogenesis in a subcutaneous implantation of iGG-MA. Altogether, the data endorse the potential use of modified gellan gum-based hydrogel as a suitable material in NP tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. PMID: 25370800 [PubMed - as supplied by publisher]
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Related Articles Mesenchymal stem cell technology in the treatment of degenerative disc disease. J Neurosurg Sci. 2014 Nov 6; Authors: Skovrlj B, Cunn G, Guzman J, Qureshi SA Abstract AIM: Low back pain is the leading cause of disability and a major health problem around the world. As of today, symptomatic relief is the best option offered to patients, with mixed results. This study will review the use of mesenchymal stem cell (MSC) technology in the conservative and surgical treatments of degenerative disc disease (DDD). METHODS: A series of PubMed--National Library of Medicine searches were performed. Only articles in English journals or with published with English language translations were included. Level of evidence of the selected articles was assessed. RESULTS: There are multiple in--vitro and animal studies describing successful intervertebral disc (IVD) repair/regeneration with the use of live MSC therapy. Only 3 human studies (22 patients) with inconsistent results have been performed using bone marrow MSCs injected into diseased IVDs in patients with DDD. There exist multiple in--vitro and animal studies describing successful bony fusion with the use of MSC--containing bone graft substitutes. Only 3 retrospective human studies (115 patients) using CBM in spinal fusion have been performed with successful fusion rates ranging from 90.2%--92.3%. However, these studies lacked power and had significant conflicts of interest. There are multiple challenges with the use of MSC technology in humans. CONCLUSION: MSCs may be a promising therapy in the treatment of DDD. However, their lack of success in human models leaves room for further research and focus in this field. Currently, there is no clinical evidence to support the use of this technology in the conservative or surgical management of patients with DDD. PMID: 25373318 [PubMed - as supplied by publisher]
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Related Articles Stem cell therapies for intervertebral disc degeneration: immune privilege reinforcement by Fas/FasL regulating machinery. Curr Stem Cell Res Ther. 2014 Nov 10; Authors: Ma CJ, Liu X, Che L, Liu ZH, Samartzis D, Wang HQ Abstract As a main contributing factor to low back pain, intervertebral disc degeneration (IDD) is the fundamental basis for various debilitating spinal diseases. The pros and cons of current treatment modalities necessitate biological treatment strategies targeting for reversing or altering the degeneration process in terms of molecules or genes. The advances in stem cell research facilitate the studies aiming for possible clinical application of stem cell therapies for IDD. Human NP cells are versatile with cell morphology full of variety, capable of synthesizing extracellular matrix components, engulfing substances by autophagy and phagocytosis, mitochondrial vacuolization indicating dysfunction, expressing Fas and FasL as significant omens of immune privileged sites. Human discs belong to immune privilege organs with functional FasL expression, which can interact with invasive immune cells by Fas-FasL regulatory machinery. IDD is characterized by decreased expression level of FasL with dysfunctional FasL, which in turn unbalances the interaction between NP cells and immune cells. Certain modulation factors might play a role in the process, such as miR-155. Accumulating evidence indicates that Fas-FasL network expresses in a variety of stem cells. Given the expression of functional FasL and insensitive Fas in stem cells (we term as FasL privilege), transplantation of stem cells into the disc may regenerate the degenerative disc by not only differentiating into NP-like cells, increasing extracellular matrix, but also reinforce immune privilege via interaction with immune cells by Fas-FasL network. PMID: 25381758 [PubMed - as supplied by publisher]
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Related Articles Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci. 2014 Nov 25; Authors: Le Maitre CL, Binch AL, Thorpe A, Hughes SP Abstract This review paper discusses the process of disc degeneration and the current understanding of cellular degradation in patients who present with low back pain. The role of surgical treatment for low back pain is analysed with emphasis on the proven value of spinal fusion. The interesting and novel developments of stem cell research in the treatment of low back pain are presented with special emphasis on the importance of the cartilaginous end plate and the role of IL-1 in future treatment modalities. PMID: 25423135 [PubMed - as supplied by publisher]
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Related Articles Phenotypic stability, matrix elaboration and functional maturation of nucleus pulposus cells encapsulated in photocrosslinkable hyaluronic acid hydrogels. Acta Biomater. 2014 Oct 29; Authors: Kim DH, Martin JT, Elliott DM, Smith LJ, Mauck RL Abstract Degradation of the nucleus pulposus (NP) is an early hallmark of intervertebral disc degeneration. The capacity for endogenous regeneration in the NP is limited due to the low cellularity and poor nutrient and vascular supply. Towards restoring the NP, a number of biomaterials have been explored for cell delivery. These materials must support the NP cell phenotype while promoting the elaboration of an NP-like extracellular matrix in the shortest possible time. Our previous work with chondrocytes and mesenchymal stem cells demonstrated that hydrogels based on hyaluronic acid (HA) are effective at promoting matrix production and the development of functional material properties. However, this material has not been evaluated in the context of NP cells. Therefore, to test this material for NP regeneration, bovine NP cells were encapsulated in 1%w/vol HA hydrogels at either a low seeding density (20×10(6)cellsml(-1)) or a high seeding density (60×10(6)cellsml(-1)), and constructs were cultured over an 8week period. These NP cell-laden HA hydrogels showed functional matrix accumulation, with increasing matrix content and mechanical properties with time in culture at both seeding densities. Furthermore, encapsulated cells showed NP-specific gene expression profiles that were significantly higher than expanded NP cells prior to encapsulation, suggesting a restoration of phenotype. Interestingly, these levels were higher at the lower seeding density compared to the higher seeding density. These findings support the use of HA-based hydrogels for NP tissue engineering and cellular therapies directed at restoration or replacement of the endogenous NP. PMID: 25448344 [PubMed - as supplied by publisher]
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