Joji Mochida

Transplantation of mesenchymal stem cells embedded in Atelocollagen® gel to the intervertebral disc: a potential therapeutic model for disc degeneration

Intervertebral disc degeneration is considered to be one of the major causes of low back pain. Despite this irreversible phenomenon, 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. Based on previous studies, we hypothesize that maintenance of proteoglycan content in the disc is achieved by avoiding the depletion of nucleus pulposus and preserving the structure of the annulus is a primary factor in decelerating disc degeneration. One novel approach to solve the dilemma of intervertebral disc degeneration is found at the stem cell level. Mesenchymal stem cells (MSCs) are known to possess the ability to differentiate into various kinds of cells from mesenchymal origin. Although the majority of cells that contribute to disc formation are known to obtain chondrocyte-like phenotypes, no reported study has emphasized the correlation with mesenchymal stem cells. To evaluate the possible potential of MSCs in disc cell research and treatment of degenerative disc disease, autologous MSCs embedded in Atelocollagen gel were transplanted into the discs of rabbits which had undergone a procedure proven to induce degeneration. The results suggest that MSC transplantation is effective in decelerating disc degeneration in experimental models and provided new hopes for treatment of degenerative disc disease in humans. Atelocollagen gel served as an important carrier of MSCs in transplantation, permitting proliferation, matrix synthesis and differentiation of MSCs. This study strengthens the viable efficacy of practical application of MSCs in treatment of intervertebral disc disease.

Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model : Potential and limitations for stem cell therapy in disc regeneration

Study Design. An in vivo study to assess the differentiation status of mesenchymal stem cells (MSCs) transplanted to the nucleus pulposus of degenerative discs in a rabbit model. Objectives. To evaluate the fate of MSCs transplanted to the nucleus pulposus of degenerative discs in a rabbit and to determine whether they are a suitable alternative for cell transplantation therapy for disc degeneration. Summary of Background Data. Although MSCs have been proposed as candidate donor cells for transplantation to treat intervertebral disc degeneration, their differentiation after transplantation has not been adequately investigated. Methods. Autologous MSCs, labeled with green fluorescent protein, were transplanted into mature rabbits. Consecutive counts of transplanted MSCs in the nucleus area were performed for 48 weeks after transplantation. Differentiation of transplanted cells was determined by immunohistochemical analysis. The proteoglycan content of discs was measured quantitatively using a dimethylmethylene blue assay, and mRNA expression of Type I and II collagen, aggrecan and versican was measured semi-quantitatively using reverse transcription polymerase chain reaction. Results. Many cells that were positive for green fluorescent protein were observed in the nucleus pulposus of cell-transplanted rabbit discs 2 weeks after transplantation. Their number increased significantly by 48 weeks. Some GFP-positive cells were positive for cell-associated matrix molecules, such as Type II collagen, keratan sulfate, chondroitin sulfate, aggrecan, and the nucleus pulposus phenotypic markers, hypoxia inducible factor 1 alpha, glutamine transporter 1, and matrix metalloproteinase 2. MSCs did not show significant expression of these molecules before transplantation. Biochemical and gene expression analyses showed significant restoration of total proteoglycan content and matrix-related genes compared with nontransplanted discs. Conclusions. MSCs transplanted to degenerative discs in rabbits proliferated and differentiated into cells expressing some of the major phenotypic characteristics of nucleus pulposus cells, suggesting that these MSCs may have undergone site-dependent differentiation. Further studies are needed to evaluate their functional role.

Nucleus pulposus allograft retards intervertebral disc degeneration

Autogenous implantation of nucleus pulposus or nucleus pulposus cells that were activated by coculture retards intervertebral disc degeneration, but harvesting such grafts causes disc degeneration at the donor site. This study examined whether nucleus pulposus allografts similarly retard disc degeneration and whether such allografting induces immunologic rejection. Japanese White rabbits served as donors and recipients for allografts. Lumbar disc degeneration was induced by aspirating the nucleus pulposus. Two weeks later, intact nucleus pulposus or nucleus pulposus cells were injected and compared with a sham procedure and normal control. The recipients’ discs were examined histologically and immunologically at intervals for 16 weeks. Discs receiving an intact nucleus pulposus showed the least degeneration, followed by discs receiving nucleus pulposus cells, both of which were better than no treatment. These findings correlated directly with the intensity of immunochemical staining for Type II collagen. Allogeneic grafts did not induce any appreciable host-versus-graft response. Injection of nucleus pulposus and nucleus pulposus cells retards intervertebral disc degeneration. However, injection of intact nucleus pulposus is more effective than injection of nucleus pulposus cells alone. The intercellular matrix plays an important, but poorly understood, role in preserving intervertebral discs.

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc

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.

Percutaneous reinsertion of the nucleus pulposus : an experimental study

Study Design. An analysis of the histologic changes in intervertebral discs after percutaneous reinsertion of the nucleus pulposus in rats. Objective. To devise a way to delay further disc degeneration resulting from spinal deformity and the adverse effects of various treatments. Summary of Background Data. The role of the nucleus pulposus of the intervertebral disc described by many investigators has not been fully clarified. Methods. Disc herniation was induced in the tails of 112 Wistar rats, using a fixation device between the 5th and 8th coccygeal vertebrae. After percutaneous nucleotomy at coccygeal vertebrae 5‐6 and 6‐7, fresh nucleus pulposus, cryopreserved nucleus pulposus, or an artificial substitute was inserted into the intervertebral disc at coccygeal vertebrae 5‐6. Two, 4, or 8 weeks after reinsertion, disc sections from each coccygeal level were studied histopathologically. Results. In the groups with reinsertion of fresh or cryopreserved nucleus pulposus, degenerative changes of the disc with the reinserted nucleus at coccygeal vertebrae 5‐6 were milder than those of the disc without reinsertion at coccygeal vertebrae 6‐7. However, no apparent benefit from reinsertion was observed in the group with artificial substitutes. Conclusions. Early reinsertion of the nucleus pulposus (fresh or cryopreserved) delays degeneration of such disc materials as the anulus fibrosus, endplate, and remaining nucleus pulposus.

A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat

The basic molecular characteristics of intervertebral disc cells are still poorly defined. This study compared the phenotypes of nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) cells using rat coccygeal discs and AC from both young and aged animals and a combination of microarray, real-time RT-PCR and immunohistochemistry. Microarray analysis identified 63 genes with at least a fivefold difference in fluorescence intensity between the NP and AF cells and 41 genes with a fivefold or greater difference comparing NP cells and articular chondrocytes. In young rats, the relative mRNA levels, assessed by real-time RT-PCR, of annexin A3, glypican 3 (gpc3), keratin 19 (k19) and pleiotrophin (ptn) were significantly higher in NP compared to AF and AC samples. Furthermore, vimentin (vim) mRNA was higher in NP versus AC, and expression levels of cartilage oligomeric matrix protein (comp) and matrix gla protein (mgp) were lower in NP versus AC. Higher NP levels of comp and mgp mRNA and higher AF levels of gpc3, k19, mgp and ptn mRNA were found in aged compared to young tissue. However, the large differences between NP and AC expression of gpc3 and k19 were obvious even in the aged animals. Furthermore, the differences in expression levels of gpc3 and k19 were also evident at the protein level, with intense immunostaining for both proteins in NP and non-existent immunoreaction in AF and AC. Future studies using different species are required to evaluate whether the expression of these molecules can be used to characterize NP cells and distinguish them from other chondrocyte-like cells.

Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 105, 106, or 107 cells per disc. The MSC‐transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC‐transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 106 MSCs transplanted disc, while less viable cells were detected in 105 MSCs transplanted and more apoptotic cells in 107 MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration. Published by Wiley Periodicals, Inc. J Orthop Res 28:1267–1275, 2010

Differential phenotype of intervertebral disc cells: microarray and immunohistochemical analysis of canine nucleus pulposus and anulus fibrosus.

Study Design. Microarray gene expression profiling, quantitative gene expression analysis, and immunohistochemistry was used to investigate molecular variations between nucleus pulposus (NP) and anulus fibrosus (AF) of the dog intervertebral disc (IVD). Objective. To identify specific molecules with differing expression patterns in NP and AF and compare their profile with articular cartilage (AC). Summary of Background Data. Although experimental and animal studies have demonstrated the potential of cell based approaches for NP regeneration, there is still a deficiency of basic knowledge about the phenotype of IVD cells. Methods. Comparative microarray analysis of beagle lumbar NP and AF was performed. Molecules of interest were evaluated by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, comparing lumbar and coccygeal NP and AF and AC. To assess interspecies variations, genes that had been found differentially expressed in rat tissues were also investigated. Results. Forty-five genes with NP/AF signal log ratio ≥1 were identified. &agr;-2-Macroglobulin, cytokeratin-18, and neural cell adhesion molecule (CD56) mRNA were higher in NP compared to AF and AC, and desmocollin-2 mRNA was higher in NP than AF. The expression profiles were similar in lumbar and coccygeal discs, although certain variations were noticed. Interspecies differences between rat and dog were evident in the expression of several genes. Immunohistochemistry confirmed differences in gene expression at the protein level. Conclusion. This study reports on the expression of molecules that have not been described previously in IVD, in non-notochordal discs comparable with human. Interspecies differences were noted between rat and dog tissues, whereas variations between caudal and lumbar discs were less prominent. The NP of the beagle as a chondrodystrophoid dog breed is potentially more similar to the human than the NP of species whose discs do not naturally degenerate. Therefore, studies on appropriate species may contribute to a better understanding of the cell types residing in the IVD.

The importance of preserving disc structure in surgical approaches to lumbar disc herniation.

Study Design This study examined the clinical and radiographic results in patients with lumbar disc herniation treated surgically with one of four procedures: two different methods of herniotomy using a posterior approach and two different methods of percutaneous nucleotomy. Objectives The authors goal was to identify the structural changes in the disc after each of the procedures and to correlate them with the clinical outcome. Summary of Background Data The details of herniotomy procedure via a posterior approach or by percutaneous nucleotomy as described by many authors have not been consistent. Furthermore, previous reviews of these studies have been less than satisfactory. Methods One hundred fifty‐seven herniotomies using a posterior approach and 94 percutaneous nucleotomies were studied with a follow‐up of at least 2 years. Each of these groups was divided into two subgroups to evaluate the role of the remaining nucleus pulposus in the central area of the disc. Results Changes seen in imaging studies, such as a decrease in disc height and an increase of intervertebral instability in plain radiographs and a decrease in signal intensity of the disc in magnetic resonance images, were significantly less common in the subgroups of herniotomy and percutaneous nucleotomy in which the nucleus pulposus in the central area of the disc was preserved when compared with the subgroups in which a complete removal of the disc was attempted. The changes seen in the imaging studies were significant in patients younger than 40 years of age and correlated closely with the clinical results. Conclusions Preserving the nucleus pulposus during the surgical treatment of lumbar disc herniation in patients younger than 40 years of age resulted in better radiographic and clinical results.

Enhancement of Intervertebral Disc Cell Senescence by WNT/β-Catenin Signaling–Induced Matrix Metalloproteinase Expression

OBJECTIVE To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis. METHODS The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription-polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway-focused microarray analysis. RESULTS We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells. CONCLUSION The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration.