Gwendolyn A. Sowa

Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation

Recent studies have shown that mesenchymal stem cell (MSC)‐based therapy might be an effective approach for the treatment of intervertebral disc degeneration (IDD). However, many unanswered questions remain before clinical translation, such as the most effective stem cell type, a reliable transplantation method, including the carrier choice, and the fate of stem cells after misdirected delivery, among others. The objective of the study was to evaluate the fate and effect of allogenic bone marrow MSCs after transplantation into an IDD model. The L2–3, L3–4 and L4–5 intervertebral discs (IVDs) of four rabbits were stabbed to create IDD. Rabbit MSCs were expanded in vitro and in part transduced with retrovirus/eGFP. After 3 weeks, 1 × 105 MSCs were injected into the IVDs. The rabbits were followed by X‐ray and MRI 3 and 9 weeks after injection. Then the animals were sacrificed and the spines analysed histologically. MRI showed no signs of regeneration. X‐ray and gross anatomy inspection demonstrated large anterolateral osteophytes. Histological analysis showed that the osteophytes were composed of mineralized tissue surrounded by chondrocytes, with the labelled MSCs among the osteophyte‐forming cells. The labelled MSCs were not found in the nucleus. Inflammatory cells were not observed in any injected IVDs. These results raise concern that MSCs can migrate out of the nucleus and undesirable bone formation may occur. While cause cannot be inferred from this study, the presence of MSCs in the osteophytes suggests a potential side‐effect with this approach. IVD regeneration strategies need to focus on cell carrier systems and annulus‐sealing technologies to avoid pitfalls. Copyright

Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration

BACKGROUND CONTEXT Destruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD). PURPOSE To summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD. METHODS A comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems. RESULTS Upregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9. CONCLUSIONS Upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.

Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.

Study Design. Changes in gene expression profile and cell fusion of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) after coculture were analyzed. Objective. To investigate the mechanisms of the interaction between NPC and MSC such us differentiation, stimulatory effect, and cell fusion. Summary of Background Data. Introduction of exogenous cells to supplement and replenish intervertebral disc cell population offers a potential approach to treat intervertebral disc degeneration (IDD). Recent evidences showed that intradiscal injection of MSC effectively alter the course of IDD in vivo, and the regenerative potential may result from up-regulated extracellular matrix protein synthesis mediated by MSC and NPC interaction. Methods. Using a double labeling cell system and flow activated cell sorting, we quantitatively analyzed changes in the gene expression profile of human male MSC and female NPC after coculture in a 3-dimensional system that allows short distance paracrine interactions typical of the nucleus pulposus. Furthermore, we analyzed for cell fusion in the cell interaction by fluorescence in situ hybridization (FISH) for X and Y chromosomes, using a 3-dimensional culture system to allow cell-to-cell interactions conducive to cell fusion. Results. Two weeks of coculture cell interaction in a 3-dimensional environment induces a change in MSCs towards a more chondrogenic gene expression profile indicating MSC differentiation, and NPC gene expression changes in matrix and chondrogenic genes demonstrating only a modest trophic effect of MSC on NPC. Moreover, FISH analysis demonstrated that cell fusion is not responsible for MSC plasticity in the interaction with NPCs. Conclusion. This study clarifies the mechanism of MSCs and NPCs interaction in a 3-dimensional environment, excluding cell fusion. These data support the use of undifferentiated MSC for stem cell therapy for IDD treatment.

p38 MAPK Inhibition Modulates Rabbit Nucleus Pulposus Cell Response to IL-1

Analysis of disc gene expression implicated IL‐1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL‐1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL‐1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL‐1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT‐PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E‐2 (PGE‐2), and proteoglycan synthesis measured after 10 days. IL‐1 upregulation of mRNA for cycloxygenase‐2 (COX‐2), matrix metalloproteinase‐3 (MMP‐3), IL‐1, and IL‐6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin‐like‐growth‐factor I (IFG‐1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase‐1 (TIMP‐1) was modestly increased by IL‐1, while those for Transforming Growth Factor‐β (TGF‐β) SOX‐9, and versican remained unchanged. Blocking p38 MAPK reduced IL‐1 induced NO and PGE‐2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF‐1, TGF, and SOX‐9) from 50% to 120%, decreased basal PGE‐2 accumulation, but had no effect on message for TIMP‐1, MMP‐3, or COX‐2. Inhibition of p38 MAPK in cytokine‐activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL‐1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL‐1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

Study Design. Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1&bgr; or TNF-&agr;, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. Objective. To determine the role of signaling through p38 MAPK in nucleus pulposus cells response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. Summary of Background Data. Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1&bgr; and tumor necrosis factor-&agr; are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. Materials and Methods. Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-&agr;, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. Results. Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-&agr; induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-&agr;. Conclusion. Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

Characterization of intervertebral disc aging: longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression.

Study Design. A cohort of young, healthy New Zealand White rabbits was followed longitudinally with serial magnetic resonance imaging (MRI) analysis and terminal analysis of histologic changes and gene expression. Objective. To examine the changes observed during normal aging in the intervertebral disc. Summary of Background Data. Although there is a correlation between aging and the onset of intervertebral disc degeneration (IDD), evidence suggests that distinct pathways are involved in these processes. Our group has characterized a reproducible rabbit model of IDD by MRI, radiograph, histology, and mRNA expression. However, no similar analysis has been performed longitudinally for intervertebral disc aging to allow comparison of these 2 important processes. Methods. Four skeletally mature female NZW rabbits were housed for 122 weeks, and lumbar spine MRIs were characterized serially. Histologic and quantitative gene expression analysis of the nucleus pulposus of these aging animals was performed, and compared with adult and young rabbits. Results. Mean MRI index decreased by <25% through 120 weeks. The histologic analysis showed changes in cell composition, with abundant notochordal cells in the young, chondrocyte-like cells and notochordal cells in the adult, and clusters of hypertrophic chondrocytes in the aging discs. The PCR analysis of the nucleus pulposus showed that gene expression of collagen decreased, whereas that for proteoglycans increased with aging. BMP-2, TIMP-1, and SOX-9 expression was significantly lower in the young compared with adult discs and TGF-&bgr;1 demonstrated lower gene expression in young and aging animals. Conclusion. Although dramatic cellular changes were observed, age-related MRI changes occurred in this rabbit model of normal aging at a much slower rate than in a previous injury model of degeneration. In addition, the gene expression analysis of the nucleus pulposus demonstrated remarkable differences between aging and injury induced degeneration. These results suggest that aging and injury contribute uniquely to the process of IDD.

Human Nucleus Pulposus Cells React to IL-6 : Independent Actions and Amplification of Response to IL-1 and TNF-α

Study Design. Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1&bgr; or TNF-&agr;. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. Objective. To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-&agr; actions in these cells. Summary of Background Data. Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. Methods. NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1&bgr; or TNF-&agr;. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as 35S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. Results. IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-&agr; stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-&agr;. MMP-13 induction by TNF-&agr; was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-&agr; fourfold. Conclusion. The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-&agr;, thus, helping preserve disc composition, structure, and function.

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro

Study Design. We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. Objective. To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. Summary of Background Data. Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. Methods. Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by 35S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. Results. Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% ± 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 ± 507.1 &mgr;m2, velocity: 0.09 ± 0.01 &mgr;m/min); giant cells (area: 12678 ± 1637.0 &mgr;m2, velocity: 0.08 ± 0.01 &mgr;m/min) which grew rapidly without cell division; polygonal cells (area: 3053 ± 751.2 &mgr;m2, 0.14 ± 0.01 &mgr;m/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 ± 235.6 &mgr;m2, 0.19 ± 0.01 &mgr;m/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. Conclusion. These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration

Oxidative damage is a well‐established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial‐derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria‐targeted reactive oxygen species (ROS) scavenger XJB‐5‐131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δ mice, previously established to be a useful in vivo model to study age‐related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial‐derived ROS contributes to age‐associated IDD in Ercc1−/Δ mice. Collectively, these data provide strong experimental evidence that mitochondrial‐derived ROS play a causal role in driving changes linked to aging‐related IDD and a potentially important role for radical scavengers in preventing IDD.

Spine degeneration in a murine model of chronic human tobacco smokers

OBJECTIVE To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.