Adipose tissue-derived stem cells in combination with xanthan gum attenuate osteoarthritis progression in an experimental rat model.

The current study explored the efficacy of an intra-articular (IA) injection of allogeneic adipose tissue-derived stem cells (ADSCs) combined with xanthan gum (XG) in a rat osteoarthritis (OA) model. We confirmed that XG significantly inproved proliferation of ADSCs in a dose dependent manner in vitro. The rat OA model was induced by an anterior cruciate ligament transection (ACLT), and at 4 weeks after surgery, rats were divided into four groups: the XG-ADSCs group, the ADSCs group, the XG group and the phosphate-buffered saline (PBS) group. A single dose of 1 × 106 allogeneic ADSCs suspended in 1% XG, ADSCs suspended in PBS, 1% XG alone or PBS alone was injected into the OA joint of rats in the respective treatment groups. Rats were sacrificed at 8 weeks after surgery. Treatment outcomes were evaluated by weight-bearing control of the hind limbs, gross morphological analysis, histological analysis and specific staining of articular cartilage, and measurement of inflammatory factors in synovial fluid. For the rats in the XG-ADSC-s and ADSCs-treated groups, the weight-bearing percentage of the right hind limb was significantly increased compared to that in the PBS group and was sustained over 4 weeks. However, the positive effect in the XG-ADSCs group was significantly greater than that in the ADSCs group. For the rats in the XG group, the efficacy decreased during the third week after surgery. The articular cartilage was relatively normal in the XG-ADSCs group, and moderate degeneration was observed in the ADSCs and XG groups. ADSCs and XG-ADSC treatments significantly decreased the concentrations of IL-1ß, TNF-α, MMP-3 and MMP-13 in synovial fluid; however, the attenuating effect of the XG-ADSCs treatment was significantly enhanced compared with that of the ADSCs treatment alone. These results indicate that a single IA injection of allogeneic ADSCs combined with XG efficiently attenuated OA progression with a therapeutic effect that was significantly greater than that of either ADSCs or XG alone. IA injection of XG-ADSCs might be an effective treatment for OA in humans.

Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model.

Mesenchymal stem cell (MSC)-based cell therapy is a promising avenue for osteoarthritis (OA) treatment. In the present study, we evaluated the efficacy of intra-articular injections of culture-expanded allogenic adipose tissue-derived stem cells (ADSCs) for the treatment of anterior cruciate ligament transection (ACLT) induced rat OA model. The paracrine effects of major histocompatibility complex (MHC)-unmatched ADSCs on chondrocytes were investigated in vitro. Rats were divided into an OA group that underwent ACLT surgery and a sham-operated group that did not undergo ACLT surgery. Four weeks after surgery mild OA was induced in the OA group. Subsequently, the OA rats were randomly divided into ADSC and control groups. A single dose of 1 × 106 ADSCs suspended in 60 µL phosphate-buffered saline (PBS) was intra-articularly injected into the rats of the ADSC group. The control group received only 60 µL PBS. OA progression was evaluated macroscopically and histologically at 8 and 12 weeks after surgery. ADSC treatment did not cause any adverse local or systemic reactions. The degeneration of articular cartilage was significantly weaker in the ADSC group compared to that in the control group at both 8 and 12 weeks. Chondrocytes were co-cultured with MHC-unmatched ADSCs in trans-wells to assess the paracrine effects of ADSCs on chondrocytes. Co-culture with ADSCs counteracted the IL-1ß-induced mRNA upregulation of the extracellular matrix-degrading enzymes MMP-3 and MMP-13 and the pro-inflammatory cytokines TNF-α and IL-6 in chondrocytes. Importantly, ADSCs increased the expression of the anti-inflammatory cytokine IL-10 in chondrocytes. The results of this study indicated that the intra-articular injection of culture-expanded allogenic ADSCs attenuated cartilage degeneration in an experimental rat OA model without inducing any adverse reactions. MHC-unmatched ADSCs protected chondrocytes from inflammatory factor-induced damage. The paracrine effects of ADSCs on OA chondrocytes are at least part of the mechanism by which ADSCs exert their therapeutic activity.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc.

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -ß, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12-14 weeks gestation, but was undetectable at 8-11 weeks. The effect of 17ß-estradiol (E2) on GPR30-mediated proliferation and interleukin-1ß (IL-1ß)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1ß-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1ß-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors.

Cartilage Derived Morphogenetic Protein-2 Induces Cell Migration and Its Chondrogenic Potential in C28/I2 Cells.

BACKGROUND: Intervertebral disc degeneration is a major cause of low back pain. Previous researches have demonstrated local administration of signalling molecules as potential biological therapies for disc regeneration. Our laboratory has published encouraging results for effectiveness of injection of the cartilage derived morphogenetic protein-2 (CDMP-2) into ovine discs following annular injury. To elucidate the mechanisms underpinning these in vivo effects, this project aimed to investigate the potential of CDMP-2 on cellular migration, proliferation and extracellular matrix production in a human chondrocytic cell line. METHODS: To evaluate cell motility, cells were seeded into Boyden chambers and CDMP-2 as a chemo-attractant or a stimulant was placed into either the bottom or top chambers respectively. Cells that had completed migration through the porous membrane were visualized by immunocytochemical staining and analysed using Image J. The effect of CDMP-2 on cell proliferation, proteoglycan and collagen production, as well as chondrogenic gene expression in human chondrocytic cell line C28/I2 was also examined. RESULTS: The results revealed that cells migrated significantly under the influence of CDMP-2 (200 ng/ml) stimulation compared to control (3-fold increase, p = 0.033) and demonstrated a significant chemotactic movement towards a solution of 200ng/ml CDMP-2 (>2-fold increase, p = 0.027). A 35% increase in C28/I2 proliferation was observed after CDMP-2 stimulation (p < 0.0001) compared to control, and in the presence of 100ng/ml CDMP-2, proteoglycan synthesis had an 8-fold increase (p = 0.048). Similarly, gene expression analysis demonstrated increased expression of aggrecan, collagen types II, X and XXVII, BMPR-1A and BMPR-2 when cells were treated with CDMP-2. CONCLUSION: The study shows that C28/I2 cells can migrate under the influence of CDMP-2 as a chemoattractant or migration stimulator, suggestive of an effect on chondrocytic cells in the intervertebral disc. Further, CDMP-2 can stimulate C28/I2 cells to proliferate and synthesize key extracellular matrix proteins.

BMP-7 in combination with estrogen enhances bone formation in a fracture callus explant culture.

In postmenopausal women, estrogen withdrawal results in decrease in bone density or osteoporosis. Osteoporosis leads to fracture and retards bone-healing response. Bone morphogenetic protein-7 (BMP-7), a member of the transforming-growth factor-beta superfamily, has been shown as a promising candidate that stimulates bone growth in its application to fracture healing. The purpose of this study was to determine whether BMP-7 could enhance bone formation in the absence of estrogen. Female rats underwent a controlled closed fracture at the midshaft of the right femur. The callus tissues were harvested from the fracture site eight days following the fracture, and were cultured in serum-free media. The explanted callus tissues were then treated with BMP-7, estrogen (E2) or both. We assessed bone formation by measuring alkaline phosphatase (AP) activity, expression of an osteogenic transcription factor, Runt-related transcription factor-2 (Runx2), production of nitric oxide (NO), and calcium mineralization. Supplementation of serum-free cultures with BMP-7 alone increased cell proliferation by twofold, caused a 6.5-fold increase in AP activity, and enhanced calcium mineralization after 48 h. Moreover, BMP-7 in combination with E2 caused a 8.2-fold increase in the AP activity. Runx2 protein expression was increased following stimulation with BMP-7 and E2. Interestingly, E2 induced the amount of NO production by twofold, whereas BMP-7 did not, either alone or with E2. Thus, BMP-7 could enhance early and late markers of bone fracture healing in callus explant cultures, except for NO. BMP-7 could be a promising growth factor in the treatment of fractures as a consequence of osteoporosis.

The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro.

This study addresses the role of bone morphogenetic protein-7 (BMP-7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP-7 in monolayer and three-dimensional cultures. After 3 days of stimulation, BMP-7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7-21 days, BMP-7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real-time PCR, Western blot, histological, and immunohistochemical staining. BMP-7 supplementation appeared to enhance upregulation of lineage-specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP-7 in the presence of TGF-beta3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP-7 increased alkaline phosphatase activity and dose-dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP-7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP-7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co-ordinating with initial lineage-specific signals to accelerate cell fate determination. BMP-7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell-based tissue repair.

BMP13 prevents the effects of annular injury in an ovine model.

Chronic back pain is a global health problem affecting millions of people worldwide and carries significant economic and social morbidities. Intervertebral disc damage and degeneration is a major cause of back pain, characterised by histological and biochemical changes that have been well documented in animal models. Recently there has been intense interest in early intervention in disc degeneration using growth factors or stem cell transplantation, to replenish the diseased tissues. Bone Morphogenetic Proteins (BMPs) have been approved for clinical use in augmenting spinal fusions, and may represent candidate molecules for intervertebral disc regeneration. BMP13 has an important role in embryonic development and recent genetic evidence shows a role in the development of the human spine. This study explores the effect of BMP13 on a damaged intervertebral disc in an ovine model of discal degeneration. We found that, when injected at the time of injury, BMP13 reversed or arrested histological changes that occurred in the control discs such as loss of extracellular matrix proteins. In addition, BMP13 injected discs retained greater hydration after 4 months, and possessed more cells in the NP. Taken together, BMP13 may be a potent clinical therapeutic agent when used early in the degeneration cascade to promote healthy disc tissue.

BMP-13 emerges as a potential inhibitor of bone formation.

Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.

Differentiation of rodent bone marrow mesenchymal stem cells into intervertebral disc-like cells following coculture with rat disc tissue.

This study aimed to evaluate whether rat mesenchymal stem cells (rMSCs) could be differentiated in vitro into disc-like cells by coculturing with intervertebral disc tissue. rMSCs were cultured with rodent intervertebral disc for up to 30 days in transwell plates. The differentiation of rMSCs was evaluated by immunostaining, Western blot, real-time RT-PCR, Northern blot, and electron microscopy. The potentials of multilineage differentiation and proteoglycan and collagen synthesis were also investigated. rMSCs underwent morphological changes to form three-dimensional micromasses and expressed collagen-2, aggrecan, and sox-9 at RNA and protein levels after 14 days of coculture. These changes were not detected in the samples of rMSCs cultured alone. Cocultured rMSCs also showed other characteristic features of disc-like cells, including the extracellular matrix formation, and proteoglycan and collagen synthesis. In addition, cellular contact between cocultured rMSCs and disc tissue was observed by electron microscopy. Committed rMSCs still retained their differentiation ability into mesoderm lineages of adipocytes or osteocytes when the local environment was altered. This study supports that MSCs are a promising source for cell therapy and tissue engineering in disc regeneration, and highlights that rMSCs can be induced into nucleus pulposus-like cells in vitro under the direct influence of intact disc tissue.

BMP-2 enhances TGF-beta3-mediated chondrogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in alginate bead culture.

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.

Long-term serial passage and neuronal differentiation capability of human bone marrow mesenchymal stem cells.

The development of methods to induce differentiation of human mesenchymal stem cells (hMSCs) has opened the possibility of using these cells in regenerative or reparative therapies. However, the low frequency of hMSCs in tissue means it is often necessary to expand these cells extensively in vitro. In this study, we evaluated the effects of long-term serial passage on the characteristics of bone marrow-derived hMSC populations. In addition, we examined the effect on subsequent hMSC neural differentiation ability, which has not been reported earlier. The hMSC population examined was found to maintain a stable phenotype during the first 6-8 passages of culture as assessed by proliferative ability, morphological appearance, and surface antigen, gene and protein expression, and also expressed pluripotency and neural lineage markers constitutively in the undifferentiated state. Long-term subcultivation neither resulted in spontaneous neural differentiation nor compromised the ability of hMSCs to develop toward an early neuronal fate. In addition, the transformation elicited in hMSC cultures in response to cytokine-based neuronal differentiation was examined by live cell microscopy. We demonstrated, for the first time, that the observed changes result from active and dynamic processes involving outgrowth and motility of cellular extensions, processes entirely distinct from the rapid epiphenomena of cytotoxicity and cytoskeleton disruption generated by chemical induction methods. Cytokine-induced differentiation of hMSCs was also associated with upregulation of early neural gene and protein expression. These findings support the neuronal differentiation capability of hMSCs, although further investigation is required to confirm the ability to attain a mature neuronal phenotype.

Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance (SPR) technology.

Pentosan polysulfate (NaPPS) and chondroitin sulfates (ChSs) have recently been shown to exhibit both symptom and disease modifying activities in osteoarthritis (OA), but their respective mechanisms of action are still the subject of conjecture. Excessive catabolism of joint articular cartilage is considered to be responsible for the initiation and progression of OA but the abilities of these drugs to mitigate this process has received only limited attention. Human neutrophil elastase (HNE) is a proteinase, which can degrade the collagens and proteoglycans (PGs) of the cartilage directly or indirectly by activating latent matrix metalloproteinases. Hyaluronidase (HAase) is an endoglycosidase, which degrades glycosaminoglycans including hyaluronan, which provides the aggregating component of the PG aggrecan complex. In the present study the molecular interactions between the NaPPS, ChSs and some other sulfated polysaccharides with immobilized HNE, HAase or lysozyme (a cationic protein implicated in PG metabolism) were studied using a SPR biosensor device-BIAcore2000. The above three enzymes were covalently immobilized to a biosensor chip CM5 separately using amine coupling. The binding affinity of each sulfated polysaccharide and the kinetics of NaPPS over the concentration range of 0.3-5.0 microg/ml were determined. The inhibition of HNE by the sulfated polysaccharides as determined using the synthetic substrate succinyl-Ala-Ala-Val-nitroanilide (SAAVNA) in a functional assay was compared with their respective binding affinities for this proteinase using the BIAcore system. The results obtained with the two independent techniques showed good correlation and indicated that the degree and ring positions of oligosaccharide sulfation were major determinants of enzyme inhibitory activity. The observed difference in order of binding affinities of the drugs to the immobilized HNE, HAase and lysozyme suggests a conformational relationship, in addition to the charge interactions between the sulfate esters of the polysaccharides and the cationic amino acids of the enzymes. Significantly, the SPR biosensor technology demonstrated that small differences among sulfated polysaccharides, even subtle variations among different NaPPS batches, could be readily detected. The SPR technology therefore offers not only a sensitive and reproducible method for ranking noncompetitive enzyme inhibitors for drug discovery but a rapid and quantitative bioassay for monitoring batch consistency of manufacture.