TNF-α and IL-1β inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells

AimsJoint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs).Main methodsIn MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct.Key findingsTNF-α (from 0.1 to 10 ng/ml) and IL-1β (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-α-treated cultures did not reverse TNF-α effects, indicating that IL-1 was not involved in TNF-α-stimulated TNAP activity. Both TNF-α and IL-1β decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures.SignificanceThe different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.     

Multipotential stromal cells in the talus and distal tibia in ankle osteoarthritis – Presence,potency and relationships to subchondral bone changes

A large proportion of ankle osteoarthritis (OA) has an early onset and is post-traumatic. Surgical interventions have low patient satisfaction and relatively poor clinical outcome, whereas joint-preserving treatments, which rely on endogenous multipotential stromal cells (MSCs), result in suboptimal repair. This study investigates MSC presence and potency in OA-affected talocrural osteochondral tissue. Bone volume fraction (BV/TV) changes for the loading region trabecular volume and subchondral bone plate (SBP) thickness in OA compared with healthy tissue were investigated using microcomputed tomography. CD271-positive MSC topography was related to bone and cartilage damage in OA tissue, and in vitro MSC potency was compared with control healthy iliac crest (IC) MSCs. A 1.3- to 2.5-fold SBP thickening was found in both OA talus and tibia, whereas BV/TV changes were depth-dependent. MSCs were abundant in OA talus and tibia, with similar colony characteristics. Tibial and talar MSCs were tripotential, but talar MSCs had 10-fold lower adipogenesis and twofold higher chondrogenesis than IC MSCs (P = .01 for both). Cartilage damage in both OA tibia and talus correlated with SBP thickening and CD271+ MSCs was 1.4- to twofold more concentrated near the SBP. This work shows multipotential MSCs are present in OA talocrural subchondral bone, with their topography suggesting ongoing involvement in SBP thickening. Potentially, biomechanical stimulation could augment the chondrogenic differentiation of MSCs for joint-preserving treatments.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Sox9-Regulated miRNA-574-3p Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.     

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis

IntroductionInflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA.MethodsExpression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses.ResultsRA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP^−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro.ConclusionsThese data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.     

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1–10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.     

Improved isolation and expansion of bone marrow mesenchymal stromal cells using a novel marrow filter device

Background aimsMesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and malignant transformation. Changes of gene expression with prolonged culture may alter the therapeutic potential of the cells. Increasing the recovery of MSCs from the freshly harvested bone marrow allowing for less ex vivo expansion would represent a major advance in MSC therapy.MethodsHuman bone marrow cells from eight healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators. The initial nucleated cell recovery and the final yield, immunophenotype and trilineage differentiation potential of second-passage MSCs were examined.ResultsThe marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after the second passage. The immunophenotype and differentiation potential of MSCs isolated using the two methods were equivalent and consistent with the defining criteria. The two independent investigators generated comparable results.ConclusionsThis novel filter device is a fast, efficient and reliable system to isolate MSCs and should greatly expedite pre-clinical and clinical investigations of MSC therapy.     

Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.     

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta

Background

Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.     

Differentiation of Rabbit Bone Mesenchymal Stem Cells into Endothelial Cells In Vitro and Promotion of Defective Bone Regeneration In Vivo

Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects.     

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods

Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results

At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion

This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.