Human adipose and synovial-derived MSCs synergistically attenuate osteoarthritis by promoting chondrocyte autophagy through FoxO1 signaling.

BACKGROUND: Human adipose-derived stem cells (ADSCs) exert a strong anti-inflammatory effect, and synovium-derived stem cells (SDSCs) have high chondrogenic potential. Thus, this study aims to investigate whether a combination of human ADSCs and SDSCs will have a synergistic effect that will increase the chondrogenic potential of osteoarthritis (OA) chondrocytes in vitro and attenuate the cartilage degeneration of early and advanced OA in vitro. METHODS: ADSCs, SDSCs, and chondrocytes were isolated from OA patients who underwent total knee arthroplasty. The ADSCs-SDSCs mixed cell ratios were 1:0 (ADSCs only), 8:2, 5:5 (5A5S), 2:8, and 0:1 (SDSCs only). The chondrogenic potential of the OA chondrocytes was evaluated in vitro with a transwell assay or pellet culture with various mixed cell groups. The mixed cell group with the highest chondrogenic potential was then selected and injected into the knee joints of nude rats of early and advanced OA stages in vivo. The animals were then evaluated 12 and 20 weeks after surgery through gait analysis, von frey test, microcomputed tomography, MRI, and immunohistochemical and histological analyses. Finally, the mechanisms underlying these findings were investigated through the RNA sequencing of tissue samples in vivo and Western blot of the OA chondrocyte autophagy pathway. RESULTS: Among the MSCs treatment groups, 5A5S had the greatest synergistic effect that increased the chondrogenic potential of OA chondrocytes in vitro and inhibited early and advanced OA in vivo. The 5A5S group significantly reduced cartilage degeneration, synovial inflammation, pain sensation, and nerve invasion in subchondral nude rat OA, outperforming both single-cell treatments. The underlying mechanism was the activation of chondrocyte autophagy via the FoxO1 signaling pathway. CONCLUSION: A combination of human ADSCs and SDSCs demonstrated higher potential than a single type of stem cell, demonstrating potential as a novel treatment for OA.

Bioinspired collagen-gelatin-hyaluronic acid-chondroitin sulfate tetra-copolymer scaffold biomimicking native cartilage extracellular matrix facilitates chondrogenesis of human synovium-derived stem cells.

The microenvironment plays a crucial role in stem cell differentiation, and a scaffold that mimics native cartilaginous extracellular components can promote chondrogenesis. In this study, a collagen-gelatin-hyaluronic acid-chondroitin sulfate tetra-copolymer scaffold with composition and architecture similar to those of hyaline cartilage was fabricated using a microfluidic technique and compared with a pure gelatin scaffold. The newly designed biomimetic scaffold had a swelling ratio of 1278 % ± 270 %, a porosity of 77.68 % ± 11.70 %, a compressive strength of 1005 ± 174 KPa, and showed a good resilience against compression force. Synovium-derived stem cells (SDSCs) seeded into the tetra-copolymer scaffold attached to the scaffold firmly and exhibited good mitochondrial activity, high cell survival with a pronounced glycosaminoglycan production. SDSCs cultured on the tetra-copolymer scaffold with chondrogenic induction exhibited upregulated mRNA expression of COL2A1, ChM-1, Nrf2, TGF-ß1, and BMP-7. Ex vivo study revealed that the SDSC-tetra-copolymer scaffold regenerated cartilage-like tissue in SCID mice with abundant type II collagen and S-100 production. BMP7 and COL2A1 expression in the tetra-copolymer scaffold group was much higher than that in the gelatin scaffold group ex vivo. The tetra-copolymer scaffold thus exhibits strong chondrogenic capability and will facilitate cartilage tissue engineering.

Role of lineage-specific matrix in stem cell chondrogenesis.

Cartilage repair in clinics is a challenge owing to the limited regenerative capacities of cartilage. Synovium-derived stem cells (SDSCs) are suggested as tissue-specific stem cells for chondrogenesis. In this study, we hypothesize that decellularized extracellular matrix (dECM) deposited by SDSCs could provide a superior tissue-specific matrix microenvironment for optimal rejuvenation of adult SDSCs for cartilage regeneration. dECMs were deposited by adult stem cells with varying chondrogenic capacities; SDSCs (strong) (SECM), adipose-derived stem cells (weak) (AECM) and dermal fibroblasts (weak) (DECM), and urine-derived stem cells (none) (UECM). Plastic flasks (Plastic) were used as a control substrate. Human SDSCs were expanded on the above substrates for one passage and examined for chondrogenic capacities. We found that each dECM consisted of unique matrix proteins and exhibited varied stiffnesses, which affected cell morphology and elasticity. Human SDSCs grown on dECMs displayed a significant increase in cell proliferation and unique surface phenotypes. Under induction media, dECM expanded cells yielded pellets with a dramatically increased number of chondrogenic markers. Interestingly, SECM expanded cells had less potential for hypertrophy compared to those grown on other dECMs, indicating that a tissue-specific matrix might provide a superior microenvironment for stem cell chondrogenic differentiation.

Effect of the small compound TD-198946 on glycosaminoglycan synthesis and transforming growth factor ß3-associated chondrogenesis of human synovium-derived stem cells in vitro.

As an alternative to chondrocytes-based cartilage repair, stem cell-based therapies have been investigated. Specifically, human synovium-derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD-198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD-198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD-198946 were examined in chondrogenic cultures of hSSC-derived cell pellets. The effect of TD-198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD-198946 dose-dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor ß3 (TGFß3), TD-198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD-198946 attenuated TGFß3-associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD-198946 as judged by safranin O staining. Thus, TD-198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD-198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFß3 + 0.25 nM TD-198946. These studies have demonstrated that TD-198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell-based cartilage therapy.

A Protocol to Prepare Decellularized Stem Cell Matrix for Rejuvenation of Cell Expansion and Cartilage Regeneration.

Traditional ex vivo expansion of adult stem cells yields an insufficient quantity of less potent cells. Here we describe the fabrication of decellularized matrix deposited by synovium-derived stem cells (SDSCs). This matrix could serve as a three-dimensional expansion system to rejuvenate cells for proliferation and tissue-specific differentiation potential, which could benefit cartilage regeneration. The decellularized stem cell matrix (DSCM) might be a powerful system for tissue engineering and regeneration.

Stromal cell-derived factor-1α and transforming growth factor-ß1 synergistically facilitate migration and chondrogenesis of synovium-derived stem cells through MAPK pathways.

The clinical translation of tissue engineering methods is confined by the limited external cell sources, which is hopefully to be addressed by the cell guidance approach as cytokine-induced homing and differentiation of the patients' autologous cells. Synovium-derived stem cells (SDSCs) are a potent cell source for cartilage restoration due to its intrinsic proximity and tissue-specific chondrogenic capacity. In this study, stromal cell-derived factor-1α (SDF-1α) in combination with transforming growth factor ß1 (TGF-ß1) were used to induce SDSCs migration and chondrogenesis in vitro. The migration capacity was evaluated by transwell assay and for chondrogenic evaluation, the expression of Sox9, ACAN and COL2A1 were assessed by quantitative RT-PCR while the expression of sulfated GAG and collagen II were evaluated by Alcian Blue stain and immunohistochemistry respectively. Our data showed that SDF-1α/CXC chemokine receptor 4 (CXCR4) was involved in SDSCs migration through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Exogenous TGF-ß1 enhanced SDF-1α-induced SDSCs migration in a concentration and time-dependent manner through CXCR4, evidenced as complete blockage by AMD3100, the CXCR4 antagonist and this effect was mediated by extracellular regulated protein kinases (ERK) activation. Moreover, the addition of SDF-1α augmented the TGF-ß1-induced SDSCs chondrogenesis, evidenced by the increased pellet sizes and the expressions of COL 2A1, ACAN and Sox9. This effect was related to c-Jun N-terminal kinase (JNK) activation. Collectively, these results suggest that SDF-1α and TGF-ß1 interacts with each other and synergistically enhance the SDSCs migration and chondrogenesis through MAPK pathways.

TGF-ß1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells.

BACKGROUND: Unlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-ß1 (TGF-ß1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components. RESULTS: TGF-ß1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-ß1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels. CONCLUSIONS: This study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects.

Applied osmotic loading for promoting development of engineered cartilage.

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300mOsM) or isotonic (330mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187kPa and 6.77±0.54%ww (GAG) for the 400mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle-van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.