Synovial mesenchymal progenitor derived aggrecan regulates cartilage homeostasis and endogenous repair capacity.

Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the "anti-proteinase" molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have demonstrated that intra-articular injection of aggrecan into OA joints inhibits cartilage degeneration and stimulates cartilage repair respectively. Furthermore, when synovial MPCs overexpressing aggrecan were transplanted into injured joints, increased cartilage regeneration was observed vs. wild-type MPCs or MPCs with diminished aggrecan expression. Overall, these results suggest that aggrecan secreted from joint-associated MPCs may play a role in tissue homeostasis and repair of synovial joints.

17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo.

Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21-/-) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and in vivo), while maintaining their chondrogenic phenotype. This study provides new insights into the regulation of chondrogenesis that might ultimately lead to new therapies for cartilage injury and/or OA.

Characterizing heterogeneity in the response of synovial mesenchymal progenitor cells to synovial macrophages in normal individuals and patients with osteoarthritis.

BACKGROUND: Resident macrophages in OA synovial tissue contribute to synovitis through pro-inflammatory mediators driving cartilage loss. What remains unknown is how these macrophages interact with synovial mesenchymal progenitor cells (sMPCs) in the joint. sMPCs have the potential to undergo chondrogenesis, but for yet unknown reasons, this ability is decreased in OA patients. In this study, we sought to identify if alteration of macrophage activity regulates the chondrogenic capacity of sMPCs. METHODS: An explant model was developed using human synovium obtained from normal individuals and OA patients. These explants were subjected to macrophage depletion and/or cytokine stimulation in order to regulate/deplete the residing macrophage population. Supernatant was collected following a 12-day treatment phase and subjected to inflammatory secretome analysis. sMPCs from the explants were subsequently placed under 21-day chondrogenic differentiation and levels of type II collagen (Col2a), Aggrecan (Acan), and Sox9 gene expression was quantified. RESULTS: Inflammatory secretome analysis from OA patients revealed the presence of pro-inflammatory analytes following pro- and anti-inflammatory cytokine stimulation and/or macrophage depletion. Additionally, chondrogenic differentiation of sMPCs was heterogeneously impacted across all OA patients following pro-/anti-inflammatory cytokine stimulation and/or macrophage depletion. CONCLUSION: Tissue resident synovial macrophages can regulate the chondrogenic differentiation of sMPCs after cytokine stimulation in a patient specific manner. The secretion profile of OA synovium was also responsive to cytokine stimulation and/or macrophage depletion as observed by the largely pro-inflammatory milieu upregulated following cytokine stimulation.

Ion channel expression and function in normal and osteoarthritic human synovial fluid progenitor cells.

Osteoarthritis (OA) is a chronic disease affecting the cartilage of over 15% of Canadians. Synovial fluid mesenchymal progenitor cells (sfMPCs) are present in joints and are thought to contribute to healing. OA sfMPCs have a greater proliferative ability but decreased chondrogenic potential. However, little is known about the factors influencing/regulating the differences between normal and OA sfMPCs. Recently, our lab has shown that sfMPC chondrogenic differentiation in vitro is favorably biased toward a similar osmotic environment as they experience in vivo. The current study now examines the expression and functionality of a variety of ion channels in sfMPCs derived from normal individuals and early OA patients. Results indicated that there is differential ion channel regulation at the functional level and expression level in early OA sfMPCs. All ion channels were upregulated in early OA compared to normal sfMPCs with the exception of KCNMA1 at the mRNA level. At the protein level, TRPV4 was over expressed in early OA sfMPCs, while KCNJ12 and KCNMA1 were unchanged between normal and early OA sfMPCs. At the functional level, the inward rectifying potassium channel was under expressed in early OA sfMPCs, however the membrane potential was unchanged between normal and early OA sfMPCs. In the synovial environment itself, a number of differences in ion concentration between normal and early OA synovial fluid were observed. These findings suggest that normal and OA progenitor cells demonstrate functional differences in how they interact with the synovial ion environment.

Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

Osmolarity regulates chondrogenic differentiation potential of synovial fluid derived mesenchymal progenitor cells.

Cartilage is one of few tissues where adult stem/progenitor cells have not been putatively identified. Recent studies have provided strong evidence that a sub-population of mesenchymal progenitor cells (MPCs) derived from the synovial fluid may be able to affect some degree of cartilage repair both in vivo and in vitro/ex vivo, however this does not appear to be the case in patients with arthritis. Previously, it has been found that synovial fluid osmolarity is decreased in patients with osteoarthritis (OA) or Rheumatoid arthritis (RA) and these changes in osmolarity have been linked to changes in chondrocyte gene regulation. However, it is yet unknown if changes in osmolarity regulate the gene expression in synovial fluid MPCs (sfMPCs), and by extension, chondrogenesis of this cell population. In the present study we have collected synovial fluid samples from normal, OA and RA knee joints, quantified the osmolarity of the fluid and modified the culture/differentiation media to span a range of osmolarities (264-375 mOsm). Chondrogenesis was measured with Alcian blue staining of cultures in addition to quantitative PCR (qPCR) using probes to Sox9, ACAN and Col2A1. Overall, sfMPCs from arthritic joints demonstrated decreased chondrogenic potential compared to sfMPCs isolated from normal synovial fluid. Furthermore, the sfMPCs retained increased chondrogenic potential if differentiated under the same osmolarity conditions for which they were initially derived within. In conclusion, it does appear the synovial fluid osmolarity regulates the chondrogenic potential of sfMPCs, however, further study is required to elucidate the mechanism by which the changes in osmolarity are sensed by the cells and regulate chondrogenic gene expression.