Autologous Platelet Lysate Does Not Enhance Chondrogenic Differentiation of Equine Bone Marrow-Derived Mesenchymal Stromal Cells Despite Increased TGF-ß1 Concentration.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are being investigated for their potential in the treatment of musculoskeletal injuries, including tendon and ligament lesions, and cartilage lesions. Culture expansion of cells has traditionally been performed in medium supplemented with fetal bovine serum (FBS), however, concerns regarding the antigenicity and potential viral or prion contamination of FBS have prompted interest in alternative medium supplements. Platelet lysate (PL) contains elevated concentrations of growth factors, including transforming growth factor-ß (TGF-ß), platelet-derived growth factors, and fibroblast growth factor, released from the α-granules of platelets; therefore, PL could be an ideal medium supplement. The effect of PL on mesenchymal stromal cell (MSC) growth and differentiation has not been fully elucidated. We hypothesized that PL medium would contain significantly higher amounts of TGF-ß1 than FBS medium and would be associated with enhanced osteogenic and chondrogenic differentiation. MSCs were isolated from bone marrow collected from five adult horses. Cells were cultured in traditional medium supplemented with FBS or in medium supplemented with fibrinogen depleted-PL (FD-PL). Immunophenotyping was performed using flow cytometry. Trilineage differentiation was assessed through histology and gene expression analysis using quantitative reverse transcription-polymerase chain reaction. TGF-ß1 was quantified in both medium types. The immunophenotypes of BM-MSCs cultured in FBS and FD-PL medium were similar with both culture types containing cells positive for stromal cell markers [cluster of differentiation 29 (CD29), CD44, CD90, CD105, and major histocompatibility complex I (MHCI)] and negative for exclusion markers (CD45, CD79α, and MHCII). Despite significantly higher TGF-ß1 concentration in FD-PL medium, chondrogenic and osteogenic differentiation were not significantly different between FBS and FD-PL supplemented cultures. PL is an appropriate alternative medium supplement for the culture of equine BM-MSCs up to passage 3. However, despite increased TGF-ß1 concentration in FD-PL medium, significant changes in chondrogenic differentiation compared with FBS medium should not be expected.

Sustained Interleukin-10 Transgene Expression Following Intra-Articular AAV5-IL-10 Administration to Horses.

Joint trauma leads to post-traumatic inflammation with upregulation of inflammatory cytokines and degradative enzymes. If severe enough, this response can lead to irreversible post-traumatic osteoarthritis. Interleukin-10 (IL-10), a cytokine with potent anti-inflammatory effects, has been shown to have chondroprotective effects. A gene therapy approach using a vector to overexpress IL-10 in the joint represents a feasible method of delivering sustained high doses of IL-10 to post-traumatic joints. We hypothesized that an AAV5 vector overexpressing IL-10 would result in rapid and sustained IL-10 expression following direct intra-articular injection and that this increase would not be reflected in systemic circulation. In addition, we hypothesized that intra-articular AAV5-IL-10 injection would not induce a local inflammatory response. Twelve horses were assigned to either treatment (AAV5-IL-10-injected) or control (PBS-injected) groups. Middle carpal joints were injected with 1012 vector genomes/joint or phosphate-buffered saline (PBS) alone (3 mL). Serial synovial fluid samples were analyzed for inflammatory changes, IL-10 concentration, and vector genome copy number. Serum samples were also analyzed for IL-10 concentration and vector genome copy number. Synovial membrane was collected on day 84. Synovial fluid IL-10 was significantly increased within 48 h of AAV5-IL-10 injection and remained increased, compared to PBS-injected joints, until day 84. Serum IL-10 was not different between groups. Vector administration did not cause a significant synovial inflammatory response. Vector genomes were detectable in the plasma, synovial fluid, and synovial membrane of AAV5-IL-10-injected horses only. IL-10 has the potential to modulate the articular inflammatory response, thereby protecting cartilage from degradation and osteoarthritis. This study demonstrates the feasibility and efficiency of intra-articular AAV5-IL-10, and future studies investigating the chondroprotective effects of IL-10 in inflamed joints in vivo are warranted.

The effect of hypoxia on chondrogenesis of equine synovial membrane-derived and bone marrow-derived mesenchymal stem cells.

BACKGROUND: Joint injury is extremely common in equine athletes and post-traumatic osteoarthritis (PTOA), a progressive and debilitating disease, is estimated to affect 60% of horses in the USA. The limited potential for intrinsic healing of articular cartilage has prompted intense efforts to identify a cell-based repair strategy to prevent progression of PTOA. Mesenchymal stem cells (MSCs) have the potential to become an ideal source for cell-based treatment of cartilage lesions; however, full chondrogenic differentiation remains elusive. Due to the relatively low oxygen tension in articular cartilage, hypoxia has been proposed as a method of increasing MSC chondrogenesis. The objective of this study was to investigate the effect of hypoxic culture conditions on chondrogenesis in equine synovial membrane-derived MSCs (SM-MSCs) and bone marrow-derived MSCs (BM-MSCs). MSCs were isolated from synovial membrane and bone marrow collected from 5 horses. Flow cytometric analysis was used to assess cell surface marker expression including CD29, CD44, CD90, CD105, CD45, CD-79α, MHCI and MHCII. MSC pellets were cultured in normoxic (21% O2) or in hypoxic (5% O2) culture conditions for 28 days. Following the culture period, chondrogenesis was assessed by histology, biochemical analyses and gene expression of chondrogenic-related genes including ACAN, COL2b, SOX9, and COL10A1. RESULTS: Both cell types expressed markers consistent with stemness including CD29, CD44, CD90, CD105, and MHCI and were negative for exclusion markers (CD45, CD79α, and MHCII). Although the majority of outcome variables of chondrogenic differentiation were not significantly different between cell types or culture conditions, COL10A1 expression, a marker of chondrocyte hypertrophy, was lowest in hypoxic SM-MSCs and was significantly lower in hypoxic SM-MSCs compared to hypoxic BM-MSCs. CONCLUSIONS: Hypoxic culture conditions do not appear to increase chondrogenesis of equine SM-MSCs or BM-MSCs; however, hypoxia may downregulate the hypertrophic marker COL10A1 in SM-MSCs.

Comparison of the Chondrogenic Differentiation Potential of Equine Synovial Membrane-Derived and Bone Marrow-Derived Mesenchymal Stem Cells.

Focal cartilage injury occurs commonly and often precipitates OA. Mesenchymal stem cells (MSCs) may be useful for repairing cartilage lesions, thereby preventing joint degeneration. Although MSCs isolated from bone marrow have been shown to have chondrogenic potential, synovial membrane-derived MSCs (SM-MSCs) may have superior chondrogenic abilities due to a common progenitor cell between synovium and cartilage. The objective of this study was to directly compare the immunophenotype, proliferative capabilities, and chondrogenic potential of equine SM-MSCs and bone marrow-derived MSCs (BM-MSCs). In order to do this, MSCs were isolated from synovial membrane and bone marrow collected from 6 adult horses. Flow cytometric analysis was used to assess cell surface marker expression including CD29, CD44, CD90, CD105, CD45, CD-79α, MHCI, and MHCII. Proliferation rates and doubling time were quantified in P1 and P2 cells. Trilineage differentiation assays were performed. MSC pellets were cultured in chondrogenic induction media for 28 days. Pellets were stained with toluidine blue to assess proteoglycan deposition. Expression of the chondrogenic-related genes ACAN, COL2b, and SOX9 was quantified using qRT-PCR. The immunophenotypes of BM-MSCs and SM-MSCs were similar with both cell types being positive for expression of stem cell markers (CD29, CD44, CD90, CD105, and MHCI) and negative for exclusion markers (CD45 and CD79α). Although SM-MSCs did not express the exclusion marker, MHCII, expression of MHCII was moderate in BM-MSCs. Overall, chondrogenic differentiation was not significantly between the cell types with histologic parameters, proteoglycan content and gene expression being similar. BM-MSCs showed enhanced osteogenic differentiation compared to SM-MSCs. Synovial membrane is a feasible source of MSCs in the horse, however, superior chondrogenesis in vitro should not be expected under currently described culture conditions.