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

Abstract

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.

Keywords: Bone marrow; Chondrogenesis; Equine; Hypoxia; Mesenchymal stem cell; Normoxia; Synovial membrane.

Fig. 1

Characterization of BM-MSCs and SM-MSCs using flow cytometric quantification of cell surface marker expression. a Expression of cell surface markers expected to be positive in MSC populations and b expression of cell surface markers expected to be negative in MSC populations. The white histograms represent isotype controls and black histograms represent respective cell surface marker staining. The mean ± SEM percentage of positive cells is in the corner of each histogram. Each histogram is a representative result of 5 horses

Fig. 2

Photomicrographs of BM-MSC and SM-MSC pellets cultured in normoxic (21% O₂) and hypoxic (5% O₂) conditions for 28 days. Pellets were stained with H&E and toluidine blue (scale bar = 100 μm)

Fig. 3

Mean ± SEM a) GAG, b) DNA, and c) GAG:DNA content in BM-MSC and SM-MSC pellets cultured in normoxic (21% O₂) and hypoxic (5% O₂) conditions for 28 days

Fig. 4

Relative expression (mean ± SEM) of chondrogenic-related genes including SOX9, ACAN, COL2b and COL10A1 for BM-MSC and SM-MSC pellets cultured in normoxic (21% O₂) and hypoxic (5% O₂) conditions for 28 days. Expression is relative to normoxic BM-MSC pellets. 18S was used as a reference gene in all analyses. * p < 0.05

Fig. 5

Photomicrographs of control and induced BM-MSCs and SM-MSCs stained with alizarin red (osteogenesis) and Oil Red O (adipogenesis). Osteogenically induced MSCs stained with alizarin red are positive for extracellular calcium consistent with osteogenesis. Adipogenically induced MSCs demonstrated positive Oil Red O staining of lipid droplets consistent with adipogenesis. (Bar = 100 μm)