Abstract
Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro “mechano-hypoxia conditioning” with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-β3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10–20% vs 30–40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8–5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.
Highlights
The knee menisci are fibrocartilaginous structures that serve to protect the articular cartilage from excessive stress
Strain accumulated to 40–45% each day in dynamic compression (DC) 1%/2 kPa with peak stresses being lower than in DC 30–40% (Supplementary Figure S4.1); this indicated that DC 1%/2 kPa was the less aggressive DC loading regime. c-FOS and VEGF were measured as loading and hypoxia (HYP)-sensitive genes, respectively, and SRY-Box transcription factor 9 (SOX9) as an indicator for hyaline cartilagetype matrix expression
There were no significant differences in gene regulation for DC 1%/ 2 kPa and cyclic hydrostatic pressure (CHP) (Figure 2B)
Summary
The knee menisci are fibrocartilaginous structures that serve to protect the articular cartilage from excessive stress. Their functional extracellular matrix (ECM) is mainly composed of type I collagen (Fox et al, 2012; Makris et al, 2011). The inner regions are avascular and unable to heal upon injury (Arnoczky and Warren, 1982). These injuries are associated with early osteoarthritis (OA) development (Lohmander et al, 2007). The objective of meniscus tissue engineering is to generate cellular replacements that prevent OA development upon implantation
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