Mechano-Hypoxia Conditioning of Engineered Human Meniscus

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.

Engineered Human Meniscus in Modeling Sex Differences of Knee Osteoarthritis in Vitro

“…The experiment is outlined in Figure 1 . Most culture methods and assays were performed identically to those described in previous work ( Liang et al, 2017 ; Szojka et al, 2021a ; Szojka et al, 2021b ).…”

Meniscus regeneration by 3D printing technologies: Current advances and future perspectives

“…The development of different bioactive scaffolds has been reported in the literature, tacking advantage from several different fabrication methods including lyophilization, solvent leaching, solvent casting and particulate leaching, lamination technique, electrospinning. 39 Repopulation of commercial collagen type I supports with MFCs 248 and preparation of decellularized meniscal ECMs 201 , 249 have also been considered. In this dynamic context, the field of 3D printing/bioprinting is gaining wide attention, highlighting the tremendous potential of these technologies.…”

“…Intriguingly, hypoxia (regardless of loading) promotes accumulation of hyaline cartilage while dynamic compression (regardless of oxygen tension) induces a matrix remodeling associated to SOX9 and COL1A1 upregulation. 248 As these two conditions were demonstrated to exert a priming role on cells behavior it descends that they are essential features to bear in mind when validating the bioactivity of a new meniscal scaffold.…”

Creating an Optimal In Vivo Environment to Enhance Outcomes Using Cell Therapy to Repair/Regenerate Injured Tissues of the Musculoskeletal System

“…There are various approaches to address the issue of the biomechanical environment with regard to the development of constructs to facilitate the repair and regeneration of damaged or diseased MSK tissues. The first is to use an artificial scaffold containing cells, with the scaffold supplying a somewhat rigid but biodegradable template after implantation to allow the cells to adapt to the in vivo conditions in a mechanically active environment [ 76 , 77 ]. A second approach is to condition the in vitro generated scaffold containing matrix molecules, such as collagen I to which cells have been added, in a biomechanically active environment in vitro, prior to implantation [ 78 ].…”