Poster BB13 - Decellularized meniscus as a biomaterial for cartilage repair that supports stem cell invasion and chondrogenesis

Introduction: Cartilage damage affects 25 million people each year, arising from trauma, sports injuries, and wear and tear. Bone marrow derived mesenchymal stem cells (MSCs) are a frequently used cell source for tissue engineered cartilage repair because of their proliferation rates, differentiation capacity, and accessibility for clinical translation. However, MSC chondrogenic capacity presents some limitations, and even when cultured in 3D hydrogels, matrix secretion outcomes can be non-uniform throughout the construct. To overcome this, the Gottardi lab has developed a new extracellular matrix-based scaffold based on porcine meniscus decellularization (MEND). MEND is created by the selective removal of elastin from meniscal cartilage which, while preserving the bulk of its mechanical properties, creates a wealth of channels for re-cellularization. In this work, we show that MEND can be recellularized with MSCs and investigate MEND’s ability to support uniform chondrogenesis of the construct.

Materials and

Methods: We first seeded MEND with varying densities of MSCs to achieve uniform recellularization. We then compared the chondrogenic response of MSCs in MEND against that of MSCs in 3D hydrogels with controlled composition, such as methacrylated type I collagen (ColMA) and methacrylated gelatin-hyaluronic acid (GelMA/HAMA). We also compared against pellet culture as a positive control. After 21 days of culture in chondrogenic medium, differentiation was assessed via histology (Alcian Blue), biochemical analysis (GAG/DNA assay), and RT-qPCR for key chondrogenic genes (SOX9, ACAN, COL1A1, COL2A1). Finally, we fluorescently labeled MSC derived matrix using functional non-canonical amino acid tagging (FUNCAT) to distinguish newly produced matrix from that of the supporting scaffolds. All outcomes were performed in biological triplicate, technical duplicate, and compared to day 0 results.

Results, Conclusions, and Discussions:

Results: 100k cells per MEND produced the most homogenous distribution of cell nuclei throughout the scaffold, as assessed by DAPI staining. Chondrogenic gene expression was robust and analogous across all scaffold conditions including pellet controls, suggesting MEND can support chondrogenesis as well as other materials proposed for cartilage engineering (Figure 1). However, cell-seeded MEND after chondrogenesis showed more uniform matrix deposition compared to ColMA, GelMA/HAMA, and pellet culture. Quantitative biochemical analysis of GAG secretion further demonstrated that MEND had the highest GAGs per cell production compared to the other conditions.

Discussion: Our data suggests that MEND has a uniform distribution of channels, which is ideal for MSC seeding. MEND retains its natural structure in the presence of MSCs, unlike ColMA, which is subjected to high levels of cell mediated contraction. Evidence of robust and uniform MSC chondrogenesis supports MEND’s relevance for cartilage engineering.

Conclusion: MEND shows promise in providing a scaffold with composition close to that of cartilage and an abundance of channels that allows for uniform recellularization, matching key requirements for clinically relevant articular cartilage engineering. Interestingly, the extent to which MEND supports a robust chondrogenic phenotype of MSCs may open the way to a matrix-assisted microfracture approach for cartilage defect repair.

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