Investigation of region-specific effects of pepsin-digested decellularized meniscus on human adipose-derived stromal cells within alginate hydrogels

Abstract

Meniscus tears are one of the most common musculoskeletal injuries, but treatment options remain limited. The meniscus can be divided into distinct inner and outer regions based on differences in the tissue structure and composition. Recognizing that the extracellular matrix (ECM) provides cell-instructive cues that can direct cell differentiation, the current study investigated the effects of incorporating region-specific ECM derived from decellularized meniscus on the viability, growth, and lineage-specific differentiation of human adipose-derived stromal cells (ASCs) encapsulated in alginate beads. The first phase of research focused on validating a novel decellularization protocol for porcine meniscus, which demonstrated the effective removal of cellular content while preserving key ECM constituents including glycosaminoglycans (GAGs) in both the inner and outer meniscus regions. Subsequently, the decellularized inner meniscus (DIM) and decellularized outer meniscus (DOM) were digested with pepsin and combined with a propriety alginate formulation to enable rapid cell encapsulation under mild conditions. Viability staining confirmed that encapsulated human ASCs remained highly viable over 28 days in culture in proliferation or chondrogenic differentiation media. Under both media conditions, the ASC density was significantly higher in the alginate beads incorporating DIM or DOM as compared to alginate alone controls at 28 days. In addition, gene expression analysis and immunofluorescence staining supported that the incorporation of the pepsin-digested ECM within the alginate enhanced fibrochondrogenic marker expression in the samples cultured in chondrogenic differentiation medium. Qualitatively, more intense staining for collagen types I and II were observed within the beads incorporating DOM as compared to DIM, supporting that the formulations had varying effects. Overall, these studies provide new insight supporting that region-specific meniscus ECM can be harnessed to direct cell phenotype and function towards the goal of developing tissue-specific bioinks for tissue-engineering applications.