Functional heterogeneity of meniscal fibrochondrocytes and microtissue models is dependent on modality of fibrochondrocyte isolation

Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology related studies. However, how these modalities may impact study outcomes are unknown. Here, we show og- and d-isolated MFC have distinct proliferative capacity, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming and migratory capacities. Our data show that microtissue models developed from og-isolated MFC display a contractile phenotype with higher expressions of α-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC designated digestion-after-outgrowth (dog). The transcriptomic prolife of dog-MFC are distinct from d-and og-MFC including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue models containing a higher frequency of smaller collagen fiber diameters. Thus, we demonstrate that the modalities of MFC isolation influences the downstream outcomes of bioengineering and mechanobiology-related studies. In details the current study aimed to deduce the differences in the phenotype of cell population isolated using different isolation methods digestion (d), outgrowth (og) and digestion-after-outgrowth (dog). Further, using the pellet based microtissue model we evaluated how different cell isolation methods effect morphology and chondrogenic differentiation capacities as measured by ECM synthesis, expression of chondrogenic markers, microtissue mechanical properties, expression of contractile markers alpha-smooth muscle actin (alpha-SMA) and Transgelin (TAGLN) and migration of the cells and detailed transcriptomics analysis of cells cultured under chondrogenic induction cultured under hypoxia (5% O2) as optimised in our previous study to promote MFC proliferation and upregulation of the expression of collagen type II and aggrecan in the expanded cells