Andarawis-Puri, N. | Search bioRxiv

bioRxiv2022

Regenerative MRL/MpJ Tendon Cells Exhibit Sex Differences in Morphology, Proliferation, Mechanosensitivity, and Cell-Matrix Remodeling

Clinical and animal studies have reported the influence of sex on the incidence and progression of tendinopathy, which results in disparate structural and biomechanical outcomes. However, there remains a paucity in our understanding of the sex-specific biological mechanisms underlying effective tendon healing. To overcome this hurdle, our group has investigated the impact of sex on tendon regeneration using the super-healer Murphy Roths Large (MRL/MpJ) mouse strain. Despite a shared scarless healing capacity, we have shown that MRL/MpJ patellar tendons exhibit sexually dimorphic regulation of gene expression for pathways involved in fibrosis, cell migration, and extracellular matrix (ECM) remodeling following an acute midsubstance injury. Moreover, we previously found decreased matrix metalloproteinase-2 (MMP-2) activity in female MRL/MpJ tendons after injury. Thus, we hypothesized that MRL/MpJ scarless tendon healing is mediated by sex-specific and temporally distinct orchestration of cell-ECM interactions. Accordingly, the present study comparatively evaluated MRL/MpJ tendon cells under two-dimensional (glass) and three-dimensional (nanofiber scaffolds) culture platforms to examine cell behavior under biochemical and biophysical cues associated with tendon homeostasis and healing. Female MRL/MpJ cells showed reduced 2D migration and spreading area accompanied with enhanced mechanosensing, 2D ECM alignment, and fibronectin-dependent cell proliferation. Interestingly, female MRL/MpJ cells cultured on 3D isotropic scaffolds showed diminished ECM deposition and alignment. Regardless of culture condition and sex, MRL/MpJ cells outperformed B6 cells and elicited a universal regenerative cellular phenotype. These results illustrate the utility of these in vitro systems for elucidating regenerative tendon cell biology, which will facilitate the long-term development of more equitable therapeutics.

bioengineering↗

bioRxiv2022

Detergent-Free Decellularization Preserves the Structural and Biological Integrity of Murine Tendon

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. Here, we adapted and established the advantages of a detergent-free decellularization method that relies on Latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Compared with previous tendon decellularization studies, our method minimized collagen denaturation while adequately removing cells and preserving bulk tissue alignment and mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/provisional), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact StatementIn this study, we present a decellularization method for tendon that does not rely on any detergents or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of tendon structural organization, collagen molecular integrity, and ECM-associated biological cues that are essential for studying physiological cell-ECM interactions. Lastly, we demonstrated the success of this method on healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

bioengineering↗