Regenerative MRL/MpJ tendon cells exhibit sex differences in morphology, proliferation, mechanosensitivity, and cell-ECM organization.

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. We have previously shown that the scarless healing capacity of MRL/MpJ patellar tendons is associated with sexually dimorphic regulation of gene expression for pathways involved in fibrosis, cell migration, adhesion, and extracellular matrix (ECM) remodeling following an acute mid-substance 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 on two-dimensional (2D; glass) and scaffold platforms to examine cell behavior under biochemical and topographical cues associated with tendon homeostasis and healing. Female MRL/MpJ cells showed reduced 2D migration and spreading area accompanied by enhanced mechanosensing, ECM alignment, and fibronectin-mediated cell proliferation compared to male MRL/MpJ cells. Interestingly, female MRL/MpJ cells cultured on isotropic scaffolds showed diminished cell-ECM organization compared to male MRL/MpJ cells. Lastly, MRL/MpJ cells elicited enhanced cytoskeletal elongation and alignment, ECM deposition and organization, and connexin 43-mediated intercellular communication compared to male B6 cells, regardless of culture condition or sex. These results provide insight into the cellular features conserved within the MRL/MpJ phenotype and potential sex-specific targets for the development of more equitable therapeutics.

Detergent-Free Decellularization Preserves the Mechanical 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. In this study, 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. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk 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 statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for 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.

Innate tissue properties drive improved tendon healing in MRL/MpJ and harness cues that enhance behavior of canonical healing cells.

Development of tendon therapeutics has been hindered by the lack of informative adult mammalian models of regeneration. Murphy Roth's Large (MRL/MpJ) mice exhibit improved healing following acute tendon injuries, but the driver of this regenerative healing response remains unknown. The tissue-specific attributes of this healing response, despite a shared systemic environment within the mouse, support the hypothesis of a tissue-driven mechanism for scarless healing. Our objective was to investigate the potential of MRL/MpJ tendon extracellular matrix (ECM)-derived coatings to regulate scar-mediated healing. We found that deviations in the composition of key structural proteins within MRL/MpJ vs C57Bl/6 tendons occur synergistically to mediate the improvements in structure and mechanics following a 1-mm midsubstance injury. Improvement in mechanical properties of healing MRL/MpJ vs C57Bl/6 tendons that were isolated from systemic contributions via organ culture, highlighted the innate tendon environment as the driver of scarless healing. Finally, we established that decellularized coatings derived from early-deposited MRL/MpJ tendon provisional extracellular matrix (provisional-ECM), can modulate canonical healing B6 tendon cell behavior by inducing morphological changes and increasing proliferation in vitro. This study supports that the unique compositional cues in MRL/MpJ provisional-ECM have the therapeutic capability to motivate canonically healing cells toward improved behavior; enhancing our ability to develop effective therapeutics.

In Vitro Evaluation of Cell Compatibility of Dental Cements Used with Titanium Implant Components.

PURPOSE: To evaluate the biocompatibility of five dental cement compositions after directly exposing human gingival fibroblast (HGF) and MC3T3-E1 preosteoblast cells to cement alone and cement applied on commercially pure titanium (cpTi) specimens. MATERIALS AND METHODS: Nanostructurally integrated bioceramic (NIB), resin (R), resin-modified glass ionomer (RMGIC), zinc oxide eugenol (ZOE), and zinc phosphate (ZP) compositions were prepared according to the respective manufacturer's instructions. Samples were prepared in cylindrical Teflon molds or applied over the entire surface of polished cpTi discs. All samples were cured for 0.5, 1, 12, or 24 hours post-mixing. Direct contact testing was conducted according to ISO 10993 by seeding 6-well plates at 350,000 cells/well. Plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 hours before individually plating samples and cpTi control discs. Plates were then incubated for an additional 24 hours. Microtetrazolium (MTT) cell viability assays were used to measure sample cytotoxicity. RESULTS: For samples that cured for 24 hours prior to direct contact exposure, only NIB and ZP cements when cemented on cpTi demonstrated cell viability percentages above the minimum biocompatibility requirement (≥70%) for both the investigative cell lines. R, RMGIC, and ZOE cements exhibited moderate to severe cytotoxic effects on both cell lines in direct contact and when cemented on cpTi specimens. For HGF cells, ZOE cemented-cpTi specimens exhibited significantly decreased cytotoxicity, whereas RMGIC cemented-cpTi specimens exhibited significantly increased cytotoxicity. CONCLUSIONS: Despite previous studies that showed enhanced cpTi corrosion activity for fluoride-containing compositions (NIB and ZP), there was no significant difference in cytotoxicity between cement alone and cemented-cpTi. In general, the MC3T3-E1 preosteoblast cells were more sensitive than HGF cells to cement composition. Ultimately, cement composition played a significant role in maintaining host cell compatibility. Results of this work help illustrate the impact of different cement formulations on host cell health and emphasize the need for understanding material properties when selecting certain formulations of dental cements, which can ultimately influence the survival of dental implant systems.