Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory.

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.

In Vivo Analysis of the Biocompatibility and Immune Response of Jellyfish Collagen Scaffolds and its Suitability for Bone Regeneration.

Jellyfish collagen, which can be defined as "collagen type 0" due to its homogeneity to the mammalian types I, II, III, V, and IX and its batch-to-batch consistent producibility, is of special interest for different medical applications related to (bone) tissue regeneration as an alternative to mammalian collagen-based biomaterials. However, no in vivo studies regarding the induction of M1- and M2-macrophages and their time-dependent ration as well as the analysis of the bone regeneration capacity of jellyfish collagen scaffolds have been conducted until now. Thus, the goal of this study was to determine the nature of the immune response to jellyfish collagen scaffolds and their bone healing capacities. Two in vivo studies using established implantation models, i.e., the subcutaneous and the calvarian implantation model in Wistar rats, were conducted. Furthermore, specialized histological, histopathological, and histomorphometrical methods have been used. As a control biomaterial, a collagen scaffold, originating from porcine pericardium, which has already been stated as biocompatible, was used for the subcutaneous study. The results of the present study show that jellyfish collagen scaffolds are nearly completely resorbed until day 60 post implantation by stepwise integration within the subcutaneous connective tissue mediated mainly by macrophages and single multinucleated giant cells. Interestingly, the degradation process ended in a vessel rich connective tissue that is understood to be an optimal basis for tissue regeneration. The study results showed an overall weaker immune response to jellyfish collagen than to porcine pericardium matrices by the induction of significantly lower numbers of macrophages together with a more balanced occurrence of M1- and M2-macrophages. However, both collagen-based biomaterials induced balanced numbers of both macrophage subtypes, which supports their good biocompatibility. Moreover, the histomorphometrical results for the calvarial implantation of the jellyfish scaffolds revealed an average of 46.20% de novo bone formation at day 60, which was significantly higher compared to the control group. Thereby, the jellyfish collagen scaffolds induced also significantly higher numbers of anti-inflammatory macrophages within the bony implantation beds. Altogether, the results show that the jellyfish collagen scaffolds allowed for a directed integration behavior, which is assumed to be in accordance with the concept of Guided Bone Regeneration (GBR). Furthermore, the jellyfish collagen scaffolds induced a long-term anti-inflammatory macrophage response and an optimal vascularization pattern within their implant beds, thus showing excellent biocompatibility and (bone) tissue healing properties.

Biophysical matrix cues from the regenerating niche direct muscle stem cell fate in engineered microenvironments.

Skeletal muscle stem cells (MuSCs) are essential for efficacious muscle repair, making MuSCs promising therapeutic targets for tissue engineering and regenerative medicine. MuSCs are presented with a diverse and temporally defined set of cues from their microenvironment during regeneration that direct stem cell expansion, differentiation, and return to quiescence. Understanding the complex interplay among these biophysical and biochemical cues is necessary to develop therapies targeting or employing MuSCs. To probe the role of mechanical cues presented by the extracellular matrix, we leverage chemically defined hydrogel substrates with controllable stiffness and adhesive ligand composition to characterize the MuSC response to matrix cues presented during early and late phases of regeneration. We demonstrate that relatively soft hydrogels recapitulating healthy muscle stiffness promote MuSC activation and expansion, while relatively stiff hydrogels impair MuSC proliferation and arrest myogenic progression. These effects are seen on soft and stiff hydrogels presenting laminin-111 and exacerbated on hydrogels presenting RGD adhesive peptides. Soluble factors present in the MuSC niche during different phases of regeneration, prostaglandin E2 and oncostatin M, synergize with matrix-presented cues to enhance stem cell expansion on soft substrates and block myogenic progression on stiff substrates. To determine if temporally varied matrix stiffness reminiscent of the regenerating microenvironment alters MuSC fate, we developed a photoresponsive hydrogel system with accelerated reaction kinetics that can be rapidly softened on demand. MuSCs cultured on these materials revealed that the cellular response to a stiff microenvironment is fixed within the first three days of culture, as subsequent softening back to a healthy stiffness did not rescue MuSC proliferation or myogenic progression. These results highlight the importance of temporally controlled biophysical and biochemical cues in regulating MuSC fate that can be harnessed to improve regenerative medicine approaches to restore skeletal muscle tissue.