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Rotator cuff tears are common musculotendinous injuries with a high risk of permanent functional disability. Following surgical repair, sub-optimal patient outcomes are directly correlated with poor muscle quality; namely, injury site fatty infiltration, fibrosis, and muscle atrophy. Muscle resident fibro-adipogenic progenitor cells (FAPs) have been identified as key regulators of post-injury skeletal muscle regeneration and repair by maintaining a pro-myogenic environment. In this work, human FAPs (hFAPs) were encapsulated into hyaluronic acid (HyA)-based hydrogels functionalized with bsp-RGD(15) cell adhesion peptide, heparin, and a matrix metalloproteinase (MMP)-cleavable crosslinker. Hydrogel-encapsulated hFAPs increased expression of the pro-myogenic marker UCP1 and production of the anti-inflammatory cytokine IL-10 while downregulating the expression of the fibrotic marker αSMA over time. A murine model of unilateral rotator cuff transection, denervation, and delayed repair was treated with the HyA hydrogel or PBS and compared to a contralateral, non-injured control limb. Muscle histology 6 weeks post-repair revealed that the hydrogel reduced fibrosis, FI, and muscle atrophy while supporting vascularization of the injured tissue region. Collectively, these results suggest that the HyA hydrogel alone can promote muscle regeneration in a clinically relevant delayed repair model of rotator cuff tear, which is hypothesized due to controlled FAP differentiation into pro-myogenic lineages., (© 2025 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Fibrotic disease is caused by the continuous deposition of extracellular matrix by persistently activated fibroblasts (also known as myofibroblasts), even after the resolution of the injury. Using fibroblasts from porcine aortic valves cultured on hydrogels that can be softened via exposure to ultraviolet light, here we show that increased extracellular stiffness activates the fibroblasts, and that cumulative tension on the nuclear membrane and increases in the activity of histone deacetylases transform transiently activated fibroblasts into myofibroblasts displaying condensed chromatin with genome-wide alterations. The condensed structure of the myofibroblasts is associated with cytoskeletal stability, as indicated by the inhibition of chromatin condensation and myofibroblast persistence after detachment of the nucleus from the cytoskeleton via the displacement of endogenous nesprins from the nuclear envelope. We also show that the chromatin structure of myofibroblasts from patients with aortic valve stenosis is more condensed than that of myofibroblasts from healthy donors. Our findings suggest that nuclear mechanosensing drives distinct chromatin signatures in persistently activated fibroblasts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Cells sense mechanical cues from the extracellular matrix to regulate cellular behavior and maintain tissue homeostasis. The nucleus has been implicated as a key mechanosensor and can directly influence chromatin organization, epigenetic modifications, and gene expression. Dysregulation of nuclear mechanosensing has been implicated in several diseases, including bone degeneration. Here, we exploit photostiffening hydrogels to manipulate nuclear mechanosensing in human mesenchymal stem cells (hMSCs) in vitro. Results show that hMSCs respond to matrix stiffening by increasing nuclear tension and causing an increase in histone acetylation via deactivation of histone deacetylases (HDACs). This ultimately induces osteogenic fate commitment. Disrupting nuclear mechanosensing by disconnecting the nucleus from the cytoskeleton up-regulates HDACs and prevents osteogenesis. Resetting HDAC activity back to healthy levels rescues the epigenetic and osteogenic response in hMSCs with pathological nuclear mechanosensing. Notably, bone from patients with osteoarthritis displays similar defective nuclear mechanosensing. Collectively, our results reveal that nuclear mechanosensing controls hMSC osteogenic potential mediated by HDAC epigenetic remodeling and that this cellular mechanism is likely relevant to bone-related diseases., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Bone marrow derived human mesenchymal stem cells (hMSCs) are a promising cell source for regenerative therapies; however, ex vivo expansion is often required to achieve clinically useful cells numbers. Recent results reveal that when MSCs are cultured in stiff microenvironments, their regenerative capacity can be altered in a manner that is dependent on time (e.g., a mechanical dosing analogous to a chemical one). It is hypothesized that epigenomic modifications are involved in storing these mechanical cues, regulating gene expression, and ultimately leading to a mechanical memory. Using hydrogels containing an allyl sulfide cross-linker and a radical-mediated addition-fragmentation chain transfer process, in situ softened hMSC-laden hydrogels at different time points are achieved and the effects of short-term and long-term mechanical dosing on epigenetic modifications in hMSCs are quantified. Results show that histone acetylation and chromatin organization adapt rapidly after softening and can be reversible or irreversible depending on time of exposure to stiff microenvironments. Furthermore, epigenetic modulators are differentially expressed depending on the culture history. Collectively, these experiments suggest that epigenetic remodeling can be persistent and might be a memory keeper.

The use of human mesenchymal stem/stromal cells (hMSCs) in most clinical trials requires millions of cells/kg, necessitating ex vivo expansion typically on stiff substrates (tissue culture polystyrene [TCPS]), which induces osteogenesis and replicative senescence. Here, we quantified how serial expansion on TCPS influences proliferation, expression of hMSC-specific surface markers, mechanosensing, and secretome. Results show decreased proliferation and surface marker expression after five passages (P5) and decreased mechanosensing ability and cytokine production at later passages (P11-P12). Next, we investigated the capacity of poly(ethylene glycol) hydrogel matrices (E ~ 1 kPa) to rescue hMSC regenerative properties. Hydrogels reversed the reduction in cell surface marker expression observed at P5 on TCPS and increased secretion of cytokines for P11 hMSCs. Collectively, these results show that TCPS expansion significantly changes functional properties of hMSCs. However, some changes can be rescued by using hydrogels, suggesting that tailoring material properties could improve in vitro expansion methods.

Valvular interstitial cells (VICs) are key regulators of the heart valve's extracellular matrix (ECM), and upon tissue damage, quiescent VIC fibroblasts become activated to myofibroblasts. As the behavior of VICs during disease progression and wound healing is different compared to healthy tissue, we hypothesized that the organization of the matrix mechanics, which results from depositing of collagen fibers, would affect VIC phenotypic transition. Specifically, we investigated how the subcellular organization of ECM mechanical properties affects subcellular localization of Yes-associated protein (YAP), an early marker of mechanotransduction, and α-smooth muscle actin (α-SMA), a myofibroblast marker, in VICs. Photo-tunable hydrogels were used to generate substrates with different moduli and to create organized and disorganized patterns of varying elastic moduli. When porcine VICs were cultured on these matrices, YAP and α-SMA activation were significantly increased on substrates with higher elastic modulus or a higher percentage of stiff regions. Moreover, VICs cultured on substrates with a spatially disorganized elasticity had smaller focal adhesions, less nuclear localized YAP, less α-SMA organization into stress fibers and higher proliferation compared to those cultured on substrates with a regular mechanical organization. Collectively, these results suggest that disorganized spatial variations in mechanics that appear during wound healing and fibrotic disease progression may influence the maintenance of the VIC fibroblast phenotype, causing more proliferation, ECM remodeling and matrix deposition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

Sensing cell adhesion by means of a colourimetric response provides an intuitive measure of cell binding. In this study polydiacetylene-containing peptide amphiphiles fibres were designed to sense cell adhesion by means of a colour change. The diacetylene-containing peptide amphiphiles were functionalised with the cell-binding motif RGDS, and subsequently mixed with non-functionalised diacetylene-containing spacer amphiphiles. The diacetylenes in the backbone of these fibres were polymerised using UV-light to give dark blue fibre solutions. Subsequent cell adhesion induced a colour change from blue to pink. The propensity of the RGDS fibres to change colour upon cell adhesion could be tuned by varying the C-terminal amino acid of the spacer amphiphile. In addition to this, by varying the RGDS density we found that the optimum colourimetric response was obtained for fibres with a 6 : 1 ratio of non-RGDS to RGDS amphiphiles.