Ford, E. M. | bioRxiv Science

bioRxiv2026

Matrix stiffening toolbox: dynamic hydrogels for three-dimensional cell culture with real-time cell response

Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (SMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset.

bioengineering↗

bioRxiv2020

Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

Tendon injuries are difficult to heal in part because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo. We have established multi-functional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironments. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multi-functional collagen mimetic peptides (mfCMPs) that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically-demanding environments like tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multi-functional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are non-cytotoxic both in 2D bolus exposure (hydrogel components) and 3D encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (two-weeks); and provide mechanical support to tendon defects at the time of injection and in situ formation. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair prior to in vivo testing and indicate the promise of multi-functional and synthetic hydrogels for continued translation. IMPACT STATEMENTTendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multi-functional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this paper, the preclinical efficacy of multi-functional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

bioengineering↗