Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro.

Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol-ene Michael addition between PEGαMA (8-arm, 10 kg mol-1) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 ± 0.24 kPa, approximating healthy lung tissue (1-5 kPa). Next, residual αMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 ± 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alpha-smooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro.

Peptide-Functionalized Hydrogels Modulate Integrin Expression and Stemness in Adult Human Epidermal Keratinocytes.

The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through ß-integrin signaling. Wound-healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)-based hydrogel formulations with mechanical properties (elastic modulus, E = 20.9 ± 0.56 kPa) and bioactive peptide sequences that mimic the epidermal ECM. These hydrogels enable systematic investigation of the influence of cell-binding domains from fibronectin (RGDS), laminin (YIGSR), and collagen IV (HepIII) on keratinocyte stemness and ß1 integrin expression. Quantification of 14-day keratin protein expression shows four hydrogels improve stemness compared to standard techniques. Three hydrogels increase ß1 integrin expression, demonstrating a positive linear relationship between stemness and ß1 integrin expression. Multifactorial statistical analysis predicts an optimal peptide combination ([RGDS] = 0.67 mm, [YIGSR] = 0.13 mm, and [HepIII] = 0.02 mm) for maintaining stemness in vitro. Best-performing hydrogels exhibit no decrease in Ki-67-positive cells compared to standards (15% decrease, day 7 to 14; p < 0.05, Tukey Test). These data demonstrate that precisely designed hydrogel biomaterials direct integrin expression and promote proliferation, improving the regenerative capability of cultured keratinocytes for basic science and translational work.

Tissue-informed engineering strategies for modeling human pulmonary diseases.

Chronic pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH), and chronic obstructive pulmonary disease (COPD), account for staggering morbidity and mortality worldwide but have limited clinical management options available. Although great progress has been made to elucidate the cellular and molecular pathways underlying these diseases, there remains a significant disparity between basic research endeavors and clinical outcomes. This discrepancy is due in part to the failure of many current disease models to recapitulate the dynamic changes that occur during pathogenesis in vivo. As a result, pulmonary medicine has recently experienced a rapid expansion in the application of engineering principles to characterize changes in human tissues in vivo and model the resulting pathogenic alterations in vitro. We envision that engineering strategies using precision biomaterials and advanced biomanufacturing will revolutionize current approaches to disease modeling and accelerate the development and validation of personalized therapies. This review highlights how advances in lung tissue characterization reveal dynamic changes in the structure, mechanics, and composition of the extracellular matrix in chronic pulmonary diseases and how this information paves the way for tissue-informed engineering of more organotypic models of human pathology. Current translational challenges are discussed as well as opportunities to overcome these barriers with precision biomaterial design and advanced biomanufacturing techniques that embody the principles of personalized medicine to facilitate the rapid development of novel therapeutics for this devastating group of chronic diseases.

Photopolymerization kinetics of methyl methacrylate with reactive and inert nanogels.

The enhanced in situ photopolymerization kinetics of methyl methacrylate (MMA) to poly(methyl methacrylate) (PMMA) through the incorporation of both inert and reactive nanogel (NG) fillers under ambient conditions has been demonstrated. In addition to the polymerization kinetics, the physical and chemical properties of the prepolymeric NG were also utilized to tune the thermoplasticity and mechanical properties of the PMMA polymer network. The protocol followed in this study imparts superior MMA photopolymerization kinetics (≥ 60% double-bond conversion within 15 min for > 35 wt% nanogel loadings and ≥ 95% double-bond conversion in < 60 min for all NG concentrations) when compared with traditional polymerization mechanisms. PMMA remained a glassy material following the incorporation of both inert and reactive NG as demonstrated by the glass transition temperature (Tg) of the ultimate networks. Network linearity is uncompromised following incorporation of inert NG additives, thereby preserving the thermoplasticity of the PMMA network. As the non-functionalized, inert NG content increases, the maintenance of thermoplasticity occurs at the expense of mechanical properties (10× reduction of maximum strength at 25 wt% loading). These effects are less pronounced when reactive nanogels are employed (no significant reduction of maximum strength at 25 wt% loading with minimal crosslinking). The incorporation of NGs enable high chemical tunability within linear polymer networks. Given the wide range of monomers available for the synthesis of NGs, the methodology detailed in this study offers a scheme for the optimization of linear networks for specific targeted applications, hitherto deemed unrealistic under established polymerization protocols.