(31h) Biologically Relevant Nonlinear Elastic Microenvironments As Tunable 3D Cell Culture Platforms

Nonlinear elastic extracellular matrix (ECM) properties are difficult to recapitulate without the use of fibrous architectures which couple strain-stiffening with stress-relaxation. Herein, bottlebrush polymer hydrogels were used to isolate and tune the strain-stiffening biomechanical feedback sensed by human mesenchymal stromal cells (hMSCs) in a non-fibrous, non-stress relaxing engineered microenvironment. Recent work has demonstrated that these covalently crosslinked bottlebrush polymer hydrogels can recapitulate strain-stiffening biomechanical microenvironments at biologically relevant stresses. By tailoring the bottlebrush polymer length in the hydrogel network, the critical stress associated with the onset of stiffening can be systematically varied to occur outside and within a biologically relevant stress regime (BRSR, < 25 Pa). When the critical stress of the bottlebrush polymer hydrogel was tuned to be within the BRSR, encapsulated human mesenchymal stromal cells (hMSCs) adopted a unique protrusion-rich morphology. This morphology was elucidated to be driven by cell-matrix interactions and regulated through actomyosin dynamics. Inspired by how strain-stiffening extracellular microenvironments play a critical role in cellular transformations in vivo, the poly(ethylene glycol)-based bottlebrush polymer hydrogels were then engineered to mimic the strain-stiffening mechanical properties found in tissue-specific niches. Specifically, the collagen I-rich osteoid supports the transition of osteoblasts into osteocytes during bone formation as the osteoprogenitor cells become encased and buried within this nonlinear elastic, fibrous matrix. In this work, bottlebrush polymer hydrogels are used as a model culture platform probe how nonlinear elastic mechanical properties can regulate cell fate. To date, there have been few successful attempts to monitor and support the hMSC-to-osteoblast-to-osteocyte transition using a three-dimensional culture platform in vitro. Using the bottlebrush polymer hydrogels as an osteoid-mimetic synthetic culture platform, a 6-week osteogenic differentiation protocol was used to differentiate encapsulated hMSCs within 3D bottlebrush hydrogel networks or collagen I gel controls. In the bottlebrush hydrogels, the cells formed dendritic protrusions which fused into a functional dendritic network without significant scaffold compaction which was a major limitation in the collagen I scaffolds. These protrusion-rich morphologies coincide with an upregulation of E11 and DMP-1 (pre-osteocyte markers) and the formation of connexin 43 positive gap junctions. Fluorescence recovery after photobleaching experiments revealed the real-time recovery of calcein in photobleached cells from surrounding cells through functional gap junctions. The nonlinear elastic mechanical properties of the bottlebrush polymer hydrogels drive the upregulation of a protrusion-rich morphotype promoting osteocytogenesis. These biologically relevant strain-stiffening synthetic extracellular microenvironments hold great potential as in vitro 3D culture models. In the future these bottlebrush polymer scaffolds could be tailored to provide both the biomechanical and biochemical cues necessary to study exciting questions within the osteochondral niche and beyond.