Robust phenotypic maintenance of limb cells during heterogeneous culture in a physiologically relevant polymeric-based constructed graft system

A major challenge during the simultaneous regeneration of multiple tissues is the ability to maintain the phenotypic characteristics of distinct cell populations on one construct, especially in the presence of different exogenous soluble cues such as growth factors. Therefore, in this study, we questioned whether phenotypic maintenance over a distinct population of cells can be achieved by providing biomimetic structural cues relevant to each cell phenotype into the construct's design and controlling the presentation of growth factors in a region-specific manner. To address this question, we developed a polymeric-based constructed graft system (CGS) as a physiologically relevant model that consists of three combined regions with distinct microstructures and growth factor types. Regions A and B of the CGS exhibited similar microstructures to the skin and soft tissues and contained rhPDGF-BB and rhIGF-I, while region C exhibited a similar microstructure to the bone tissue and contained rhBMP-2. Primary rat skin fibroblasts, soft tissue fibroblasts, and osteoblasts were then cultured on regions A, B, and C of the CGS, respectively and their phenotypic characteristics were evaluated in this heterogenous environment. In the absence of growth factors, we found that the structural cues presented in every region played a key role in maintaining the region-specific cell functions and heterogeneity during a heterogeneous culture. In the presence of growth factors, we found that spatially localizing the growth factors at their respective regions resulted in enhanced region-specific cell functions and maintained region-specific cell heterogeneity compared to supplementation, which resulted in a significant reduction of cell growth and loss of phenotype. Our data suggest that providing biomimetic structural cues relevant to each cell phenotype and controlling the presentation of growth factors play a crucial role in ensuring heterogeneity maintenance of distinct cell populations during a heterogeneous culture. The presented CGS herein provides a reliable platform for investigating different cells responses to heterogeneous culture in a physiologically relevant microenvironment. In addition, the model provides a unique platform for evaluating the feasibility and efficacy of different approaches for simultaneously delivering multiple growth factors or molecules from a single construct to achieve enhanced cell response while maintaining cellular heterogeneity during a heterogenous culture.