Osteochondral Differentiation of Fluorescent Multireporter Cells on Zonally-Organized Biomaterials

Abstract

The regeneration of functional osteochondral tissue is mired by the difficulty of recapitulating the zone-dependent differences in tissue composition and constituent cell phenotype. The objective of the current work is to study the ability of multidirectional zonal scaffolds to promote the differentiation of murine bone marrow stromal cells (BMSCs) and chondrocytes along the osteogenic and chondrogenic lineages, respectively, by analyzing dual or triple transgenic fluorescent reporter expression in vitro and in vivo. BMSCs containing fluorescent reporters for bone sialoprotein (BSP) and dentin matrix protein (DMP-1) were seeded on zonal scaffolds in vitro, where successful differentiation was determined from a continued progression of BSP to DMP1-exclusive fluorescence and was corroborated by gene expression and histological analyses. Zonal chondrogenesis was then separately studied using articular chondrocytes containing fluorescent reporters for types 1, 2, and 10 collagens. The majority of fluorescent cells (65.1%) displayed exclusive fluorescence of the type 2 collagen reporter, where a significant portion (27.7%) cofluoresced type 2 and type 10 collagen reporters, indicating a progression to hypertrophy in vitro. Zonal gene expression, histological, and immunohistochemical analyses revealed prehypertrophic chondrocytes embedded in zonally-organized matrix. Upon ectopic implantation, the prehypertrophic chondrocytes formed mineralized tissue through an endochondral ossification pathway. Fluorescent multireporter cells accurately monitored the differentiation behavior of BMSCs and articular chondrocytes on zonal scaffolds and revealed subtle differences in tissue and chondrocyte phenotype.

Impact Statement

This study represents significant advancement in the use of biomimetic scaffolds to direct zonal osteochondral tissue formation. We describe the use of a novel fluorescent reporter system that enables the real-time evaluation of cellular differentiation in a nondestructive manner. In this study, we use this tool to confirm the osteogenic and chondrogenic capabilities of our scaffold alongside control scaffolds, and use cryohistological methods to probe zone-specific differences in cell and tissue quality. We believe this approach can be widely adopted by others for a variety of biomaterial and cell systems in the development of tissue engineered therapeutics.