Optimization of in vitro Osteogenesis and Evaluation of Polymeric Three-Dimensional Tissue Engineering Scaffolds for Bone Healing in the Canine Model

Abstract

Tissue engineering holds much promise for treatment of large bone defects. The objective of the work presented herein describes the manipulation of progenitor cells known as mesenchymal stromal cells (MSCs) and three-dimensional scaffolds as potential regenerative strategies for critically sized bone defects using the dog as a translational model. First, in vitro osteogenesis of autologous and induced pluripotent (iPS)-derived canine MSCs (cMSCs) was evaluated in response to two osteogenic induction agents, dexamethasone and bone morphogenic protein (BMP)-2. Results demonstrated that dexamethasone decreased early-stage osteogenesis in the presence of BMP-2. In contrast, inclusion of dexamethasone and BMP-2 resulted in the greatest degree of late-stage osteogenesis for both autologous cMSC preparations but was inhibitory to mineralization of the iPS-derived cMSCs. To examine potential explanations for these differences, real-time quantitative PCR (qPCR) was performed to assess osteogenic gene expression. Results demonstrated that expression of Runx2, osterix, and osteocalcin were increased in response to BMP-2 in the autologous cMSCs. This work demonstrates that cMSC osteogenesis can be optimized, but that optimal conditions for early- and late-stage differentiation may differ. Second, porous shape memory polymer (SMP) scaffolds were assessed for cytocompatibility and in vitro osteogenesis in combination with cMSCs. Two compositions of SMPs were evaluated: (1) poly(e-caprolactone) [PCL], and (2) PCL-poly(L-lactic acid) [PCL:PLLA]. Canine MSCs attached, proliferated, and exhibited minimal cytotoxicity on both SMP compositions. Furthermore, both SMP compositions supported robust in vitro osteogenesis in response to appropriate osteogenic induction agents (e.g. dexamethasone and BMP-2). This work demonstrates that SMPs provide a cytocompatible scaffold suitable for osteogenic differentiation of cMSCs. Lastly, these SMP compositions were evaluated for in vivo biocompatibility and osteogenesis using rat subcutaneous and rabbit femoral condylar implantation models, respectively. Results demonstrated scaffolds to be biocompatible as evidenced by formation of fibrovascular tissue within SMP pores with minimal inflammation. SMPs also exhibited osteogenesis, as a substantial amount of new bone was formed within the defects and SMPs. Collectively, the work presented herein represents a significant contribution to the field by evaluating a novel tissue engineering construct and by developing strategies for improved osteogenesis in the context of a biomechanically relevant canine translational model.