Bone substitutes are required for the healing of large bone defects, including orthopedic, craniofacial, and dental reconstructions. Bone tissue engineering has emerged as a promising strategy to address the limitations of currently used bone substitutes. The central paradigm of these approaches is the incorporation of cells, a scaffold, and bioactive factors into an implantable construct. However, the efficacy of these strategies has been limited by the availability of a readily accessible, controlled, and sustained mineralizing cell source. This work aims to meet this need by inducing osteoblastic differentiation of primary skeletal muscle cells by genetic engineering with the Runx2 osteoblastic transcription factor. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype for bone tissue engineering applications.

We first tested the central hypothesis by retrovirally overexpressing a constitutive Runx2 transgene in primary skeletal muscle cells in monolayer culture. Runx2 inhibited myogenic differentiation in these cells, as evident by downregulated muscle-specific gene expression and suppressed myotube formation. Runx2 stimulated osteoblastic differentiation, as demonstrated by upregulation of osteoblastic gene expression and alkaline phosphatase biochemical activity. Furthermore, Runx2-engineered myoblasts showed significant deposition of a hydroxyapatite mineral. Interestingly, Runx2 overexpression had a more potent effect on osteoinduction relative to treatment with soluble BMP-2 protein, which may have been the result of bypassing BMP-2-stimulated autoregulatory factors. Collectively, these results demonstrate that Runx2 stimulates transdifferentiation of primary skeletal muscle cells into an osteoblastic phenotype.

These cells were further examined in the environment of a three-dimensional tissue-engineered construct. Runx2-engineered myoblasts or control cells were seeded onto fibrous collagen scaffolds and cultured up to 8 weeks in static conditions in vitro. Cells were viable on these scaffolds for prolonged culture times. Runx2-engineered cells upregulated osteogenic and downregulated myogenic gene expression on the scaffolds, similar to cells in monolayer culture. Finally, the Runx2-overexpressing myoblasts produced a hydroxyapatite mineral coating on these scaffolds. Although mineral deposition was confined to the construct periphery, colocalizing with cell viability, the mineral coating was sufficient to improve the mechanical properties of the constructs 30-fold. These results established Runx2-engineered skeletal myoblasts as a promising cell source for bone tissue engineering.

Further characterization of these cells involved an in vitro and in vivo comparison with myoblasts retrovirally engineered to overexpress BMP-2, a well-characterized osteogenic factor used in bone tissue engineering strategies. Retroviral delivery of BMP2 or Runx2 stimulated differentiation into an osteoblastic phenotype, as demonstrated by the induction of osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in monolayer culture and on collagen scaffolds both in vitro and in an intramuscular site in vivo. In general, BMP-2 stimulated osteoblastic markers faster and to a greater extent than Runx2, although experimental conditions were identified under which these two factors produced similar effects. Additionally, Runx2-engineered cells did not utilize paracrine signaling via secreted osteogenic factors, in contrast to cells overexpressing BMP-2, as demonstrated by conditioned media studies and activation of Smad signaling. These results emphasize the complexity of gene therapy-based orthopedic therapeutics as an integrated relationship of differentiation state, proliferative capacity, construct maturation, and paracrine signaling of osteogenic cells. This study was significant in evaluating proposed therapeutic systems and defining a successful strategy for integrating gene medicine and orthopedic regeneration.

Although ex vivo gene therapy is a promising approach to orthopedic regenerative medicine, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, we developed a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant in developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.

In summary, this work has established Runx2-engineered primary skeletal myoblasts as a promising cell source for bone tissue engineering. These cells have been characterized in monolayer culture and in tissue engineered constructs in vitro and in vivo. The degree of Runx2-stimulated osteoblastic differentiation compares favorably with that of BMP-2-based strategies, the current standard in orthopedic gene therapy. Finally, we have engineered a system to exogenously regulate Runx2-stimulated osteoblastic differentiation. This work has successfully identified strategies to address cell sourcing limitations of bone tissue engineering and alternate methods for orthopedic gene therapy, as well as enhance our understanding of the molecular biology of osteoblastic differentiation.