Large bone defects, such as those resulting from trauma or tumor resection, are currently repaired using autografts as the gold standard. However, major limitations of this therapeutic strategy, including restricted tissue availability and donor site morbidity, have necessitated the development of cell- and protein-based approaches. Cell-based bone tissue engineering strategies can enable localization of both an osteoprogenitor cell source and differentiation stimulus directly to the defect space. This reduced dependency on endogenous cell migration as well as the prospective benefit of delivered cell paracrine signaling position cell-based approaches as a promising alternative to the delivery of osteoinductive protein alone. However, challenges to control delivered cell behavior, including viability and differentiation, remain a significant barrier to clinical translation.
Our research objectives were to evaluate a bioluminescent imaging (BLI) technique for the longitudinal monitoring of delivered mesenchymal stem cell (MSC) number and subsequently evaluate the effect of two MSC delivery strategies on cell survival and facilitated bone regeneration. Despite the widespread employment of BLI for tracking delivered cells in vivo, this technique had not yet been validated for large volume, multicomponent vehicles. To accomplish this goal, we developed and evaluated a BLI protocol using our alginate/mesh delivery platform implanted subcutaneously within an immunocompromised rat model. Correlation between BLI signal and viable MSC number was observed to persist through 1 week in vivo. While this work culminated in a powerful tool for subsequent studies, it also highlighted the potential role of confounding factors, including fibrotic and vascular tissue development, on BLI signal correlation strength. Next we evaluated the utility of human platelet lysate (hPL), an enriched cytokine mixture, for the maintenance of delivered MSC viability and promotion of construct vasculature. Although hPL when implemented as either a pretreatment or co-delivery strategy had no influence on either outcome measure, limitations in the applicability of hPL pre-clinical testing via rodent testbeds, both immunocompromised and syngeneic, were identified through complementary in vitro analysis.
Finally, we determined the effect of MSC aggregation, a strategy shown to enhance cell immunomodulatory properties, on delivered MSC survival and cell-based bone regeneration. Delivery of MSC spheroids was initially investigated within an immunocompromised rodent model (Nude rats) and found to have no effect on cell survival, construct vascularization, nor critically-sized bone defect repair. Interestingly, animal-to-animal variability within this model was found to be a significant predictor of cell-based bone regeneration, prompting the decision to conduct further studies within a syngeneic model. When examined within a syngeneic rodent model (Lewis rats), rMSC aggregates elicited a surviving cell fraction and construct vasculature comparable to that of single cell delivery. Despite in vitro observation that the osteoinductive potential of alginate/mesh constructs was increased with rMSC seeding, delivery of rMSC-containing treatments to the femoral defect space attenuated bone repair. In comparison to acellular treatment, regenerated bone volume and biomechanics were reduced with either single cell or aggregate delivery.
The studies presented here explore two stem cell delivery strategies for large volume tissue regeneration. This research implemented a novel imaging platform to relate key cell-based tissue regeneration metrics, namely delivered cell survival, construct vasculature, and functional outcomes, in an effort to elucidate fundamental principles for development of an effective MSC-based large bone defect therapeutic strategy. Importantly, this body of work also drew attention to several aspects of rodent model selection including xenogenicity, cross-reactivity, and biological variability.