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Description
Symbrachydactyly is a rare congenital upper limb anomaly, that occurs in 1/30,000- 1/40,000 live births resulting in children born with short boneless fingers. Nowadays, these pediatric patients are treated with phalangeal bone transfer from the foot. However, morbidities are occurring at the donor site which result in unstable toes with significant disfigurations that worsen with the child growth.
In this project, we used a developmentally-inspired strategy to engineer osteogenic grafts for phalanx reconstruction via endochondral ossification (ECO). Human adipose-derived stem cells (ASC) isolated from the stromal vascular fraction (SVF) were seeded into collagen sponges and exposed to chondrogenic and hypertrophic factors to generate in vitro clinically-pertinent osteogenic grafts (100-200 mm3) in the form of hypertrophic cartilage templates (HCTs).
Specifically, we evaluated in vitro the impact of (i) the cell source (freshly isolated SVF cells or expanded ASCs), (ii) the scaffolding material (collagen type I sponge crosslinked or not), (iii) the cell seeding density (3x105 to 3x106 cells/construct) and (iv) the duration of exposure to chondrogenic (3 to 5 weeks) and hypertrophic factors (1 to 2 weeks) on the maturation of the HCTs generated. The mineralization (by alizarin red staining) and the cartilage maturation (evidenced by the cell morphology and the uniformity and intensity of glycosaminoglycan (GAG) revealed by Safranin-O staining) of these HCTs were evaluated on histological sections. Next, the bone forming capacity of these HCTs was assessed in vivo in an ectopic nude (immunocompromised) mouse model for up to 12 weeks, reflecting the clinical scenario of phalangeal soft tissue pocket
In vitro, we were able to generate HCTs of pertinent clinical sizes. As expected, the maturation of the HCTs was dependent on the duration of exposure to chondrogenic and hypertrophic factors. In addition, we observed that the cartilage formation was obtained more rapidly when using freshly isolated SVF cells rather than expanded ASCs. Overall, the best in vitro outcome was obtained for the crosslinked collagen sponge loaded with SVF cells at the highest cell density.
In vivo, we observed that the bone formation was correlated with the degree of hypertrophic cartilage maturation. Interestingly, even the HCTs that had very limited cartilage at the time of implantation were capable of generating bone once implanted, suggesting that the cells primed in vitro are capable of forming cartilage before the bone remodeling occurs in the early stages of the implantation. Similarly, across all the conditions tested, the quantity of bone tissue obtained in vivo was superior to the quantity of cartilage tissue obtained in vitro. Finally, while inferior to the freshly isolated SVF cells-based constructs, ASCs-based constructs remained capable of generating clinically pertinent bone tissue in vivo.
Taken together, these results demonstrate the feasibility of using SVF cells or expanded-ASCs to generate osteogenic grafts of pertinent clinical size in the context of symbrachydactyly. Moreover, despite the limited amount of donor-tissue available in pediatric patients, the data obtained for the expanded ASCs suggest that an autologous approach to generate osteogenic phalanx grafts for children born with symbrachydactyly would remain possible.
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