Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing

Abstract Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. Statement of significance In the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.