Marble-derived microcalcite improves bone healing in mice osteotomy.

Approximately 10% of all fractures result in delayed healing or non-unions. Bone healing can be improved by the application of osteoconductive and osteoinductive biomaterials. Microcalcite (MCA) as a naturally available calcium carbonate-based biomaterial derived from marble may have the potential to improve bone healing. Herein, we studied for the first time, if MCA in combination with platelet-rich plasma (PRP) can be used as a bone graft material for bone healing in vivo. For this purpose, osteotomies were induced in CD-1 mice (n = 60). Animals received into the osteotomy gap either MCA-loaded PRP (MCA + PRP; n = 20), PRP alone (PRP; n = 20) or no application (NONE; n = 20). Bone healing was evaluated at two and five weeks after osteotomy by micro-computed tomography (µCT), histomorphometric, immunohistochemical and Western Blot analyses. µCT of MCA + PRP femurs revealed more bone volume and an increased polar moment of inertia, indicating a higher biomechanical stability when compared to PRP and NONE femurs. Histomorphometry revealed an increased total callus area after two weeks and a reduced callus tissue area after five weeks in MCA + PRP and PRP animals compared to NONE animals, indicating an accelerated process of bone healing and remodeling over the study period. Moreover, histomorphometric analyses demonstrated an increased fraction of osseous tissue within the callus in MCA + PRP femurs when compared to PRP and NONE femurs. Immunohistochemical analyses showed increased numbers of Ki67+ cells in callus tissue of MCA + PRP femurs. Of interest, Western Blotting revealed a significantly reduced expression of BMP-4 in MCA + PRP animals, while the expression of BMP-2 did not reveal any significant differences between the groups. This indicates a modified balance between angiogenesis and osteogenesis due to MCA. In conclusion, the application of MCA with PRP improved bone healing in a murine osteotomy model and, thus, might be a promising novel bone graft material which may be of interest for clinical fracture treatment.

In vitro osteogenic differentiation of adipose-derived mesenchymal stem cell spheroids impairs their in vivo vascularization capacity inside implanted porous polyurethane scaffolds.

Undifferentiated adipose-derived mesenchymal stem cell (adMSC) spheroids are attractive vascularization units for tissue engineering. Their osteogenic differentiation further offers the possibility of directed generation of bone constructs. The aim of this study was to analyze how this differentiation affects their in vivo vascularization capacity. Green fluorescent protein (GFP)-positive adMSCs were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of undifferentiated and differentiated spheroids using the liquid overlay technique. Subsequently, polyurethane scaffolds were seeded with these spheroids and successful osteogenic differentiation was proven by von Kossa staining and high-resolution microtomography. The scaffolds were then implanted into dorsal skinfold chambers of C57BL/6 wild-type mice to analyze their vascularization and incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with differentiated spheroids exhibited a markedly impaired vascularization. Immunohistochemical analyses revealed that this was caused by the lost ability of differentiated spheroids to form GFP-positive microvascular networks inside the scaffolds. This was associated with a reduced tissue incorporation of the implants. Moreover, they no longer exhibited a mineralized matrix after the 14day implantation period, indicating the dedifferentiation of the spheroids under the given in vivo conditions. These findings indicate that osteogenic differentiation of adMSC spheroids markedly impairs their vascularization capacity. Hence, it may be reasonable to combine adMSC spheroids of varying differentiation stages in scaffolds for bone tissue engineering to promote both vascularization and bone formation.