Inhibition of angiogenesis impairs bone healing in an in vivo murine rapid resynostosis model.

Biologics can improve bone formation, but may diffuse away from sites of therapeutic need. We developed a click-chemistry hydrogel that rapidly polymerizes in situ to control delivery of biologics during post-suturectomy resynostosis in 21-day-old male mice. Here, we used this model to determine the role of angiogenesis in post-suturectomy resynostosis and examine whether controlled release of angiogenesis inhibitors could delay bone regeneration. Hydrogels [DB-co-PEG/poly (TEGDMA)-co-(N3-TEGDMA)] were produced containing anti-angiogenic compounds [anti-VEGFA-antibody or hypoxia inducible factor 1α-inhibitor topotecan]. Bioactivity in vitro was assessed by tube length and branching points of endothelial cells in hydrogel-conditioned media. In vivo effects were examined 14 day post-suturectomy, based on the temporal analysis of angiogenic mRNAs during resynostosis following posterior frontal suture removal. MicroCT was used to quantify angiogenesis in contrast-agent-perfused blood vessels and bone defect size in defects receiving hydrogel, anti-VEGFA/hydrogel, or topotecan/hydrogel. Shorter endothelial tube length and less branching were seen in inhibitor-conditioned media (topotecan > AbVEGFA). In vivo, both compounds inhibited angiogenesis compared with hydrogel-only. Anti-VEGFA/hydrogel reduced resynostosis compared with empty defects, but topotecan/hydrogel blocked bone regeneration. We demonstrate that anti-angiogenic compounds can be incorporated into a spontaneously polymerizing hydrogel and remain active over 14 days in vitro and in vivo. Moreover, bone formation can be delayed by inhibiting neovascularization, suggesting possible use as a therapeutic to control resynostosis following suturectomies and potential applications in other conditions where rapid osteogenesis is not desired. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2742-2749, 2017.

Spag17 deficiency results in skeletal malformations and bone abnormalities.

Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.