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Objectives and questions: Neoangiogenesis drives the enchondral ossification during bone growth regulated by molecules like e.g. VEGF. The heparan-sulfate-proteoglycan Syndecan-1 (Sdc1) plays a role in communication of cells by acting as a co-receptor for ligands like VEGFa-165 produced by hypertrophic chondrocytes. In previous analysis we showed that Sdc-1-deficient bones have reduced blood vessel invasion at the chondro-osseous interfaces like bone epiphysis and fracture callus in vivo, but the mechanism behind that remained unknown.

Objective: To understand the role of Sdc-1 in the interaction of chondrocytes (CHO) and endothelial cells (EC) we used deep mRNA expression analysis in native and fractured bones in mice.

Material and methods: Native femora of 10 weeks old, female WT (n=5) mice were used for single cell sequencing analysis as described by Tikhonova et al. (Nature 2019). Additionally, mice (WT n=5) underwent midshaft femur fracture (Ethic-app: 81-02.04.2019.A164). Fractured femurs were analysed after 14 days of healing (PFD14) using single cell sequencing with the focus on genes expressed at the chondro-osseous interface and cellular interaction.

Results: Single cell mRNA expression analysis revealed changes in the femur tissue composition with regard to increased cell numbers in almost all cell clusters identified and chondrocytes (controls: 1,813 cells), PFD14: 14,851cells) outstanding. Our data could confirm the increase of VEGFa in hypertrophic chondrocytes in native bones and to a high extent (higher cell numbers) during bone healing (PFD14). These cells also revealed expression of genes important for matrix calcification like Bglap/Osteocalcin and Spp1/Osteopontin. Specifically, these cells show Sdc-1 expression and in fractured bones higher cell number of Sdc-1 expressing late hypertrophic chondrocytes were found. However, VEGFR2, main receptor of VEGFa in bone, was expressed only in endothelial cells. The expression pattern of these target genes was similar in either, native and fractured bones, but at PFD14 the extent of chondrocytes, especially hypertrophic chondrocytes, and endothelial cells were highly increased.

Discussion and conclusions: Our results stress the importance of the interaction of hypertrophic chondrocytes producing VEGFa most likely bound to Sdc-1 and presenting the ligand to invading endothelial cells. This close proximity of CHO cells enables activation of EC cells to further migrate and induce enchondral ossification. Sdc-1 facilitates the signal transduction between adjacent cells at the chondro-osseous interface and therefore has a significant impact on neoangiogenesis at the chondro-osseous border in bone growth as well as during bone healing. Increased understanding of regulatory processes of the enchondral ossification process, like the interaction of adjacent cells, produced chemokines, receptors and co-receptors in bone growth and bone healing is important for clinical problems like growth disturbances of bones or impaired bone healing.