Blood vessel invasion at chondro-osseous interfaces is impaired in absence of Syndecan-1 in bone growth and fracture healing

Introduction: Neoangiogenesis drives the replacement of mineralized cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) while interacting with VEGF and OPG, plays a role in the communication of osteoclasts and osteoblasts and in the development of blood vessels. To understand the function of Sdc1 in enchondral ossification we analysed bone structure and vessel development in bone growth and fracture healing in mice deficient in Sdc1.

Methods: Femora of C57BL/6 WT (n=11) and Sdc1-/- (n=13) mice (male/female) were used for native bone analysis at 4 month age. Female mice (WT n=6-14, Sdc1-/- n=6-8, per time point) underwent midshaft femur fracture stabilized using an intramedullary nail and healed for up to 28 days. Bone structure was analysed using microCT scans with a resolution of 9µm. Fracture callus composition was quantified after Alcian Blue staining as callus area, fibrous tissue, cartilage and trabecular bone in 5µm thick slices. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. Bone marrow isolates (WT/Sdc1-/-) were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel.

Results: Bone structure in 4 month old male Sdc1 deficient mice was significantly reduced compare to age matched male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice showed a significant less number of vessel buds at the chondro-osseous border at the growth plate at the age of 4 month compared to WT mice in male and female mice. During fracture healing, callus development was delayed with regard to cartilage area at day 7 and trabecular bone area at day 14. A decreased number of vessel buds invading at the borderline of cartilage to bone in the callus were counted in Sdc1 deficient callus tissue. Quantification of microvessel outgrowth of endothelial cells from bone marrow in matrigel revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT.

Discussion: Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone as well as during bone healing in the callus area. This emphasises the importance to further characterise the mechanism, how Syndecan-1 regulates the process of endothelial invasion during enchondral ossification.

Keywords: Fracture healing, vascularization, Syndecan-1, regeneration

Korrespondenzadresse: Richard Stange, University Hospital Münster, Institute of Musculoskeletal Medicine, Department of Regenerative Musculoskeletal Medicine, Waldeyer Straße 1, 48149 Münster, Deutschland, E-Mail: [e-mail]

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Artikel online veröffentlicht:
13. März 2024