gms | German Medical Science

Objectives: Bone is a highly vascularized organ with many different pathways that effect bone regeneration and remodeling. Recently, we demonstrated that the heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction between osteoclasts and osteoblasts. Furthermore, Sdc1 is well known as an important player in vascularization of tissues during several modification processes e.g. the multiple myeloma.

Since osteogenesis and angiogenesis are closely related in bone, we expected Sdc1 to have an influence on vessel structure during aging. Therefore, angiogenesis of metaphyseal bone in young mice with a high active bone remodeling was compared to aged mice in which bone resorption overbalances bone formation and the influence of Syndecan-1 deficiency was characterized.

Methods: Animals: Growth plates of 36 mice were investigated (C57BL/6 (WT) and Sdc1-/-, 12 mice/timepoint, 4, 12 and 18 months, 6 males and 6 females). Both femura of each mouse were dissected, cryoprotected and embedded.

Sectioning: At -23°C the embedded bones were sectioned into 80 µm thick slices such that the 3D network of the vascularization of the bone could be visualized.

Staining: For each bone two slices from the periphery and two slices from the center of the bone were stained with Endomucin antibody (sc-65495, Santa Cruz Biotechnology) and DAPI as counter staining.

Imaging & Quantification: 3D imaging was performed with Olympus IX83 fluorescence and 2-photone microscopy. The number of bulbs which build the borderline of the blood vessels at the growth plate in bone represents angiogenic activity which was investigated at the interfaces of the growth plate. For the first time, we used a custom made software to quantify vessel number semi-automatically, reproducible and investigator independent.

Results and Conclusion: We verified our custom-made software using slices of WT mice and showed that there is no variation in the number of vascular bulbs related to the width of the growth plate in periphery versus the central zone of bones in all age groups. This indicates a homogeneously distributed angiogenesis throughout this area. Furthermore, in both, WT and Sdc1 deficient mice the number of bulbs decreased significantly with age. However, comparing WT and Sdc1 knockout mice a highly significant decrease in the active sprouting angiogenesis in 4 months and 12 months old Sdc1-/- mice was found whereas 18 months old mice showed no significant differences.

We could demonstrate for the first time that Syndecan-1 has a significant impact on active sprouting angiogenesis during osteogenesis and bone aging. As mice age, the impact of Sdc1 on angiogenesis is decreased and overall angiogenesis is reduced during aging. Using 3D imaging and semi-automated quantification of angiogenesis in long bones, we could extend the possibility of research in this field.