Vascularization of primary and secondary ossification centres in the human growth plate

Abstract

Background: The switch from cartilage template to bone during endochondral ossification of the growth plate requires a dynamic and close interaction between cartilage and the developing vasculature. Vascular invasion of the primarily avascular hypertrophic chondrocyte zone brings chondroclasts, osteoblast- and endothelial precursor cells into future centres of ossification. Vascularization of human growth plates of polydactylic digits was studied by immunohistochemistry, confocal-laser-scanning-microscopy and RT-qPCR using markers specific for endothelial cells CD34 and CD31, smooth muscle cells α-SMA, endothelial progenitor cells CD133, CXCR4, VEGFR-2 and mesenchymal progenitor cells CD90 and CD105. In addition, morphometric analysis was performed to quantify RUNX2and DLX5hypertrophic chondrocytes, RANKchondro- and osteoclasts, and CD133progenitors in different zones of the growth plate. Results: New vessels in ossification centres were formed by sprouting of CD34endothelial cells that did not co-express the mature endothelial cell marker CD31. These immature vessels in the growth plate showed no abluminal coverage with α-SMAsmooth muscle cells, but in their close proximity single CD133precursor cells were found that did not express VEGFR-2, a marker for endothelial lineage commitment. In periosteum and in the perichondrial groove of Ranvier that harboured CD90/CD105chondro-progenitors, in contrast, mature vessels were found stabilized by α-SMAsmooth muscle cells. Conclusion: Vascularization of ossification centres of the growth plate was mediated by sprouting of capillaries coming from the bone collar or by intussusception rather than by de-novo vessel formation involving endothelial progenitor cells. Vascular invasion of the joint anlage was temporally delayed compared to the surrounding joint tissue.