Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone

Vascularization and the influence of growth hormone on this process were studied during endochondral bone differentiation. Vascular invasion was monitored by immunofluorescent localization of two vascular basement membrane proteins, type IV collagen and laminin, a recently described glycoprotein. In addition, endothelial cell invasion was identified by localization of Factor VIII. New bone formation was induced by subcutaneous implantation of a coarse powder of demineralized rat bone matrix. On days 1 through 9, no vascular elements were detected in the plaque. Mesenchymal cells appeared on day 3, proliferated, and differentiated into cartilage on day 7, while the capillaries proliferated at the periphery of the plaque. Beginning on day 9 with capillary incursion into the center of the plaque, type IV collagen, laminin, and Factor VIII were localized in the invading vascular endothelial cells. Type IV collagen and laminin appeared synchronously in the capillary basement membranes and later in the endothelial lining of cavernous sinusoids. Their distribution pattern was identical. The vascular invasion was prominent by day 14. In hypophysectomized rats, cartilage differentiated normally but vascularization was delayed and reduced. Bone formation was scanty as indicated by ⁴⁵Ca incorporation. Administration of bovine growth hormone to hypophysectomized recipients restored vascularization and bone formation to the level observed in controls.