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Four different cell populations--designated PF, OB, OC, and PC--were isolated from calvaria of 18-day-old chick embryos for analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods. Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between the osteoblastic layer and calcified matrix. These cells were provisionally called osteocytic osteoblasts. They represent the "transition state" between osteoblasts and osteocytes. On the basis of histological studies with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), the PF population was considered to originate primarily from the periosteal fibroblasts, the OB population from the osteoblasts and osteocytic osteoblasts. The population of cells still present in calvaria from removal of periosteal fibroblasts and osteoblasts was called the OC population. This cell population was very much enriched with osteocytes. The fourth isolated population (PC) was a mixed population of fibroblasts, osteoblasts, and preosteoblasts. On exposure to parathyroid hormone (PTH), all four cell populations showed increased lactate production, but only the OB and OC populations displayed increased cAMP production. Prostaglandin E1 (PGE1) stimulated cAMP production in both OB and PF cells. From the results of this study it was concluded that PTH receptors are present on all of the cell types studied, but that occupancy of the receptor induces adenylate cyclase stimulation only in osteocytes and fully differentiated osteoblasts.

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The presence of proteoglycans (PGs) was studied in compact lamellar rat and human bone at the electron microscopic level. With the cationic dye cuprolinic blue (CB1), PGs could be demonstrated in the mineralized bone matrix. The amounts of PGs appeared to be equal in the different lamellae and osteons. More CBl-positive material was found in the outermost lamella of the cortex, in the perilacunar matrix around the osteocyte lacunae, and around the canaliculi. Enzyme digestion with chondroitinase ABC demonstrated that the CBl-positive rods consisted of PGs. These observations amplify biochemical studies in which PGs have been isolated from the mineralized bone matrix. The presence of CBl-positive rods in the mineralized matrix suggest that PGs do not have to be removed completely to make the matrix calcifiable.