Enzymatic isolation of osteoblasts from fetal rat calvaria

A microanatomical study of the immature rat calvarium was performed in order to develop a method for the isolation of osteoblasts by enzymatic means. Although generalized osteogenesis was evident in fetal rats, differential growth patterns were observed beginning at 19 days in utero. Considerable portions of the endocranial periosteal bone surface were lined by flattened, less active cells; discrete areas also contained multinucleated osteoclasts. Cell counts of whole calvaria revealed that one fifth of the total cell population were osteoblasts, most of which were located in the central portions of the frontal and parietal bones. Prior excision of these segments permitted the subsequent removal of virtually all periosteal tissues. Cleaned 19-day fetal bones, incubated in crude collagenase for two hours, released about 40,000 cells/calvarium, consisting of 85-90% osteoblasts and lesser amounts of connective tissue and bone marrow elements. Because of the relatively small sizes of most extraneous cells, purity on a cell volume basis was approximately 95%. It is predicted this preparation will be useful in the investigation of certain aspects of osteoblastic function.