Enzymatic isolation of an osteoblastic preparation suitable for the investigation of cellular calcium transport.

A micro-anatomical 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 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 2 hr, released about 40,000 cell/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%. Measurements of the calcium content in isolated osteoblasts were found to be adversely affected by 3 major factors, including: 1) the presence of calcium-containing particles in the crude collagenase preparation utilized for cell isolation; 2) a calcium-accumulating effect of prolonged collagenase exposure on cell isolates; and 3) the existence of pounced osseous fragment contamination. These detrimental influences were eliminated respectively by: 1) filtration of collagenase solutions prior to bone incubation; 2) reduction of the collagenase exposure time; and 3) utilization of a mild acid incubation to solubilized osseous fragment mineral. These procedures, in concert, reduced the apparent osteoclastic calcium concentration from approximately 160 to 10 mM. The latter value should be considered only a maximal limit at this time since it is not clear if mild acid exposure removes all osseous calcium deposits.