Cultures of human bone cells were established, maintained, and characterized with respect to several metabolic parameters. These studies were undertaken with a view to using the bone culture system as a means of studying mechanisms of bone metabolism. The donor patients' ages ranged from 1 to 90 years and their disease states included congenital limb anomalies, exostosis, and osteo- and rheumatoid arthritis. Cultures were maintained up to 5 months. The osteoblast-like character of these cells was confirmed with the use of measurements applied to bone cells from other systems. Analyses showed that (a) the cells' appearance resembled that of cultured osteoblasts from other animal sources, b) intracellular cAMP was stimulated by human parathyroid hormone, c) osteocalcin was detected in the medium of all tested bone cell cultures and its production was found to be stimulated by 1,25-dihydroxycholecalciferol, and d) newly synthesized collagen was almost exclusively type I. In contrast, cultures of human fibroblasts, established in one instance from tissue specimens of the same donor patient, grew faster, reached a higher limiting density, and produced a greater proportion of type III collagen than the corresponding bone cells. Furthermore, fibroblasts did not accumulate osteocalcin in their culture medium. The conditions described in this report to maintain human bone cells in culture should provide a suitable test system to study the regulation of human bone metabolism.

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A B S T R A C T A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglyeans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue.Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate.The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibition.Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred.These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix.

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The development of systems for the growth of osteoblasts on bioerodible polymeric matrices was explored. Three classes of bioerodible polymers were studied as possible matrix supports for osteoblast growth: the poly(anhydrides), poly(phosphazenes) and poly(1actic acid/glycolic acid) copolymers. Neonatal calvarial cells from Sprague-Dawley rats were seeded onto polymer disks at a density of 1 x lo4 cells/cm2. Initial attachment and spreading, rate of growth and morphology were determined, and retention of osteoblast-like phenotype was assessed through measurements of alkaline phosphatase activity in the presence and absence of 2,25(OH)2 vitamin 0 3 . All results were considered relative to tissue culture polystyrene.Cells were found to attach to all polymers at 8 hr post-seeding. By 24 hr, cell numbers on all polymers were found to be decreased, except for poly(1actic acid/ glycolic acid). Rat calvarial osteoblasts seeded on poly-(lactic acid/glycolic acid) reached confluency and retained their phenotype.Successful construction of viable osteoblastbioerodible polymer composite materials, as presented in our study, may find their usefulness as grafts for atrophic non-unions of bone, for healing craniofacial and other defects and for use as prosthetic implants or coatings. 'To whom correspondence should be addressed, at M.I.T., Building 56, Room 141, Cambridge, Massachusetts 02139, U.S.A.Composite systems of osteoblast cultures may also find their usefulness in furthering our understanding of bone differentiation, maturation and metabolism in a matrix environment.