A Collagen/DBP Sponge System Designed for in Vitro Analysis of Chondroinduction
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A COLLAGEN/DBP SPONGE SYSTEM DESIGNED FOR IN VITRO ANALYSIS OF CHONDROINDUCTION SHUICHI MIZUNO, CHRIS LYCETTE, CHARLENE QUINTO, JULIE GLOWACKI Orthopedic Research, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115
ABSTRACT In response to subcutaneous implants of demineralized bone powder (DBP), cells are attracted to the DBP, are converted to chondroblasts, and produce a cartilage matrix that is resorbed and replaced by bone. In order to define the cellular mechanisms of this induction, we developed a collagen sponge model for simulating the in vivo environment and for promoting the ingrowth and viability of cells cultured in them in vitro. Reconstituted pepsin-digested type I collagen from bovine hide was neutralized. Rat DBP (75-250 gim) was added into the collagen mixture (20 mg/ml). In order to simulate the connective tissue environment, modified chondroitin sulfate, heparan sulfate, or hyaluronic acid was added into the mixture. Aliquots (0.2 ml) were placed in 3/8 inch diameter molds and freeze-dried. Human dermal fibroblasts were cultured from minced fresh tissue and inoculated at 1.5 x 105 cells/sponge. Fifteen hours later, some sponges were transferred to medium which contained growth factors (PDGF or TGF-13). At intervals, samples were examined histologically. The inoculated cells attached to the collagen fibers and migrated into the sponge. Eventually the sponges contracted and acquired an oval shape. Cells on or near DBP were ovoid or stellate in shape. Cell morphology was modulated by glycosaminoglycan composition of the sponge. Increasing doses of PDGF or TGF-P promoted cellularity within the sponges. In conclusion, this system simulates the in vivo environment but allows accessibility for analysis. This three-dimensional matrix culture system will enable investigation of mechanisms of chondroinduction by morphogenic material.
INTRODUCTION Urist reported that demineralized bone and dentin are postembryonic osteoinductive stimuli when implanted in muscle pouches of rodents; the demineralized matrices stimulate the formation of Mat. Res. Soc. Symp. Proc. Vol. 252. 91992 Materials Research Society
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ectopic bone [1]. Reddi and Huggins found that implantation of particles of demineralized bone resulted in a synchronous induction of endochondral bone (2]. We reported that connective tissue cells are attracted to the demineralized bone powder (DBP), converted to chondroblasts, and produce a cartilage matrix that becomes calcified 11-12 days after subcutaneous implantation in a highly synchronized process in CD rats [3]. After mineralization, the cartilage is invaded by vessels, chondrolysis begins, and bone formation follows. More recently, we reported that human dermal fibroblasts produce cartilage-specific matrix in vitro when grown on DBP [4]. Several groups have reported that bone-inductive activity can be extracted from demineralized bone matrix. Urist et al. [5] reported that an 18 K protein derived from bovine bone called bone morphogenetic protein (BMP) in
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