In Vivo Effects of an Osteogenic Bone Extract on Cartilage of the Chick Embryo
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IN VIVO EFFECTS OF AN OSTEOGENIC BONE EXTRACT ON CARTILAGE OF THE CHICK EMBRYO Sally R. Frenkel, Ph.D.* Ann B. Prewett, Ph.D.t Richard D. Finkelman, Ph.D.: OBJECTIVES Growth and differentiation of animal cells are influenced by a number of interacting local and circulating protein factors. Several such growth factors, or morphogenetic proteins, that affect hard tissue regeneration and repair have been isolated from bone [1-3]. Bone matrix is a complex milieu consisting of serum, cell, and hydroxyapatite binding proteins, bioactive agents, and other proteins whose functions are not yet known. Most of the growth factors can be dissociated from the decalcified collagen matrix only by using chaotropic solvents such as guanidine hydrochloride or urea [4]. This extraction removes virtually all noncollagenous proteins from the matrix, producing a nonselective portfolio of proteins, of which growth factors are only a small percentage. A series of chromatographic separations are then required to isolate the morphogenetic proteins from the mixture [5]. Recently, a process has been developed that permits selective extraction of certain growth factors from demineralized bone and produces a water-soluble extract of collagenous and noncollagenous proteins. When placed in an orthotopic site in the rat, the extract exhibits pronounced osteogenic activity and has a surface-adherent property that may be exploited in the coating of allograft bone or osteoprosthetic implants to facilitate osseointegration. The objective of this study is to observe the in vivo effects of this extract on proliferation and synthetic activity of cartilage cells of the chick embryo. MATERIALS AND METHODS
Rabbit bone was cleaned, ground to a coarse particulate, washed, and freeze-dried. Particles were exhaustively decalcified in 0.6N HCI and were treated with an inorganic acid solution at an elevated temperature to solubilize and remove proteins loosely associated with the collagen matrix that have no identifiable mitogenic activity. The soluble proteins were separated from the active fraction by filtration. The active fraction was solubilized in acidic solution and concentrated by precipitation using 95% ethanol. The precipitate was resuspended in 0.9% saline at a concentration of 1.0 mg/ml. White Leghorn chick embryos were incubated at a temperature of 37 0C and 60% humidity. On day 12 of incubation, 10 gg of concentrated extract in 0.1 ml saline was injected into the air space of experimental eggs, via a window cut in the shell. Control eggs were injected with saline only. Two additional injections were administered on days 14 and 16. To determine the level of mitotic activity, one-half the eggs were injected on day 17 with 10 tCi 3H-thymidine and sacrificed 90 min after absorption of the isotope. Remaining eggs were injected with 10 .iCi of 3 H-proline to monitor protein synthesis and were sacrificed 24 hr later. The 24-hr time period was chosen so that the isotopic label would appear in secreted matrix products (such as collagen) rather than over cell
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