Differentiation of Cartilage on Three Substrata Under the Influence of an Aggregate of Morphogenetic Protein and Other Bone Tissue Noncollagenous Proteins (BMP/iNCP)

Abstract

A cellulose acetate membrane was fashioned into a cone to serve as a substratum for an outgrowth of connective tissue from normal neonatal muscle, and a container for diffusion of bone morphogenetic protein (BMP) of an aggregate of BMP and cold water insoluble noncollagenous protein (BMP/iNCP). The BMP/iNCP was prepared by dissociative extraction and differential precipitation with other bone matrix proteins that slowly become soluble and diffusible in culture media at 37 degrees. The BMP/iNCP was applied either on, within, or beneath the surface of the explants or suspended in the culture medium. Under the influence of BMP in tissue cultures, without any bone matrix or bone collagen in the system, connective tissue outgrowths of muscle differentiate into cartilage on three substrata: (1) cellulose acetate membranes with pore size of 0.45-5.0 micron; (2) remnants of undissolved BMP/iNCP; and (3) degenerating myofibers. The cartilage developed in the interior of muscle, possibly by phenotypic cell transformation, when the pore size of the membrane was 0.1-0.22 micron too small to sustain anchorage of the explant. Cartilage developed on particle surfaces when the muscle tissue and BMP/iNCP particle were minced and mixed before explantation. The cartilage preferentially grew out directly onto the cellulose acetate membrane when the pore size was optimal for anchorage and the BMP/iNCP was suspended on the surface of the explant to either simultaneously percolate through the explant or diffuse through the culture medium. The biosynthetic activity of cells proliferating before and associated with cell differentiation was measured by 35S uptake in total glycosaminoglycan (GAG) per microgram of DNA. When the pore size was 8.0 micron, large enough to permit cells to migrate across the membrane, a thick plate of fibrous connective tissue developed on the undersurface of the membrane without any evidence of cartilage cell differentiation in any location. Repeated doses of BMP/iNCP with each change of culture medium produced a greater incidence and quantity of cartilage than a single dose, but the 35S incorporation into GAG always reached peak levels, in the interval between four and ten days, irrespective of the schedule of administration or dosage. These observations suggested that the exogenous or endogenous noncollagenous proteins are a carrier for BMP and can substitute for whole bone matrix or bone collagen.