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Bone morphogenetic protein (BMP) induces cartilage and bone development in embryonic and post-fetal life. Bone morphogenetic protein-induced chondrogenesis in outgrowths of muscle connective tissues on various furrowed and unfurrowed fibronectin-coated substrata was observed by biochemical and histologic methods. The substrata consisted of cellulose acetate membranes, atelocollagen, denatured autoclaved demineralized bone matrix, bone matrix deactivated by extraction of BMP with guanidine hydrochloride (GuHCl), and aggregates of insoluble BMP and associated noncollagenous proteins (BMP/NCP) prepared from bovine bone. The furrows, which were mechanically cut into the substrata, were intended to increase surface area and provide extra spaces for cell proliferation under compression. The extent to which extracellular attachments were required for induced cartilage development was reflected in the quantity of cartilage formed when BMP/NCP, either in insoluble or in water-soluble form, was introduced was greatest on GuHCl-extracted bone matrix. On cellulose acetate and atelocollagen, BMP-induced cartilage development was relatively scanty. A substratum of bone matrix, denatured by autoclaving at a minimum of 125 degrees, permitted cell-to-cell adhesion but not cell-to-substratum attachments; the end product was loose fibrous connective tissue only. In contrast, cartilage development occurred on surfaces of undissolved particles of BMP/NCP. Water-soluble human BMP induced development of masses of amorphous cartilage. Even after it was extracted with GuHCl, rat bone matrix may have retained trace amounts of endogenous BMP. Thus, when the requirements of cells for cell-to-cell adhesion and cell-to-substratum attachment, including mechanical factors such as furrows to enlarge surface area, were met, cartilage development was a manifestation of temporal, spatial, and BMP distribution patterns. In situ hybridization and immunofluorescent microscopy with the aid of antirecombinant BMP antibiotics may provide new information about morphogenesis.

An allogenic aggregate of bone morphogenetic protein (BMP) and insoluble non-collagenous proteins (NCP) as well as a crude GuHCl extract were isolated from rats diaphyseal bones. Intramuscular implantation of 5 mg and 10 mg rat BMP/NCP in rats formed new ossicles, whereas 20 mg GuHCl extract failed to induce heterotopic bone formation. When 6 samples of inactivated rat bone matrix gelatin (BMG) were reconstituted with 0.75 mg of either BMP/NCP or GuHCl extract all 3 matrices reconstituted with BMP/NCP but only 1 out of 3 samples reconstituted with GuHCl extract induced heterotopic bone formation. Inactivated BMG alone did not show any osteoinductive activity. The small amount of BMP/NCP necessary for osteoinduction when recombined with inactivated BMG suggests that growth factors in bone matrix without inherent bone-forming activity enhance BMP activity. In vitro, connective tissue outgrowths of neonatal rat muscle on a substratum of inactivated rat BMG differentiated into cartilage in response to 0.05 microgram/ml, 0.5 microgram/ml and 5.0 micrograms/ml allogenic BMP/NCP added to the medium during the incubation period of 2 weeks. On day 14 of cultivation S35-sulphate incorporation into glycosaminoglycans (GAG) and H3-thymidine incorporation into DNA were measured, and the results related to the DNA content and the weight of the incubated muscle tissue, respectively. All doses of BMP/NCP increased GAG synthesis statistically significantly (p less than 0.05 to p less than 0.001). In contrast to that, DNA synthesis rate was not influenced by BMP/NCP. This suggests that GAG synthesis was not caused by cell proliferation but by cell differentiation.

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.

Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG--modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage.

The differentiation of connective tissue outgrowths of adult bone marrow in response to the organic matrix of bone was observed in tissue culture by correlated histologic, electron microscopic, biochemical, and radioisotope-labeling methods. On a substratum of bone matrix gelatin, myelogenous cells degenerate and disappear while stromal and perivascular cells proliferate and differentiate into mesenchymal-type, monocytoid, and giant cells. From primary cultures of bone marrow cells on bone matrix, cartilage differentiates in isolated areas but only in small islets. With continuous subculture through 25 generations, the proportions of two functionally different populations of chondrogenetic and matrix-resorbing cells gradually emerge. Up to the time of the ninth generation of subculture, the chondrogenetic population predominates. After the 14th to the 25th generation, clones of matrix-resorbing large monocytoid cells predominate and rapidly digest the matrix substratum. Measurements of 35S uptake demonstrate that control cultures of muscle-derived mesenchymal-type cells produce about twice as much cartilage as marrow-derived mesenchymal-type cells. A decline in the chondrogenetic cell population and corresponding rise in the matrix-resorbing cell population is demonstrable by a progressive increase in the quantity of hydroxyproline-containing peptides in the culture medium. This decline is not attributable to conditions in culture because there was progressive loss of chondrogenetic activity of the eighth and 20th passage even when the cells were transplanted in diffusion chambers back into an isologous host. The problem is how to account for the competence of marrow stromal cells to differentiate into bone without bone matrix in vivo but not in vitro. Mixed cultures of muscle and marrow outgrowths produce only half as much cartilage (measured by 36S-uptake/microgramDNA in the system) as muscle outgrowths alone. These observations suggest that a bone marrow-derived matrix-resorbing cell population, by some unknown mechanism, inhibits proliferation of cartilage-bone precursor cell populations. The nature of the inhibition requires investigation by detailed biochemical analyses of marrow cell culture media and chemical extracts of whole bone marrow tissue.

Fetal rat calvarium in an optimum (CMRL-1066) culture medium (with respect to amino acids. Tween 80, DNA precursors and hydrogen acceptors used in oxidative metabolism) produces new lamellar bone. The explanted osteoprogenitor cells survive, proliferate on living membrane bone surfaces and remodel membrane bone into lamellar bone. In the same culture medium, calvarial connective tissue and osteoprogenitor cells grow out of the membrane bone interstices onto a substratum consisting of bone matrix gelatin (BMG) and differentiate not into bone but into hyaline cartilage. Outgrowths from either bone or muscle onto a substratum of BMG prepared from bone autodigested in neutral buffer solutions, produce only fibrous connective tissue. In BGJ, a culture medium containing suboptimal ingredients for cell proliferation, calvarial bone produces neither lamellar bone nor new cartilage but is gradually resorbed and replaced by fibroblasts. In CMRL culture medium, outgrowths of mesenchymal-like cells from muscle onto a substratum of living membrane bone produce an epiphyseal plate-like columnar deposit of new hyaline cartilage. These observations suggest that the function of BMG is to evoke mesenchymal cell differentiation into prechondroblasts during the latent or migratory morphogenetic phase while the effect of the culture medium is to provide the bionutritional requirements for synthesis of hyaline cartilage matrix by chondrocytes during the patent phase of development.

Connective tissue outgrowths of neonatal muscle onto a substratum of bone matrix differentiate into cartilage in response to a bone morphogenetic protein (BMP). The BMP can be separated from bone matrix by extraction with 4 M guanidine hydrochloride (GuHCl) or degraded in situ by endogenous proteolytic enzymes to deactivate the matrix. Rat triceps muscle was minced in a suspension of noncollagenous bone matrix proteins including BMP (BMP/NCP) in culture medium. To investigate the possible synergistic interactions in induced chondrogenesis, six biosynthesized, highly purified growth factors were similarly added to the culture alone or in combination with BMP. Human interleukin-1 (IL-1) and Forskolin were also introduced to test the effects on BMP/NCP-induced chondrogenesis. On Day 14 of cultivation, [3H]thymidine incorporation into DNA and [35S]sulfate incorporation into glycosaminoglycans (GAG) were measured, and the values were expressed as percentages of the control. The quantity of induced cartilage formation was estimated by a histomorphometric scoring system. Under the influence of BMP/NCP, cultures grew on deactivated matrix, incorporated 55% more [3H]thymidine into DNA, incorporated 115% more [35S]sulfate into GAG than control cultures, and differentiated into cartilage. Without BMP/NCP, growth factors, IL-1, and Forskolin did not produce a comparable incorporation of either [3H]thymidine or [35S]sulfate, and they induced differentiation of fibrous tissue only. In the presence of BMP/NCP, cartilage developed in nearly all cultures. When the media were supplemented with growth factors, measurable increases in uptake of [3H]thymidine occurred with human epidermal growth factor (h-EGF), insulin-like growth factor-1 (IGF-1), nerve growth factor (NGF), transforming growth factor-beta (TGF-beta), bovine acidic fibroblast growth factor (baFGF), IL-1, bovine basic fibroblast growth factor (bbFGF), and Forskolin. Measurable increases in uptake of [35S]sulfate into GAG occurred with IL-1, baFGF, TGF-beta, h-EGF, IGF-1, bbFGF, NGF, and Forskolin. Synergistic interaction with BMP was considered when the quantity of cartilage developed (on a scale of 0-12 scores) in excess of the quantity of Score 4 induced by BMP/NCP alone. A cytokine, IL-1, had the greatest effect (Score 9). TGF-beta (Score 7), baFGF (Score 6), and NGF (Score 6) had relatively little effect. h-EGF, IGF-1, bbFGF, and Forskolin had no effect on cartilage development.(ABSTRACT TRUNCATED AT 400 WORDS)

Outgrowths of mesenchyme-type cells from explants of allogeneic rat muscle onto a substratum of bone matrix containing bone morphogenetic protein (BMP) differentiate into cartilage. When BMP is chemically extracted from the bone matrix, the explanted cells develop only into fibrous tissue. When exogenous bovine BMP is introduced into the culture medium, either as a microsuspension or as a layer of particles between the matrix and the muscle cell tissue, cartilage develops at the interface between the matrix and the mesenchymal cell outgrowth. The chondrogenetic response is induced by as little as 2 micrograms of BMP; the optimum dose is 10 micrograms/40 mg (wet weight) of explant. The endogenous BMP equivalent for a comparable chondrogenetic response is about 0.6 micrograms/mg of allogeneic matrix. The minimum time for transfer of BMP to mesenchymal cell receptors is 1.0 hour, adequate time is 2.5 hours, and optimum time is approximately 5.0 hours. Measured in terms of incorporation of 3H-thymidine into DNA and of 35S sulfate into glycosaminoglycan, there is a latent period of one to three days preceeding the differentiation of mesenchyme-type cells into cartilage. During this latent period BMP-modulated mesenchymal cells disaggregate, migrate, reaggregate, and proliferate on new surfaces and constitute the morphogenetic phase of bone development. By the fourth day cells simultaneously undergo mitotic division, synthesize extracellular cartilage matrix, and establish the cytodifferentiation phase of development.

Bone matrix gelatin, prepared by chemical extraction of soluble noncollagenous proteins, was half digested with a chromatographically purified collagenase. The residue was placed on one side and autologous muscle on the other side of cellulose acetate membranes in diffusion chambers and tissue cultures. In this avascular system, the muscle septa connective tissue proliferated and differentiated into cartilage. Muscle tissue cultured in media conditioned with matrix residues and then transferred into a vascularized muscle pouch differentiated into cartilage and bone. These observations form the basis for a working hypothesis that myositis ossificans is a response of new populations of proliferating intramuscular connective tissue cells to a bone matrix-derived diffusible molecule.

Urea and guanidine extracts of demineralized beef and rabbit bone matrix were assayed both in vivo and in vitro. One month following intramuscular implantation into mouse thighs, these extracts induced ectopic cartilage and bone. Seven days following continuous in vitro exposure to the same extracts, stage 24 chick limb bud mesenchymal cells in cultures had differentiated into greater numbers of chondrocytes than controls. These results suggest the feasibility of using limb bud mesenchymal cell cultures as an in vitro assay for bone matrix derived, extractable bioactive factors which effect the conversion of mesenchymal cells into chondrocytes as a requisite step in in vivo osteogenesis.

A bone morphogenetic protein (BMP) fraction obtained from the organic matrix of dentin is extractable from human osteosarcoma. A fresh en-bloc specimen excised from the femur of a 16-year-old boy was extracted with 4 M guanidine hydrochloride. The GuHCl-soluble, water-insoluble protein fraction induced new bone formation when implanted into the thigh muscles of athymic nude mice.

The ultrastructure of post fetal chondrogenesis was investigated in chondrogenetic implants and explants of bone matrix gelatin: (A) in direct contact with living cells; (B) separated from living cells by cellulose acetate membranes; (C) in serum or in a culture medium with no contact with living cells. Ruthenium red (RR) staining coarse granules free of interconnecting filaments aggregated in interstitial fluid and inside the surface of the bone matrix gelatin within 72 hours in systems with either direct or indirect contact with living connective tissue cells. No RR granules formed when control denatured or autolysed bone matrix gelatin were substituted for undenatured bone matrix gelatin. No RR granules formed in bone matrix gelatin incubated in serums or media in the absence of living cells in the system. These observations suggest the possibility that 4 days before overt transmembrane cartilage cell differentiation, covert (presumptive) chondroblasts synthesize and secrete precursor substance for aggregation of filament-free RR granules. The relationship between chondromorphogenesis and filament-free RR granules requires further investigation.

Connective tissue outgrowths of muscle occur in vitro onto a substratum of bone matrix gelatin (BMG) chemically modified by various inorganic and organic cations or anions. Outgrowths onto BMG denatured by lyotropic concentrations of Ca2+ or complexed by only 1 mmole/1 of Cu2+ differentiate into fibrous tissue only. Onto BMG complexed with either positively or negatively charged organic molecules, the product was also fibrous tissue. Outgrowths onto undenatured and partially recalcified MBG differentiated into cartilage. These observations substantiate the view that differentiation of cartilage in tissue culture occurs in response to the undenatured form of a bone matrix protein molecular and not an unspecific surface net electrochemical charge.

Dunn osteosarcomas synthesize 2 times more alkaline phosphatase than do Ridgeway osteosarcomas, 3 times more than do HeLa cells, and 4 to 5 times more than do rat or mouse fibroblast cell cultures. Implants of killed freeze-dried Dunn cell cultures into the thigh muscles are resorbed and replaced by normal cartilage, bone, and bone marrow tissue, while implants of freeze-dried Ridgeway cells are resorbed and replaced by fibrous tissue only. Outgrowths of normal muscle septum connective tissue cells onto the stroma of Ridgeway tumors differentiate into fibrous tissue. Cultures of either tumor on a substratum of bone matrix stroma prepared from normal bone proliferate, assume a spherical shape, and perpetuate the transformed osteoblast-like cell without forming attachments or adapting to the contour of the substratum. Outgrwoths of muscle mesenchymal cells on the Dunn tumor stroma differentiate into cartilage. Dunn osteosarcoma cell cultures proliferate on the inside and produce deposits of normal bone (not tumorous bone) on the outside of diffusion chambers. Killed freeze-dried cell cultures produce transfilter deposits of normal bone and bone marrow, but the quantity is significantly lower. On a substratum of cellulose acetate, outgrowths of muscle connective tissue will differentiate into cartilage when cell-free Dunn stroma is present under the organ culture grid. Tumorigenesis and normal cartilage and bone morphodifferentiation are antithetic, but tumor cells transfer a bone morphogen similar to the bone morphogenetic protein (BMP) of normal bone matrix. BMP recruits mesenchymal cells to proliferate and differentiate into cartilage and bone.

The bull shark, Carcharhinus leucas, employing archaic but effective means of regulating the physical-chemical composition of its body fluids, thrives in tropical fresh-water rivers and lakes. The ionic strength of the serum and the concentrations of total solutes, calcium, urea, and other ions are below the levels found in marine elasmobranchs but higher than the levels in teleosts. The patterns of the calcium deposits of the vertebrae are identical in marine and fresh-water subspecies.