Internalization and intracellular processing of bone morphogenetic protein (BMP) in rat skeletal muscle myoblasts (L6).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily capable of inducing bone and cartilage formation in ectopic extraskeletal sites and transducing their effects through binding to serine-threonine kinase receptors. In this study, the fate of 125I-labelled native BMP after binding to cell surface receptors on L6-myoblasts was examined with both continuous and intermittent exposure of the ligand. BMP was readily internalized in L6 cells at +37 degrees C, and the internalization reached a plateau in 2 h. Intracellular degradation of 125I-labelled BMP was established, and degradation products were also detected in binding buffer, indicating exocytosis of the processed ligands. BMP receptors were shown to be subject to acute down-regulation by the ligand, and receptors were completely recycled in 3 h. Hence, we conclude that BMP receptors, like receptors for various other polypeptide ligands, have the ability to mediate intracellular delivery and degradation of the ligand.

Use of myoblasts in assaying the osteoinductivity of bone morphogenetic proteins.

A novel, time- and BMP-saving in vitro method for the detection and quantitation of bone morphogenetic protein (BMP) activity was developed based on the measurable effects of BMP on rat skeletal muscle myoblasts (L6). Calcium incorporation, stimulation of alkaline phosphatase activity and production of osteocalcin were used as markers of bone cell metabolism and on-going morphogenesis. The morphological change was confirmed by Chlorantine fast red and von Kossa staining. The response of various BMPs was purity-dependent and consistent with intramuscular implantations of the same materials. Neither TGF-beta1 nor insulin could induce the same actions. The data from this study indicate that at least in part in vivo implantations of BMP extracts can be replaced by in vitro measurement of osteoinductivity. Considerable saving of time, BMP and experimental animals can be achieved using cell culture conditions for the determination of bone-forming activity.

The role of transforming growth factor-beta on retarded osteoblastic differentiation in vitro.

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.

Purification of monocomponent bovine bone morphogenetic protein in a water-soluble form.

Noncollagenous protein material was extracted from HCl-demineralized bovine bone particles in 4 M guanidinium hydrochloride. Water- and citrate buffer-insoluble material was collected, solubilized in 6 M urea and fractionated by preparative isoelectric focusing using a running voltage of 5000 V. The material removed from the area between pH 4.7 and 5.7 of the isoelectric focusing gel was osteoinductive (identified by its capacity to induce bone development). This was solubilized in 6 M urea and dialyzed against 0.2 M Tris buffer. The Tris buffer-soluble material was fractionated by HPLC gel filtration. The water- and citrate buffer-insoluble material contained mainly high-molecular-weight protein complexes which were osteoinductive, and < 5% of the material was osteoinductive monocomponent bone morphogenetic protein. The Tris buffer-soluble material contained only two polypeptides: an osteoinductive peptide of molecular weight 18,500 and a non-osteoinductive peptide of molecular weight 8,000. The very high voltage used during the isoelectric focusing caused a slow break-down of the urea-soluted protein complexes, which significantly increased the yield of monocomponent bone morphogenetic protein. By the present method it is possible to prepare Tris buffer solution containing up to 2 mg/ml of pure monocomponent bone morphogenetic protein.

High yield of osteoinductivity can be derived from demineralized bone matrix using collagenase digestion.

A bone morphogenetic protein purification method for minor quantities of bone material was developed based on collagenase splitting of bone connective tissue. Our aim was to remove and characterize the osteoinductive protein preparation in native form without using strongly dissociative agents. We started from 80 g of HCl-demineralized reindeer bone material which was treated with type I collagen splitting collagenase. The solution was dialyzed against 10 mM glycine-HCl buffer, pH 5.2. The formed precipitate was found to be osteoinductive. After fractionation of the material using HPLC gel filtration it was observed that the high-molecular-weight component of the precipitate was biologically active. Isoelectric focusing revealed that the component consisted of at least eight different protein molecules. Lower-molecular-weight components induced no bone formation. These preliminary findings suggest that in native form at least one part of BMP is in a complex form and other extracellular matrix components bound to the osteoinductive protein complex are significant for BMP action and may act synergistically or as carriers for the BMP.

Partially purified reindeer (Rangifer tarandus) bone morphogenetic protein has a high bone-forming activity compared with some other artiodactyls.

Noncollagenous proteins, including bone morphogenetic protein (BMP), were extracted in 4 mol/l guanidinium hydrochloride (GuHCl) from the pulverized and HCl-demineralized matrix of reindeer, bovine, sheep and porcine bone. To remove water-soluble material, the GuHCl solution was dialyzed against water and water-insoluble material and redissolved in 4 mol/l GuHCl. Gelatin peptides were removed by extraction in 0.25 mol/l citrate buffer (pH 3.1). The yield consisted mostly of large complexes and protein molecules of molecular weight less than 35,000 daltons. Isoelectric focusing of the material showed three to four different protein molecules: three acidic and one neutral. Bone-forming activity was investigated by implanting 0.6-15.0 mg of partially purified protein preparation into the thigh muscles of BALB mice. Radiologically detectable formation of new bone required 0.6 mg of reindeer BMP, 2.5 mg of bovine BMP, 5.1 mg of sheep BMP, and 8.0 mg of porcine BMP. A rough estimate of the area of the deposits showed that reindeer BMP had the highest bone formation activity, and porcine had the lowest. The formation of new bone was confirmed histologically. It is suggested that the differences in osteogenic activity are due to quantitative differences in BMP constituents or in the degree of complex formation in the protein preparations. Also immune and other defense mechanisms may generate differences in osteogenic response.

Measurement of total and local bone morphogenetic protein concentration in bone tumours.

Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.