Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a polyanhydride delivery vehicle.

Controlled release delivery vehicles for water-soluble osteogenic proteins from demineralized bovine bone matrix were constructed using polyanhydride polymers. The water-soluble proteins were isolated from a 4 M guanidine hydrochloride extract of bone matrix. The water-soluble proteins possessed Chondrogenic Stimulating Activity (CSA) when tested in stage 24 chick limb bud cell cultures, but were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice by themselves. The polyanhydride polymers alone were also incapable of inducing ectopic cartilage or bone. However, when the water-soluble proteins were incorporated into the polymeric delivery vehicle, the combination was capable of inducing cartilage and bone up to 50% of the time. These results demonstrate that it is possible to use polyanhydride polymers as controlled-release delivery vehicles for soluble bioactive factors that interact with a local cell population.

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Heterogeneity of latent transforming growth factor-beta isolated from bone matrix proteins.

Transforming growth factor-beta (TGF beta) is a family of 25-kDa peptides that appear to be important regulators of cell proliferation, differentiation, and differentiated function in many tissues. TGF beta is present in platelets and serum and is released by cultured cells as several distinct large mol wt complexes of TGF beta with other proteins. These complexes are biologically inactive and are generically called latent TGF beta (L-TGF beta). Large quantities of TGF beta are present in bone matrix. This study was undertaken to determine whether the TGF beta in bone matrix was present as a free 25-kDa peptide or a large mol wt L-TGF beta complex. TGF beta activity was determined by inhibition of [3H]methylthymidine incorporation in mink lung epithelial cells. The specificity of inhibition was determined by treating fractions with a polyclonal rabbit antiporcine TGF beta-blocking antibody before assay. Latency was examined by assaying untreated and acid-treated fractions for TGF beta activity. Acid treatment of EDTA extracts of the bovine bone matrix proteins increased TGF beta activity from a mean of 0.8 pg/microgram protein to 56 pg/micrograms. Under native conditions L-TGF beta eluted from S400 between the 600-400 kDa mol wt standards. No activity eluted in the fractions with authentic 25-kDa TGF beta. Eighty-five percent of the L-TGF beta bound to lentil lectin, and this separated into four discrete L-TGF beta peaks (I-IV) at 0.22, 0.25, 0.35, and 0.42 M NaCl with Mono-Q anion exchange chromatography. Mono-Q pools II and III were reseparated by molecular size on Superose-12 under native and dissociative conditions. Under native conditions TGF beta activity was latent and eluted in the large mol wt fractions. No 25-kDa TGF beta was present. With dissociating conditions (4 M GuHCl) all TGF beta activity eluted in the small mol wt fractions identical to the elution position of authentic 25-kDa TGF beta. The active fractions from the dissociative separation of Mono-Q pool III were separated by C4 reverse phase HPLC. There were three discrete peaks of TGF beta activity corresponding to TGF beta 1, TGF beta 2, and an unidentified form of TGF. Maximum activation of L-TGF beta in each Mono-Q peak occurred at pH 3-3.5. There was partial activation at pH 4.5, but no additional activation at pH 1.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells.

The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.

Regional distribution of mineral and matrix in the femurs of rats flown on Cosmos 1887 biosatellite.

We combined biochemical measurements with novel techniques for image analysis in the rat femur to characterize the location and nature of the defect in mineralization known to occur in growing animals after spaceflight. Concentrations of mineral and osteocalcin were low in the distal half of the diaphysis and concentrations of collagen were low with evidence of increased synthesis in the proximal half of the diaphysis of the flight bones. X-ray microtomography provided semiquantitative data in computer-generated sections of whole wet bone that indicated a longitudinal gradient of decreasing mineralization toward the distal diaphysis, similar to the chemistry results. Analysis of embedded sections by backscattered electrons in a scanning electron microscope revealed distinct patterns of mineral distribution in the proximal, central, and distal regions of the diaphysis and also showed a net reduction in mineral levels toward the distal shaft. Increases in mineral density to higher fractions in controls were less in the flight bones at all three levels, with the most distal cross-sectional area most affected. The combined results from these novel techniques identified the areas of femoral diaphysis most vulnerable to the mineralization defect associated with spaceflight and/or the stress of landing.

A simple and efficient scheme for the purification of insulin-like growth factor II from human bone matrix extract.

Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10-15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence. From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source of IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.

Purification of bone morphogenetic protein derived from bovine bone matrix.

Bone morphogenetic protein (BMP) was extracted from the bovine bone matrix and purified by liquid chromatography. The molecular weight of the BMP was 18 kDa by SDS-PAGE, and its pI value was 4.9. Amino acid analysis suggested that the BMP is a polypeptide containing 163 amino acids. In the present study, telopeptide-free type I collagen was used as a carrier of BMP.

Immunoreactive calbindin-D9K localization in matrix vesicle-initiated calcification in rat epiphyseal cartilage: an immunoelectron microscope study.

Calbindin-D9K immunoreactivity was localized by electron microscopy in rat calcifying epiphyseal plate cartilage. Antigen-antibody reaction sites were visualized by the presence of protein A-gold complex particles on undecalcified material embedded in Lowicryl K4M. Immunoreactive calbindin-D9K was found in the hyaloplasm of hypertrophic chondrocytes and inside and at the ends of their cell processes. It was localized outside the cells, inside matrix vesicles (MVs), often against the inner face of the delimiting membrane, and inside the trilaminar membrane. Immunoreactive calbindin-D9K appeared to be extruded from the chondrocytes into the matrix vesicles when the latter were formed during the budding of cell processes. In calcifying MVs, gold particles were detected over the needle-shaped crystallites and often over the crystallites lying against the inner leaflet of the vesicular membrane. At a later stage of matrix vesicle calcification after MV membrane disruption, the number of gold particles remained unchanged over the clusters of crystallites at the loci from which the crystallites appeared to have grown and radiated. At a yet more advanced stage of calcification, they remained in the same areas, which were limited to the lateral edges of calcified cartilage longitudinal septa. These results suggest that immunoreactive calbindin-D9K plays a role in calcium input to matrix vesicles and may be involved in matrix vesicle calcification, perhaps in the initial event of matrix vesicle crystal nucleation.

Separation of bone matrix proteins by calcium-induced precipitation.

It was found that significant precipitation occurred immediately after calcium, at a concentration as low as 2 mM, was added to a desalted solution of EDTA extract of adult bovine femur. The maximal yield of the precipitates was observed at a calcium concentration of 30 mM. These precipitates were dissolved in 0.5 M EDTA, desalted, and characterized by Sepharose CL-6B gel filtration chromatography and high performance gel-exclusion chromatography. Results revealed that the precipitates were enriched in a 40 K protein and a higher molecular weight fraction as compared with the original extract of bone proteins. The 40 K fraction was isolated and identified as osteonectin, as judged from amino acid analysis, electrophoresis, and immunodetection. The supernatant after calcium-induced precipitation predominantly contained osteocalcin and a 50 K protein that was tentatively identified as alpha 2HS protein. Osteonectin was purified from the calcium-induced precipitates from the EDTA extract of bovine bone. By calcium titration using fluorescence spectrometry, the isolated osteonectin showed high affinity to calcium ions with an apparent dissociation constant (K0.5) of 8 x 10(-7) M. Thus, the use of calcium to separate bone proteins, especially osteonectin, was proved to be a useful technique. In addition, calcium-induced precipitation of osteonectin suggested a possible in vivo mechanism via which osteonectin might interact with calcium ions and participate in the initial immobilization of calcium to induce the nucleation of calcification in bone tissue.

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a collagenous delivery vehicle.

A controlled-release delivery vehicle for water-soluble osteogenic proteins from demineralized bone matrix was constructed using purified type I collagen. The water-soluble proteins were isolated from a 4 M GdnHCl extract of bone matrix. Although the water-soluble proteins were capable of inducing cartilage formation in vitro, they were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice in the absence of an appropriate delivery vehicle. The collagen-based delivery vehicle alone was also incapable of inducing osteogenesis in vivo. However, when the water-soluble proteins were incorporated into the delivery vehicle, the combination was capable of inducing cartilage and bone 76% of the time. These results demonstrate that it is possible to formulate a controlled-release delivery vehicles for soluble bioactive factors which upon release interact with local responsive cells.

Thrombospondin is an osteoblast-derived component of mineralized extracellular matrix.

Thrombospondin, the most abundant protein of platelet alpha granules, is a biosynthetic product of a variety of connective tissue cells and a component of many extracellular matrices. In this study, thrombospondin distribution in bone was investigated using a monoclonal antibody specific for the human protein. Thrombospondin was localized in osteoid of undemineralized, frozen sections of fetal subperiosteal bone, and identified as a component of mineralized bone matrix of neonatal and/or young (growing) bone of many animal species by Western blot analysis. Adult human bone cells were demonstrated to contain mRNA for thrombospondin by hybridization of a cDNA thrombospondin probe to a 6.1 kb mRNA. Pulse-chase experiments indicated that the protein was synthesized and the majority was secreted from osteoblastic cells. Treatment of the cells with TGF-beta (0.01-10 ng/ml) slightly decreased total thrombospondin synthesis, but caused an increase in the retention on newly synthesized thrombospondin in the cell layer/matrix fraction. In cell attachment assays, thrombospondin mediated adhesion, but not spreading of adult human bone cells.

A comparative ultrahistochemical study of glycosaminoglycans with cuprolinic blue in bone formed in vivo and in vitro.

Histochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization process in vivo but such studies have not yet been conducted in vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formed in vivo and bone nodules formed in vitro from osteoblastic cells in culture. For this investigation, we used a cationic phthalocyanin dye, cuprolinic blue, in a critical electrolyte concentration which simultaneously stained the glycosaminoglycans and demineralized the bone. This histochemical technique demonstrated (1) osteoblast cells in vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix both in vivo and in vitro. In the uncalcified matrix, PG were either free with a granular or rodlike structure or tightly connected to the periphery of the collagen fiber. Contrarily, in the calcified matrix, PG formed dense filamentous reticular patches between the collagen fibers. (3) Similarities in localization, organization, and morphology were noted in PG of bone formed de novo in vitro and in vivo with the exception of the mineralization front, where the staining in vivo compared with in vitro was faint or absent.

Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone.

Immunocytochemistry utilizing the protein A-gold technique was used to examine the ultrastructural cellular and extracellular distribution of a major phosphoprotein in chick bone. HCl-extracts of embryonic and neo-natal chick bones contain a major 66kD phosphoprotein (BPP) which was purified and used to raise polyclonal antibodies in rabbits. The mid-diaphyseal regions of 8-, 12- and 18-day embryonic chick tibiae were fixed with 1% glutaraldehyde and embedded in Epon or Lowicryl. Electron microscopy following incubation of tissue sections with the antibody and the protein A-gold complex revealed specific immunolabeling over the rER and Golgi apparatus of osteoblasts and over those areas of bone matrix containing Ca and P as determined by electron probe x-ray microanalysis. These included extracellular areas in the matrix undergoing early mineralization and electron dense patches occurring at the mineralization front and extending throughout the more mature bone regions. Biochemical analyses of bone tissue processed similarly to that used for immunocytochemistry confirmed the retention of phosphoprotein in the tissue. The spatial correlation of phosphoprotein in the extracellular matrix with Ca-P mineral deposits confirms an earlier report using 33Pi and radioautography and may indicate a role for phosphoproteins in calcification.

A study of the mechanical strength of long bone defects treated with various bone autograft substitutes: an experimental investigation in the rabbit.

This study was designed to determine which of several bone grafting materials would be the most efficacious substitute for autogenous bone graft in the treatment of segmental long bone defects. The experimental model was a 1-cm defect in the rabbit ulna. The control group had nothing implanted in the defect. The six grafts tested were: (a) autogenous iliac crest bone, (b) autogenous cortical bone (ulna), (c) hydroxylapatite, (d) hydroxylapatite-demineralized bone matrix (allograft) composite graft, (e) freeze-dried bone (allograft), and (f) demineralized bone matrix (allograft). At 6 weeks postoperatively, the ulnas were harvested, examined radiographically, and tested mechanically in torsion. The radiographic examination proved to be of little value because some materials were radiodense at the time of implantation. The rates (percentage) of union, torques at failure, and energy to failure values were statistically significantly higher than control for all groups except hydroxylapatite. We concluded that demineralized bone matrix and hydroxylapatite-demineralized bone matrix composite graft compare favorably with cortical replacement (autograft) in mechanical strength and rate of union and therefore may be satisfactory substitutes for bone grafting. Freeze-dried bone did not appear to be as satisfactory because of its low mean energy to failure, but statistical analysis failed to confirm this opinion. Hydroxylapatite graft, when used alone, does not appear to be a suitable material for grafting segmental bone defects.

Matrix collagen of devitalized bone is resistant to osteoclastic bone resorption.

The resorption of devitalized bone by isolated osteoclasts in culture was studied by scanning electron microscopy. Osteoclasts attached to the bone and resorbed mineral but left most of matrix collagen undigested in the resorption pits. Postculture-treatment of bone substrate with collagenase completely removed the matrix collagen in the resorption pits, whereas trypsin did not. The results suggest that native matrix collagen is resistant to osteoclastic attack in this in vitro model using devitalized bone.

Chains of matrix-derived type X collagen: size and aggregation properties.

Type X collagen was isolated from extracts of embryonic chick cartilages by immunoprecipitation and subsequently analyzed by SDS-PAGE. Most of the chains migrated with a molecular weight of 59 kDa, suggesting that the matrix form of type X collagen has not undergone post-secretory proteolytic processing. Minor amounts of material were also observed at 120 kDa, 70 kDa and 50 kDa. These were dimers or limited proteolytic products of type X chains.

Collagen and non-collagenous proteins in different mineralization stages of human femur.

Mineralized tissues exhibit varying degrees of mineralization in different areas within the same bone. Using the technique of density gradient fractionation, bone powder from the diaphysis of human femur has been separated in different fractions corresponding to the degree of mineralization. Isolated bone fractions were analysed for their content in collagen and non-collagenous proteins. The results showed marked differences between compact and spongy bones, this latter containing higher proportions of little mineralized bone particles than the former (p less than 0.01). The ethylenediaminetetra-acetic acid (EDTA) extractability and the bone matrix size decreased relative to the decrease in specific gravity of bone particles. Among the matrix components of different fractions, sialoprotein consistently increased with the increase in specific gravity while proteoglycan decreased in reverse manner to the increase in collagen. However, in the most mineralized fraction (specific gravity: 2.33 g/cm3), the proteoglycan amount increased while collagen decreased. In conclusion, this study of bone maturation in human femur confirms the suitability of the technique of density gradient fractionation in the studies of bone matrix-mineral interactions. Apart from the fraction with the highest specific gravity, the analytical results obtained in fractions are similar to those observed in age-related bone changes, suggesting that the increase in mineralization degree of bone particles may be related to their age.

[Relevant laboratory diagnostic methods for the evaluation of the osteoinductivity of bone matrix implants]. / Relevanz labordiagnostischer Methoden zur Bewertung der Osteoinduktivität von Knochenmatriximplantaten.

For the valuation of the osteoinductive efficacy of different bone grafts bears upon increasing the determination of the alkaline phosphatase, the content of calcium, hydroxyproline and protein. We could prove the quantitative differentiation of different prepared bone matrix implants being possible, nevertheless the histological examination is an unavoidable, full of good sense completion.