Affinity of osteogenin, an extracellular bone matrix associated protein initiating bone differentiation, for concanavalin A

Subcutaneous implantation of demineralized bone matrix results in bone differentiation. The bone inductive protein, osteogenin, was isolated recently by heparin affinity chromatography. The affinity of osteogenin for various lectins was examined to attain further purification and characterization. Osteogenin extracted from bovine bone matrix binds to concanavalin A (Con A) but not to wheat germ agglutinin or soybean lectin. The present data indicate that the bone inductive protein, osteogenin, is a glycoprotein. The use of a Con A Sepharose affinity column followed by preparative gel electrophoresis resulted in a greater than 250,000 fold purification of osteogenin.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation

Osteogenin was purified from bovine bone matrix and its activity monitored by an in vivo bone induction assay. The purification method utilized extraction of the bone-inducing activity with 6 M urea, followed by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Active fractions were further purified by preparative sodium dodecyl sulfate gel electrophoresis without reduction. Osteogenin activity was localized in a zone between 30 and 40 kDa. The amino acid sequences of a number of tryptic peptides of the gel-eluted material were determined. Reduction and alkylation of purified osteogenin in 7 M guanidine hydrochloride resulted in the total loss of biological activity. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a broad band with an apparent molecular mass of 22 kDa.     

Osteogenin, a bone morphogenetic protein, adsorbed on porous hydroxyapatite substrata, induces rapid bone differentiation in calvarial defects of adult primates.

Osteogenin, a bone morphogenetic protein, in conjunction with insoluble collagenous bone matrix initiates local endochondral bone differentiation by induction in vivo. This study, by exploiting the affinity of native osteogenin for hydroxyapatite, was designed to construct a delivery system for the expression of the biologic activity of osteogenin in nonhealing calvarial defects of adult primates. After exposure of the calvaria, 64 cranial defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). Defects were implanted with disks of porous nonresorbable and resorbable hydroxyapatite substrata obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. In each animal, one disk of each hydroxyapatite preparation was treated with osteogenin isolated and purified from baboon bone matrix after sequential chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200 gel filtration columns. The remaining two defects were implanted with one disk of each hydroxyapatite preparation without osteogenin as control. Histomorphometry on decalcified sections prepared on days 30 and 90 showed superior osteogenesis in osteogenin-treated nonresorbable hydroxyapatite specimens as compared with controls. On day 90, substantial bone formation also had occurred in control nonresorbable hydroxyapatite specimens. On day 90, but not on day 30, significantly greater amounts of bone had formed in osteogenin-treated resorbable specimens as compared with resorbable controls. Overall, resorbable substrata performed poorly when compared with nonresorbable substrata, perhaps due to a premature dissolution of the implants. These results provide evidence that the biologic activity of osteogenin can be restored and delivered by a substratum other than the organic collagenous matrix, inducing rapid bone differentiation in calvarial defects of adult nonhuman primates. The adsorption strategy of osteogenin on porous inorganic nonimmunogenic substrata may help to design appropriate osteogenic delivery systems for craniofacial and orthopedic applications in humans.     

Importance of geometry of the extracellular matrix in endochondral bone differentiation

Subcutaneous implantation of coarse powders (74-420 micron) of demineralized diaphyseal bone matrix resulted in the local differentiation of endochondral bone. However, implantation of matrix with particle size of 44-74 micron (Fine matrix) did not induce bone. We have recently reported that the dissociative extraction of coarse matrix with 4 M guanidine HCl resulted in a complete loss of the ability of matrix to induce endochondral bone; the total loss of biological activity could be restored by reconstitution of extracted soluble components with inactive residue. To determine the possible biochemical potential of fine matrix to induce bone, the matrix was extracted in 4 M guanidine HCl and the extract was reconstituted with biologically inactive 4 M guanidine HCl-treated coarse bone matrix residue. There was a complete restoration of the biological activity by the extract of fine matrix upon reconstitution with extracted coarse matrix. Polyacrylamide gel electrophoresis of the extract of fine matrix revealed similar protein profiles as seen for the extract of coarse matrix. Gel filtration of the 4 M guanidine HCl extract of fine powder on Sepharose CL-6B and the subsequent reconstitution of various column fractions with inactive coarse residue showed that fractions with proteins of 20,000-50,000 mol wt induced new bone formation. These observations demonstrate that although fine bone matrix contains, osteoinductive proteins, matrix geometry (size) is a critical factor in triggering the biochemical cascade of endochondral bone differentiation. Mixing of coarse matrix with Fine results in partial response and it was confined to areas in contact with coarse particles. The results imply a role for geometry of extracellular bone matrix in anchorage-dependent proliferation and differentiation of cells.     

Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons.

A major goal of the combined effort of basic scientists and plastic and reconstructive surgeons is the development of novel bone substitutes based on osteogenic growth and differentiation factors with optimal delivery systems for skeletal repair. Osteogenin is a protein initiator of bone differentiation. The present study examined the osteogenic potential of osteogenin in combination with porous hydroxyapatite replicas obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. Bovine osteogenin, with an apparent molecular weight of 28 to 42 kDa, purified by hydroxyapatite-Ultrogel adsorption chromatography, heparin-Sepharose affinity chromatography, and HR Sephacryl S-200 molecular sieve chromatography, was delivered into rods of nonresorbable and resorbable hydroxyapatite replicas with an average porosity of 600 microns. A total of 48 rods were bioassayed for osteogenic activity by intramuscular implantation into eight adult baboons (Papio ursinus) as a prerequisite for clinical trials in humans. Bovine osteogenin fractions reconstituted with baboon insoluble collagenous bone matrix were implanted in an additional four adult baboons. Specimens were harvested at 30 and 90 days after implantation and subjected to histomorphometry and alkaline phosphatase activity determination. Differentiation of bone occurred in nonresorbable hydroxyapatite rods, both osteogenin-treated and controls. However, no bone formation was observed in resorbable rods, even in the presence of osteogenin. These results demonstrate that the surface and chemical characteristics of the substratum, independent of the osteogenic stimulus, have a profound influence on the morphogenesis of bone. The demonstration of bone induction in nonhuman primates with porous nonresorbable hydroxyapatite replicas and baboon insoluble collagenous bone matrix reconstituted with bovine osteogenin establishes the therapeutic potential of the principle of bone induction in craniofacial, periodontal, and orthopedic reconstructive surgery.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.     

Osteogenin inhibits proliferation and stimulates differentiation in mouse osteoblast-like cells (MC3T3-E1)

Osteogenin, a novel bone differentiation factor, was recently purified and characterized. We examined its effect on the proliferation and differentiation of MC3T3-E1 osteoblast-like cells. Cell proliferation was inhibited the first 48 h after addition of osteogenin, and this effect was independent of serum. Osteogenin did not influence the cell morphology. Alkaline phosphatase promptly increased in a dose and time-dependent manner and appeared to be specific. Treatment with TGF-beta 1 resulted in inhibition of alkaline phosphatase activity, and was reversed by osteogenin within 48 h. Cell cultures treated with osteogenin for 72 h after confluence became responsive to parathyroid hormone. Synthesis of collagenous proteins was stimulated by osteogenin. The present results demonstrate a significant influence of osteogenin on the differentiation of osteogenic phenotype in MC3T3-E1 cells in vitro.     

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.     

Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development.     

Osteogenin promotes reexpression of cartilage phenotype by dedifferentiated articular chondrocytes in serum-free medium

Chondrocytes lose their phenotypic traits, including type II collagen, after serial passage in monolayer cultures. Osteogenin, a bone morphogenetic protein, induces cartilage and bone in nonskeletal sites. This investigation examined the ability of osteogenin to promote the reexpression of cartilage phenotype by dedifferentiated chondrocytes obtained from rabbit articular cartilage. The results revealed that osteogenin, in synergism with selected growth factors, promoted the reexpression of type II collagen and proteoglycans by dedifferentiated chondrocytes in agarose. Insulin, a constituent of the basal medium, appeared to be essential for the colony-forming aspect of this phenomenon, since when insulin was replaced by insulin-like growth factor-1 colony formation did not occur. Epidermal growth factor, platelet-derived growth factor (PDGF), and basic fibroblast growth factor appeared to be an optimal combination for the action of osteogenin. Neutralizing antibodies to transforming growth factor-beta did not influence the response to osteogenin. It is noteworthy that, compared to freshly passaged cells, those stored in liquid nitrogen were not as responsive to osteogenin and growth factors. A higher concentration of fibroblast growth factor in conjunction with osteogenin and PDGF, increased the responsiveness of frozen cells only in part, as the Alcian blue-positive proteoglycan matrix was not restored completely.     

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.     

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16--18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4 m guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)--polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an Mr of approximately 73 000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation. © 1998 Chapman & Hall     

Isolation and characterization of insulin-like growth factor-II from human bone

Human bone was sequentially extracted with 4 M guanidine hydrochloride to remove nonmineralized tissue components, 0.5 M EDTA to dissolve the mineral phase, 4 M guanidine hydrochloride to remove matrix associated proteins and finally a combination of 4 M guanidine hydrochloride and 0.5 M EDTA to remove residual proteins. The extracts were examined for the presence of factors that were able to stimulate the incorporation of [3H] thymidine into DNA and [14C] leucine into protein in a cloned rat bone cell culture system. The majority of the bioactivity was found in the first guanidine hydrochloride extract (59 +/- 12%) while the second guandine hydrochloride extract contained 27 +/- 8%. In addition to several known growth factors already reported to be present in bone (transforming growth factor-beta and insulin-like growth factor-I) insulin-like growth factor-II was identified by its chromatographic, electrophoretic and immunological properties as well as by N-terminal sequence data. The insulin-like growth factor-II levels (802 +/- 112 micrograms/kg wet weight bone) were 10 fold higher than that found for insulin-like growth factor-I (84 +/- 23 micrograms/kg wet weight).     

Biosynthesis and fate of proteoglycans in cartilage and bone during development and mineralization

Subcutaneous implantation of demineralized bone matrix in rats induces migration of host cells into the site and results in the sequential development of cartilage and bone. The biosynthesis and metabolic fate of proteoglycans in the plaques at the bone matrix implantation site were investigated by [35S]sulfate labeling in vivo. 35S-Labeled proteoglycans were extracted with 4 M guanidine HCl and purified by DEAE-Sephacel chromatography. Analysis of proteoglycans on Sepharose CL-2B chromatography showed two major peaks at Kd = 0.28 and 0.68 (peaks I and II, respectively). Peak I proteoglycan has a high buoyant density and contains chondroitin sulfate chains of average Mr = 20,000. Peak II proteoglycan has a lower average buoyant density and contains dermatan sulfate chains of average Mr = 33,000. Throughout the endochondral bone development sequence, peak II proteoglycan predominates. Peak I was low on Day 3, became prominent on Day 7 (approximately 30% of the total radioactivity), and declined after Day 9. The calculated half-lives of peak I and II proteoglycans labeled on Day 7 were about 1.8 and 2.8 days, respectively. After the initiation of osteogenesis, a species of mineral-associated proteoglycan was extracted with a 4 M guanidine HCl solvent containing 0.5 M EDTA. This proteoglycan has a small hydrodynamic size (Kd = 0.38 on Sepharose CL-6B chromatography) and shows a long half-life, about 6 days.     

Purification and characterization of a unique osteoinductive factor from bovine bone

A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.     

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.     

The isolation and partial characterization of a rat incisor dentin matrix polypeptide with in vitro chondrogenic activity

In vivo implants of demineralized dentin matrix into muscle induce the formation of bone within the muscle. As with bone matrix implants, the bone induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to bone matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble proteins extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-100 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-10,000, based on globular protein standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-10 ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the bone morphogenetic protein family of proteins in these essential features.     

Intact growth factors are conserved in the extracellular matrix of ancient human bone and teeth: a storehouse for the study of human evolution in health and disease

For the first time we have extracted, solubilized and identified growth factors, such as insulin growth factor II (IGF-II), bone morphogenetic protein-2 (BMP-2), and transforming growth factor-beta (TGF-beta), from archaeological compact human bone and tooth dentin dating from the late pre-ceramic pottery Neolithic (late PPNB) and the early Middle Ages. These factors are typical of special physiological or pathological situations in the metabolism of bone. The extracellular matrix proteins from bone and teeth of individuals from the late PPNB and early Middle Ages were separated by 2-D electrophoresis and more than 300 different protein spots were detected by silver staining. The matrix protein patterns of compact bone and tooth from the same individual (early Middle Ages) are very different and only 16% of the protein spots were detected in both compact bone and tooth dentin.