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.     

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.     

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.     

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.     

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.     

Comparison of bone inductive proteins of rat and porcine bone matrix

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.     

Chondrogenic potential of mesenchymal cells elicited by bone matrix in vitro

Demineralized bone matrix (DBM) induces development of bone in vivo via the endochondral mode of development. Early events in this inductive process involve the appearance of mesenchymal cells (day 3) followed by chondrogenic differentiation (day 7) after subcutaneous implantation of DBM. In this investigation the chondrogenic potential in vitro of day 3 and day 4 mesenchymal cells from a DBM-induced implant has been explored. Immunofluorescent examination of day 3 cell cultures maintained for 4 days revealed the presence of type II collagen and cartilage-specific proteoglycans only in spherical or polyhedral cells. Micromass cultures and agarose suspension cultures showed toluidine-blue metachromasia in only a small population of cells. Biochemical estimation of 35SO4-labeled proteoglycans from suspension cultures of day 3 and day 4 cells maintained for 3 days indicated the presence of 29% and 38% large cartilage-specific proteoglycans, respectively. Addition of bone-inductive guanidine extract of DBM to the cultures did not significantly increase the percentage of large proteoglycans. These observations suggest that day 3 and day 4 cells can undergo chondrogenic differentiation in vitro without the continued presence of the bone-inductive guanidine extract. The presence of guanidine extract in cultures did not enhance chondrogenic expression or promote the recruitment of mesenchymal cells and their transformation to the chondrogenic phenotype.     

Mechanism and specificity of reconstitution of dimeric lactate dehydrogenase from Limulus polyphemus

D-Lactate dehydrogenase (EC 1.1.1.28) from Limulus polyphemus is a homodimer which is composed of identical subunits of Mr = 35 000. The enzyme may be reversibly denatured and dissociated at acid pH or in 6M guanidine X HCl. The sigmoidal time course of reactivation obeys a consecutive uni-bimolecular mechanism with k1 = 6 X 10(-4) S-1 and k2 = 1.3 X 10(-4) M-1 S-1 (20 degrees C) as first- and second-order rate constants. Cross-linking experiments with glutaraldehyde prove that reactivation and dimer formation run parallel. Joint "synchronous" reconstitution of the enzyme with dimeric porcine mitochondrial malate dehydrogenase (after denaturation in 6M guanidine X HCl) does not yield active hybrids. The unchanged kinetics of reactivation in the absence and presence of the prospective partner of hybridization prove that inactive hybrid intermediates may also be excluded. The absence of hybrids upon synchronous reconstitution of the two closely related dimeric NAD-dependent dehydrogenases clearly suggests that the assembly of nascent oligomeric proteins must be highly specific.     

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).     

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.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Proteoglycans of guinea-pig coastal cartilage. Fractionation and characterization.

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea-pigs. Three solvents were used in sequence: 0.4 M guanidine - HCl, pH 5.8, 4 M guanidine - HCl, pH 5.8, and 4 M guanidine - HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. Amino acid analyses of dissociative fractions were inaccordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6-sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts. The results indicate that the proteoglycans of guinea-pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.     

Correlation of the appearance of γ-carboxyglutamic acid with the onset of mineralization in developing endochondral bone

γ-Carboxyglutamic acid (Gla) is a constituent of the non-collagenous bone protein osteocalcin. The appearance of γ-carboxyglutamic acid during $\text{de}\text{novo}$ differentiation and development of endochondral bone has been correlated with the onset of mineralization. Discrete stages of endochondral bone development were studied by subcutaneous implantation of demineralized rat diaphyseal bone matrix. Residual Gla in acid-demineralized bone matrix was lost rapidly on implantation. Gla levels were basal during mesenchymal cell proliferation (day 3) and chondrogenesis (days 5–7). Gla and calcium levels began to increase during cartilage mineralization (day 9) and continuously increased after day 10 concomitant with bone differentiation.     

A water-soluble fraction from adult bone stimulates the differentiation of cartilage in explants of embryonic muscle

A water-soluble fraction of a 4 M guanidine HCl extract of demineralized adult bovine bone stimulated the differentiation of cartilage in explants of minced skeletal muscle from embryonic chick legs; cartilage was also induced by a semipurified protein preparation. Cartilage could be identified in treated cultures at 1 week with muscle from day-9 embryos, not before 2 weeks with muscle from day-12 embryos, and not before 3 weeks with muscle from day-19 embryos. The ability to respond to this water-soluble fraction by exhibiting cartilage differentiation was dose-dependent, but not confined to any particular muscle region of the day-12 embryonic leg. These observations indicate that bone-derived soluble chondroinductive agents act on cells in minced embryonic muscle preparations. The induction of cartilage is dependent upon the accessibility of the responding cells to the agents, on the concentration of inductive agents, and on the developmental age of the responsive tissue.     

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.     

The occurrence of low buoyant density proteoglycans in embryonic chick cartilage

Two proteoglycan fractions, PCS-H and PCS-L, have previously been isolated from 4 M guanidine HCl extract of embryonic chick cartilages. This communication reports further studies with PCS-L indicating that this fraction contains several different forms, of which one differs from hitherto known cartilage proteoglycans in 1) markedly lower buoyant density, 2) susceptibility to reduction with 2-mercaptoethanol, 3) aggregate-forming ability in 4 M guanidine HCl, and 4) presence of dermatan sulfate-chondroitin sulfate copolymer chains. Also isolated from the PCS-L fraction is a keratan sulfate-rich proteoglycan which represents the smallest molecular size species in cartilage proteoglycan populations.     

Effects of low concentrations of guanidine . HCl on the reconstitution of lactic dehydrogenase from pig muscle in vitro. Evidence for guanidine binding to the native enzyme

The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.     

Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Extracellular bone matrix-derived growth factor

Demineralized extracellular bone matrix, when implanted subcutaneously into allogeneic rats, induces an invariant sequence of events resulting in de novo cartilage, bone and bone marrow formation. We have recently demonstrated the dissociative extraction and successful biological reconstitution of boneinducing molecule(s) in demineralized bone matrix. As mesenchymal cell proliferation precedes differentiation of endochondral bone, we have examined the bone-inductive molecules for mitogenic activity on human and rat fibroblasts and bovine endothelial cells. The results revealed that the molecular fraction obtained by molecular sieve chromatography on Sepharose CL-6B responsible for endochondral bone induction is capable of stimulating human and rat fibroblasts proliferation by 2500 and 300 % respectively, as compared to cells grown in a serum-free medium. Endothelial cells do not seem to share this response. This factor exhibits a significant effect on growth-promoting activity for both human and rat fibroblasts at a dose of 0.5 ng protein/ml of culture medium. These results demonstrate the presence of a tightly bound growth factor in the extracellular bone matrix.