Tricalcium phosphate and osteogenin: a bioactive onlay bone graft substitute.

The disadvantages of autogenous bone grafts has prompted a search for a dependable onlay bone graft substitute. A combination of tricalcium phosphate, a resorbable ceramic, and osteogenin, an osteoinductive protein, was evaluated as an onlay bone graft substitute in a rabbit calvarial model. Twenty-eight tricalcium phosphate implants (15 mm diameter x 5 mm; pore size, 100-200 microns) were divided into experimental and control groups and placed on the frontal bone of 14 adult New Zealand White rabbits. In the experimental animals, 185 micrograms of osteogenin was added to each implant. In the control animals, the implants were placed untreated. Implants were harvested at intervals of 1, 3, and 6 months, and evaluated using hematoxylin and eosin histology, microradiography, and histomorphometric scanning electron microscope backscatter image analysis. At 1 month there was minimal bone ingrowth and little tricalcium phosphate resorption in both the osteogenin-treated and control implants. At 3 months, both the osteogenin-treated and control implants showed a modest increase in bone ingrowth (8.85 percent versus 5.87 percent) and decrease in tricalcium phosphate (32.86 percent versus 37.08 percent). At 6 months, however, the osteogenin-treated implants showed a statistically significant increase in bone ingrowth (22.33 percent versus 6.96 percent; p = 0.000) and decrease in tricalcium phosphate (27.25 percent versus 37.80 percent; p = 0.004) compared with the control implants. The bone within the control implants was mostly woven at 6 months, whereas the osteogenin-treated implants contained predominantly mature lamellar bone with well-differentiated marrow. All implants maintained their original volume at each time interval studied. The tricalcium phosphate/osteogenin composite, having the advantage of maintaining its volume and being replaced by new bone as the tricalcium phosphate resorbs, may be applicable clinically as an onlay bone graft substitute.

Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3.

We have identified a new member of the transforming growth factor-beta (TGF-beta) superfamily, growth/differentiation factor-10 (GDF-10), which is highly related to bone morphogenetic protein-3 (BMP-3). The nucleotide sequence of GDF-10 encodes a predicted protein of 476 amino acids with a molecular weight of approximately 52,000. The GDF-10 polypeptide contains a potential signal sequence for secretion, a putative RXXR proteolytic processing site, and a carboxy-terminal domain with considerable homology to other known members of the TGF-beta superfamily. In the mature carboxy-terminal domain GDF-10 is more homologous to BMP-3 (83% amino acid sequence identity) than to any other previously identified TGF-beta family member. GDF-10 also shows significant homology to BMP-3 (approximately 30% amino acid sequence identity) in the pro- region of the molecule. Based on these sequence comparisons, GDF-10 and BMP-3 define a new subgroup within the larger TGF-beta superfamily. By Northern analysis, GDF-10 mRNA was detected primarily in murine uterus, adipose tissue, and brain and to a lesser extent in liver and spleen. In addition, GDF-10 mRNA was present in both neonatal and adult bone samples, with higher levels being detected in calvaria than in long bone. These results suggest that GDF10 may play multiple roles in regulating cell differentiation events, including those involved in skeletal morphogenesis. Gdf10 was mapped to the proximal region of mouse chromosome 14 close to a region known to contain a spontaneous recessive mutation that is associated with a craniofacial defect.

Osteoclast recruiting activity in bone matrix.

An activity that recruits osteoclasts has been identified and partially characterized from bone matrix. Bone-derived osteoclast recruiting activity (BORA) was co-purified with osteogenin, a bone inductive protein. Osteogenin was extracted from bovine bone with 6 M urea and purified by chromatography on hydroxyapatite, heparin-Sepharose and Sephacryl S-200 gel filtration. The biologically active osteoclast formation-stimulating material was further purified by C18 reverse phase HPLC. BORA is obviously distinct from osteogenin and transforming growth factor beta (TGF-beta), since further purified osteogenin and pure TGF-beta did not stimulate the formation of osteoclast-like cells. BORA (0.1-10 micrograms/ml) stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in a dose-dependent manner. These multinucleated cells resorbed bone when cultured on bovine bone slices. The effect of BORA is primarily directed to differentiate osteoclast precursors, since it did not stimulate osteoclast function in in vitro resorption assay where disaggregated rat osteoclasts were cultured on bovine bone slices. However, after 24 h preincubation with 50 nM PTH in the mouse calvaria assay, BORA at 10 micrograms/ml significantly stimulated bone resorption.

Initiation and promotion of bone differentiation by bone morphogenetic proteins.

The presence of growth and differentiation factors in bone has been demonstrated by subcutaneous implantation of demineralized bone matrix that initiates new cartilage and bone morphogenesis. The genes for bone morphogenetic proteins (BMPs) have been cloned and expressed. Recombinant BMPs induce endochondral bone formation in vivo. The multistep sequential developmental cascade consists of chemotaxis, mitosis, and differentiation of cartilage and bone. The pleiotropic response has been well characterized. BMPs stimulate osteogenic and chondrogenic phenotypes. Natural bovine osteogenin (BMP-3) and recombinant BMP-4 are equipotent in chemotaxis, limb bud chondrogenesis, cartilage maintenance, and in vivo bone induction. There are multiple isoforms of BMPs, raising the biologic relevance of the redundancy. The mode of action and second messengers are not clear. BMPs appear to have cognate receptors as demonstrated by iodinated BMP-2B (BMP-4). Other novel members of the BMP family include osteogenic protein 1 (BMP-7) and osteogenic protein 2 (BMP-8). Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily and include three distinct subfamilies: BMP-2, BMP-3, and BMP-7. Native BMP-3 and recombinant BMP-4 bind type IV collagen of the basement membrane. This novel connection may be the long elusive mechanistic explanation for the requirement of angiogenesis and vascular invasion for bone morphogenesis. BMPs may have a role in fracture repair, periodontal regeneration, and alveolar ridge augmentation.

Reconstruction of the bone--bone marrow organ by osteogenin, a bone morphogenetic protein, and demineralized bone matrix in calvarial defects of adult primates.

Information concerning the efficacy of osteogenin, a bone morphogenetic protein, and demineralized bone matrix in orthotopic sites in nonhuman primates is a prerequisite for potential clinical application in humans. After exposure of the calvaria, 84 cranial defects, 25 mm in diameter, were prepared in 26 adult male baboons (Papio ursinus). Defects were implanted with insoluble collagenous bone matrix (ICBM, the inactive collagenous residue after dissociative extraction of bone matrix with 4 M guanidine hydrochloride) reconstituted with osteogenin fractions isolated from baboon bone matrix by chromatography on heparin-Sepharose and hydroxyapatite-Ultrogel (Og Hep-HA) or osteogenin further purified using Sephacryl S-200 gel filtration chromatography (Og S-200). Baboon osteogenin with the highest biologic activity in a rodent bioassay, as determined by alkaline phosphatase activity, calcium content, and histologic analysis, was used for orthotopic implantation in baboons. Additional defects were implanted with baboon demineralized bone matrix (DBM) or ICBM without osteogenin as control. Defects also were grafted with corticocancellous bone harvested from the iliac crest or left ungrafted to monitor the spontaneous regeneration potential of the adult baboon calvaria. Undecalcified bone sections at 7 microns were prepared from the harvested specimens 30 and 90 days after surgery. Histomorphometry demonstrated that Og S-200 induced copious amounts of bone and osteoid as early as day 30 (P < 0.01 versus ICBM, autogenous grafts and untreated defects). At day 90, in implants of Og S-200, Og Hep-HA, and DBM, bone and marrow formation was extensive, culminating in complete regeneration of the craniotomies. In implants of DBM, bone formed with an intervening phase of cartilage development. This provides the phenotypic evidence of endochondral bone differentiation by induction in defects of membranous calvarial bone in adult primates. These results establish the potential therapeutic application of osteogenin and demineralized bone matrix for the architectural reconstruction of the bone-bone marrow organ in humans.

Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor beta 1 mRNA expression.

Subcutaneous implantation of demineralized bone matrix initiates a sequence of developmental events, which culminate in endochondral bone formation. During early stages of development of matrix-induced implants, ED1, Ia-positive monocytes-macrophages were observed, suggesting that in the initial phases of the endochondral bone formation cascade, the bone-inductive protein osteogenin and related bone morphogenetic proteins (BMPs) might serve as potent chemoattractants to recruit circulating monocytes. In this investigation, we demonstrate that at concentrations of 10-100 fg/ml (0.3-3 fM), native bovine osteogenin and recombinant human BMP-2B (rhBMP-2B) induce the directed migration of human blood monocytes in vitro. This chemotactic response was associated with expression of BMP binding sites (receptors) on monocytes. About 750 receptors per cell were detected with an apparent dissociation constant of 200 pM. Both osteogenin and rhBMP-2B at higher concentrations (0.1-30 ng/ml) stimulated mRNA expression for an additional regulatory molecule, type beta 1 transforming growth factor (TGF-beta 1) in human monocytes. TGF-beta 1, in turn, is known to induce a cascade of events leading to matrix generation. Monocytes stimulated by TGF-beta are known to secrete a number of chemotactic and mitogenic cytokines that recruit endothelial and mesenchymal cells and promote their synthesis of collagen and associated matrix constituents. TGF-beta 1 in concert with these other cytokines and matrix components regulates chemotaxis, mesenchymal proliferation, differentiation, angiogenesis, and controlled synthesis of extracellular matrix. Our results demonstrate that osteogenin and related BMPs through their profound effects on monocyte recruitment and cytokine synthesis may promote additional successive steps in the endochondral bone formation cascade.

Initiation of bone regeneration in adult baboons by osteogenin, a bone morphogenetic protein.

Osteogenin, and related bone morphogenetic proteins, induce endochondral bone differentiation through a cascade of events which include formation of cartilage, hypertrophy and calcification of the cartilage, vascular invasion, differentiation of osteoblasts, and formation of bone. These events have been studied in a postnatal model of bone development in rodents. Information concerning the morphogenetic potential of osteogenin in primates is a prerequisite for potential clinical application in man. The efficacy of allogeneic osteogenin in primates was investigated in both extraskeletal and skeletal sites in 19-Chacma baboons (Papio ursinus). Osteogenin was isolated from demineralized baboon bone matrix and purified by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Protein fractions with a molecular mass range of 26-42 kDa induced cartilage and bone differentiation in the subcutaneous space of rats. Final purification to homogeneity was obtained by electroendosmotic elution from a preparative sodium dodecyl sulphate (SDS) polyacrylamide gel, resulting in a single band on a SDS-polyacrylamide gel with an apparent molecular mass of 30-34 kDa, with biological activity in rats. The osteoinductive potential of osteogenin in primates was tested first in intramuscular sites in baboons and found to be active. The bone regeneration potential was investigated in nonhealing calvarial defects surgically prepared in adult male baboons. Baboon osteogenin induced complete regeneration of the cranial wound. These findings in adult primates establish a primary role for osteogenin in initiation and promotion of osteogenesis, and imply a potential therapeutic application based on cell biology of extracellular matrix-cell interactions.

Advances in osteogenin and related bone morphogenetic proteins in bone induction and repair.

Bone matrix is a repository of growth and differentiation factors as demonstrated by the induction of local cartilage and bone formation in rats. The bone inductive activity, termed osteogenin, can be dissociatively extracted, and it was isolated by heparin affinity, hydroxyapatite and molecular sieve chromatography. Osteogenin has been purified to homogeneity from bovine bone matrix and the sequences of several tryptic peptides have been determined. The sequences were similar to portions of the amino acid sequence deduced from the cDNA clone of bone morphogenetic protein-3 (BMP-3). The carboxyl-terminal quarter of osteogenin has sequence identity to the corresponding regions of two related proteins BMP-2A and BMP-2B. The bone inductive proteins are members of the TGF-beta superfamily, by virtue of the location of the highly conserved cysteines in their carboxyl-terminal region. Osteogenin and related BMPs initiate cartilage and bone formation in vivo. The study of the mechanism of action of these proteins will add considerable new information on the molecular signals controlling endochondral bone formation. In vitro data indicate that osteogenin stimulates the expression of the osteogenic and chondrogenic phenotypes. Our results demonstrate their profound influence on proteoglycan synthesis and degradation in bovine cartilage explant cultures. High affinity specific binding sites have been identified in both MC3T3 cells and articular chondrocytes. In vivo experiments demonstrate the efficacy of primate osteogenin in restoring large calvarial defects in adult baboons, establishing a primary role for osteogenin in therapeutic initiation and promotion of osteogenesis.

Recent progress in bone induction by osteogenin and bone morphogenetic proteins: challenges for biomechanical and tissue engineering.

Implantation of demineralized bone matrix results in local bone induction. Bone induction is a sequential biological chain reaction that consists of chemotaxis and proliferation of mesenchymal cells and differentiation of bone. Osteogenin, a bone morphogenetic protein has been purified and the amino acid sequence determined. Recently a family of bone morphogenetic proteins have been cloned and expressed by recombinant DNA technology. The availability of growth and morphogenetic factors will permit the rational design of new bone. The challenge for the biomechanical engineer is to attain mechanically optimal and functionally adaptive new bone for various skeletal prostheses. We are on the threshold for fabrication of new bone based on sound architectural design principles of tissue engineering based on cellular and molecular biology of growth and differentiation factors.

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.

Bone induction by osteogenin and bone morphogenetic proteins.

Bone induction is a multistep process that includes chemotaxis and attachment of mesenchymal stem cells, proliferation of progenitor cells and differentiation into cartilage and bone. Endochondral bone formation is the predominant sequence. Bone induction can be operationally divided into the following phases: initiation, promotion and maintenance. The initiation of bone induction is primarily regulated by osteogenin and bone morphogenetic proteins. Recent work has led to the isolation, purification and cloning of osteogenin and other bone morphogenetic proteins. Other growth factors such as platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta isoforms, insulin like growth factors (IGF 1 and 2), and fibroblast growth factor (FGF) may promote and perhaps maintain the newly induced bone.

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.

Initiation of bone development by osteogenin and promotion by growth factors.

The cellular and molecular basis of bone development and its regulation by differentiation and growth factors is an exciting area of current research. This article briefly reviews the historical progress in the isolation of osteogenin, a novel bone differentiation factor, and its modulation by well known growth factors. Endochondral bone development is a multistep sequential cascade and the process must be operationally dissected. It has been accomplished with the demineralized bone matrix-induced bone formation model. The reproducible development of cartilage and bone in an extraskeletal site permits the study of the initiation of the first cycle of endochondral bone formation and mineralization. Recent progress in the isolation of osteogenin, a specific bone differentiation factor, by heparin affinity chromatography permits the further investigation of the commitment and clonal expansion of the putative osteoprogenitor stem cells. Once initiated, bone formation is promoted by growth factors such as platelet derived growth factor, fibroblast growth factor, insulin like growth factor, transforming growth factor beta and a plethora of non specific cytokines. Finally bone development is further modulated by systemic hormones and nutrition and a host of physical signals including electrical, gravitational and mechanical forces.

The number of 1,25-dihydroxyvitamin D3 receptors is decreased in both intestine and kidney of genetically diabetic db/db mice.

In previous studies on calcium homeostasis in diabetes, drug-induced diabetic rats have generally been used, and various alterations have been demonstrated in several parameters of the vitamin D-endocrine system. It is, however, still questionable whether the drug-induced diabetic rat is the most appropriate animal model for the investigation of calcium and vitamin D metabolism because of the toxicity of diabetogenic agents toward the principle organs of vitamin D metabolism, such as liver and kidney. Therefore, in the present study, we examined the strain of genetically diabetic mice, C57BL/KsJ db/db, to evaluate 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in intestine and kidney and to investigate the alterations of calcium and vitamin D metabolism. In both control and diabetic mice, intestinal and renal 1,25-(OH)2D3 receptors were demonstrated; they had a sedimentation coefficient of 3.4S. The number of specific 1,25-(OH)2D3-binding sites in intestine was 118 +/- 11 fmol/mg protein in diabetic mice, significantly lower than the value of 199 +/- 11 fmol/mg protein in controls (P less than 0.01). Moreover, the renal concentration of specific 1,25-(OH)2D3-binding sites of 34.6 +/- 7.1 fmol/mg protein in diabetic mice was also significantly reduced compared to the value of 63.3 +/- 5.7 fmol/mg protein in controls (P less than 0.01). There were no significant differences in the equilibrium dissociation constants (Kd) of intestinal and renal receptors between control and diabetic mice. Significant hypocalcemia was demonstrated in the diabetic mice (P less than 0.01), suggesting the development of a negative calcium balance. Diabetic mice showed a significant decrease in renal 24,25-(OH)2D3 production (P less than 0.02), whereas renal 1,25-(OH)2D3 production was significantly increased in the diabetic group (P less than 0.05) compared to the control value. It is probable from these results that the genetic/endogenous diabetes may be directly associated with the alterations of mineral homeostasis. The altered calcium and vitamin D metabolism in diabetic mice is suggested to be derived, at least in part, from the decreased number of the 1,25-(OH)2D3 receptors in both intestine and kidney in the diabetic state.

Induction and maintenance of new bone formation by growth and differentiation factors.

The cell biology of bone formation can be better understood by dissecting the complex multistep process into individual steps. It is well known that demineralized matrix has the potential to initiate new bone formation locally at a heterotopic site of implantation. The sequential development of bone in response to bone matrix is reminiscent of the cellular lineages in the epiphyseal growth plate. The developmental cascade has permitted the operational distinction of the major phases of new bone formation such as: migration of progenitor cells; mitosis of mesenchymal stem cells; differentiation to cartilage and bone; mineralization and remodelling; and finally hematopoietic marrow differentiation. Thus the initiation of bone formation can be investigated as opposed to maintenance of already formed bone as in the orthotopic sites. Recent work has resulted in the identification and isolation of osteogenin, a bone-inductive protein. The newly formed bone is then maintained by a variety of polypeptide growth factors which have a regulatory role. The local action of initiation and maintenance factors is further modulated in a collaborative manner by systemic factors such as hormones and nutrition and the extracellular matrix.

The renal mitochondrial metabolism of 25-hydroxyvitamin D-3: a possible role for phospholipids.

The effect of exogenous phospholipids on chick kidney mitochondrial 25-hydroxyvitamin D-3 metabolism was examined. Phosphatidylserine, phosphatidylcholine and phosphatidylinositol had no effect on either the 1- or 24-hydroxylation of 25-hydroxyvitamin D-3. Phosphatidylethanolamine and cardiolipin both brought about a dose-dependent decrease in the 1-hydroxylase activity in mitochondria from vitamin D-deficient chicks but not from vitamin D-replete chicks. There were no major differences in the phospholipid composition of mitochondria from vitamin D-deficient and -replete chicks nor in the fatty acid composition of these phospholipids. Preliminary kinetic studies suggest that cardiolipin acts as a noncompetitive inhibitor of the 1-hydroxylase in mitochondria isolated from vitamin D-deficient chicks. It does not appear to exert its effect by virtue of altering the distribution of substrate or products. Investigation of the effect of fatty acid methyl esters on the hydroxylase activities suggests that it may be the fatty acid moiety of the phospholipid, rather than the phosphate moiety in the polar head group, that is involved in the phospholipid effect on the hydroxylation of 25-hydroxyvitamin D-3.

Homologous desensitization of cultured chick kidney cells to parathyroid hormone.

The development of refractoriness of the cAMP response to PTH in primary cultures of chick kidney cells and recovery from the refractory state was investigated. When cells were preincubated with bovine PTH1-34, complete refractoriness to a subsequent challenge with the hormone developed within 2 h and at hormone concentrations as low as 5 ng/ml. The ability of PTH to stimulate activation of cAMP-dependent protein kinase was also abolished by preincubation with the hormone. When cells were desensitized and then incubated in hormone-free medium, recovery of the cAMP response began within an hour and was maximal, but not complete (80%) after 16 h. Cycloheximide did not affect either desensitization or the rate or extent of recovery from the refractory state. Low concentrations of forskolin (2.5 X 10(-7) M) greatly enhanced cAMP production stimulated by PTH and higher concentrations (10(-6) - 10(-4) M) stimulated rates of cAMP production 50 times those obtained with PTH alone. Preincubation with forskolin did not bring about desensitization to PTH nor did preincubation with PTH affect the subsequent response to forskolin. The half-life of biologically active bovine PTH1-34 in chick kidney cell culture was approximately 12 h and the rate of its removal was not significantly altered during a 20-h incubation period. The results suggest that desensitization of chick kidney cells to PTH is not suggest that desensitization of chick kidney cells to PTH is not brought about by cAMP generation itself, is not primarily dependent on protein synthesis, and does not involve a change in the rate of removal of biologically active hormone from the medium. In addition, recovery of the cAMP response to PTH also does not require new protein synthesis. These results are compatible with a mechanism of desensitization which occurs at the level of the receptor or hormone-receptor coupling to adenyl cyclase.