Repair of bone defect in primate mandible using a bone morphogenetic protein (BMP)-hydroxyapatite-collagen composite

The purpose of this study was to evaluate a hydroxyapatite (HA)-collagen (Col)-bone morphogenetic protein (BMP) composite as an osteoinductive bone substitute. Partially-purified BMP from bovine bone was mechanically mixed with highly purified type I collagen from calf dermis and then subsequently mixed with pure synthetic hydroxyapatite granules or block. The HA-Col-BMP composite, or the HA-Col composite as a control, was implanted in a surgically-induced mandible bone defect (6 x 7 x 10 mm) in an adult Japanese monkey. The mandible was excised three months after implantation and studied histologically. The BMP-containing implant induced much more new bone than the control implant in all experimental animals of each group, with either HA granules or HA block. Newly formed bone was attached tightly to HA and infiltrated deeply into the pores of the HA of the BMP-containing implant, while fibrous tissue existed between the host bone and HA in the control implant. Thus, we conclude that an HA-Col-BMP composite could be a superior biomaterial for a bone substitute.     

An experimental study on the relationship between BMP's activity and its carrier

To find out the relationship between human bone morphogenetic protein's activity and its carrier and to document the clinical application of BMP, we investigated six kinds of BMP carrier's influence upon hBMP's osteogenic activity with small sample size at first. Then three kinds of carriers selected from the six, added with the seventh carrier, were investigated with large sample size. The result showed that the compound of hBMP and hDDBM had best osteogenic ability. The human bone matrix had been formerly demineralized and extracted without hBMP. The hDDBM showed porous structure under SEM, its mean pore's diameter was 127 +/- 34 microns. It was demonstrated that the function of demineralized bone matrix extracted without BMP (i.e. DDBM) was related not only to its pore's diameter, but to the fibrolike matter in it as well. With the method of BMP bioassay, BMP was composited with carrier, then the compound was injected into the calf of mouse. This made BMP's bioassay become simple and reliable.     

An experiment study of the guided bone regeneration

BMP is one of important factors in the pathophysiology of bone regeneration. Fifteen New Zealand rabbits were used in this experiment. We studied the distribution and effectiveness of endogenic BMP on a 10 mm bone defect of radius, by utilizing immunohistochemistry of BMP and quantitative computer imaging system. On the 3rd day, death of osteocytes and BMP positive blood clot were observed. The mesenchymal cells from periosteum and endoosteum, and osteoblast were also BMP positive. By quantitative study, we found there was a gradient distribution of BMP in bone defect, i.e, the value of BMP decreased gradually along the distance from the fracture ends. The maximal value of BMP was noted at the 1st week postoperation. In conclusion, two sources of endogenic BMP were found, one was from the absorption of necrotic tissue of fracture ends, the other was from the secretion of osteogenic mesenchymal cells during the process of bone regeneration. Nonunion of bone defect was caused in part by the gradient distribution of BMP. Accordingly, the concept of effective quantity of endogenic BMP was drawn rosen. It might be a new method in the treatment of bone defect by increasing the concentration of endogenic BMP and improving its distribution.     

Effect(s) of the demineralization process on the osteoinductivity of demineralized bone matrix

Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. The present study was directed at establishing in vivo and in vitro models for a quantitative assessment of the osteoinductivity of DBM before clinical use. Athymic mice were used in an in vivo assay to overcome the species limitations (for human DBM) found in xenogeneic animal models. Calcium contents of explants, as an indicator of new bone formation, were assayed and expressed as a change in the weight percent calcium in the explant as compared to the weight percent of calcium in the implanted material. A total of 82 mice (2 implants per mouse) were used in this study. Significant amounts of new bone were induced in this animal model in response to implantation of DBM. Muscular implantation was found to be more osteoinductive (increases of 10.0 +/- 0.4 calcium weight percent of explant) than subcutaneous implantation (increases of 1.62 +/- 0.27 calcium weight percent of explant) and new bone formation in muscular implantation sites of athymic mice mimics endochondral bone formation. Between weeks 1 to 4, the weight of explanted materials did not significantly differ from the weight of the implanted material; however, by week 5 the explant weight began to increase. Calcium deposition over the 5 weeks of implantation increased in a nearly linear fashion. Consequently week 4 was chosen as the optimum time for explantation in the in vivo assay in that sufficient calcium levels had been achieved without a significant increase in explant dry weight. Aliquots of 10, 20, 30, and 40 mg per implantation site were used in dose response studies in the in vivo bioassay. Dose response curves with DBM exhibited maximal activity at the 20 mg DBM implant dose in the in vivo bioassay. An in vitro bioassay was also developed where human periosteal (HPO) cells were chosen because osteoprogenitor cells found in bone repair typically come from periosteal tissue. Alkaline phosphatase (ALP) activity in confluent cell cultures of HPO cells exposed to DBM, as an indicator of osteoblast induction, reached its highest level on day 5 of DBM treatment. Aliquots of 2, 5, 10, 20, 30, and 40 mg DBM per flask were chosen in dose response studies using the in vitro bioassay. These dose response studies with DBM revealed that quantities approximating 5 to 10 mg DBM in the in vitro model provided for maximal levels of ALP in cell extracts. A linear correlation (R2 = 0.7397) was demonstrated between the in vivo calcium remineralization assay and the in vitro ALP assay of osteoinductivity of DBM, suggesting that the in vitro assay can be used to quantitatively assess the osteoinductive potential of DBM where production and distribution of clinically usable DBM dictates rapid analysis.     

Purification of bone morphogenetic protein and investigation of its effects on osteoblastic cell line UMR108

It is well known that the bone matrix contains proteins which can induce ectopic endochondral bone formation in vivo. One class of these proteins is the bone morphogenetic protein (BMP). In order to investigate the physiological function of the BMP, its purification was attempted from an extract of demineralized bone matrix and its actions on the osteoblastic cell line were investigated. To isolate the BMP, a demineralized bone matrix was extracted with 4M guanidine-HCl. A water-insoluble fraction (G-WI) was separated from the demineralized bone extract by dialysis against distilled water and centrifugation. The BMP was purified from G-WI by gel filtration on Sephacryl S-200 HR, cation exchange with Mono-S, heparin affinity column and finally by C1/8 reverse phase chromatography. Peptide sequence analysis revealed that the purified BMP fraction contained "BMP-3" reported by Wozney et al. (1988). In order to investigate its function, the BMP was applied to the rat osteogenic sarcoma cell line UMR108. The BMP inhibited the growth of the UMR108 cells and enhanced the alkaline phosphatase activity in a dose-responsive manner.     

A histological study on tissue responses to titanium implantation in rat maxilla: the process of epithelial regeneration and bone reaction

The present study aimed to establish a titanium implantation model using rat maxillae as well as demonstrate the chronological tissue responses to implantation. Pure titanium implants were inserted in the upper first molar extraction sites of Wistar rats 1 month after tooth extraction. The animals were sacrificed at 1 to 30 days postimplantation, and prepared tissue specimens were processed for light microscopy. The removal of implants from tissue blocks was done using 2 methods: mechanical removal or a cryofracture technique. In the early stages, peri-implant tissues showed severe damage to the oral epithelium and collagen bundles with significant inflammatory cell infiltration. The peri-implant epithelium grew apically along the implant by 10 days postimplantation, and regenerated to show a similar feature of junctional epithelium seen in normal rats at 15 days postimplantation, at which time no signs of inflammation were observed. The regenerated collagen bundles in the connective tissue were arranged circumferentially to the implants in the horizontal sections. New bone formation first appeared around the implants at 5 days postimplantation, covering the entire perimeter of implants by 30 days postimplantation. Scanning electron microscopic observations of the surface texture of the removed implants suggest the probability of an adhesive mechanism between the implants and the peri-implant epithelium and/or the alveolar bone. These findings indicate that this experimental model is useful for detailed analysis of peri-implant tissue because of its easy implantation procedure.     

Cell differentiation in response to partially purified osteosarcoma-derived bone morphogenetic protein in vivo and in vitro

Bone morphogenetic protein (BMP) and associated noncollagenous proteins (NCP) were isolated from human osteosarcoma tissue. Implantation of 5- and 10-mg samples induced heterotopic ossification in the mouse quadriceps. Osteosarcoma-derived BMP/NCP induced the same process of osteogenesis as human BMP/NCP isolated from bone matrix in vivo. In vitro continuous perfusion of neonatal rat muscle tissue with 5 micrograms/ml osteosarcoma-derived BMP/NCP increased glycosaminoglycan (GAG) synthesis significantly whereas DNA synthesis was relatively unchanged. Similar results were found when muscle tissue was preincubated with 200 micrograms of osteosarcoma-derived BMP/NCP for four hours followed by an incubation period of 14 days in BMP-free medium: GAG synthesis increased significantly, whereas DNA synthesis did not change. The increase in GAG synthesis coincided with cell differentiation but not cell proliferation. Histologic findings confirmed chondrogenesis in vitro. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that osteosarcoma-derived BMP/NCP included a prominent component with a molecular weight of 18,000 d.     

Enhancement of ectopic bone formation in mice with a deficit in Fas-mediated apoptosis

Bone formation is under the control of cytokines as well as growth factors such as bone morphogenetic proteins (BMP). This suggests the possibility that osteogenesis might be modulated by factors which also modulate the immune system. To test whether immune disorders in the host may influence bone formation, we studied BMP-induced bone formation in a C3H/HeJ strain of mice bearing a mutant gene, the lymphoproliferation gene (lpr) or the generalized lymphoproliferative disease gene (gld), both of which are known to be a Fas deletion mutant and a Fas ligand mutant, respectively, and to induce immune disorders via a deficit in Fas-mediated apoptosis. Crude BMP derived from bovine bone were injected into the muscular tissue in the femur of adult C3H/HeJ mice or C3H/HeJ mice bearing an lpr or gld gene. Quantitative analysis of the resulting ectopic bone formation by X-ray photography 2 weeks after injection revealed that the presence of either the lpr or gld gene caused a bone mass significantly larger in dimension than that seen in the wild type mice. Histological examination also revealed the different influence between these mutant genes on the level of bone formation exhibited by hyaline cartilage and bone trabeculae. Based on these results, we discussed the possible mechanisms of the enhanced ectopic bone formation under the deficit in Fas-mediated apoptosis.     

Functional neuromuscular stimulation for combined control of elbow extension and hand grasp in C5 and C6 quadriplegics

Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.     

Efficacy of perichondrium and a trabecular demineralized bone matrix for generating cartilage

A pedicled auricular perichondrial flap wrapped around trabecular demineralized bovine bone matrix can generate an autologous cartilage graft. In earlier experimental studies, it was demonstrated that this graft could be used for nasal and cricoid reconstruction. It was assumed that the vascularization of the perichondrial flap was obligatory, but it was never proven that the flap should be pedicled. Moreover, for clinical use, the dimensions of the auricle would set restrictions to the size of the graft generated. Therefore, the possibility to generate cartilage with a composite graft of a free perichondrial flap wrapped around demineralized bovine bone matrix, by using young New Zealand White rabbits, was studied. This composite graft was implanted at poorly (subcutaneously in the abdominal wall; n = 12), fairly (subcutaneously in the pinna; n = 12), and well-vascularized sites (quadriceps muscle; n = 12). As a control, trabecular demineralized bovine bone matrix was implanted without perichondrial cover. Half of these grafts (n = 6) were harvested after 3 weeks, and the remaining grafts (n = 6) after 6 weeks of implantation. In histologic sections of these grafts, the incidence of cartilage formation was scored. Furthermore, the amount of newly formed cartilage was calculated by computerized histomorphometry. Trabecular demineralized bovine bone matrix without perichondrial cover demonstrated early resorption; no cartilage or bone was formed. In demineralized bovine bone matrix wrapped in perichondrium, early cartilage formed after 3 weeks at well- and fairly vascularized sites. No cartilage could be detected in grafts placed at a poorly vascularized site after 3 weeks; minimal cartilage formed after 6 weeks. In summary, the highest incidence of cartilage formed when trabecular demineralized bovine bone matrix was wrapped either in a pedicled auricular perichondrial flap or in a free perichondrial flap, which was placed at a well-vascularized site. Second, a significantly higher percentage of the total area of the graft was cartilaginized at well-vascularized sites after 3 weeks. The newly generated cartilage contained collagen type II and proteoglycans with hyaluronic acid binding regions, whereas collagen type I was absent, indicating the presence of hyaline cartilage. This study demonstrates that new cartilage suitable for a graft can be generated by free perichondrial flaps, provided that the site of implantation is well vascularized. Consequently, the size of such a graft is no longer limited to the dimensions of the auricle.     

Poly-(L-lactic) acid membranes in palatal surgery in beagle dogs: clinical and histologic evaluation

This article reports on the histologic findings from a larger study that was designed to investigate whether the attachment of scar tissue to underlying bone, which is normally found after palatal surgery, can be prevented by using biodegradable poly-(L-lactic) acid membranes. Von Langenbeck's procedure was simulated in 12-week-old beagle dogs without clefts. In one group normal wound healing was allowed. In two groups, membranes were inserted immediately after surgery or 3 weeks thereafter. Sham and control groups were also included. Histologic evaluation was carried out at regular intervals. Reports have been published on other aspects, such as clinical wound healing, contraction and maxillary arch development in beagle dogs following this treatment. After direct implantation of membranes, wound healing was retarded. Disintegration of the membranes started soon after implantation and remaining particles were surrounded by a fibroblastic sheath and a fibrous capsule. At sites where membrane particles persisted, attachment of the scar tissue to the underlying bone by Sharpey's fibers was prevented.     

Effects of short-term treatment with the bisphosphonates zoledronate and pamidronate on rat bone: a comparative histomorphometric study on the cancellous bone formed before, during, and after treatment

To study the anti-resorptive effects of zoledronate and pamidronate on growing long bones we have performed a histomorphometric analysis of the three regions of the proximal tibial cancellous bone of bone formed before, during, and after drug treatment. Male rats (190-220 g) were treated subcutaneously for 10 days with zoledronate (0.028-2.8 microg/kg) or pamidronate (3.7-370 microg/kg) and sacrificed 5 days later. To delineate the three regions of cancellous bone, and for dynamic bone histomorphometry, calcein and demeclocycline were injected at various times. Both bisphosphonates caused a dose-dependent suppression of cancellous bone turnover and resorption to produce an increase in cancellous bone, but zoledronate was 100 times more potent than pamidronate. The increase in the bone amount and connectivity was more pronounced in the bone formed during treatment where transient bone resorption and normal bone formation led to a positive bone balance. In the bone formed before treatment, inhibition of bone resorption associated with reduced bone formation produced a net gain in amount of bone. Although both bone regions showed a positive bone balance, more bone accumulated in the bone formed during treatment probably because its trabecular bone surface was three times greater. In the primary spongiosa formed after treatment, a moderate increase in the bone amount and connectivity was observed only at the highest dose of both bisphosphonates. The bone formed before, during, and after treatment with bisphosphonates responds differently due to differences in bone architecture, rates of modeling and remodeling, and period of drug exposure.     

Use of myoblasts in assaying the osteoinductivity of bone morphogenetic proteins

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

Composition, constitution, and interaction of bone with hydroxyapatite coatings determined by FT Raman microscopy

Transfer of steroidal and nonsteroidal compounds across guinea pig amnion and chorion laeve was investigated as a function of stage of gestation, tissue orientation, steroid specificity, and molecular size. Each fetal membrane was examined at early and late stages of gestation, before and after pubic symphysis relaxation. Early amnion was impermeable to macromolecules and small charged molecules while [3H]estrone and [3H]pregnenolone were transferred, the latter depending on tissue orientation and involving conjugation at the basolateral interface. After symphysis dilation, amnion transferred all substrates tested with the exception of BSA; the molecular weight cutoff was approximately 5,000. Unlike amnion, early chorion transferred both free and conjugated steroids as well as inorganic sulfate. Transfer of estrone involved conjugation and depended on tissue orientation. Transfer of [3H]estrone-sulfate, [3H]estrone-glucuronide, and [3H]pregnenolone-sulfate was similar despite selective deconjugating activity toward estrone-sulfate. Near term, chorion was impermeable to inorganic sulfate and transfer of estrone-glucuronide depended on tissue orientation, involving deconjugation in the maternal to fetal direction. At no stage of gestation did chorion transfer macromolecules. These results suggest that the transfer of free and conjugated steroids across fetal membranes is differentially regulated by tissue, its stage of development, and direction of transfer.     

Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1

Osteogenic protein-1 (OP-1, BMP-7), a member of the transforming growth factor-beta supergene family, induces cartilage and bone formation when implanted in intra- and extraskeletal sites in vivo. The human OP-1 gene has been cloned and biologically active recombinant OP-1 homodimers (hOP-1) produced. The amount of bone induced by hOP-1 in vivo is related to the amount of protein implanted. Dentine possesses bone morphogenetic protein (BMP) activity. Impure material from allogenic bone with BMP activity induced reparative dentine formation in dogs. The objective of this study was to determine if the amount of reparative dentine stimulated by hOP-1 is related to the amount of protein utilized in direct pulp-capping experiments. Freshly exposed molar and premolar pulps were treated with varying amounts of a complex comprising hOP-1 and a carrier matrix of purified bovine type-1 collagen powder (CM) moistened with sterile saline. Reparative dentine was present in all hOP-1/CM treated teeth (12 of 15) that remained sealed for the 6 weeks' healing. Substantially more new dentine was present in teeth treated with hOP-1/CM than in those treated with Ca(OH)2 paste and the amount of reparative dentine formed was proportional to the amount of hOP-1/CM (P < 0.05). No reparative dentine formed in collagen carrier or untreated teeth. The appearances of the new tissue suggested that much of the mass of the hOP-1/CM was replaced first by a pulp-like connective tissue, which mineralized to form reparative dentine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.     

Recombinant human bone morphogenetic protein (rhBMP) induced heterotopic bone development in vivo and in vitro

In vivo, recombinant human bone morphogenetic protein (rhBMP-2) with deactivated bone matrix as a carrier, implanted in muscle in adult rates, induced development of heterotopic bone, including bone marrow. The volume of bone was proportional to the dose of rhBMP-2 in a range of 0.2-150 microg. In vitro, in response to 50-microgram dose range, subcutis- and brain-derived outgrowths differentiated into loosely woven connective tissues composed of spindle-shaped fibroblasts, adipocytes, and cartilage. Muscle-derived connective tissues cultivated first in culture media supplemented with 50 microgram of rhBMP-2 for 72 hr, then enclosed in a diffusion chamber, and immediately transplanted into a rectus abdominous muscle pouch in an autogenic rat for 28 days, induced cartilage development on the inside and transmembrane hetertoptic bone development including bone marrow on the outside. These experiments are interpreted to show that muscle derived connective tissue cells have the competence of embryonic cells to develop de novo in response to BMP in postfetal life.     

Effects of bioabsorbable and non-resorbable barrier membranes on bone augmentation in rabbit calvaria

The aim of this study was to compare the effects of bioabsorbable and non-resorbable membranes on experimental guided bone augmentation in 8 Japanese white rabbits. A cutaneous flap was demarcated and raised from the forehead of each animal, the periosteum was lifted, and the calvarial bone on both sides of the midline was exposed. A titanium screw was inserted into the bone on each side of the midline and one screw was covered with a bioabsorbable (polylactic acid) membrane and the other with a non-resorbable (expanded polytetrafluoroethylene) membrane. The implanted screws and membranes were then covered with the periosteum and cutaneous flap. After healing for 6 months, the animals were euthanized and the experimental area was prepared for histological investigation. New bone had formed under both membranes with no sign of infection or membrane exposure. The amount of newly generated bone (89.0 +/- 17.3% versus 54.7 +/- 14.0%, P <0.05) and the percentage of newly generated bone height (81.5 +/- 6.3% versus 58.9 +/- 7.8%, P <0.05) in the space beneath the non-resorbable membrane was greater than that beneath the bioabsorbable membrane. However, there were no statistically significant differences between the bioabsorbable and non-resorbable membranes with respect to the percentage areas of mineralized bone (52.3 +/- 11.3% versus 47.1 +/- 6.7%, P = 0.8658) and bone marrow (47.7 +/- 11.3% versus 52.9 +/- 6.7%, P = 0.4838) and bone contact with the screw (88.3 +/- 6.9% versus 89.2 +/- 7.3%, P = 0.9999). In conclusion, at least within the limitations of this rabbit model, we suggest that non-resorbable membranes with sufficient stiffness should be used to obtain greater bone volume and height instead of bioabsorbable membranes for the GBR procedure, and that this will facilitate predictable bone augmentation in spaces beyond the bone surface. Therefore, the bioabsorbable membrane could not replace the non-resorbable membrane used in this model.