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This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

This Classic Article is a reprint of the original work by Hiroshi Nogami and Marshall R. Urist, A Morphogenetic Matrix for Differentiation of Cartilage in Tissue Culture. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI10.1007/s11999-009-1068-3; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1970 by the Society for Experimental Biology and Medicine and is reprinted with permission from Nogami H, Urist MR. A morphogenetic matrix for differentiation of cartilage in tissue culture. Proc Soc Exp Biol Med. 1970;134;530-535.

This Classic Article is a reprint of the original work by Marshall R. Urist, A Morphogenetic Matrix for Differentiation of Bone Tissue. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1068-3; and a third Classic Article is available at DOI 10.1007/s11999-009-1069-2. The Classic Article is copyright 1970 by Springer and is reprinted with permission from Urist MR. A morphogenetic matrix for differentiation of bone tissue. Calc Tiss Res. 1970:4(Suppl);98-101.

This case report documents the first use of bone morphogenetic protein (BMP) as an adjuvant to revascularisation with a first dorsal metacarpal arterio-venous pedicle in the treatment of a patient with Stage III Kienbock's disease. The patient had complete relief of her symptoms of wrist pain by 8 months postoperatively, when X-rays showed no further evidence of lunate collapse and an MRI scan demonstrated islands of revascularisation. It is impossible to prove unequivocably that BMP contributed to the result seen in this one patient, but this adjuvant concept is based on experimental evidence demonstrating that optimal bioengineering of vascularised bone is dependent on four factors - a structural matrix, progenitor cells, BMP and a vascular supply, and BMP may play a future role in promoting new bone formation in Kienbock's disease.

A chronic nonunion of a proximal pole fracture of the scaphoid was treated by curettage of the nonunion, single K-wire fixation, and implantation of 50 mg of human bone morphogenetic protein followed by 12 weeks of cast immobilization without any conventional corticocancellous bone grafting or rigid screw fixation. Radiographs showed signs of bony healing by 12 weeks and a magnetic resonance imaging scan 6 years after surgery showed no signs of avascular necrosis. The potential future applications of human bone morphogenetic protein in hand surgery are discussed.

A retrospective evaluation was done of 15 patients (17 hips) with symptomatic osteonecrosis of the hip treated with core decompression combined with an allogeneic, antigen-extracted, autolyzed fibula allograft and 50 mg of partially purified human bone morphogenetic protein and noncollagenous proteins. The average duration of clinical followup of the patients was 53 months (range, 26-94 months). The osteonecrotic involvement of the hip was classified by plain radiographs using a modification of the Ficat staging system and MRI evaluations. Fifteen hips were classified as Ficat Stage IIA, one hip (one patient) was classified as Ficat Stage IIB, and one hip (one patient) was classified as Ficat Stage III. Fourteen hips had involvement of 50% or less of the femoral head and 2/3 or less involvement of the weight-bearing surface of the femoral head, based on a magnetic resonance imaging evaluation. The procedures were a clinical success in 14 of 15 hips (93%; 13 patients) with Stage IIA disease. Three of 17 hips (three patients) had radiographic progression (Ficat Stages IIA, IIB, and III) of the femoral head and were converted to total hip replacements. Only one of seven hips (six patients) with 50% or less involvement of the femoral head and between 1/3 and 2/3 of the weightbearing surface of the femoral head developed radiographic progression of the femoral head. There was no radiographic progression in the 3 hips with less than 1/3 involvement of the weightbearing surface of the femoral head. Further evaluation of the potential efficacy of bone morphogenetic protein is required in randomized trials.

A bone morphogenetic protein bioimplant was used for primary reconstruction of a 6-cm mandibular discontinuity defect, after a segmental resection of an ameloblastoma. Radiographic evidence of new bone induction was seen at 3 and 9 months, postoperatively. A biopsy was taken at 9 months demonstrated viable new bone formation at the bioimplant site. This is the first reported case using a bone morphogenetic protein bioimplant in a human, followed by histological confirmation of new bone.

Clinicians await the availability of synthetic bioimplants that will replace the need for autogeneic bone grafts in bone reconstructive surgery. For more than a decade, researchers have evaluated delivery vehicles for the tissue morphogen bone morphogenetic protein. The object of this investigation was to measure induced bone development when bone morphogenetic protein was delivered by human tendon collagen, human demineralized bone matrix, hydroxyapatite, a composite of human tendon collagen and human demineralized bone matrix (tendon collagen + demineralized bone matrix), Poloxamer 407, and a composite of human demineralized bone matrix and Poloxamer 407. Sixty-three adult male Swiss Webster mice (Harlan Sprague-Dawley, Indianapolis, Ind.) received 126 implants. The animals were divided into seven groups of nine animals, depending on carrier (six carriers plus the positive control group) used. Each animal received a bone morphogenetic protein-enhanced carrier in one hindquarter muscle mass, with the contralateral leg being implanted with the carrier alone. Implants were evaluated by quantitative radiomorphometry validated by histologic methods. Radiographically, no significant differences were identified among any of the implants evaluated (p > 0.05). Histomorphometric analysis demonstrated that Poloxamer 407 was significantly (p < 0.05) better at delivering bone morphogenetic protein than the other carriers involved in this investigation. The new bone developed in a tubular or spherical shape. Interaction of endogenous and exogenous delivery systems seems to be essential for optimal transmission of bone morphogenetic protein. The importance of the excipient to deliver bone morphogenetic protein and develop a bone morphogenetic protein concentration gradient has been emphasized by other investigators and confirmed by our research on poloxamer. With further research on the physicochemical mechanisms of localization and transmission of bone morphogenetic protein, it may be possible to avoid hazardous operations with autogeneic bone.

A composite inductive allograft consisting of an allogeneic, autolysed, antigen-free cortical bone carrier lyophilized with partially purified human bone morphogenetic protein was implanted in 30 consecutive femoral reconstructions that resulted from failure of fracture healing. There were 24 atrophic shortened femoral nonunions, four equal length femoral nonunions, and two femoral malunions. There were 10 men and 20 women with an average age of 47 years (range, 28-75 years). Allogeneic, autolysed antigen-free cortical bone was used as a structural alloimplant and as a delivery system for partially purified human bone morphogenetic protein. The composite implant of human bone morphogenetic protein/allogeneic, autolysed antigen-free cortical bone was used in conjunction with one-stage lengthening of the extremity, restoration of mechanical axis and rotational alignment. In 26 of 30 femurs, the human bone morphogenetic protein/allogeneic autolysed antigen-free cortical bone consisted of an allogeneic cortical bone implant incorporated into a one-stage lengthening of atrophic femoral nonunion. In four patients with equal length femoral nonunions, the human bone morphogenetic protein/allogeneic, autolysed antigen-free implant was placed as an medical femoral shaft onlay graft. Internal remodeling of the implant occurred within 8 to 12 weeks after implantation. Lengthening defects greater than 2 cm were supplemented with intercalary autogeneic bone graft. Twenty-four femurs healed at an average of 6 months at an average followup of 55 months. Four of six plate fatigue failures were salvaged with repeat plating. Two patients were lost to followup. The human bone morphogenetic protein/allogeneic, autolysed antigen-free bone allograft is an excellent structural and delivery system that induces host bone formation and implant remodeling allowing salvage of difficult femoral nonunions.

The purpose of this study was to compare extraction socket healing in 8 patients after implantation with either xenogenic bovine bone (n=5 sites), demineralized freeze-dried bone (DFDBA) (n=3 sites), autologous bone (n=3 sites), or human bone morphogenetic proteins in an osteocalcein/osteonectin carrier (hBMP/NCP) (n=2 sites). Three of the patients received 6 commercially pure micro screws which were fixed into extraction sockets, after which the sockets were implanted with either bovine bone (n=3 sites), DFDBA (n=2 sites) or intraoral autologous bone (n=1 site). Biopsies of the extraction sockets were taken from 3 to 6 months after treatment (average, 4.6 months). For comparison of healing between the implanted materials, histologic evaluation and bone scores were determined. Bone scores of 0 indicated an absence of new bone, with dead implanted bone particles entrapped within connective tissue, while a score of 3 indicated the entire field consisted of vital bone. Biopsies from bovine bone sockets revealed dead implanted particles surrounded by connective tissue. Isolated sections showed host bone in contact with the bovine bone particles. Bone scores ranged from 0 to 3. Biopsies from DFDBA-implanted sites revealed dead particles entrapped with dense connective tissue. The bone scores ranged from 0 to 1. Biopsies from sites implanted with hBMP/NCP revealed a combination of woven and lamellar bone with bone scores of 3. Five of the 6 micro screws were processed and evaluated. One screw was mobile at the time of removal and was not evaluated. Bone scores were used to compare new bone formation adjacent to the micro screws. Bone scores ranged from 0 to 2. A score of 0 indicated non-vital implant material in contact with host bone and connective tissue in contact with implant; 2 indicated vital bone in contact with the majority of the implant surface. Retrieved sockets with micro screws implanted with bovine bone (n=2) demonstrated a connective tissue interface between the screws and the surrounding tissues (bone score 0). The adjacent tissues showed dead bovine particles entrapped within fibrous tissue. Retrieved screws implanted with DFDBA (n=2) were surrounded by connective tissue, with dead bone particles enmeshed within fibrous tissue (bone score 0). The screw implanted with intra-oral autologous bone was primarily surrounded by vital bone with a connective tissue interface (bone score 1). Three implant threads were in contact with bone. The results of this study indicate that bovine bone, DFDBA, and intraoral autologous bone do not promote extraction socket healing. Sockets implanted with hBMP/NCP contained vital woven and lamellar bone. Xenogenic bovine bone and DFDBA did not contribute to bone to micro screw contacts and are not recommended for enhancement of vital bone to implant contacts. Intraoral autogenous bone also does not appear to significantly contribute to bone to implant contacts. Intraoral autologous bone, xenogenic bone, and DFDBA appear to interfere with normal extraction socket healing.

Fifteen patients with posttraumatic shortened atrophic femoral nonunions were treated with one-stage lengthening. The alloimplant was composed of allogeneic antigen extracted autolyzed human bone perfused with partially purified human cortical bone morphogenetic protein associated with noncollagenous protein and used as graft. The composite was lyophilized and sterilized with ethylene oxide. All 15 nonunions were atrophic diaphyseal and were lengthened through intercalary segmental defects bridged with the human bone morphogenetic protein composite alloimplants stabilized to the medial femoral cortex through plate osteosynthesis and lag screw fixation. One lengthened proximal femur had fatigue failure of the plate and was treated successfully by exchange plating. The average increase in length was 2.8 cm (range, 1.5-5 cm) and an average percentage increase in length of 8% (range, 4%-132%) of the residual shortened femur. The human bone morphogenetic protein composite produced an immediate reactive bone formation in the host bone and progressive remodeling of the donor recipient interfaces. There were no infections, allergic reactions, clinical rejection of the human bone morphogenetic protein composite alloimplants, or evidence of malignant disease. One-stage femoral lengthening augmented with human bone morphogenetic protein composite graft bridged the intercalary defect, remodeled the atrophic host bone and restored bone continuity within 1 to 2 years. Human bone morphogenetic protein composite alloimplants are a substitute of autogeneic bone graft and offer an alternative to iliac crest bone without the associated morbidity.

Bone morphogenetic proteins (BMP) were identified in full-thickness autogeneic implants of the urinary bladder by monoclonal antibody BMP immunohistochemistry and by in situ hybridization of BMP-2, -4, and -5 mRNAs. Within the first week after transplantation, BMP mRNAs were expressed in uroepithelial cytoplasm and basement membranes of proliferating cells. About the second week, BMP-2, -4, and -5 mRNA expression and monoclonal antibody BMP-2 synthesis were colocalized in the sites of conversion of transitional to columnar epithelium. In close association with basement membrane, mesenchymal-type cells migrated, proliferated, and formed osteogenetic condensations. The areas of condensation were composed of densely packed cells with high nucleocytoplasmic ratios and high mitotic activity, and resembled periosteum of growing membrane bone. For the first 3 to 4 weeks, the bone deposits grew rapidly in size on surfaces of columnar uroepithelial basement membrane. After 4 to 5 weeks, the growth was constrained by enclosure in a thick, dense, periosteum-like fibrous tissue, completely separated from basement membrane. Serial observations are submitted herewith as circumstantial evidence that bone development is a product of mutual interaction of uroepithelium and mesenchymal-type cells across basement membranes in response to endogenous BMP-2, -4, and -5. Neither BMP-2, -4, -5 mRNA nor monoclonal antibody BMP-2 was detected in the lamina propria, smooth muscle, or fibrous tissue.

Dysfunctional teeth, donated by community dental clinics, were recycled for research on bone morphogenetic protein (BMP) in health and disease. The crown remnants were trimmed away, and the roots were washed in 70% alcohol, demineralized, lyophilized, and prepared in one of two forms: (1) human partially demineralized root dentin matrix (PDM) or (2) autolysed, antigen-extracted root matrix (AAAD), including attached cementum. Composites of either PDM or antigen-extracted, autolysed, delipidized allogenic dentin matrix (AAAM) and recombinant human bone morphogenetic protein (rhBMP-2) were implanted in either normal or athymic mice. The percentage of muscle replaced by heterotopic bone was estimated by computer-assisted random point analysis. Implants of PDM and AAAD made from dysfunctional teeth exhibited little or no endogenous BMP activity and failed to induce new bone formation. Composites of 1 microgram of rhBMP-2 per 70 mg of PDM carrier induced 61% replacement of muscle by bone formation; 1 microgram of rhBMP-2 in 70 mg of AAAM matrix induced 78% replacement of muscle mass by bone; 2 or 5 micrograms of rhBMP-2 and 70 mg of AAAM carrier induced 100% replacement of thigh muscle mass by bone. In athymic mice, the areas of new bone were only slightly greater than those in normal mice. These observations suggest that root dentin prepared from extracted teeth may be recycled for use as a carrier of rhBMP-2 because it induces new bone formation in the periodontium.

In adult rabbits, mid-diaphyseal segments of the radius or ulna were excised to produce defects greater than the critical size for spontaneous bone repair. The defects were enveloped in sleeves composed of nonbiodegradable expanded polyfluoroethylene (ePTFE), pore size 30, 60, 90 microns, and compared with sleeves of three biodegradable materials. Bone morphogenetic protein and associated noncollagenous bone matrix protein (BMP/NCP) or recombinant human morphogenetic protein (rhBMP-2) were implanted inside the sleeves. Albumin was implanted for a control system. Without intracompartmental BMP, only about 10%-15% of the defect was repaired by bone growth extending from the bone ends into the sleeves composed of ePTFE, pore size 30 microns. With sleeves with pore size 60 or 90 microns and intracompartmental BMP/NCP, 54%-96% regeneration occurred within 8 weeks after the operation. Sleeves of biodegradable nonimmunogenic materials such as polyorthoester (POE) and polylactic-polyglycollic acids (PLA/PGA) permitted 86%-98% restoration of bone continuity, but only when BMP was present in the lumen. With puncture holes (0.5 mm in diameter), implants of BMP/NCP in the 30-micron PTFE sleeve produced transmembrane external callus formation and bone regeneration to 147%. Sleeves composed of aorta first calcified, then induced complete intracompartmental bone regeneration. Atelocollagen sleeves incited a low-grade inflammatory cell reaction and did not promote complete regeneration. Under conditions presently undisclosed segments of the ulna bridged with ePTFE, were incompletely paired, even with intracompartmental BMP/NCP. Puncture holes of 0.5 mm admitted ingrowth of capillaries and introduced local conditions favorable for the response to BMP/NCP. BMP/NCP may promote proliferation of nutrient vessels and differentiation of bone marrow stroma cells between the open bone ends. For further investigation, the hypothesis to be examined is that the optimum response to BMP/NCP and rhBMP-2 would emerge in compartments containing first a high concentration gradient and second proliferating perivascular cells.

In the process of separation of bone morphogenetic proteins from bone matrix, lipids were found in unexpected amounts closely associated with noncollagenous proteins soluble in guanidine hydrochloride. Lipids representing 33.7-49.9% by weight were recovered with various solvents. Composites of noncollagenous proteins and lipids soluble in either chloroform- methanol or acetone implanted in the hindquarter muscles of mice induced the formation of large deposits of heterotopic bone. The protein-lipid aggregates formed microspherules which were stained by Sudan Black B. Implants of bone morphogenetic proteins and noncollagenous proteins-lipid microspherules stained with Sudan Black B induced bone development in the same manner as unstained delipidized bone morphogenetic proteins associated with noncollagenous proteins. Lipid-free osteocalcein, osteonectin, albumin and other bone matrix proteins did not induce bone formation or bind Sudan Black B. The more highly purified the noncollagenous proteins, with or without activity of bone morphogenetic proteins, the lower the level of binding with Sudan Black B. Acetone-soluble bone matrix lipids consisted chiefly of triglycerides, cholesterol and saturated short chain fatty acids, and included little or no phospholipids or monounsaturated fatty acids. Composites of recombinant bone morphogenetic proteins-2 and acetone-soluble lipids induced larger deposits of bone than implants of recombinant bone morphogenetic proteins-2 without acetone-soluble lipids. The hypothesis that an association of bone lipids with protein facilitates the local transport of bone morphogenetic proteins warrants further investigation.

The SENCAR (sensitive to carcinogenesis) mouse is a unique tool for investigating the interaction between a specific defect in intracellular signaling, dietary calcium, and metabolic bone disease. The SENCAR mouse was developed by selective breeding for enhanced sensitivity to two-stage carcinogenesis. Its major genetic defect, which renders it exquisitely sensitive to stimulation with diacylglycerol or phorbol esters, is in the regulatory domain of protein kinase C, one of the primary intracellular mediators of hormonal effects. At sexual maturity, SENCAR mice are large and have big bones, but our previous pharmacokinetic studies showed that they accumulate less calcium under normal conditions and lose more calcium under adverse conditions than do other, standard strains of mice. To histologically define the effect of low dietary calcium on bone metabolism, we performed histomorphometric analysis of tetracycline-labeled sections of femoral bone from male SENCAR mice maintained on calcium-sufficient and calcium-deficient diets during the critical period from 10 to 14 weeks of age. The bone volume, absolute osteoid volume, and mineral apposition rate were lower at 14 than at 10 weeks of age in SENCAR mice fed 0.02 or 0.6% calcium diets. Calcium deficiency increased the architectural disarray and the probability of observing focal discontinuities in the growth plate. Thus, characteristic features of impaired bone metabolism (low bone volume and apposition rate) develop early in SENCAR mice and are exacerbated by low dietary calcium. Detailed examinations of the histology and biochemistry of SENCAR mouse bone will provide insights into the mechanisms by which specific defects in the signal transduction of protein kinase C contribute to impaired bone metabolism.

The calpain-calpastatin system, which consists of calpains I and II (two ubiquitously distributed calcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain-calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain-calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Native bone morphogenetic protein and associated noncollagenous proteins induced the formation of heterotopic bone in the hindquarter muscles of osteopetrotic (op/op) mice and those of their phenotypically normal littermates (+/?). In op/op mice, the heterotopic bone consisted of a disorganized, densely packed mixture of irregular calcified cartilage, osteoid, chondro-osteoid, and fibrous tissue. Injections of recombinant human macrophage colony-stimulating factor initiated bone resorption that began in the peripheral vascularized regions of the metaphyses and continued in central areas of uncalficified avascular chondro-osteoid. On vascularized surfaces, osteoclasts were stained with tartrate-resistant acid phosphatase. In op/op mice treated with macrophage colony-stimulating factor, the osteoclasts were small, with only two or three nuclei, and they did not exhibit tartrate-resistant acid phosphatase staining. In untreated op/op mice, surgical blood clots persisted in the heterotopic sites as late as 3 weeks after the operation, whereas in treated op/op mice, the blood clots were absorbed almost as rapidly as in normal mice. Histologically, recombinant human macrophage colony-stimulating factor restored normal macrophage functions: absorption and organization of blood clot, osteoclastogenesis, synthesis of tartrate-resistant acid phosphatase, bone remodeling, islands of myelopoiesis, and construction of an ossicle complete with a cortex and a medulla filled with functioning hematopoietic bone marrow.

By means of monoclonal anti-bone morphogenetic protein 2 immunohistochemical methods, endogenous bone morphogenetic protein was observed in the process of generation of heterotopic bone in experimental punch holes in the rabbit's ear. In repair of the punch hole, dermis, subcutaneous connective tissue, and perichondrium proliferated, hypertrophied, and differentiated in the rim within 2 weeks. By 3 to 4 weeks, epidermis grew centripetally down into and across the dorsal and ventral openings and sealed the punch hole. A blastema-like structure consisting of a condensation of the mesenchymal type cells covered the cut ends of the elastic cartilage. The condensation differentiated into chondro-osteoprogenitor cells and hyaline cartilage within 4 to 5 weeks. Within 4 to 6 weeks, sprouting capillaries, macrophages, and monocytes resorbed and replaced hyaline cartilage with a perichondral ring of bone. Anti-bone morphogenetic protein 2 appeared first in the perichondrium, then in the condensation, and later in the chondro-osteoprogenitor cells. A basic assumption was that latent non-reactive bone morphogenetic protein was converted to the anti-bone morphogenetic protein 2-reactive form by injury, inflammation, and proteolysis. The reactive form and various other local factors contributed the temporal and spatial constraints of a morphogenetic field for development of heterotopic bone. The receptors and mechanism of bone morphogenetic protein signal transduction are unknown.

Bone morphogenetic protein is an important molecule whose bioactivity depends on the carrier. Squalane is used in the formulation of various kinds of cosmetics because it is easily emulsified and has the property of spreading well. Thus, squalane might be effective as a bone morphogenetic protein delivery system. As a test for this possibility, gelatin capsules containing squalane and bone morphogenetic protein (bovine derived partially purified) composite were implanted under the hind-quarter perimuscular membrane of ddY mice. Control capsules containing only bone morphogenetic protein were used for controls. The implants were radiographically and histologically examined at 1 to 4 weeks after the operation. According to the radiographic analysis, squalane and bone morphogenetic protein composite and bone morphogenetic protein only control specimens formed widespread heterotopic bone tissues. The amount of heterotopic bone formation in the composite experimental specimens was approximately 40% greater than that in the controls. Histologic examination of experimental and control specimens revealed varying amounts of perichondral ossification by 2 weeks. By 3 and 4 weeks, the bone deposits were colonized by hematopoietic bone marrow. Squalane was effective for the slow local release of bone morphogenetic protein. Furthermore, the squalane and bone morphogenetic protein composite was a reliable osteoinductive biomaterial.

An extensive literature on bone morphogenetic protein (BMP) induced generation and regeneration shows general agreement about one observation. The incidence and quantity of bone were greatest when BMP was delivered with a carrier of various biologic and non biologic polymers. In the present research, neutral lipids either endogenous in demineralized bone matrix (DBM) or exogenous in orign were employed as a delivery system for induced heterotopic bone formation in the rectus muscle in rats. Total neutral lipids including cholesterol were measured by correlated gravimetric and Sudan Black B dye binding methods. The heterotopic bone was measured by computer assisted radiomorphometric and histologic methods. Bone formation was measured by total calcium, DNA-P, and alkaline phosphatase activity. Composites of BMP and neutral lipids, separable from phospholipids by extraction with absolute acetone, were consistently osteoinductive. A significant quantity of the total bone lipid was closely associated with and extractable from the bone matrix non-collagenous protein fraction which had high levels of BMP activity. Lathyritic matrix was very low both in dye binding and osteoinductive activity. These observations suggest the possibility that lipids may serve as a BMP carrier in the process of induced bone development.

A composite implant consisting of a coral cylinder, moose bone morphogenetic protein and type IV collagen was used to repair a segmental tibial defect in sheep. Healing, related variance in mechanical strength and immune responses were evaluated. In comparison with a coral control, a larger amount of newly formed external callus was observed in the composite group at 6 weeks. The maximal torque capacity, maximal angular deformation at failure and bone stiffness of a healed osteotomised tibia recovered 113%, 117% and 120% in the coral controls and 67%, 92% and 79% in the composite implants against the corresponding contralateral tibia at 16 weeks respectively. A significantly elevated anti-BMP antibody was detectable in the composite group at 3 and 6 weeks. Augmented bone formation at an early stage and weakened torsional performance at a later stage in the composite implants may indicate the phase-specific osteoinduction and the immune response of xenogenic BMP with time.

The objectives of the present research on the osteopetrotic mouse are to investigate the factors influencing heterotopic bone development. The osteopetrotic mutant was deficient in macrophage colony stimulating factor and failed to activate functioning monocytes, macrophages, and osteoclasts. Macrophage colony stimulating factor deficiency also caused a heretofore undescribed delay in organization and absorption of hematomas resulting from surgical operations. Surgically implanted in a heterotopic site, bone morphogenetic protein induced approximately 10% more bone in osteopetrotic than littermate+/? mice. Radiographically, the heterotopic bone was at least 50% denser than new bone. The new bone was metachromatic or slightly basophilic rather than eosinophilic and undermined with large deposits of hypercalcified hypertrophic cartilage. Bone mineral in the osteopetrotic mouse was deposited in an apatite-like form with a higher calcium/phosphorus ratio than the bone of +/? littermates. High levels of alkaline phosphatase synthesis were sustained longer in the osteopetrotic mouse than in the +/? littermate. Tartrate resistant acid phosphatase synthesis was almost nil in osteopetrotic mice during the first 4 weeks, and thereafter appeared coincidental with spontaneous remission of osteopetrosis at 6 weeks. Implants of the mineralized cortical bone matrix of the osteopetrotic mouse showed minimal if any bone morphogenetic protein activity of matrix of +/? littermate or otherwise normal mice. The cause of the remission of the bone disorder in the osteopetrotic mouse is not known but is of great interest to students studying the problem of coupling of bone formation to bone resorption.

Bone morphogenetic protein-2 is a low molecular weight glycoprotein, classified as a morphogen. The sine qua non of bone morphogenetic protein is consistently reproducible induction of bone development in heterotopic sites. Bone morphogenetic proteins belong to the expanding transforming growth factor-beta superfamily. Bone morphogenetic protein-2 has pleiotropic functions that range from extraskeletal and skeletal organogenesis to bone generation and regeneration. Bone morphogenetic protein induced bone formation in postfetal life recapitulates the process of embryonic and endochondral ossification. Through recombinant gene technology, human bone morphogenetic protein-2 is available in almost unlimited amounts for basic research and clinical trials. Human bone morphogenetic protein-2 induces structurally sound orthotopic bone in a variety of experimental systems, including femoral defects in rats, tibial and ulnar defects in rabbits, femoral defects in sheep, mandibular defects in dogs, spinal fusion in dogs, and porous ingrowth in rats. Human bone morphogenetic protein-2 research extends to the fields of developmental biology, genetics, and evolution. Bone morphogenetic protein has been used successfully at the authors' institution to heal clinical nonunions and to achieve spinal fusion. This report reviews the current understanding of bone morphogenetic proteins in general and BMP-2 in particular and summarizes their potential applications.

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.

Four commercially available preparations of demineralized freeze-dried human bone powders (DFDB) were investigated for endogenous bone morphogenetic protein (BMP) as observed by osteoinductive activity. Composites of DFDB without and with 1 microgram or 10 micrograms of recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted into the hindquarter muscles of Swiss-Webster mice. The four batches of DFDB without rhBMP-2 were placed also in hindquarter muscles of athymic mice. Three weeks after implantation, the area of induced bone and cartilage formation was measured by radiographic and histomorphometric methods. In normal mice, without rhBMP-2, DFDB implants induced development of dense fibrous connective tissue with minimal, if any, new bone. In athymic mice, DFDB induced development of small patches of appositional new bone. In contrast, in normal mice, composites of DFDB and rhBMP-2 induced development of large areas of heterotopic bone and bone marrow formation. The bone morphogenetic response occurred with a statistically significant difference (p < 0.0001) between implants of 1 microgram and the 10-microgram rhBMP-2 composites. Thus, DFDB from all four bone banks demonstrated comparable capacity to serve as a carrier for rhBMP-2.

Few studies have been performed to investigate the characteristics of native Bone Morphogenetic Protein (BMP). We derived 5.85 g of native moose BMP from 102 kg of fresh moose bone and performed chromatographic studies to characterise its structure. We found the BMP molecule to have a molecular weight of 11-700 kDa. To investigate the ideal local delivery system for BMP we used porous discs as a carrier. These were implanted into 132 mice. The volume of new bone formed was evaluated by means of histological specimens, radiographs and isotope studies. The best results were obtained by implanting porous discs (coral and ceramic) with type IV collagen and BMP. We suggest that this is the best delivery system for future experiments regarding the acton of BMP.

The purpose of this study was to test the osteoinductive properties of demineralized freeze-dried bone (DFDBA) randomly purchased from four commercial bone banks. Twenty-five (25) milligrams of bone from each of the banks was implanted into the hindquarter muscles of athymic mice. Two samples from each of the banks were compared with samples from the other banks. A total of 16 implants were grafted into 8 mice. Two additional mice served as controls. One mouse received an implantation of deactived human cortical bone matrix (DBM) (negative control). The other mouse received an implant of human bone morphogenetic protein/non-collagenous proteins (hBMP/NCP) infused to surface demineralized human cortical bone (positive control). At 21 days the mice were killed, the hindquarters were photographed, and the tissues were prepared for histologic evaluation. Of the 16 commercial DFDBA implants, 12 were available for histologic evaluation. There was no radiographic evidence of bone formation for the DFDBA implanted mice or the DBM implants. Small bone ossicles were scarcely visible in the hindquarters of the mouse which received the hBMP/NCP infused bone. Histomorphometric analysis was used to determine the percentage of new and dead bone. The bone was measured in pixels. The predominant histologic feature of the DFDBA implants was non-vital bone chips with minimal amounts of new bone. The average amount of non-vital bone ranged from 78.4% to 92.5%. There was no evidence of bone formation for the DBM implants. The average amount of bone for the mouse which received hBMP/NCP was 96%. The results of this pilot study indicate that commercially-available DFDBA induced clinically insignificant amounts of bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Demineralized matrix was delipidized with chloroform methanol before and after demineralization and implanted in muscle of allogeneic rats. Because lipids are difficult to separate completely from bone collagen, another preparation was gelatinized and delipidized with either chloroform methanol or acetone or both. Bone matrix demineralized without delipidization induced formation of a spherical-shaped deposit of new bone, which was remodeled to form a shell of cortical bone and central pool of normal hematopoietic bone marrow. When the bone was delipidized, only 20% to 25% was resorbed and replaced by new bone; unresorbed matrix failed to recalcify. Gelatinized bone matrix delipidized before implantation was even less well resorbed or replaced by new bone, but 12% to 44% of the matrix residue recalcified. The new deposits of bone were colonized by blood-borne bone marrow-derived stem cells and developed central pools of normal hematopoietic bone marrow. Recalcified residual matrix did not develop bone marrow. Additional investigations are required to determine whether the host bed adipocytes provide the phospholipids for recalcification of bone matrix. There was no preliminary recalcification in matrix-induced bone development, even though the 2 processes may occur simultaneously under specified experimental and pathologic conditions. Additional investigations are in progress to determine whether certain acetone soluble lipids may form the endogenous delivery system for bone morphogenetic protein and induced bone development.

Bovine bone morphogenetic protein and associated noncollagenous proteins (bBMP/NCP) were implanted in skull trephine defects, 14 and 22 mm in diameter, in adult minipigs. The defects were larger than the critical size for repair of cranial bones in pigs observed for a period of six to 16 weeks. The trephines were placed either in the nasal bone at eye level, or in the frontal bone. In minipigs the repair was more advanced in trephines in the frontal bone than in the nasal region, but was incomplete in either bone bed. As in dogs, implants of bBMP/NCP in heterotopic sites were resorbed before a competent cell population could respond. Serum anti-BMP antibody was elevated. The IgG levels of the antibody titer showed a clearcut dose-response; the level was significantly higher in one pig with implants of 85 mg of bBMP/NCP proteins in a frontal bone trephine.

Bone morphogenetic protein (BMP) induces cartilage and bone development in embryonic and post-fetal life. Bone morphogenetic protein-induced chondrogenesis in outgrowths of muscle connective tissues on various furrowed and unfurrowed fibronectin-coated substrata was observed by biochemical and histologic methods. The substrata consisted of cellulose acetate membranes, atelocollagen, denatured autoclaved demineralized bone matrix, bone matrix deactivated by extraction of BMP with guanidine hydrochloride (GuHCl), and aggregates of insoluble BMP and associated noncollagenous proteins (BMP/NCP) prepared from bovine bone. The furrows, which were mechanically cut into the substrata, were intended to increase surface area and provide extra spaces for cell proliferation under compression. The extent to which extracellular attachments were required for induced cartilage development was reflected in the quantity of cartilage formed when BMP/NCP, either in insoluble or in water-soluble form, was introduced was greatest on GuHCl-extracted bone matrix. On cellulose acetate and atelocollagen, BMP-induced cartilage development was relatively scanty. A substratum of bone matrix, denatured by autoclaving at a minimum of 125 degrees, permitted cell-to-cell adhesion but not cell-to-substratum attachments; the end product was loose fibrous connective tissue only. In contrast, cartilage development occurred on surfaces of undissolved particles of BMP/NCP. Water-soluble human BMP induced development of masses of amorphous cartilage. Even after it was extracted with GuHCl, rat bone matrix may have retained trace amounts of endogenous BMP. Thus, when the requirements of cells for cell-to-cell adhesion and cell-to-substratum attachment, including mechanical factors such as furrows to enlarge surface area, were met, cartilage development was a manifestation of temporal, spatial, and BMP distribution patterns. In situ hybridization and immunofluorescent microscopy with the aid of antirecombinant BMP antibiotics may provide new information about morphogenesis.

Bone morphogenetic protein (BMP) reproducibly induces chondrogenesis and osteogenesis when implanted into skeletal muscle. The exact identity of the cell that responds to BMP is not known. Furthermore, controversy exists regarding the possibility that myoblastic cells may transdifferentiate to chondrocytes and osteoblasts under the influence of BMP. We have therefore, undertaken studies on the effects of BMP on differentiation in L6 and C2C12 cells, two rodent myoblastic cell lines. To gain insights into the mechanisms of action of BMP, we have studied the effects of BMP on the levels of expression of the four known myogenic determination genes: myogenin, Myo D, herculin, and myf-5. BMP inhibited myogenesis in myoblastic cells. Convincing evidence of transdifferentiation of myoblasts to chondrocytes or osteoblasts was not seen. BMP inhibited the expression of all four myogenic determination genes.

Titanium sponges were infused with bone morphogenetic protein (BMP-Ti), and the osteoinductivity of the resultant composite was measured. New bone formation occurred three weeks after implantation and was identified by soft x-ray analysis. Quantitative analysis showed no significant difference between BMP-Ti composites and control samples (BMP only). Consequently, pure titanium neither inhibited nor promoted BMP activity. Chondrocytes and new bone formation occurred in direct contact with the surfaces of the titanium. X-ray microanalysis demonstrated new bone formation inside the pores of the titanium sponges. The BMP-Ti composite has interesting properties as an osteoinductive implant and has potential practical clinical applications.

To promote a high concentration gradient, syngeneic bone marrow and muscle connective tissue cell cultures and subcultures were exposed to bone morphogenetic protein and associated noncollagenous bone matrix proteins (BMP/NCP) in vitro, transferred to diffusion chambers, and then transplanted into the anterior abdominal wall of isogeneic rats. Both muscle- and marrow-derived cells differentiated into cartilage and chondroosteoid on the inside of diffusion chambers. New bone developed on the vascularized outside surfaces in juxtaposition to avascular tissue on the inside. Levels of glycosaminoglycans (GAGs) and DNA synthesis in tissues inside the chambers were sharply elevated. These data, correlated with histologic observations, demonstrate that the BMP/NCP recruits mesenchymal-type cells for a skeletal tissue pathway of development. Muscle-derived cells, as distinguished from marrow-derived cells, originate in a nonosseous environment but differentiate into osteoprogenitor (osteogenic stem) cells and display levels of DNA synthesis almost as high as marrow stroma-derived cells. The rise in DNA synthesis was maximal in the interval from two to three weeks after transplantation. Whether the bone marrow stroma mesenchymal cell target for BMP is only the osteogenic stem cell population or also includes colony-forming units fibroblasts (CFU-F) remains to be investigated by experiments on established cell lines.

Twenty-five patients with resistant nonunions including partial or complete segmental defects were treated with a composite alloimplant of human bone morphogenetic protein (h-BMP) and autolyzed, antigen-free, allogeneic bone (AAA). The series consisted of 16 females and nine males; average age was 45 years. Preoperative symptoms averaged 30 months (range, five to 83 months); 22 of 25 patients had failed multiple attempts at electrical stimulation. Twenty-three of 25 patients had an average of three prior failed surgical attempts at union (range, one to ten). There were ten segmental defects with an average length of 4 cm (range, 2-9 cm). The composite implant was incorporated as an onlay in 15 extremities and as an inlay graft supported by internal fixation in ten extremities. Seven patients received supplementary autogeneic cancellous bone grafting. Average healing time was six months (range, three to 14 months). Average follow-up time was 21 months (range, five to 82 months). Functional results were rated as excellent, 14; good, five; and fair, five. One failed to unite because of a recurrent infection. Union was obtained in 24 of 25 patients. There were five failures of the original operation that required reoperations; union eventually occurred in four of five extremities by repeat composite grafting and replacement of the failed internal fixation. Bony union between host bone and the composite implant began at an average of eight weeks postoperatively. Present results indicate that h-BMP/AAA composite implants represent adjunctive treatment of difficult nonunions. The h-BMP/AAA composite implants may be implanted in either partial or complete segmental defects of long bones.(ABSTRACT TRUNCATED AT 250 WORDS)

An allogenic aggregate of bone morphogenetic protein (BMP) and insoluble non-collagenous proteins (NCP) as well as a crude GuHCl extract were isolated from rats diaphyseal bones. Intramuscular implantation of 5 mg and 10 mg rat BMP/NCP in rats formed new ossicles, whereas 20 mg GuHCl extract failed to induce heterotopic bone formation. When 6 samples of inactivated rat bone matrix gelatin (BMG) were reconstituted with 0.75 mg of either BMP/NCP or GuHCl extract all 3 matrices reconstituted with BMP/NCP but only 1 out of 3 samples reconstituted with GuHCl extract induced heterotopic bone formation. Inactivated BMG alone did not show any osteoinductive activity. The small amount of BMP/NCP necessary for osteoinduction when recombined with inactivated BMG suggests that growth factors in bone matrix without inherent bone-forming activity enhance BMP activity. In vitro, connective tissue outgrowths of neonatal rat muscle on a substratum of inactivated rat BMG differentiated into cartilage in response to 0.05 microgram/ml, 0.5 microgram/ml and 5.0 micrograms/ml allogenic BMP/NCP added to the medium during the incubation period of 2 weeks. On day 14 of cultivation S35-sulphate incorporation into glycosaminoglycans (GAG) and H3-thymidine incorporation into DNA were measured, and the results related to the DNA content and the weight of the incubated muscle tissue, respectively. All doses of BMP/NCP increased GAG synthesis statistically significantly (p less than 0.05 to p less than 0.001). In contrast to that, DNA synthesis rate was not influenced by BMP/NCP. This suggests that GAG synthesis was not caused by cell proliferation but by cell differentiation.

Isolated from an osteosarcoma specimen from a 15-year-old girl and assayed by implantation, partly purified lyophilized bone morphogenetic protein (BMP) induced heterotopic ossification in the mouse quadriceps. The apparent molecular weight was 18 kDa. Osteosarcoma-derived BMP induced the same process of bone development as human BMP from normal bone matrix.