Synergistic effect of IGF-1 and OP-1 on matrix formation by normal and OA chondrocytes cultured in alginate beads.

OBJECTIVE: Growth factor therapy may be useful for stimulation of cartilage matrix synthesis and repair. Thus, the purpose of our study was to further understand the effect of combined insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) treatment on the matrix synthesized by human adult normal and osteoarthritic (OA) chondrocytes. DESIGN: Chondrocytes were isolated post-mortem from articular cartilage from tali of normal human donors and femoral condyles of OA patients undergoing knee replacement surgery. Cells were cultured in alginate beads for 21 days in four experimental groups: (1) "mini-ITS" control; (2) 100 ng/ml IGF-1; (3) 100 ng/ml OP-1; (4) IGF-1+OP-1, each at 100 ng/ml. Beads were processed for histological (Safranin O and fast green), morphometrical and immunohistochemical (aggrecan, decorin, type I, II, VI, and X collagens, and fibronectin accumulation) analyses. RESULTS: Histology showed that IGF-1 alone did not induce substantial matrix production. OP-1 alone caused a considerable matrix formation, but the highest matrix accumulation by normal and OA chondrocytes was found when OP-1 and IGF-1 were added together. Morphometrical analysis indicated larger matrices produced by OA chondrocytes than by normal cells under the combined treatment. All tested matrix proteins were more abundant in the combination group. Type X collagen was detected only under the combined OP-1 and IGF-1 treatment and was present at very low levels. Type I collagen was found only in OA chondrocytes. CONCLUSIONS: The results obtained in the current study suggest that combined therapy with IGF-1 and OP-1 may have a greater potential in treating cartilage defects seen in OA than use of either growth factor alone.

Regulation of anabolic and catabolic gene expression in normal and osteoarthritic adult human articular chondrocytes by osteogenic protein-1.

OBJECTIVE: Osteoarthritis is characterized by dramatic changes in chondrocyte metabolism including the overexpression of catabolic enzymes, but also a lack of anabolic activity. In this respect, osteogenic protein 1 (OP-1) appears to be one of the most potent anabolic factors of chondrocytes. In this study, we were interested in: (1) whether recombinant human OP-1 exerts its anabolic effects also on osteoarthritic chondrocytes, (2) whether OP-1 modulates the expression of catabolic genes, and (3) whether the BMP effects are related to the expression levels of its intracellular mediators (R- and I-Smads). METHODS: Chondrocytes were isolated from cartilage of either normal (n = 5) or osteoarthritic (n = 8) human knee joints and cultured in short-term high-density monolayer cultures with and without recombinant OP-1. RNA was isolated and analyzed for mRNA expression levels of anabolic (aggrecan, collagen type II), catabolic (MMP-1, -3, -13, ADAMTS-4), and intracellular signaling mediators (Smad 1, 4, 5, 6, 7, and 8) by quantitative online PCR. RESULTS: After OP-1 stimulation, the anabolic genes were significantly up-regulated in osteoarthritic chondrocytes in comparison to normal chondrocytes. Neither in normal nor osteoarthritic chondrocytes were significant changes observed for the matrix degrading enzymes. Smads were also expressed in both normal and osteoarthritic cells at roughly the same level with and without stimulation with OP-1. CONCLUSION: Osteoarthritic chondrocytes are not hypo-responsive to anabolic stimulation by OP-1. Thus, human recombinant OP-1 could be a suitable anabolic activator of osteoarthritic chondrocytes. This might be of particular interest as chondrocytes themselves showed very low levels of OP-1 expression.

Acceleration of callus maturation using rhOP-1 in mandibular distraction osteogenesis in a rat model.

The aim of this study was to assess if the application of rhOP-1 induces accelerated consolidation of the callus in mandibular distraction osteogenesis. In seven adult Wistar rats a bilateral osteotomy of the horizontal ramus of the mandible was performed in the molar region and a custom designed distractor was mounted to the mandible. With a rate of 0.7 mm per day the device was activated bilaterally after the seventh postoperative day. After seven days of distraction two times 50 microg rhOP-1 were injected on two subsequent days directly into the callus. The contralateral side received an injection of placebo solution. The animals were killed four weeks after the end of distraction. A three-point bending test revealed a significantly higher strength of the distracted mandible in the rhOP-1 side (66.3 N vs. 30.4 N, P=0.034, paired t-test). Undecalcified histological sections were examined using microradiography and fluorescence microscopy after sequential intravital polychromic labelling. A continuous bony bridging was seen in all rhOP-1 sites and in none of the control sites. The data indicate that rhOP-1 may be an option to accelerate callus maturation in mandibular distraction osteogenesis.

Periodontal tissue regeneration by combined applications of recombinant human osteogenic protein-1 and bone morphogenetic protein-2. A pilot study in Chacma baboons (Papio ursinus).

Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue engineering in clinical contexts.

Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates.

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.

Long-term evaluation of bone formation by osteogenic protein 1 in the baboon and relative efficacy of bone-derived bone morphogenetic proteins delivered by irradiated xenogeneic collagenous matrices.

To investigate the long-term efficacy of irradiated recombinant human osteogenic protein 1 (hOP-1) in bone regeneration and morphogenesis, hOP-1 was combined with a bovine collagenous matrix carrier (0, 0.1, 0.5, and 2.5 mg hOP-1/g of matrix), sterilized with 2.5 Mrads of y-irradiation, and implanted in 80 calvarial defects in 20 adult baboons (Papio ursinus). The relative efficacy of partially purified bone-derived baboon bone morphogenetic proteins (BMPs), known to contain several osteogenic proteins, was compared with the recombinant hOP-1 device in an additional four baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 90 and day 365 showed that gamma-irradiated hOP-1 devices induced regeneration of the calvarial defects by day 90, although with reduced bone area compared with a previous published series of calvarial defects treated with nonirradiated hOP-1 devices. One year after application of the irradiated hOP-1 devices, bone and osteoid volumes and generated bone tissue areas were comparable with nonirradiated hOP-1 specimens. Moreover, 365 days after healing regenerates induced by 0.5 mg and 2.5 mg of irradiated hOP-1 devices showed greater amounts of bone and osteoid volumes when compared with those induced by nonirradiated hOP-1 devices. On day 90, defects treated with 0.1 mg and 0.5 mg of bone-derived baboon BMPs, combined with irradiated matrix, showed significantly less bone compared with defects receiving irradiated devices containing 0.1 mg and 0.5 mg hOP-1; 2.5 mg of partially purified BMPs induced bone and osteoid volumes comparable with the 0.1-mg and 0.5-mg hOP-1 devices. Control specimens of y-irradiated collagenous matrix without hOP-1 displayed a nearly 2-fold reduction in osteoconductive bone repair when compared with nonirradiated controls. These findings suggest that the reduction in bone volume and bone tissue area on day 90 may be caused by a reduced performance of the irradiated collagenous matrix substratum rather than to a reduction in the biological activity of the irradiated recombinant osteogenic protein. This is supported by the results of in vitro and in vivo studies performed to determine the structural integrity of the recovered gamma-irradiated hOP-1 before application in the baboon. Recoveries by high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS/PAGE)/immunoblot analyses indicated that doses of 2.5-3 Mrads of gamma-irradiation did not significantly affect the structural integrity of the recovered hOP-1. Biological activity of the recovered hOP-1 was confirmed in vitro by showing induction of alkaline phosphatase activity in rat osteosarcoma cells (ROS) and in vivo by de novo endochondral bone formation in the subcutaneous space of the rat. These findings in the adult primate indicate that a single application of gamma-irradiated hOP-1 combined with the irradiated xenogeneic bovine collagenous matrix carrier is effective in regenerating and maintaining the architecture of the induced bone at doses of 0.5 mg/g and 2.5 mg/g of carrier matrix.

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)

Immunolocalization of Bone Morphogenetic Protein-2 and -3 and Osteogenic Protein-1 during murine tooth root morphogenesis and in other craniofacial structures.

The distribution of Bone Morphogenetic Protein-2, and -3 (BMP-2 and BMP-3) and Osteogenic Protein-1 (OP-1, also known as BMP-7) during root morphogenesis and in other craniofacial structures was examined in sections of 12- to 18-d-old mouse heads using polyclonal and monoclonal antibodies. BMP-3 and OP-1 were localized in alveolar bone, cementum, and periodontal ligament, whereas BMP-2 was only localized in the alveolar bone of periodontium. All three BMPs were localized in predentine, dentine, odontoblasts, osteoblasts, osteocytes, osteoid, cartilage, chondrocytes and spiral limbus. BMP-2 and OP-1 were also localized in spiral ligament and interdentate cells of the cochlea, whilst BMP-3 was restricted to the spiral ganglion. BMP-3 was also localized in ducts of submandibular and sublingual salivary glands, acini of the lacrimal gland, Purkinje cells in the cerebellum, nerve fibres of the cerebellum and brain, afferent cells of the dorsal root ganglia, inferior alveolar nerve, and peripheral processes of the vestibulocochlear nerve. OP-1 was also localized in hair and whisker follicles, sclera of the eye and in ameloblasts. The demonstration of BMP-3 in the nervous system suggests that this protein may be neurotrophic during development and maintenance of the nervous system. The composite expression of BMPs/OPs during periodontal tissue morphogenesis suggests that optimal therapeutic regeneration may entail the combined use of different BMPs/OPs.

Sinus floor augmentation with simultaneous placement of dental implants using a combination of deproteinized bone xenografts and recombinant human osteogenic protein-1. A histometric study in miniature pigs.

Maxillary sinus floor augmentation with autogenous bone has become a widely accepted procedure in implant dentistry. The use of osteoconductive bone substitutes in this indication is controversial, since their use can lead to a prolonged healing time, inhomogenous ossification, foreign body reaction, migration of particles and low bone-implant contact (BIC). The purpose of this study was to examine whether the combination of an osteoinductive protein (recombinant human osteogenic protein-1 (rhOP-1 = bone morphogenetic protein-7) with natural bovine bone mineral (BioOss) would improve ossification and the bone-implant contact (BIC) in a sinus floor augmentation with simultaneous placement of implants. In this study, the maxillary sinus floors in 5 miniature pigs were augmented with 3 ml BioOss containing 420 micrograms rhOP-1 on the test side and 3 ml BioOss alone on the control side. At the time of augmentation a titanium implant (ITI) was inserted from a laterocaudal direction. After 6 months of healing the sites of augmentation were removed and examined in non-decalcified sections by microradiography, fluorescence microscopy of sequentially labelled specimens and by histometry. On both sides, significant amounts of newly-formed bone were observed. However, on the test sites, the percentage of BIC in the augmented area was 80.0% versus 38.6% on control sites. It can be concluded that the application of bone morphogenetic proteins caused a more rapid and enhanced osseointegration of simultaneously placed implants when compared to the bone substitute alone. Therefore recombinant human osteogenic protein-1 delivered by natural bone mineral has the potential to become a clinical alternative for autogenous bone grafts in sinus floor augmentation.

Use of an osteoinductive biomaterial (rhOP-1) in healing large segmental bone defects.

OBJECTIVE: To assess the radiographic, histologic, and mechanical characteristics of new bone formation in large segmental bone defects treated with a new osteoconductive material, recombinant human osteogenic protein-1 (rhOP-1). DESIGN: In vivo animal study. INTERVENTION: Sixteen dogs (thirty-two limbs) with an ulna segmental defect (2.5 centimeters) were randomized to three treatment groups: rhOP-1, collagen alone, and no implant. MAIN OUTCOME MEASUREMENTS: Radiographic evidence of defect healing, mechanical testing (torsional strength) as compared with thirty-one control intact dog ulnas, and histologic analysis. RESULTS: At twelve weeks, complete radiographic healing was observed in twenty-five of twenty-eight defects (89 percent) treated with rhOP-1. The mechanical strength of the rhOP-1-treated defects at twelve weeks was 65 percent of that of intact ulnas. Histologic analysis revealed that defects treated with rhOP-1 were bridged with lamellar and woven bone that was in continuity with the host bone. CONCLUSIONS: The results indicate that osteoinductive materials, which have the ability to quickly fill and heal large defects, may have advantages over osteoconductive materials, which are typically used to fill smaller non-load-bearing bone voids.

Recombinant human osteogenic protein 1 in the rat mandibular augmentation model: differences in morphology of the newly formed bone are dependent on the type of carrier.

Recombinant human osteogenic protein 1 (rhOP1), also known as bone morphogenetic protein 7, was investigated in a newly developed experimental model, the rat lateral mandibular augmentation model. Algipore and Bio-Oss Block were applied as carrier materials. Extensive induction of newly formed bone was demonstrated on the test side containing rhOP1 and did not occur on the control side without rhOP1. The interstitial bone formation was limited to the augmented area and was not observed in the surrounding masseter muscle. The morphology of the newly formed bone differed in both carrier systems, which may be an important characteristic for the clinical indication of carrier systems in combination with bone morphogenetic proteins.

Identification of a microtubule-associated motor protein essential for dendritic differentiation.

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end-distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93- 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370-4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.

Osteogenic protein-1: biology and applications.

Osteogenic proteins, also referred to as BMPs, are a family of bone matrix polypeptides isolated from a variety of mammalian species. These proteins are members of the transforming growth factor-beta superfamily of molecules that contain a highly conserved 7 cysteine transforming growth factor-beta domain in their C-termini. Use of recombinantly produced human osteogenic protein-1, also referred to as BMP-7, implanted in conjunction with bovine bone derived Type 1 collagen or various non-proteinaceous biodegradable carriers into surgically created large diaphyseal segmental defects in animals leads to the regeneration of new bone that is fully functional biologically and biomechanically. Further study has shown that osteogenic protein-1 can be used as a bone graft substitute to promote spinal fusion and to aid in the incorporation of metal implants. Finally, osteogenic protein-1 shows promise as an agent for repair of osteochondral defects.

Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily.

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.

Conditions which promote mineralization at the bone-implant interface: a model in vitro study.

This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue.

Induction of cementogenesis by recombinant human osteogenic protein-1 (hop-1/bmp-7) in the baboon (Papio ursinus).

Recombinant human osteogenic protein-1 (hOP-1), a member of the bone morphogenetic protein family, was examined for its efficacy in periodontal regeneration. Twelve furcation defects, surgically prepared in the first and second mandibular molars, were treated with bovine insoluble collagenous matrix in conjunction with 0.0 (control), 100 and 500 mu g of recombinant hOP-1 per g of matrix. After 60 days of healing, histological and histometric analyses on serial, undemineralized sections cut at 7 mu m showed substantial cementogenesis on the exposed dentine of furcations treated with both doses of hOP-1 (p < 0.01 vs control). Foci of nascent mineralization were seen within the newly deposited cementoid along the coronal areas of hOP-1-treated defects. Within the furcations, there were substantial amounts of residual collagenous carrier, interspersed with a mineralized matrix having histological features of cementum. This mineralized cementum-like material was predominantly deposited around the carrier, and blended into newly formed cementum along the root surfaces. In the apical area, the cementum-like material and the remaining alveolar bony housing were not connected; indeed the two components were separated by a fibrovascular tissue that had numerous features of the periodontal ligament space. Formation and insertion of Sharpey's fibres into newly formed root cementum were also observed. It is likely that the expression of specific cell phenotypes by hOP-1 is regulated, in part, by the extracellular matrix microenvironment, including dentine. Thus, exposed dentine, in the presence of exogenous hOP-1 at the doses tested, may preferentially modulate the expression of the cementogenic phenotype. These findings in a non-human primate show that hOP-1, at the doses tested, induced cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues.

Complete regeneration of bone in the baboon by recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7).

We examined the efficacy of a single application of recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7) for its ability to regenerate large calvarial defects in adult male baboons (Papio ursinus). Recombinant hOP-1, in conjunction with baboon or bovine guanidinium-extracted insoluble collagenous bone matrix (0.1, 0.5 and 2.5 mg per g of collagenous matrix as carrier), was implanted in 46 calvarial defects surgically prepared in 14 baboons, whilst 18 defects were implanted with the carrier matrix without hOP-1. Specimens were harvested on d 15, 30, 90 and 365 and subjected to histomorphometry on serial undecalcified sections cut at 7 microm to study the temporal sequence of tissue morphogenesis after the single application of hOP-1. Histological analysis indicated that the induction of new bone formation proceeded from the periphery to the central core of hOP-1 treated specimens after rapid angiogenesis and mesenchymal cell migration in apposition to the collagenous matrix. Whilst chondrogenesis was limited, newly formed bone has already filled with fully differentiated bone marrow elements as early as d 15, even with the 0.1 mg dose of hOP-1. On d 30 and 90, doses of 0.1 and 0.5 mg of hOP-1 showed greater amounts of bone than controls, and on d 90, they induced complete regeneration of the defects. Doses of 2.5 mg hOP-1 per g of matrix induced extensive osteogenesis initially with heterotopic ossification and displacement of the temporalis muscle above the defects. One year after implantation of hOP-1 there was restoration of the internal and external cortices of the calvaria. These results show that hOP-1 induces complete regeneration of calvarial bone in the adult primate, and suggest that the optimal activity of hOP-1 to achieve regeneration is between 100 and 500 microg of hOP-1 per g of matrix. These results in the primate may form the scientific basis for future clinical applications of hOP-1.

Number of adrenergic and islet-1 immunoreactive cells is increased in avian trunk neural crest cultures in the presence of human recombinant osteogenic protein-1.

OP-1, also known as BMP-7, is a member of the TGF-beta superfamily of proteins and was originally identified on the basis of its ability to induce new bone formation in vivo. OP-1 mRNA is found in the developing kidney and adrenal gland as well as in some brain regions (Ozkaynak et al. [1991] Biochem. Biophys. Res. Commun. 179:116-123). We have tested the effect of recombinant human OP-1 on quail trunk neural crest cultures. The number of catecholamine-positive cells which developed after 7 days in vitro in the presence of OP-1 was increased in a dose-dependent manner, with a greater than 100-fold maximal stimulation observed. The increase in the number of catecholamine-positive cells in the presence of OP-1 was paralleled by an increase in the number of tyrosine hydroxylase (TH)-positive cells. In contrast, total and melanocyte cell number were unaffected by the presence of OP-1. The number of Islet-1-immunoreactive cells was also increased by OP-1, but to only about half the value seen for TH. Double label experiments revealed these Islet-1-positive cells were a subset of the TH-positive cells. Inhibitors of DNA synthesis prevented the OP-1-mediated increase in adrenergic cell number, indicating that OP-1 does not act on a postmitotic cell population. However, labeling studies with bromodeoxyuridine indicated that OP-1 did not increase the proportion of the cell population engaged in DNA synthesis. Thus, the OP-1-mediated increase in adrenergic cell number most likely occurs as a result of the enhanced survival of a subpopulation of adrenergic precursors or an increase in their probability of adrenergic differentiation, but not by increasing the mitotic rate of adrenergic precursors or adrenergic cells themselves. In contrast to OP-1, TGF-beta 1 decreased adrenergic cell number. When OP-1 and TGF-beta 1 were added simultaneously, TGF-beta 1 antagonized the OP-1-mediated increase in adrenergic cell number in a dose-dependent manner.

Effect of recombinant human osteogenic protein-1 on healing of segmental defects in non-human primates.

The effect of recombinant human osteogenic protein-1 on the healing of segmental bone defects was studied in twenty-eight African green monkeys (Cercopithecus aethiops). A 2.0-centimeter osteoperiosteal defect was created in the middle of the ulnar shaft in fourteen animals and in the diaphysis of the tibia in the other fourteen. The ulnar defect was filled with an implant consisting of 1000 micrograms of recombinant human osteogenic protein-1 in 400 milligrams of bovine bone-collagen carrier in six animals, with collagen carrier alone in two animals, and with autogenous cancellous bone graft from the contralateral tibia and femur in six animals. The tibial defect was filled with 250, 500 (two tibiae), 1000, or 2000 micrograms of recombinant human osteogenic protein-1 in 400 milligrams of collagen carrier in five animals, with collagen carrier alone in one animal, and with autogenous cancellous bone graft in six animals; in the two remaining animals (controls), the tibial defect was left unfilled. The tibial defects were stabilized with an intramedullary Steinmann pin. All animals were killed at twenty weeks postoperatively. Healing of the defects was evaluated with biweekly radiographs, with histological examination, and with mechanical testing. Radiographically, all of the defects that had been treated with recombinant human osteogenic protein-1 exhibited new-bone formation, but they differed in the degree of healing and remodeling. Five of the six ulnae treated with recombinant human osteogenic protein-1 and four of the five tibiae treated with this substance exhibited complete healing at six to eight weeks, with bridging of the defect by new bone first observed at four weeks. The two unhealed defects both exhibited new-bone formation but incomplete union, which precluded mechanical testing. No defect that had been filled with collagen carrier or that had been left unfilled exhibited any signs of healing or major new-bone formation. None of the six ulnae that had been filled with autogenous bone graft exhibited complete healing, compared with five of the six tibiae that had been so treated. Histological evaluation of the defects treated with recombinant human osteogenic protein-1 revealed the formation of new cortices with areas of woven and lamellar bone and normal-appearing marrow elements at twenty weeks postoperatively. The tibial defects that had been treated with autogenous bone graft had a similar appearance. All control ulnar and tibial defects and all ulnar defects that had been treated with autogenous bone graft had fibrous union with little new-bone formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia.

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50 micrograms of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 +/- 3.3% in vehicle-injected controls to 29 +/- 4.9%. Treatment of 14-day-old rats with 20 micrograms of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.