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.

IL-1beta, but not BMP-7 leads to a dramatic change in the gene expression pattern of human adult articular chondrocytes--portraying the gene expression pattern in two donors.

Anabolic and catabolic cytokines and growth factors such as BMP-7 and IL-1beta play a central role in controlling the balance between degradation and repair of normal and (osteo)arthritic articular cartilage matrix. In this report, we investigated the response of articular chondrocytes to these factors IL-1beta and BMP-7 in terms of changes in gene expression levels. Large scale analysis was performed on primary human adult articular chondrocytes isolated from two human, independent donors cultured in alginate beads (non-stimulated and stimulated with IL-1beta and BMP-7 for 48 h) using Affymetrix gene chips (oligo-arrays). Biostatistical and bioinformatic evaluation of gene expression pattern was performed using the Resolver software (Rosetta). Part of the results were confirmed using real-time PCR. IL-1beta modulated significantly 909 out of 3459 genes detectable, whereas BMP-7 influenced only 36 out of 3440. BMP-7 induced mainly anabolic activation of chondrocytes including classical target genes such as collagen type II and aggrecan, while IL-1beta, both, significantly modulated the gene expression levels of numerous genes; namely, IL-1beta down-regulated the expression of anabolic genes and induced catabolic genes and mediators. Our data indicate that BMP-7 has only a limited effect on differentiated cells, whereas IL-1beta causes a dramatic change in gene expression pattern, i.e. induced or repressed much more genes. This presumably reflects the fact that BMP-7 signaling is effected via one pathway only (i.e. Smad-pathway) whereas IL-1beta is able to signal via a broad variety of intracellular signaling cascades involving the JNK, p38, NFkB and Erk pathways and even influencing BMP signaling.

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.

Prefabrication of vascularized bone grafts using recombinant human osteogenic protein-1--part 3: dosage of rhOP-1, the use of external and internal scaffolds.

In a previous study vascularized bone grafts were prefabricated with recombinant human osteogenic protein-1 (rhOP-1) using blocks of xenogenic bone mineral (BioOss) as scaffolds. The present study addressed the dosage of rhOP-1 and the combination of an external (mould) and internal scaffold (granular BioOss). In five Göttingen minipigs six prefabrication sites in the latissimus dorsi muscles were randomly assigned to groups a-f. Moulds were prepared by shaping collagen/polylactide membranes in a cylindrical form which was filled with 1g BioOss granules and rhOP-1 (a: 0; b: 50; c, f, e: 250; d: 1000 microg of rhOP-1, a-e: cylinder open to muscle, e cylinder perforated, f: cylinder open to subcutaneous fat). After 6 weeks a dose dependency of bone density (a-d: 0%; 9.4%; 15.8%; 31.1%) and vessel density (a-d: 0.3; 2.4; 7.9; 25.4 counts/view) was observed histomorphometrically. Muscular surrounding was advantageous to subcutaneous tissue. Perforations of the membranes increased vessel density and did not impair bone formation. Bone density decreased in the proximity of the polylactide membranes. The membrane material was too soft and partly collapsed and therefore needs not to be reconsidered. The use of BioOss granules with 1000 microg rhOP-1 per gram proved to be a suitable concept for prefabrication of bone transplants.

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.

Mandibular reconstruction with a prefabricated vascularized bone graft using recombinant human osteogenic protein-1: an experimental study in miniature pigs. Part I: Prefabrication.

Osteogenic protein-1 (rhOP-1), also called bone morphogenetic protein-7 (BMP-7), is osteoinductive. It may be possible to reconstruct a mandible by local application of rhOP-1. However, in tumour patients this can be impossible due to scars or preceding radiotherapy. Usually vascularized bone grafts are indicated. The aim of this study was to prefabricate a vascularized bone graft in the latissimus dorsi muscle for microsurgical transplantation. In nine minipigs 600 microgram rhOP-1 were used with 8 ml xenogenic bone mineral (BioOss, Geistlich, Waldenburg, Germany) as a carrier and inserted into a pouch prepared in the M. latissimus dorsi. After 6, 12, and 24 weeks the grafts were harvested. The results were evaluated using computed tomography, histology, macro- and microangiography. A high yield of newly formed bone was obtained on the osteoconductive scaffold of the xenogenic bone. It was possible to create a vascularized osseous graft in the given shape of the BioOss blocks. In cross-sections, 68% of the scaffold was coated with new bone. The amount of new bone did not differ between the prefabrication times. Bone overgrowth was 2.1% of the graft volume. In conclusion, this study has shown that it can be possible to prefabricate a neomandible within a muscle graft, which then could be transferred for microvascular reconstruction of the mandible. Further research is required before this technique can be refined for clinical use.

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.

[Sinus floor augmentation with simultaneous implant insertion using recombinant human osteogenic protein-1]. / Sinusbodenaugmentation mit simultaner Implantatinsertion unter Verwendung von rekombinantem humanem Osteogenic Protein-1.

INTRODUCTION: The purpose of this study was to examine whether the combination of an osteoinductive protein (recombinant human osteogenic protein-(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. MATERIAL AND METHODS: In this study, the maxillary sinus floors in five 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. RESULTS: 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 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. DISCUSSION: 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.

Mandibular reconstruction with prefabricated vascularized bone grafts using recombinant human osteogenic protein-1: an experimental study in miniature pigs. Part II: transplantation.

Osteogenic Protein-1 (rhOP-1), also called bone morphogenetic protein-7 (BMP-7), is osteoinductive. The aim of this study was to present a new surgical technique: the prefabrication of a vascularized bone graft using rhOP-1 and its microsurgical transplantation. During 6 weeks, osteomuscular grafts were prefabricated in the latissimus dorsi muscle of five adult minipigs. Six hundred micrograms rhOP-1 on a carrier of xenogenic bone mineral in block form were used. The grafts were transplanted into defects of the mandibular angles performing a microsurgical anastomosis and using miniplates for fixation. Identical defects of the contralateral side were treated by direct application of 600 microg rhOP-1 and xenogenic bone mineral. A polychrome sequential labelling was applied. After transplantation the bone stayed viable, demonstrated by continuous apposition of fluorochromes (non-decalcified histologic sections) and bone scintigraphy. The reconstructive result was significantly superior in the prefabrication technique, assessed by histology and computerized tomography (CT). In conclusion, the method has a potential to become a clinical alternative for conventional vascularized bone grafts.

Regeneration of articular cartilage chondral defects by osteogenic protein-1 (bone morphogenetic protein-7) in sheep.

The efficacy of osteogenic protein-1 (OP-1; BMP-7) in regeneration of articular cartilage was examined by creating knee chondral defects in sheep. With a specially designed instrument in both knees, two 10 mm (diameter) chondral defects were created: one in the trochlea and the other on the femoral condyle. The recombinant BMP was delivered via an extra-articulary positioned mini-osmotic pump, which was fixed to the femoral diaphysis above the knee joint, and connected by a polyethylene tubing to the articular space. Prior to use, the compatibility of OP-1 with mini-osmotic pumps was tested in vitro by measuring aggregation/precipitation and modification of the released protein by size exclusion and reversed phase HPLC. The average amount of aggregation was 15% and about 5% of OP-1 was modified. However, the biological activity of OP-1 released from pumps over a period of 2 weeks at 37 degrees C was equal to ROS cell assay OP-1 standard. Following surgery, a total of 55 microg (low dose) or 170 microg (high dose) OP-1 in acetate buffer (pH 4.5) was slowly released from the pump over a period of 2 weeks. The pumps connected to control knees were filled with acetate buffer as a vehicle. Twelve animals were operated, six of which were treated with the low OP-1 dose, and six with the high OP-1 dose. Three sheep of each group were killed either at 3 or 6 months following surgery, based on arthroscopical evaluation. The chondral defects in the control knees remained empty during the observation period. At 3 months following surgery, defects treated with both OP-1 doses were filled with connective tissue and cartilage. At 6 months following surgery, both doses of OP-1 stimulated regeneration in treated knees. The boundaries between new and old cartilage were well fused and mechanically resisted animals' weight bearing. The regenerated cartilage was rich in proteoglycans and type II collagen, as demonstrated by toluidine blue staining and immunohistochemistry. No signs of endochondral bone formation above the bony tidemark were observed. We suggest that a recombinant bone morphogenctic protein stimulates ingrowth of mesenchymal cells into the chondral defects which then transform into newly formed articular cartilage-like tissue.

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)

Mandibular reconstruction in miniature pigs with prefabricated vascularized bone grafts using recombinant human osteogenic protein-1: a preliminary study.

A new technique of prefabrication of vascularized bone grafts in the latissimus dorsi muscle of miniature pigs, using recombinant human osteogenic protein-1 (rhOP-1) and xenogenic bone mineral as a carrier, is presented. The grafts were used to reconstruct a mandible using microsurgical anastomosis of the thoracodorsal vessels of the flap with a branch of the external carotid artery and the external jugular vein. The technique provided enough bone to reconstruct a mandible. The bone marrow stayed viable after transplantation, demonstrated by fluorescence microscopy and scintigraphy. Compared with identical defects of the contralateral side, which were treated with directly applied rhOP-1/bone mineral, a significantly better result was obtained with the prefabrication technique. The prefabrication technique may provide a means to treat irradiated patients with bone grafts produced by osteoinductive proteins and has a potential to become a clinical alternative to conventional vascularized bone grafts.

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.

Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons.

Osteogenic protein-1 (OP-1) is expressed in the developing nervous system and it has been found to induce dendritic growth in sympathetic neurons. To further characterize this phenomenon, the effects of OP-1 were compared to those of other members of the bone morphogenetic protein (BMP) family of growth factors. Recombinant human OP-1, BMP-6, BMP-2 and the Drosophila 60A protein induced dendritic growth in rat sympathetic neurons in a concentration-dependent manner with EC50-values of 1.8, 1.0, 1.7 and 2.7 ng/ml, respectively. In contrast, BMP-3 and cartilage-derived morphogenetic protein-2 (CDMP-2) as well as other classes of growth factors were inactive at concentrations up to 50 ng/ml. The dendritic growth induced by OP-1, BMP-6, BMP-2 and 60A was accompanied by increased expression of microtubule-associated protein-2 (MAP2) without changes in the expression of the phosphorylated forms of the M and H neurofilament subunits. These results suggest that several members of the BMP family have the capacity to regulate the morphological development of sympathetic neurons and that they may act by induction of specific cytoskeletal proteins.

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.