Carrier For Bone Morphogenetic Protein-2 Research Articles

Initiation of Heterotopic Osteogenesis in Primates after Chromatographic Adsorption of Osteogenin, a Bone Morphogenetic Protein, onto Porous Hydroxyapatite

The chromatographic adsorption of partially purified osteogenin, a bone morphogenetic protein (BMP), onto porous hydroxyapatites for the construction of an osteogenic delivery system was investigated. Osteogenin fractions, with osteogenic activity in rats, were chromatographically adsorbed onto porous hydroxyapatites and implanted intramuscularly in baboons (Papio ursinus). Histology showed extensive bone differentiation within the porous spaces as early as 30 days after implantation in baboons. The unbound eluate from the porous hydroxyapatites showed lack of osteogenic activity when bioassayed in rats. These findings in primates may help to construct custom made porous ceramic as carriers for BMPs resulting in predictable bone formation for treatment of human bone defects.

Osteogenesis in Sintered Bone Combined With Bovine Bone Morphogenetic Protein

Bone morphogenetic protein (BMP) was combined with true bone ceramic (TBC) in an attempt to produce a bone grafting material possessing the cancellous structure, osteoconductivity, and osteoinductivity of biologic bone. To determine the osteogenetic ability of this material (BMP-TBC) within bone marrow, BMP-TBC and TBC alone were implanted in the femoral condyles of Japanese White rabbits, and histographs of these implants, taken serially, were compared. Woven bone formation was observed both at the periphery and within surface pores of the BMP-TBC implants one week after implantation; these implants thus became secured to the host bone bed at an earlier stage than did TBC alone. The amount of new bone formation increased at a significantly higher rate with BMP-TBC than with TBC alone during the first four weeks. Therefore the BMP in BMP-TBC implants acts at an early stage on BMP-responding cells entering the pores of the TBC. These data also suggest that TBC, as a carrier for BMP, provides a favorable environment for the promotion of new bone formation.

Squalane as a possible carrier of bone morphogenetic protein

Gelatin capsules containing squalane partially purified bone morphogenetic protein (BMP) complex were placed on the perimuscular membrane of rats. Two kinds of control, gelatin capsules containing only BMP and those bearing squalane only, were used. The embedded areas were histopathologically examined at 3 and 6 wk after the operation. The observations revealed that the squalane/BMP complex elicited wide heterotopic bone formation with bone marrow tissue, suggesting that squalane is a possible carrier of BMP for clinical applications.

Evaluation of Polylactic Acid Homopolymers as Carriers for Bone Morphogenetic Protein

Polylactic acid (PLA) homopolymers with molecular weights of 105,000; 21,000; 3300; 650; and 160d (PLA105000, PLA21000, PLA3300, PLA650, and PLA160, respectively) were prepared and evaluated as carriers for bone morphogenetic protein (BMP). Composites consisting of 4 mg of water-soluble, semipurified BMP and 100 mg of one of the PLA homopolymers were implanted into the dorsal muscles of mice. PLA105000/BMP, PLA21000/BMP, PLA3300/BMP, and PLA160/BMP composites failed to induce new bone formation; PLA105000, PLA21000, and PLA3300 elicited foreign-body reactions or chronic inflammation (or both), and PLA160 produced tissue necrosis. PLA650/BMP composites induced cartilage formation within one week and induced bone with hematopoietic marrow at three weeks postimplantation. PLA650/BMP composites were completely absorbed and replaced by new bone. The results suggest that within this group of PLA homopolymers, PLA650 may be the only type suitable for use as a BMP carrier.

ＢＭＰ‐チタニア（ＴｉＯ２）複合体の骨髄腔内，骨欠損腔内での骨形成能

To investigate new biomaterial possessing active osteogenetic competence in hard tissue, half-purified bone morphogenetic protein (BMP) was combined with sintered titanium dioxide (titania). BMP-titania complex was implanted in bone marrow cavities and bone defects.In this experiment titania was used as a carrier of BMP, therefore the mechanical properties of titania were investigated initially, and then the effects of titania on BMP activity were studied in vitro. The biaxial flexure strength of titania was 133.8±9.33MPa, diametral tensile strength was 29.9±7.48MPa, compressive strength was 262.7±33.04MPa, and Vicker's hardness was 628.1±45.13Hv. Titania neither enhanced BMP activity, nor suppressed it. This may indicate possible use as a carrier of BMP.For the implantation of BMP-titania complex, three mg of BMP bound into a hollow titania cylinder with an external diameter of 4mm, an internal diameter of 2mm, and a length of 5mm, was deeply implanted into the marrow cavities and bone defects of the femur of Japanese white rabbits. Four weeks after implantation, all animals were sacrificed, and the tissue in the hollow part of the titania cylinder was observed histologically using H-E., toluidine blue, and tetrachrome staining.The results showed BMP-titania complex to have osteogenetic competence in both bone marrow cavities and bone defects. As a result of the histologic observation of the ossification process, it is suggested that ossification by BMP in marrow cavities is not via chondrogenesis. In conclusion, the BMP-titania complex can act as a bone substitute.

Ultrastructural observation of calcification preceding new bone formation induced by demineralized bone matrix gelatin.

Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley rats, and the resulting ultrastructural changes of the BMG were examined 3, 5, 7, 10, 15 and 20 days later. Most of the implanted BMG became calcified 7-20 days after implantation. Calcification ('acellular mineral deposition') was first observed as needle-shaped crystalline deposits in the BMG matrix on day 7 after implantation, which gradually increased in size and fused with one another in some deposits. They appeared to be divided into small partitions consisting of denatured collagen fibers unlike those of noncalcified BMG. Some deposits had electron-lucent areas in their center adjacent to well-calcified peripheral areas, and the central area contained many collagen-like fibers and spherical vacuoles. Osteoblast-like cells were not present around these calcified deposits 7 days after implantation. After that, new bone formation was often seen near the area of acellular mineral deposition, and the fused calcified deposits remained until day 15 after implantation. The noncalcified BMG was mainly absorbed by macrophages, and the sites of acellular mineral deposition were absorbed by multinucleated cells resembling osteoclasts which were considered to be activated by the implants. Acellular mineral deposition probably started as calcium and phosphate deposits on some materials in the BMG matrix, and by heterogenic nucleation without osteoblasts or matrix vesicles, inducing bone formation. Thus the BMG may be not only a carrier of bone morphogenetic protein, gradually supplying it to the surrounding tissue but also a storage site for minerals that are indispensable for bone formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Telopeptide‐depleted bovine skin collagen as a carrier for bone morphogenetic protein

The telopeptide of type I collagen is thought to be responsible for causing immunogenic response when introduced into xenogenic hosts. To eliminate this problem, solubilized bovine skin collagen was filtered through a millipore membrane, digested repeatedly with pepsin to remove telopeptides, and used as a carrier for a water-soluble, partially-purified fraction of bone morphogenetic protein (BMP) in mice. Composite implants of this telopeptide-depleted collagen and the partially-purified BMP fraction consistently elicited ectopic bone formation in mice 3 weeks postimplantation. When implanted alone, collagen or BMP failed to show this response. Collagens, prepared by use of conventional methods (acid-solubilized collagen, or collagen-digested once with pepsin) were also assessed as carriers for BMP, but were found to be inferior in terms of consistency of bone formation and amount of induced bone mass. The results suggest that telopeptide-depleted collagen permitted a gradual release of purified BMP for induction of bone, with minimal immunogenic interference. Consequently, this collagen carrier represents an important development for future clinical application of BMP.

Purification of bone morphogenetic protein derived from bovine bone matrix

Bone morphogenetic protein (BMP) was extracted from the bovine bone matrix and purified by liquid chromatography. The molecular weight of the BMP was 18 kDa by SDS-PAGE, and its pI value was 4.9. Amino acid analysis suggested that the BMP is a polypeptide containing 163 amino acids. In the present study, telopeptide-free type I collagen was used as a carrier of BMP.

骨形成因子‐ハイドロキシアパタイト複合体の骨形成能について

Bone morphogenetic protein (BMP) is known to be a protein which induces new bone at heterotopic sites. Purification of BMP has not been perfected, and obtaining large amounts of BMP is very difficult, so it seems better to use some carrier or frame material for BMP to work effectively. Various kinds of hydroxyapatite (HAP) have been used to repair periodontal osseous defects, but they do not have osteogenetic or osteoinductive properties. If osteoinductive proteins such as BMP could retain their biologic properties after being implanted into living tissue with HAP, it would be an advantage in repairing periodontal osseous defects. In this experiment, we prepared BMP-HAP complex and investigated its osteoinductive activity. BMP was extracted from bovine cortical bones in accordance with the Urist's procedure. The ability of this BMP to stimulate new bone growth was ensured by implantation in the muscle pouch of mice. HAP was synthesized by the wet method. The BMP-HAP complex was implanted in the muscle pouch of mice, and osteoinduction was examined 3, 7, 14, and 21 days after implantation to assess its osteo-inductive ability. New bone formation was studied by roentgenographic and histologic observation. In the BMP-HAP group, new bone formation was seen on the roentgenograms and new cartilage and bone were observed histologically in the tissue surrounding the apatite. In the HAP group, no new cartilage or bone formation was noted.

Ectopic Bone Induction on and in Porous Hydroxyapatite Combined with Collagen and Bone Morphogenetic Protein

Porous hydroxyapatite (HA-P) discs (5 mm in diameter; 1.5 mm thick; porosity, 80%; mean pore size, 200 micron) were impregnated with purified bovine skin collagen (1 mg/disc) and a small amount of semipurified bone morphogenetic protein (BMP) of sarcoma origin (100 micrograms/disc) and implanted into dorsal muscles of ddY mice. Within one week new ectopic cartilagenous tissue was consistently formed on the surface of the discs adjacent to the host tissue. The cartilage was resorbed and replaced by normal bone containing hematopoietic bone marrow four weeks after implantation and the discs became encased in the newly formed bone. HA-P discs impregnated with only collagen (HA-P/collagen) or only BMP (100 micrograms/disc; HA-P/BMP) did not evoke formation of new cartilage or bone. These results indicate that collagen is effective as a carrier of BMP for expression of the biologic activity of the latter in vivo and that it may be of practical use as a carrier of BMP with synthetic biomaterials.

Differentiation of Cartilage on Three Substrata Under the Influence of an Aggregate of Morphogenetic Protein and Other Bone Tissue Noncollagenous Proteins (BMP/iNCP)

A cellulose acetate membrane was fashioned into a cone to serve as a substratum for an outgrowth of connective tissue from normal neonatal muscle, and a container for diffusion of bone morphogenetic protein (BMP) of an aggregate of BMP and cold water insoluble noncollagenous protein (BMP/iNCP). The BMP/iNCP was prepared by dissociative extraction and differential precipitation with other bone matrix proteins that slowly become soluble and diffusible in culture media at 37 degrees. The BMP/iNCP was applied either on, within, or beneath the surface of the explants or suspended in the culture medium. Under the influence of BMP in tissue cultures, without any bone matrix or bone collagen in the system, connective tissue outgrowths of muscle differentiate into cartilage on three substrata: (1) cellulose acetate membranes with pore size of 0.45-5.0 micron; (2) remnants of undissolved BMP/iNCP; and (3) degenerating myofibers. The cartilage developed in the interior of muscle, possibly by phenotypic cell transformation, when the pore size of the membrane was 0.1-0.22 micron too small to sustain anchorage of the explant. Cartilage developed on particle surfaces when the muscle tissue and BMP/iNCP particle were minced and mixed before explantation. The cartilage preferentially grew out directly onto the cellulose acetate membrane when the pore size was optimal for anchorage and the BMP/iNCP was suspended on the surface of the explant to either simultaneously percolate through the explant or diffuse through the culture medium. The biosynthetic activity of cells proliferating before and associated with cell differentiation was measured by 35S uptake in total glycosaminoglycan (GAG) per microgram of DNA. When the pore size was 8.0 micron, large enough to permit cells to migrate across the membrane, a thick plate of fibrous connective tissue developed on the undersurface of the membrane without any evidence of cartilage cell differentiation in any location. Repeated doses of BMP/iNCP with each change of culture medium produced a greater incidence and quantity of cartilage than a single dose, but the 35S incorporation into GAG always reached peak levels, in the interval between four and ten days, irrespective of the schedule of administration or dosage. These observations suggested that the exogenous or endogenous noncollagenous proteins are a carrier for BMP and can substitute for whole bone matrix or bone collagen.