Comparison of bone regeneration in a rabbit skull defect by recombinant human BMP-2 incorporated in biodegradable hydrogel and in solution

The objective of this study is to compare bone regeneration induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) incorporated into a biodegradable gelatin hydrogel with that by rhBMP-2 in aqueous solution. After treating rabbit skull defects of 6 mm diameter with the two rhBMP-2 dosage forms, both of them increased the bone mineral density (BMD) at the skull defects with implantation time to a significantly higher extent than a rhBMP-2-free aqueous solution and a rhBMP-2-free empty gelatin hydrogel (p < 0.05). There was no quantifiable difference in BMD between the two dosage forms of rhBMP-2. Histological examination revealed that the integrity of newly generated bone increased with the rhBMP-2 dose, irrespective of the dosage form. The bone defect was filled with regenerated bone 21 days after treatment.     

Osteogenic evaluation of calcium/magnesium-doped mesoporous silica scaffold with incorporation of rhBMP-2 by synchrotron radiation-based μCT

The regenerative treatment of large osseous defects remains a formidable challenge in orthopedic surgery today. In the present study, we have synthesized biodegradable calcium/magnesium-doped silica-based scaffolds with hierarchically macro/mesoporous structure (CMMS), and incorporated recombinant human bone morphogenetic protein-2 (rhBMP-2) into the scaffolds to obtain a hybrid system for osteogenic factor delivery in the functional repair of bone defects. The developed CMMS/rhBMP-2 scaffolds presented interconnected porous network, macropores (200-500 μm) and mesopores (5.7 nm), as well as good bioactivity and biocompatibility and proper degradation rate. Combined with the capacity to deliver ions and growth factors, the CMMS/rhBMP-2 scaffolds significantly promoted the in vitro osteogenic differentiation of bone marrow stromal cells (bMSCs), as evidenced by the enhanced expression of Runx-2, osteopontin, osteocalcin and bone sialoprotein, and induced the ectopic bone formation in the thigh muscle pouches of mice. We further assessed the in vivo effects of CMMS/rhBMP-2 scaffolds in a rabbit femur cavity defect model by using synchrotron radiation-based μCT (SRμCT) imaging and histological analysis, indicating that the CMMS/rhBMP-2 scaffolds resulted in more bone regeneration compared to that observed with the CMMS scaffolds without rhBMP-2. Moreover, scaffolds with or without rhBMP-2 underwent gradual resorption and replacement with bone and almost disappeared at 12 weeks, while the dense CMMS/rhBMP-2 material showed slower degradation rate and promoted the least extensive neo-bone formation. This study suggested that the hybrid CMMS/rhBMP-2 scaffolds system demonstrates promise for bone regeneration in clinical case of large bone defects.     

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.     

Repair of long intercalated rib defects in dogs using recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate

Long intercalated defects in canine ribs can be repaired successfully using porous beta-tricalcium phosphate (beta-TCP) cylinders, infused with a biodegradable polymer (poly D,L-lactic acid-polyethylene block copolymer) containing recombinant human bone morphogenetic protein-2 (rhBMP-2). We previously reported the successful regeneration of bony rib and periosteum defects using beta-TCP cylinders containing 400 microg of rhBMP-2. To reduce the amount of rhBMP-2 and decrease the time required for defect repair, we utilized a biodegradable polymer carrier, in combination with rhBMP-2 and the porous beta-TCP cylinders. An 8 cm long section of rib bone was removed and replaced with an implant comprised of the porous beta-TCP cylinders and the polymer containing 80 microg of rhBMP-2. Six weeks after surgical placement of the beta-TCP cylinder/polymer/BMP-2 implants, new rib bone with an anatomical configuration and mechanical strength similar to the original bone was regenerated at the defect site. The stiffness of the regenerated ribs at 3, 6, and 12 weeks after implantation of the composite implant was significantly higher than that of ribs regenerated by implantation of rhBMP-2/beta-TCP implants. Thus, addition of the synthetic polymer to the drug delivery system for BMP potentiated the bone-regenerating ability of the implant and enabled the formation of mechanically competent rib bone. This new method appears to be applicable to the repair of intercalated long bone defects often encountered in clinical practice.     

Efficiency of magnetic liposomal transforming growth factor-beta 1 in the repair of articular cartilage defects in a rabbit model

We evaluated the efficacy of a magnetic liposomal delivery system of transforming growth factor (TGF)-beta(1) in the treatment of articular cartilage defects in a rabbit model. Articular cartilage defects were created in the patellar groove of rabbits, and a permanent magnet or a nonmagnetic alloy was implanted in the defect site. Magnetic liposomal drugs, prepared by the conventional film method and sonication, were injected into the defect site 1 week after surgery. First, the efficacy of the magnetic liposomal delivery system was evaluated by using a model compound fluorescence-labeled dextran 40,000 (FD-40). Then, the therapeutic efficiency of magnetic liposomal TGF-beta(1) was evaluated by cartilage histological scoring at 4, 8, and 12 weeks after surgery. The injected magnetic liposomal FD-40 accumulated at the target site where a permanent magnet had been implanted. The histological score showed that the injection of magnetic liposomal TGF-beta(1) under magnetic force was significantly effective in the repair of the defect site over 12 weeks after surgery. Injection of TGF-beta(1) into the cartilage defect was effective as a magnetic liposomal preparation under magnetic force, resulting in acceleration of the cartilage repair, probably because of the desirable accumulation of TGF-beta(1) at the target site.     

Repair of a proximal femoral bone defect in dogs using a porous surfaced prosthesis in combination with recombinant BMP-2 and a synthetic polymer carrier

Total hip arthroplasty (THA) has become an almost standard procedure for the treatment of various hip lesions. However, one of the limitations has been the mechanical loosening of the prosthesis, a condition termed peri-prosthetic osteolysis. Consequently, at revision surgery, various grades of bone defect are often noted. Alternative approaches aimed at overcoming this problem have included a special design of the revision prosthesis and allo- or autogeneic bone grafting in combination with or without biomaterials. In a further attempt to address the loosening of the prosthesis, we have combined human bone morphogenetic protein-2, produced by DNA recombination (rhBMP-2) with a new synthetic biodegradable polymer (poly-D,L-lactic-acid-para-dioxanone-polyethyleneglycol block co-polymer; PLA-DX-PEG). We present data on the efficacy of the rhBMP-2 laden prosthesis to reconstruct a bone defect in a canine model. In this model, medial half of the proximal femur was surgically resected to create a bone defect that was repaired with the rhBMP-2/PLA-DX-PEG composite. Twelve weeks after implantation, the original bone defects in the rhBMP-2 treatment groups had been repaired. Thus, this type of 'hybrid' prosthesis may provide a new modality to repair bone defects or restore lost bone mass encountered in revision arthroplasty.     

Enhanced regeneration of rabbit mandibular defects through a combined treatment of electrical stimulation and rhBMP-2 application

We evaluated the new bone regeneration of a rabbit mandibular defect using hBMSCs under electrical stimulation combined with rhBMP-2 in this study. An inner scaffold prepared by setting a collagen sponge with hBMSCs and hydrogel was placed into a polycaprolactone (PCL) outer box, and an electrical stimulation device was installed between the inner scaffold and the outer box. There were three experimental groups depending on electrical stimulation and application of rhBMP-2. The experimental group was divided into the following three groups. Group 1, in which rhBMP-2 (5 μg/defect) was added to hydrogel and electrical stimulation was not applied; Group 2, in which rhBMP-2 (5 μg/defect) was added as in Group 1 and electrical stimulation was applied; and Group 3, in which electrical stimulation was applied and rhBMP-2 (5 μg/defect) was injected directly into defect site. The delivered electrical stimulation was charge-balanced bi-phasic electric current pulses, and electrical stimulation was conducted for 7 days. The stimulation parameters of the bi-phasic electrical current set at an amplitude of 20 μA, a duration of 100 μs and a frequency of 100 Hz. Four weeks after surgery, new bone formation in each group was evaluated using radiography, histology, and micro-computed tomography (μCT). Groups 2 and 3 exhibited a significant increase in new bone formation compared to Group 1, while Group 3 showed the highest level of new bone regeneration. In a comparison between two groups, Group 2 showed a higher bone volume (BV) by 260 % (p < 0.01) compared with Group 1, and Group 3 showed a higher BV by 442 % (p < 0.01) compared with Group 1. The trend of the bone surface density (ratio of new bone to the real defect volume, BS/TV), trabecular number, and connectivity was identical to that of the BV. The total bone mineral density (BMD) of Groups 2 and 3 showed values higher by the ratios of 103 % (p < 0.01) and 107.5 % (p < 0.01) compared with Group 1, respectively. Part BMD for Groups 2 and 3 showed higher values by the ratios of 104.9 % (p < 0.01) and 122.4 % (p < 0.01) compared with Group 1, respectively. These results suggest that the combined treatment of electrical stimulation, hBMSCs, a collagen sponge, hydrogel, and rhBMP-2 was effective for bone regeneration of large-size mandibular defects. The application of rhBMP-2 with an injection following electrical stimulation demonstrated better efficiency as regards bone regeneration.     

Development of a novel biomaterial, hydroxyapatite/collagen (HAp/Col) composite for medical use

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nano-crystals along collagen molecules, has been synthesized. The biocompatibility, osteoconductivity and efficacy as an rhBMP-2 carrier of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants adsorbing 0 or 400 microg/ml of rhBMP-2 were implanted into bone defects of tibiae in 3 beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm remaining in 2 beagle dogs and the dogs were allowed to walk using a Ilizarov external skeletal fixator. The radiological and histological findings suggest that the implants induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation. As a next step, anterior fusion was carried out on 6 beagle dogs with the implants adsorbing 400 microg/ml of rhBMP-2, and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate. Histological and radiographical analysis suggest that enhancement of callus formation and bone bridging by rhBMP-treatment is effective to prevent collapse of the implant.     

The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora

Scaffolds prepared from biodegradable polyurethanes (PUR) have been investigated as a supportive matrix and delivery system for skin, cardiovascular, and bone tissue engineering. While previous studies have suggested that PUR scaffolds are biocompatible and moderately osteoconductive, the effects of encapsulated osteoinductive molecules, such as recombinant human bone morphogenetic protein (rhBMP-2), on new bone formation have not been investigated for this class of biomaterials. The objective of this study was to investigate the effects of different rhBMP-2 release strategies on new bone formation in PUR scaffolds implanted in rat femoral plug defects. In the simplest approach, rhBMP-2 was added as a dry powder prior to the foaming reaction, which resulted in a burst release of 35% followed by a sustained release for 21 days. Encapsulation of rhBMP-2 in either 1.3-micron or 114-micron PLGA microspheres prior to the foaming reaction reduced the burst release. At 4 weeks post-implantation, all rhBMP-2 treatment groups enhanced new bone formation relative to the scaffolds without rhBMP-2. Scaffolds incorporating rhBMP-2 powder promoted the most extensive new bone formation, while scaffolds incorporating rhBMP-2 encapsulated in 1.3-micron microspheres, which exhibited the lowest burst release, promoted the least extensive new bone formation. Thus our observations suggest that an initial burst release followed by sustained release is better for promoting new bone formation.     

Implantation study of a novel hydroxyapatite/collagen (HAp/col) composite into weight-bearing sites of dogs

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nanocrystals along collagen molecules, has been prepared. The biocompatibility, osteoconductive activity, and efficacy as a carrier of rhBMP-2 of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants (15 mm in diameter and 20 mm in length) with a surface cross-linked layer containing rhBMP-2 (0 or 400 microg/ml) were implanted into bone defects of tibiae in three beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm in two beagle dogs did not receive implants, and the dogs were allowed to walk using an Ilizarov extraskeletal fixator. The specimens were removed from one dog in each group after 12 weeks. Also, the Ilizarov fixators in the rhBMP-treated dogs were removed after 12 weeks, after which full weight bearing started. The specimens were further taken out after 18 and 24 weeks in the rhBMP-treated and non-rhBMP-treated dogs, and after 24 weeks in the control group. The change of bone mineral density, as well as radiological and histological findings, suggest that the implants are able to induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation.     

Healing of segmental bone defects in rats induced by a beta-TCP-MCPM cement combined with rhBMP-2.

A beta-tricalcium phosphate-monocalcium phosphate monohydrate (beta-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 x 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 microg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 microg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for beta-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 microg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the beta-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects.     

Comparative study between coral-mesenchymal stem cells-rhBMP-2 composite and auto-bone-graft in rabbit critical-sized cranial defect model

Tissue engineered bone has become a bone substitute for the treatment of bone defects in animal research. This study investigated the osteogenesis capacity of coral-MSCs-rhBMP-2 composite with the auto-bone-graft as control. Coral-MSCs-rhBMP-2 composite were fabricated by coral (as main scaffold), rhBMP-2 (as growth factor), and MSCs (cultured from iliac marrow as seed cells). Critical-sized defects (d = 15 mm) were made on forty rabbits crania and treated by different composite scaffolds: iliac autograft (n = 8), coral (n = 8), rhBMP-2/coral (n = 8), and MSCs/rhBMP-2/coral (n = 8). The defects were evaluated by gross observation, radiographic examination, histological examination, and histological fluorescence examinations after 8 and 16 weeks. The results showed that repair of bone defect was the least in coral group, and significant ingrowth of new bone formation and incorporation could be seen with 77.45% +/- 0.52% in radiopacity in MSCs/rhBMP-2/coral group, which was similar to that in iliac autograft group (84.61% +/- 0.56% in radiopacity). New bone formation in MSCs/rhBMP-2/coral group was more than that in rhBMP-2/coral group. And osteogenesis rate in MSCs/rhBMP-2/coral group (10.23 +/- 1.45 microm) was much faster than that in rhBMP-2/coral group (5.85 +/- 2.19 microm) according to histological fluorescence examination. Newly formed bone partly came from induced MSCs in composite scaffold according to bromodeoxyuridine immunohistochemical examination. These data implicated that MSCs could produce synergic effect with coral-rhBMP-2, and the tissue engineered bone of coral-MSCs-rhBMP-2 is comparable to auto-bone-graft for the repair of critical-sized bone defect.     

Fibroblast Growth Factor-2 Augments Recombinant Human Bone Morphogenetic Protein-2-Induced Osteoinductive Activity

The osteoinductive activity induced by recombinant human BMP-2 (rhBMP-2) blunts proportionately as the recipient ages. In order to compensate for this bluntness administration of fibroblast growth factor-2 (FGF-2) has been considered. The aim of this study was to determine whether FGF-2 administration augments osteoinductive activity caused by rhBMP-2 and to evaluate the effect of aging on bone formation induced by coadministration of rhBMP-2 and FGF-2. Sixty-four Wistar strain male rats of 8-week-old (prepubertal) and 16-week-old (postpubertal) received bone defects bilaterally in the parietal bone and the defects were filled by a polylactic acid polyglycolic acid copolymer/gelatin sponge (PGS) impregnated with rhBMP-2 plus 0 ng, 25 ng, and 250 ng FGF-2 (n=10 in each). At 2 weeks after grafting, the new bone volume seemed to be larger in the rhBMP-2+FGF-2 groups than in the rhBMP-2 alone group. At 4 weeks, the new bone formation was linked to the adjacent original bone. In the prepubertal rats, all newly formed bone was similarly calcified. In the postpubertal rats, only the rhBMP-2+25 ng FGF-2 group showed this higher degree of calcification. At 2 weeks, alkaline phosphatase (ALP) activity in the rhBMP-2+25 ng FGF-2 group was significantly (p<0.05) larger than that in the rhBMP-2 group in both prepubertal and postpubertal rats. This result shows that low-dose administration of FGF-2 enhanced the degree of calcification and ALP activity in the rhBMP-2 grafting site especially in the postpubertal rats. Therefore, FGF-2 would be a candidate to compensate for the reduction of osteoinductive activity of rhBMP-2 with aging.     

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 μg rhBMP-2 + monoolein; II: 3 μg rhBMP-2 + monoolein; III: 7 μg rhBMP-2 + monoolein; IV: 1 μg rhBMP-2 + poloxamer; V: 3 μg rhBMP-2 + poloxamer; VI: 7 μg rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 μg) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.     

Modulation of stromal cell-derived factor-1/CXC chemokine receptor 4 axis enhances rhBMP-2-induced ectopic bone formation

Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.     

Mineral apposition rates provide significant information on long-term effects in BMP-induced bone regeneration

In this study, a CaP biomaterial was used as a carrier for rhBMP-2. Biomaterials were investigated in calvarial and femoral defects using a rabbit animal model, with unloaded biomaterials serving as control. Fluorochrome labels were administered at days 14 and 70. Specimens were retrieved after 12 weeks for histological analysis. When area fractions were assessed by conventional histomorphometry, no significant effect of rhBMP-2 on the amounts of regenerated bone and residual biomaterial were seen by 12 weeks. After mineral appositional rate (MAR) measurement using double labels, calculation yielded significantly higher MARs for defects at both implantation sites, when compared with surrounding bone, whether or not biomaterials were loaded with rhBMP-2. Analyzing the effect of rhBMP-2, both defect sites showed significantly higher MARs in the rhBMP-2 group. MARs of bone surrounding the defects had also been elevated significantly by rhBMP-2 at calvarial and femoral implantation sites. It is concluded that MAR measurement is suitable to identify long-term effects of rhBMP-2 on bone formation at a time when conventional histomorphometry using fractional area determination is inadequate. Also, by MAR assessment, effects of rhBMP-2 on surrounding bone can be documented.     

Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2

We have investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta tricalcium phosphate (beta-TCP). The purpose of this study was to evaluate these BCP ceramics as carriers for rhBMP-2. BCP ceramics impregnated with the different doses of recombinant human bone morphogenetic protein 2 (rhBMP-2) (1, 5 and 10g) were used for the experimental purpose and the ceramics without rhBMP-2 were used as control. The pellets were placed into subcutaneous pockets on the dorsum of 4-week-old male Wistar rats. The animals were sacrificed 2 and 4 weeks after implantation. Bone induction was estimated by alkaline phosphatase (ALP) activity measured at 2 weeks after implantation. Pellets were also examined radiologically, histologically and histomorphometrically. The results showed that all experimental pellets exhibited new bone formation whereas the control pellets produced only fibrous connective tissue. Here, 100% HAP ceramic showed most amount of bone formation, whereas 25% HAP to 75% TCP ceramic produced the bone least in amount among different BCP ceramics at the end of 4 weeks. This study indicates that formation of new bone depends on the ceramic content with high HAP-TCP ratio and high dose of rhBMP-2.     

Recombinant human bone morphogenetic protein-2 promotes osteogenesis within atelopeptide type I collagen solution by combination with rat cultured marrow cells

We evaluated the combination effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and cultured rat bone marrow mesenchymal stem cells (MSCs) in atelopeptide type I collagen (AC) solution on osteogenesis in a diffusion chamber (DC) to develop a bone substitute having consistent osteogenic capability for clinical applications. The cultured MSCs were obtained by 10-day primary culture of fresh bone marrow cells of Fischer rats. We prepared three groups of DCs: AC solution with rhBMP-2, AC solution with cultured MSCs, and AC solution with rhBMP-2 and cultured MSCs. The prepared combined solutions were injected into DCs, which were subcutaneously implanted into the backs of syngeneic rats. DCs were harvested after 2, 4, or 8 weeks and analyzed for bone-forming capability by determining histological and osteoblastic biochemical markers. De novo bone formation was observed both inside and outside of the membrane filter of DCs in the group of AC solution with rhBMP-2 and cultured MSCs. The alkaline phosphatase activity and osteocalcin content in the group of AC solution with rhBMP-2 and cultured MSCs were significantly higher than those in the group of AC solution with cultured MSCs at any time. These findings indicate that AC aqueous solution is a useful material not only as a carrier of rhBMP-2 but also as a cell-anchorage for differentiation and proliferation of MSCs. Therefore, this study suggests that clinical repairs of bone defects are feasible using injectable AC solution with rhBMP-2 and cultured MSCs as a bone substitute.     

Evaluation of carboxymethylcellulose, hydroxypropylmethylcellulose, and aluminum hydroxide as potential carriers for rhBMP-2

Conventional iliac crest nonvascularized corticocancelous bone grafts and bone flaps have been used to treat bony defects. However, these treatments have some limitations, namely, the availability of donor tissue, donor site morbidity, difficulty to shape the bone flap to the defect, and complexity of the surgery. The bone morphogenetic protein (rhBMP-2) is osteoinductive. However, its implantation requires a matrix (carrier) in order to define the shape of the resulting bone and to retain the protein at the site for the time required for induction to occur. When the ideal carrier is found, an unlimited supply of material would be available for all applications where bone is needed. In this in vitro study, we evaluated the suitability of some potential carriers for rhBMP-2 by measuring the alkaline phosphatase (ALP) activity of fibroblast cultures. Either rhBMP-2 or sodium carboxymethylcellulose significantly increased the ALP activity, when used alone. When sodium carboxymethylcellulose was combined with rhBMP-2, there was an increase in the ALP activity, but lower than those obtained when the products were used alone. Hydroxypropylmethylcellulose alone did not affect ALP activity. However, the combination of rhBMP-2 with hydroxypropylmethylcellulose did not increase the ALP activity, despite the presence of rhBMP-2. Aluminium hydroxide proved to be an unsuitable rhBMP-2 adsorbent.     

Comparative study of biphasic calcium phosphate ceramics impregnated with rhBMP-2 as bone substitutes

We investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta-tricalcium phosphate (beta-TCP) to evaluate these ceramics as bone substitutes. BCP ceramics impregnated with different doses of recombinant human bone morphogenetic protein 2 (rhBMP-2) (1, 5, and 10 microg) were used for experimental purposes and ceramics without rhBMP-2 were used for control. The pellets were implanted under the pericranium in adult Wistar male rats and were harvested 8 weeks after implantation. The retrieved pellets were then examined radiologically, histologically, and histomorphometrically. The results revealed that the pellets treated with rhBMP-2 exhibited new bone and bone marrow, whereas control pellets produced fibrous connective tissues. The formation of new bone induced by rhBMP-2 was dose dependent. The extent of bone and bone marrow formation and the degree of resorption of the ceramic particles were significantly higher in the pellets composed of 25% HAP-75% TCP. In this study, bioresorption of the ceramic produced favorable conditions for rhBMP-2-induced bone formation.