Hae-Ri Baek

In vitro and in vivo evaluation of the bioactivity of hydroxyapatite-coated polyetheretherketone biocomposites created by cold spray technology.

Polyetheretherketone (PEEK) is a material that is widely used in medicine because its mechanical properties show excellent similarity to those of human bone. However, because it is bioinert, PEEK shows limited ability to bind to natural bone tissue. Here, we applied a cold spray method to make a hydroxyapatite (HA)-coated PEEK hybrid material and evaluated its osteointegration in vitro and in vivo. With the cold spray method, the HA coating formed a homogeneous layer and adhered strongly to the PEEK disk implant. When the material was tested in vitro, early cell adhesion and viability improved. Alkaline phosphatase (ALP) activity and calcium concentration were also higher in cells cultured on HA-coated PEEK disks. In addition, the expression of osteoblast differentiation markers, such as ALP, bone sialoprotein and runt-related transcription factor 2, increased in these cells. For the in vivo test, we designed and implanted HA-coated PEEK cylinders into a rabbit ilium model by the press-fit method. The bone-implant contact ratio, trabecular number and trabecular thickness were determined using either three-dimensional microcomputed tomography or general two-dimensional histomorphometric analysis. This report demonstrates that the HA coating on the PEEK implant added with the cold spray method increased biocompatibility in vitro and promoted osteointegration in vivo, which suggests that the HA coating may improve the biofunctionality of various medical devices used in clinical applications.

Comparative study of fusion rate induced by different dosages of Escherichia coli–derived recombinant human bone morphogenetic protein-2 using hydroxyapatite carrier

BACKGROUND CONTEXT Hydroxyapatite (HA) is considered to be useful because of its high affinity for recombinant human bone morphogenetic protein (rhBMP), mechanical resistance to compressive force, and possible reduction of rhBMP dose. PURPOSE To evaluate the osteoinductivity of Escherichia coli-derived rhBMP-2 and the suitability of porous HA as an rhBMP-2 carrier. STUDY DESIGN In vivo study using microcomputerized tomography (micro-CT) scanning. PATIENT SAMPLE Seventy-six New Zealand white male rabbits were randomized into a single control group (n=14) without rhBMP-2 and four experimental groups (10 μg, 50 μg, 200 μg, and 500 μg of rhBMP-2; n=14 in each group). The subjects were divided into 3- and 6-week groups. OUTCOME MEASURES Outcome was evaluated by radiography, bending test, three-dimensional micro-CT, and histologic examinations. METHODS Bilateral posterolateral fusion was carried out, and rhBMP-2 (0, 10, 50, 200, 500, 1,000, and 2,000 μg) was implanted into the bilateral transverse processes using HA as a carrier. RESULTS The fusion rates of the 3-week group were 83.3% for 50 and 200 μg of rhBMP-2 and 100% for 500 μg. The improved fusion rates of the 50 μg or higher groups compared with those of control were statistically significant. The fusion rates of the 6-week group were 75% for 10 μg of rhBMP-2 and 100% for 50 μg or higher. Similarly, the improved fusion rates of the 10 μg or higher groups compared with those of control were statistically significant. Significantly higher percent volumes were observed in the 3-week 200 μg of rhBMP-2 group and 6-week 200 μg of rhBMP-2 group than the 3-week HA group and 6-week HA group, respectively. Trabecular thickness was significantly higher in the 3-week 200 μg of rhBMP-2 group than the 3-week HA group. Histologic analysis of the 10 μg group showed bone tissues within the pores from 3 weeks, and this was observed more vividly in the 50, 200, and 500 μg groups. The 6-week 10 μg and 50 μg of rhBMP-2 groups had lower amounts of new tissue but higher portions of complete bone tissue within the HA specimen, along with higher formation of completely reconstituted bone tissues outside HA. CONCLUSIONS Injection of 50 μg or more of E. coli-derived rhBMP-2 into a HA carrier induced earlier bone fusion in the intertransverse process of rabbits, which confirms the excellent bone forming ability of E. coli-derived rhBMP-2 and the suitability of HA as a carrier of rhBMP-2.

Combined effects of porous hydroxyapatite and demineralized bone matrix on bone induction: in vitro and in vivo study using a nude rat model.

Hydroxyapatite (HA) is an osteoconductive material used as a bone graft extender and demineralized bone matrix (DBM) has been used as a source of osteoinductive factors. A combination of DBM and HA is expected to create a composite with both osteoconductive and osteoinductive properties. This study examined the effect of a combination of DBM and HA on osteogenesis both in vitro and in vivo using an athymic nude rat abdominal muscle pouch model, and evaluated the possibility of HA as a carrier of DBM. Alkaline phosphatase (ALP) staining, ALP assay and measurements of the mRNA expression of ALP and Runx2 by RT-PCR were performed by transplanting human mesenchymal stem cells onto a plate. Five athymic nude rats each were assigned to one of two experimental groups (DBM/HA putty and only HA, i.e. 15 pouches per group). The muscle pouches were filled with DBM/HA putty or only HA. Radiographs were obtained at weeks 4 and 8, postoperatively. The animals were sacrificed at week 8 postoperatively and high resolution microCT was used to confirm the newly formed mineralized tissue. Each pouch was fixed, embedded, sectioned and processed for hematoxylin and eosin staining. The ALP value of the DBM/HA putty was higher than those of HA and control (p < 0.05, each). The expression of ALP mRNA appeared higher on the DBM/HA putty than on HA and control. MicroCT and histology examinations of the DBM/HA putty demonstrated the presence of newly generated mineralized tissues but there was no mineralized tissue in the HA cases. In conclusion, the DBM/HA putty indicated osteoblastic differentiation in vitro and showed ectopic mineralized tissue formation in the rat abdominal pouch model. These findings indicate that the DBM/HA putty can retain its oteoinductivity and HA can be used as a carrier of DBM.

Fabrication and Evaluation of Porous Beta-Tricalcium Phosphate/Hydroxyapatite (60/40) Composite as a Bone Graft Extender Using Rat Calvarial Bone Defect Model

Beta-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) are widely used as bone graft extenders due to their osteoconductivity and high bioactivity. This study aims to evaluate the possibility of using porous substrate with composite ceramics (β-TCP: HA = 60% : 40%, 60TCP40HA) as a bone graft extender and comparing it with Bio-Oss. Interconnectivity and macroporosity of β-TCP porous substrate were 99.9% and 83%, respectively, and the macro-porosity of packed granule after crushing was 69%. Calvarial defect model with 8 mm diameter was generated with male Sprague-Dawley rats and 60TCP40HA was implanted. Bio-Oss was implanted for a control group and micro-CT and histology were performed at 4 and 8 weeks after implantation. The 60TCP40HA group showed better new bone formation than the Bio-Oss group and the bone formation at central area of bone defect was increased at 8 weeks in micro-CT and histology. The percent bone volume and trabecular number of the 60TCP40HA group were significantly higher than those of Bio-Oss group. This study confirms the usefulness of the porous 60TCP40HA composite as a bone graft extender by showing increased new bone formation in the calvarial defect model and improved bone formation both quantitatively and qualitatively when compared to Bio-Oss.

The Effects of Recombinant Human Bone Morphogenetic Protein-2-Loaded Tricalcium Phosphate Microsphere–Hydrogel Composite on the Osseointegration of Dental Implants in Minipigs

Bone formation in tooth defect areas and the osseointegration of dental implants are very important for successful dental implant surgery. The aim of the present study was to assess the strengthening effect of a β-TCP microsphere-hydrogel composite containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone healing and implant osseointegration. The molars and premolars on the left and right sides of the maxilla were extracted from six male minipigs, and dental implants were placed using either the β-TCP microsphere-hydrogel carrier alone or the carrier loaded with rhBMP-2 (500 μg). The animals were kept alive for a further 8 weeks. The molars and premolars from the left and the right sides of the mandibles of another six minipigs were extracted, and the animals were kept alive for 4 weeks. Two 5-mm-diameter bone defects were then made on both sides of the mandible. The defects were filled with saline, β-TCP microsphere-hydrogel carrier, or the carrier loaded with rhBMP-2 (300 μg), and dental implant fixtures were inserted. The animals were kept alive for a further 4 weeks. Bone formation was examined using plane radiographs, micro-CT, and the histology of undecalcified specimens. The group treated with the rhBMP-2-loaded carrier composite showed a significantly higher percentage bone volume and a greater trabecular thickness for the newly formed bone in the tooth defect areas when compared to the group treated with the carrier alone. The rhBMP-2 group had a significantly higher osseointegration, a larger percentage bone volume, greater trabecular thickness in the newly formed bone in tooth defect areas, a larger newly formed bone fraction in the fixture pitch, and a greater number of newly formed trabecular bones when compared to the other groups. We confirmed that the rhBMP-2-loaded carrier composite promotes new bone formation after tooth extraction and strengthens osseointegration of dental fixtures by improving the degree of osseointegration around the dental implant fixture.

Synergistic induction of early stage of bone formation by combination of recombinant human bone morphogenetic protein-2 and epidermal growth factor

This study evaluates whether the combination of the rhBMP‐2 and various types of growth factors including EGF, FGF, PDGF and VEGF increases osteoinductivity compared to the single use of rhBMP‐2 through in vitro and in vivo study. Cultured human MSCs were treated with rhBMP‐2 only or in combination with growth factors. For in vivo evaluation, rhBMP‐2 only or with growth factors was implanted into the calvarial defect made on SD rats. Both EGF and PDGF significantly increased both ALP activity and expression level in hMSCs when treated in combination with rhBMP‐2 at 3 and 7 days of differentiation and significantly raised the accumulation of the calcium at day 14. Furthermore, micro‐CT scanning revealed that the EGF an FGF groups show significantly increased new bone surface ratio compared to the rhBMP‐2 only group and, the EGF treatment significantly up regulated percent bone volume and trabecular number at two weeks after the surgery. VEGF treatment also significantly raised trabecular number and FGF treatment significantly increased the trabecular thickness. Histological examination revealed that the EGF combination group showed enhanced bone regeneration than the rhBMP‐2 only group two weeks after the implantation. Even though the treatment of rhBMP‐2 with PDGF and FGF failed to show enhanced osteogenesis in vitro and in vivo simultaneously, these results suggest that the positive effect of the combination of EGF and rhBMP‐2 is expected to induce the bone formation earlier compared to the single use of rhBMP‐2 in vitro and in vivo.

Fabrication and evaluation of osteoblastic differentiation of human mesenchymal stem cells on novel CaO-SiO2-P2O5-B2O3 glass-ceramics.

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender.

The inhibitory effect of zoledronate on early-stage osteoinduction by recombinant human bone morphogenetic protein 2 in an osteoporosis model

Abstract This study evaluated the effect of the combined treatment of intravenous zoledronic acid (ZA, 0.08 mg/kg) and rhBMP-2 (5 µg) on osteogenesis in a calvarial defect model of ovariectomized SD rats. New bone formation was evaluated 4 or 8 weeks after calvarial defect implantation using micro-CT and histology. Micro-CT results revealed that the rhBMP-2 group showed significantly higher calvarial defect coverage ratio compared with the ZA + rhBMP-2 group at 4 weeks. In addition, bone formation indices were significantly lower in ZA + rhBMP-2 group when compared with the rhBMP-2 group after 4 weeks, which indicates a negative effect of ZA on the initial bone formation and the bone quality. At 8 weeks, the negative effect induced by ZA treatment was alleviated as time passed. Histological examination showed similar results to the micro-CT measurements. In conclusion, although ZA treatment lowered the new bone formation induced by rhBMP-2 initially, as time passed, the negative effect was decreased.

Posterolateral lumbar fusion using Escherichia coli-derived rhBMP-2/hydroxyapatite in the mini pig.

BACKGROUND CONTEXT Hydroxyapatite (HA) is used as a bone graft extender for posterolateral spinal fusion in human. It is also useful as a recombinant human bone morphogenetic protein (rhBMP)-2 carrier because of its high affinity for rhBMP-2. PURPOSE To assess the osteoinductivity of Escherichia coli-derived rhBMP-2 (E-BMP-2) using HA granules as a carrier and to evaluate the bone-forming ability depending on the different dosages of E-BMP-2. STUDY DESIGN A mini-pig lumbar posterolateral fusion model using microcomputed tomography (μCT) scanning. PATIENT SAMPLE Thirty-one adult male mini pigs were randomized into a single control group (n=8) without E-BMP-2 and two experimental groups with two different doses of E-BMP-2 (1 mg per side, n=8 and 3 mg per side, n=15). OUTCOME MEASURES Outcome was measured by plain radiography, manual palpation, CT, three-dimensional μCT, and histologic examinations. METHODS Bilateral intertransverse process arthrodesis was performed, and E-BMP-2 (0, 1.0, 3.0 mg per side) was implanted into the intertransverse space using HA granules as a carrier. RESULTS Three mini pigs were removed because of death. Among 28 experimental subjects, 19 animals achieved solid bony union. The fusion rates were 37.5% for control group, 71.4% for 1 mg group, and 84.6% for 3 mg group. Fusion rates were significantly different among groups (p=.031). However, there was no statistically significant difference in fusion rates between 1 and 3 mg groups (p=.587). Thirty-eight intertransverse fusion masses of 19 subjects underwent μCT scanning. The bone volumes determined by μCT were 12,603±3,240 mm(3) for control group, 18,718±3,000 mm(3) for 1 mg group, and 26,768±7,256 mm(3) for 3 mg group, and the difference between groups was statistically significant (p<.001). CONCLUSIONS This study shows that E-BMP-2 has osteoinductive activity in dose-dependent fashion, and porous HA granule is suitable for E-BMP-2 carrier in a porcine posterolateral fusion model. These preliminary findings suggest that E-BMP-2-adsorbed porous HA granules could be a novel effective bone graft substitute.

Generation of an rhBMP-2-loaded beta-tricalcium phosphate/hydrogel composite and evaluation of its efficacy on peri-implant bone formation

Dental implant insertion on a site with low bone quality or bone defect should be preceded by a bone graft or artificial bone graft insertion to heal the defect. We generated a beta-tricalcium phosphate (β-TCP) and poloxamer 407-based hydrogel composite and penetration of the β-TCP/hydrogel composite into the peri-implant area of bone was evaluated by porous bone block experiments. The maximum penetration depth for porous bone blocks and dense bone blocks were 524 μm and 464 μm, respectively. We report the in-vivo performance of a composite of β-TCP/hydrogel composite as a carrier of recombinant human bone morphogenetic protein (rhBMP-2), implanted into a rabbit tibial defect model. Three holes drilled into each tibia of eight male rabbits were (1) grafted with dental implant fixtures; (2) filled with β-TCP/hydrogel composite (containing 5 μg of rhBMP-2), followed by grafting of the dental implant fixtures. Four weeks later, bone-implant contact ratio and peri-implant bone formation were analyzed by radiography, micro-CT and histology of undecalcified specimens. The micro-CT results showed a significantly higher level of trabecular thickness and new bone and peri-implant new bone formation in the experimental treatment compared to the control treatment. Histomorphometry revealed a significantly higher bone-implant contact ratio and peri-implant bone formation with the experimental treatment. The use of β-TCP/poloxamer 407 hydrogel composite as a carrier of rhBMP-2 significantly promoted new bone formation around the dental implant fixture and it also improved the quality of the new bone formed in the tibial marrow space.