• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.4
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• pp.293-300
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• 2011

Purpose: Substance P is a well known neurotransmitter and has been known to mediate pain. Recently, it has been unveiled that substance P is involved in the recruitment of mesenchymal stem cells to wound sites. The purpose of this study was to exam bone formation when a combination of substance P and silk fibroin was used in a bone defect model. Methods: Twenty rabbits were used and 40 calvarial defects were formed. They were divided as 4 groups (unfilled control, silk only, silk+$10{\mu}g$/ml substance P; Sub10, and silk+$100{\mu}g$/ml substance P; Sub100). All animals were humanely sacrificed 4 or 8 weeks after grafting. The specimens were analyzed by micro-computerized tomography and histological analysis. Results: When compared to the unfilled control to silk only group, there was significant difference in bone mineral density (BMD) and the attenuation coefficient (AC) at 4 weeks ($p$=0.037 and 0.038, respectively). When compared Sub10 group to Sub100 group, there was significant difference in BMD and AC at 8 weeks ($p$=0.004 for all). Residual graft amounts were $52.1{\pm}15.8$%, $15.2{\pm}9.2$% and $9.0{\pm}3.3$% for silk only, Sub10, and Sub100 groups, respectively. When comparing the residual graft amount of silk only to sub10 or sub100, the differences were statistically significant ($p$ <0.001). Conclusion: The silk fibroin scaffold showed higher BMD and AC than the unfilled control. The combination graft with substance P and silk fibroin scaffold showed a faster graft degradation than with a silk fibroin scaffold only.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.1
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• pp.1-14
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• 2000

The purpose of this study was to evaluate the efficacy of adding autogenous bone to the toothash-plaster mixture in the healing process of bone. Full-thickness round osseous defects with the diameter of 20mm were made at the calvarial bone of adult dogs (n=19) bilaterally, which were thought to be critical size defect. The right defects were repaired with the toothash-plaster mixture plus autogenous bone (compressed volume 0.3cc) and the left defects with only toothash-plaster mixture. At 2-, 4-, 8-, 12- and 20- week after implantation, dogs were sacrificed and evaluated the osseous healing of bony defects clinically, radiographically, and microscopically. The results were as follows; 1. At the clinical observation, the wound healed very well without any problem except severe swelling in the early period after operation. Slight depression was recognized at the both sides when the portions of cranial defect were palpated. 2. There were statistically significant differences between toothash-plaster mixture groups and autogenous bone added groups at the same period, and among the groups in the bone density of the digital radiograms (P<0.001). There was a tendency that bone density was increasing with time. 3. In light microscopic examination, new bone formation was more active in the autogenous bone added groups than toothash-plaster mixture groups at the early period after implantation but there is little difference at 20-week after implantation. 4. In fluorescent microscopic examination, the fluorescent band could be observed at the area of active bone formation and the band was more distinct in the autogenous bone added groups then toothash-plaster mixture groups. 5. In transmitted electron microscopic examination, organelles such as rER, Golgi complex and secretory granule and osteoblast were observed. In summary higher volume ratio of autogenous bone is needed to improve the bone healing in that there is little difference between toothash-plaster mixture group and autogenous bone added group at the 20-week after implantation in spite of new bone formation was more active in the autogenous bone added groups than toothash-plaster mixture groups at the early period after operation.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.39
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• pp.8.1-8.8
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• 2017

Background: For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor $(TGF)-{\beta}1$, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration. Methods: Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used. Results: The concentrations of IGF-1, VEGF, and $TGF-{\beta}1$ in MSC-CM were $1515.6{\pm}211.8pg/mL$, $465.8{\pm}108.8pg/mL$, and $339.8{\pm}14.4pg/mL$, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group. Conclusions: VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.4
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• pp.360-373
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• 2006

The purpose of this research was to investigate whether pulsed electromagnetic field (PEMF) stimulation applied to the rabbit cranial defects grafted with ${\beta}$-tricalcium phosphate (${\beta}$-TCP) could affect the new bone formation. With 16 New Zealand white rabbits under the same condition, bilateral calvarial bone defects were formed around the sagittal suture line. The defect on the left side was grafted with ${\beta}$-TCP, while on the right side was grafted by harvested autogenous bone. PEMF was applied to 8 rabbits for 8 hours per day. The bony specimen were divided into 3 groups, the group 1 was autogenous bone grafted specimen, the group 2 was ${\beta}$-TCP grafted with PEMF, and the group 3 was ${\beta}$-TCP grafted without PEMF. We investigated the bone regeneration & growth factor expression at 2, 4, 6, and 8 weeks. As a result, BMP 2 was expressed in the group 1 from 2 weeks, the group 2 from 4 weeks, and the group 3 from 6 weeks. BMP 4 was expressed in the group 1 from 2 weeks, in the group 2 and the group 3 from 4 weeks. 4. There was no significant difference in expression pattern of BMP 7, PDGF, VEGF, and TGF-${\beta}$1 during grafted bone regeneration in group 1, 2, and 3. According to our results, PEMF stimulation could be effective on the new bome formation in animal study, and have a feasibility of clinical use.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.34 no.1
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• pp.1-11
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• 2012

Purpose: The purpose of this study was to evaluate the bone regeneration capacity of silk fibroin (SF) when combined with beta tricalcium phosphate (${\beta}$-tricalcium phosphate [TCP]) and rh-bone morphogenetic protein (BMP) in vivo by micro-computed tomography (CT), soft x-ray, and histological analysis. Methods: A total of 56 critical size defects formed by a trephine bur made on 28 adult female Spague-Dawley rats were used for this study and the defect size was 5.0 mm in diameter. The defects were transplanted with (1) no graft material (raw defect), (2) autogenous bone, (3) SF ($10{\mu}g$), (4) SF-BMP ($10{\mu}g$, $0.8{\mu}g$ each), and (5) SF+${\beta}$-TCP ($10{\mu}g$). At 4 and 8 weeks after operation, the experimental animals were sacrificed. Samples were evaluated with soft x-ray, histological examinations and 3-dimensional micro-CT analysis. Results: In the 3-dimensional micro-CT evaluation, bone volume and bone surface data were higher in the SF-BMP ($12.8{\pm}1.5$, $138.6{\pm}45.0$ each) (P<0.05) and SF-TCP ($12.3{\pm}1.5$, $144.9{\pm}30.9$ each) group than in the SF group ($6.1{\pm}3.3$, $77.2{\pm}37.3$ each) (P<0.05), except for the autogenous group ($15.0{\pm}3.0$, $190.7{\pm}41.4$ each) at 4 weeks. At 8 weeks, SF-BMP ($16.8{\pm}3.5$, $173.9{\pm}34.2$ each) still revealed higher (P<0.05) bone volum and surface, but SF-TCP ($11.3{\pm}1.5$, $1132.9{\pm}52.1$ each) (P=0.5, P=0.2) revealed the same or lower amount compared with the SF group ($13.8{\pm}2.7$, $127.5{\pm}44.8$ each). The % of bone area determined by radiodensity was higher in the SF-TCP ($31.4{\pm}9.1%$) and SF-BMP ($36.2{\pm}16.2%$) groups than in the SF ($19.0{\pm}10.4$) group at the period of 4 weeks. Also, in the histological evaluation, the SF-BMP group revealed lower inflammation reaction, lower foreign body reaction and higher bone healing than the SF group at postoperative 4 weeks and 8 weeks. The SF-TCP group revealed lower inflammation at 4 weeks, but accordingly, as the TCP membrane was absorbed, inflammatory and foreign body reaction are increased at 8 weeks. Conclusion: The current study provides evidence that the silk fibrin can be used as an effective grafted material for tissue engineering bone generation through a combination of growth factor or surface treatment.

• The Journal of Korean Academy of Prosthodontics
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• v.48 no.2
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• pp.158-165
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• 2010

Purpose: The objective of the present study was to histologically evaluate durability and bone regeneration capacity of new synthetic membranes in comparison to clinically available collagen membrane. Material and methods: To the skulls of 12 rabbits, we created 4 bone defects of 6 mm in diameter on each of them. Each of defects were covered with at least one of 5 membranes; No membrane, Collagen ($Ossix^{TM}$), PLGA, HA-coated-PLGA and HA-PLGA/PLGA. After 4, 8, 12 weeks, we cut the skulls and dyed with H-E. And then, the histologic observation was done. Results: In current study, the control group which did not use the membrane showed bone regeneration at 12 weeks and covered the bone defect partially. New bones were formed through the underneath of endocranium, and the upper defect was filled with connective tissues and fats. Collagen membrane ($Ossix^{TM}$) showed new bones after 4 weeks, and they were formed through the membrane which maintained until 12 weeks. PLGA, HA-coated-PLGA, HA-PLGA/PLGA showed bone regeneration after 4 weeks and after 8 weeks, they mostly filled defects. At 12 weeks, we could find new bones and previous bones almost look alike and also, they united well. Membranes were unnoticeable after 4 weeks and were absorbed. Conclusion: Bone formation and maturation of PLGA, HA-coated-PLGA and HA-PLGA/PLGA were faster than the control group. They showed no difference on the application of HA and after 4 weeks, they were absorbed.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.4
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• pp.293-298
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• 2010

Purpose: The objective of the present study was to determine the capability of electrospun silk fibroin as a biomaterial template for bone formation when mixed with nano-hydoxyapatite in vivo. Materials and Methods: Ten New Zealand white rabbits were used for this study and bilateral round shaped defects were formed in the parietal bone (diameter: 8.0 mm). The electrospun silk fibroin was coated by nano-hydroxyapatite and grafted into the right parietal bone (experimental group). The left side (control group) did not receive a graft. The animals were sacrificed at 6 weeks and 12 weeks, humanly. The microcomputerized tomogram (${\mu}CT$) was taken for each specimen. Subsequently, they were undergone decalcification and stained for the histological analysis. Results: The average value of all measured variables was higher in the experimental group than in the control at 6 weeks after the operation. BMC in the experimental group at 6 weeks after operation was $48.94{\pm}19.25$ and that in the control was $26.17{\pm}16.40$ (P = 0.027). BMD in the experimental group at 6 weeks after operation was $324.59{\pm}165.24$ and that in the control was $173.03{\pm}120.30$ (P = 0.044). TMC in the experimental group at 6 weeks after operation was $19.50{\pm}6.00$ and that in the control was $10.52{\pm}6.20$ (P = 0.011). TMD in the experimental group at 6 weeks after operation was $508.88{\pm}297.57$ and that in the control was $273.54{\pm}175.91$ (P = 0.06). Gross image of both groups showed higher calcification area at 12 weeks than them in 6 weeks. The average value of ${\mu}CT$ analysis was higher at 12 weeks than that in 6 weeks in both groups. BMC in the experimental group at 12 weeks after operation was $51.21{\pm}8.81$ and that in the control was $33.47{\pm}11.13$ (P = 0.010). BMD in the experimental group at 12 weeks after operation was $323.39{\pm}21.54$ and that in the control was $197.75{\pm}76.23$ (P = 0.012). TMC in the experimental group at 12 weeks after operation was $21.44{\pm}5.30$ and that in the control was $13.31{\pm}4.17$ (P = 0.008). TMD in the experimental group at 12 weeks after operation was $524.47{\pm}19.37$ and that in the control was $299.60{\pm}136.20$ (P = 0.016). Conclusion: The rabbit calvarial defect could be successfully repaired by electrospun silk nano-fiber combined with nano-hydroxyapatite.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.4
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• pp.313-323
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• 2010

Purpose: This study was conducted to evaluate the effect of beta-tricalcium phosphate (Cerasorb$^{(R)}$, Germany) and deproteinized bovine bone (Bio-Oss$^{(R)}$, Switzerland) grafted to the defect of rat calvaria artificially created and the effect of use of absorbable membrane (BioMesh$^{(R)}$, Korea) on new bone formation. Materials and Methods: Transosseous circular calvarial defects with diameters of 5 mm were prepared in the both parietal bone of 30 rats. In the control group I, no specific treatment was done on the defects. In the control group II, the defects were covered with absorbable membrane. In the experimental group I, deproteinized bovine bone was grafted without absorbable membrane; in the experimental group II, deproteinized bovine bone was grafted with absorbable membrane; in the experimental group III, beta-tricalcium phosphate was grafted without absorbable membrane; in the experimental group IV, beta-tricalcium phosphate was grafted with absorbable membrane. The animals were sacrificed after 3 weeks and 6 weeks respectively, and histologic and histomorphometric evaluations were performed. Results: Compare to the control groups, the experimental groups showed more newly formed bone. Between the experimental groups, beta-tricalcium phosphate showed more resorption than deproteinized bovine bone. Stabilization of grafted material and interception of the soft tissue invasion was observed in the specimen treated with membrane. There was no statistical difference between the experimental group I, III and experimental group II, IV classified by graft material, but statistically significant increase in the amount of newly formed bone was observed in the experimental group I, II and II, IV classified by the use of membrane (P<0.05). Conclusion: Both beta-tricalcium phosphate and deproteinized bovine bone showed similar osteoconductibility, but beta-tricalcium phosphate is thought to be closer to ideal synthetic graft material because it showed higher resorption rate in vivo. Increased new bone formation can be expected in bone graft with use of membrane.

• Journal of Periodontal and Implant Science
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• v.27 no.1
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• pp.129-150
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• 1997

The purpose of this study was to evaluate the effects of tetracycline(TC}, flurbiprofen, and PDGF-BB loaded biodegradable membranes on the cell-attachment, the activity of loaded PDGF-BB, in vivo release kinetics, and guided bone regenerative potentials. To evaluate the cell attachment to membranes, the number of gingival fibroblasts attached to each membrane(10% TC, 10% flurbiprofen, $200ng/cm^2$ PDGF-BB loaded membranes, drug-unloaded membrane) was counted by coulter counter and the morphologic pattern of attached cells was examined under SEM. To determine whether the activity of loaded PDGF-BB is sustained, the cellular growth and survival rate of gingival fibroblasts was used for both standard PDGF-BB and loaded PDGF-BB. For evaluation of in vivo release kinetics, drug-loaded membranes were implanted on the dorsal skin of the rats. On 1, 3, 7, 10, 14, 21, and 28 days after implantation, the amount of remaining drugs were measured by HPLC assay for TC and flurbiprofen, and by ${\gamma}-scintillation$ counter for $PDGF-BB^{1125}$. For evaluation of guided regenerative potential, the amount of new bone in the calvarial defect(5mm in diameter) of the rat was measured by histomorphometry 1 and 2 weeks after implantation of membranes. The number of cells attached to the PDGF-BB loaded membrane was largest as compared with the other mernbranes.(p< 0.05) The activity of loaded PDGF-BB was not significantly different from the activity of standard PDGF-BB.(p<0.05) After initial burst release of drug during the first 24 hours, drugs were gradually released for 4 weeks. Especially the release rate of PDGF-BB was nearly constant during 4 weeks. PDGF-BB loaded membranes(200, $400ng/cm^2$) were effective in guided bone regeneration as compared with drug-unloaded membrane. These results implicate that drug-loaded biodegradable membranes might be a useful for guided bone regeneration.

• Journal of Periodontal and Implant Science
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• v.27 no.1
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• pp.165-177
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• 1997

The purpose of this study was to perform on the biological activity of Magnolia and Zizyphi fructus extract mixtures on the wound healing of defected rat calvaria. For the determination of the mixture ratio of two extracts for oral administration, preliminary experiments were performed with the mixture combination of 2000 and $3000{\mu}g/ml$ of Magnolia extract, and also 20, 30, 200, 300, 2000 and $3000{\mu}g/ml$ of Zizyphi fructus extract, respectively and divided into 6 groups. The combination of extracts mixture were tested on the enhancing effect of cellular activity. The effect of the extracts mixture on the cellular activity was evaluated using MTT method and measured on the results with optical density by ELISA reader. The ability to tissue regeneration of the extracts mixture was performed by measuring new bone and new connective tissue regeneration on the 5mm defected rat calvaria for 1, 2 and 3 weeks after oral administration of 2 different dosages groups : 10:1(0.1g/kg) and 10:1(0.5g/kg). It was employed the same dosages of unsaponifiable fraction of Zea Mays L as positive controls. Each group of rat was sacrificed and en bloc section for histological examination. The effect on the cellular activity of each mixture ratio showed significantly higher in $2000{\mu}g/ml$ of Magnolia extract and $200{\mu}g/ml$ of Zizyphi fructus extract group to compare with other groups. These preliminary results showed that appropriate mixture ratio of two extracts was 10:1 of Magnolia and Zizyphi fructus extract. Histological examination on the activity of tissue regeneration of each group showed that 2weeks and 3weeks specimens of 0.5g/kg of 10:1 extract mixture of Magnolia and Ziziphi fructus administrated rat calvaria revealed significantly more osteoid and new bone formation of defected calvaria with unification of defected area than the specimens of any other negative and positive controls. Even though the specimen administrated the same dosages of unsaponifiable fraction of Zea Mays L, positive controls, showed the trend that they promote significantly the repair of calvarial defect, their bone reparative activities were less inductive than the same dosages of Magnolia and Ziziphi fructus extract mixture. These results implicated that the mixture of Magnolia and Zizyphi fructus extracts should be highly effective on the wound healing of bony defected site and might have potential possibilities as an useful drug to promote periodontal tissue regeneration.