• 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.299-305
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• 2010

Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages. Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 ${\mu}m$ nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-$\beta$, heparin, ascorbic acid and hydrocortisone at a density of $1{\times}10^5$ cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells. Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 ${\mu}l$ microbeads per 108 cells was performed for 30 minutes at $4^{\circ}C$ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel. Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.4
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• pp.281-286
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• 2009

Introduction: Enamel matrix derivative (EMD) is a protein which is secreted by Hertwig root sheath and plays a major role in the formation of cementum and attachment of peridontium. Several studies have shown that EMD promoted the proliferation and differentiation of preosteoblasts, osteoblasts and periodontal ligament cells in vitro: however, reports showing the inhibition of osteogenic differentiation by EMD also existed. This study was designed to simultaneously evaluate the effect of EMD on the two cell lines (human mesenchymal stem cells: hMSC, human periodontal ligament derived fibroblasts: hPDLCs) by means of quantitative analysis of some bone related matrices (Alkaline phosphatase : ALP, osteopontin ; OPN, osteocalcin ; OC). Materials and Methods: hMSCs and hPDLCs were expanded and cells in the 4${\sim}$6 passages were adopted to use. hMSc and hPDLCs were cultured during 1,2,7, and 14 days with 0, 50 and 100 ${\mu}g/ml$ of EMD, respectively. ALP activity was assessed by SensoLyte ALP kit and expressed as values of the relative optical density. Among the matrix proteins of the bony tissue, OC and OPN were assessed and quantification of these proteins was evaluated by means of human OC immunoassay kit and human OPN assay kit, respectively. Results: ALP activity maintained without EMD at $1,2^{nd}$ day. The activity increased at $7^{th}$ day but decreased at $14^{th}$ day. EMD increased the activity at $14^{th}$ day in the hPDLCs culture. In the hMSCs, rapid decrease was noted in $7^{th}$ and $14^{th}$ days without regard to EMD concentrations. Regarding the OPN synthesis in hPDLCs, marked decrease of OPN was noted after EMD application. Gradual decrease tendency of OPN was shown over time. In hMSCs, marked decrease of OPN was also noted after EMD application. Overall concentration of OPN was relatively consistent over time than that in hPDLCs. Regarding the OC synthesis, in both of hPDLCs and hMSCs, inhibition of OC formation was noted after EMD application in the early stages but EMD exerted minimal effect at the later stages. Conclusion: In this experimental condition, EMD seemed to play an inhibitory role during the differentiation of hMSCs and hPDLCs in the context of OC and OPN formation. In the periodontium, there are many kinds of cells contributing to the regeneration of oral tissue. EMD enhanced ALP activity in hPDLCs rather than in hMSCs and this may imply that EMD has a positive effect on the differentiation of cementoblasts compared with the effect on hMSCs. The result of our research was consistent with recent studies in which the authors showed the inhibitory effect of EMD in terms of the differentiation of mineral colony forming cells in vitro. This in vitro study may not stand for all the charateristics of EMD; thus, further studies involving many other bone matrices and cellular attachment will be necessary.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.2
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• pp.206-216
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• 2000

Predictional study for lateral change between pre- and post-orthognathic surgery has been emphasized mainly on anterior area of lateral profile; upper lip, lower lip and chin et al. So interest for lateral profile change has been less in posterior area of lateral profile and literature analyzing gonial angle change is rare. The purpose of this study is to make prediction for gonial angle change possible and to offer somewhat treatment guidance for gonial angle to be improved by investigating overall gonial angle change between pre- and post-orthognathic surgery and inquiring into factors influencing on pattern of genial angle change. For this study 35 patients were selected retrospectively. Lateral cephalometric radiographs were taken in just pre-op time, pod 1 day, pod 1 year. They were analyzed and genial angles were measured. The results were as follows : 1. Gonial angle at pod 1 day was decreased about $9.3^{\circ}$ than pre-op and gonial angle at pod 1 year was increased about $4.0^{\circ}$ than pod 1 day. So genial angle at pod 1 year was decreased about $5.3^{\circ}$ than pre-op genial angle(p<0.01). 2. Mean pre-op gonial angle was $129.4^{\circ}$, showing significantly high value than normal and mean gonial angle at pod 1 year was $124.1^{\circ}$, showing value near to normal. 3. Mean gonial angle change between pre-op and pod 1 year was decreased about $5.4^{\circ}$ in female and $5.3^{\circ}$ in male. There was no statistically significant difference between male and female(p>0.05). 4. Principal factor influencing on decreased gonial angle in gonial angle change between pre-op and pod 1 year was amount of mandibular setback. 5. Principal factor influencing on increased gonial angle in gonial angle change between pod 1 day and pod 1 year was % horizontal relapse, and it was thought that resorption and bone remodelling on posterior area in mandibular distal segment also were related to increased gonial angle. 6. It is thought that sagittal split ramus osteotomy in mandibular prognathic patients with high value of gonial angle is effective to improvement of gonial angle, and In patients who have normal range of gonial angle and are required with excessive mandibular setback, short lingual cut method, additional resection of posterior margin of distal segment, Obwegeser II method will be considerd. 7. More prudent operation and careful post-op management will be responsible for maintenance of postoperative stable gonial angle.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.2
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• pp.123-132
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• 2000

$TGF-{\beta}_1$ is a potent chemotactic factor for inflammatory cells and fibroblasts. It also stimulates the celluar source and components of extracellular matrix and the production of proteinase inhibitors. Collectively, these biologic activities lead to the accumulation and stabilization of the nascent matrix, which is vital to infection control. The objective of this study is to investigate production of $TGF-{\beta}$ in vitro fibroblast culture in the presence of Staphylococcus enterotoxin B(SEB) and/or lipopolysaccharide(LPS) and to elucidate the role of $TGF-{\beta}_1$ which may be responsible for infection control. The fibroblasts were originated from gingiva and facial dermis in 26 year-old male patient. In the presence of LPS($0.01{\mu}g$, $0.1{\mu}g$, $1.0{\mu}g$), SEB($0.01{\mu}g$, $0.l{\mu}g$, $1.0{\mu}g$) respectively, $cells(5{\times}10^3ml)$ were cultivated in vitro. At 1, 3, and 5 days after incubation, cells were counted. Also, $cells(2.5{\times}10^5ml)$ were cultivated in EMEM with LPS(0.01, 0.1 and $1.0{\mu}g$), SEB(0.01, 0.1 and $1.0{\mu}g$) respectively and $LPS(0.1{\mu}g)$ and $SEB(0.1{\mu}g)$ in combination for 24, 48, and 72 hours respectively. Culture supernatants were harvested at 1, 2, and 3 days after incubation period and triplicate culture supernatants were pooled and $TGF-{\beta}_1$ was assayed in duplicate. The results were as follows. 1. In gingival fibroblast induced with SEB and LPS respectively or in combination, the suppression of cell Proliferation occurred very significantly since 3 days after incubation, compared with the control and the production of $TGF-{\beta}_1$ occurred very significantly at 1 day after incubation, compared with the control. 2. In facial dermal fibroblast induced with SEB and LPS respectively or in combination, the suppression of cell proliferation occurred very significantly at 1 day after incubation, compared with the control. In SEB exposure, the production of $TGF-{\beta}_1$ was decreased very significantly at 1 day after incubation, compared with the control. However, in LPS, SEB and LPS exposure, the production of $TGF-{\beta}_1$ was increased very significantly at 1 day after incubation, compared with the control. In conclusion, the concentration of bacterial toxins and the incubation period correlated with cell proliferation and production of $TGF-{\beta}_1$ very significantly. The gingival and facial dermal fibroblasts have different phenotype each other The orchestrated understanding of fibroblast proliferation and $TGF-{\beta}_1$ production play an important part in host defense against the bacterial Infection and may prevent tissue necrosis such as necrotizing fasciitis and life-threatening syndrome such as multiple organ failure.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.2
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• pp.164-173
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• 2000

The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.3
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• pp.199-206
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• 2010

This study investigated the effects of strontium on osteoblastic phenotypes of cultured human periostealderived cells. Periosteal tissues were harvested from mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the periostealderived cells were further cultured for 28 days in an osteogenic induction DMEM medium supplemented with fetal bovine serum, ascorbic acid 2-phosphate, dexamethasone and at a density of $3{\times}10^4$ cells/well in a 6-well plate. In this culture medium, strontium at different concentrations (1, 5, 10, and 100 ${\mu}g$/mL) was added. The medium was changed every 3 days during the incubation period. We examined the cellular proliferation, histochemical detection and biochemical measurements of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and von Kossa staining and calcium contents in the periostealderived cells. Cell proliferation was not associated with the addition of strontium in periosteal-derived cells. The ALP activity in the periosteal-derived cells was higher in 5, 10, and 100 ${\mu}g$/ml strontium-treated cells than in untreated cells at day 14 of culture. Among the strontium-treated cells, the ALP activity was appreciably higher in 100 ${\mu}g$/ml strontium-treated cells than in 5 and 10 ${\mu}g$/ml strontium-treated cells. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in strontium-treated cells than in untreated cells at day 14 of culture. Their levels were increased in a dose-dependent manner. Von Kossa-positive mineralization nodules were strongly observed in the 1 ${\mu}g$/ml strontium-treated cells at day 21 and 28 of culture. The calcium content in the periosteal-derived cells was also higher in 1 ${\mu}g$/ml strontium-treated cells at day 28 of culture. These results suggest that low concentration of strontium stimulates the osteoblastic phenotypes of more differentiated periosteal-derived cells, whereas high concentration of strontium stimulates the osteoblastic phenotypes of less differentiated periosteal-derived cells. The effects of strontium on osteoblastic phenotypes of periosteal-derived cells appear to be associated with differentiation-extent.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.3
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• pp.218-228
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• 2010

Components of dental resin-based restorative materials are reported to leach from the filling materials even after polymerization. Hydroquinone (HQ) is one of the major monomers used in the dental resin and is known as a carcinogen. Thus, carcinogenic risk of HQ leaching from the dental resin becomes a public health concern. The present study attempted to examine the carcinogenic potentials of HQ on the human epithelial cell, which is the target cell origin of the most of oral cancers. Cytotoxicity of HQ was observed above 50${\mu}M$ as measured by LDH assay, indicating a relatively low toxicity of this substance in human epithelial cells. The parameters of neoplastic cellular transformation such as cell saturation density, soft agar colony formation and cell aggregation were analyzed to examine the carcinogenic potential of HQ. The study showed that 2-week exposure of HQ showed the tendency of increase in the saturation density and the significant enhancement of soft agar colony formation at the highest dose, 50 ${\mu}M$ only. It is suggested that HQ has a weak potential of carcinogenicity. When cells were treated with HQ and TPA, a well-known tumor promoter, the parameters of neoplastic cellular transformation was significantly increased. This result indicates that the potential risk of carcinogenicity from HQ is largely dependent upon the presence of promoter. Exposure of 50 ${\mu}M$ HQ increased the time-dependent apoptosis as measured by the ELISA kit. This concentration coincides with a dose of neoplastic transformation, indicating a possible link between apoptosis and HQ-induced cellular transformation. Hydroquinone generated Reactive Oxygen Species (ROS) which was evidenced by the treatment of antioxidants such as trolox and N-acetyl cysteine and the GSH depleting agent, BSO. Antioxidants blocked the generation of ROS and the GSH depleting agent, BSO dramatically increased the ROS production. Since HQ is known to increase ROS production thru activation of transcriptional factor such as c-Myb and Pim-1, it is speculated that ROS generation by HQ plays a role in the activation of oncogene, which may lead to neoplastic transformation. In addition, ROS is involved in the alteration of signal transduction, which regulates the apoptosis in many cellular systems. Thus, ROS-mediated apoptosis may be involved in the HQ-induced carcinogenic processes. Protein kinase C (PKC) is known to play pivotal roles in neoplastic transformation of cells and its high expression is often found in a variety of types of tumors including oral cancer. PKC translocation of PKC-${\alpha}$ was observed following HQ exposure. Altered signaling system may also play a role in the transformation process. Taken together, HQ leached from the dental resin does not pose a significant threat as a cancer causing agent, but its carcinogenic potential can be significantly elevated in the presence of promoter. The mechanism of HQ-induced carcinogenesis involved ROS generation, apoptosis and altered signaling pathway. The present study will provide a valuable data to estimate the potential risk of HQ as a carcinogen and understand mechanism of HQ-induced carcinogenesis in human epithelial cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.6
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• pp.469-477
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• 2009

Purpose: This study was performed to evaluate the effect of various graft materials used with a titanium cap on the ability of new bone formation in the rabbit calvarium. Materials and Methods: A total of 32 sites of artificial bony defects were prepared on the calvaria of sixteen rabbits by using a trephine bur 8 mm in diameter. Each rabbit had two defect sites. 0.2 mm deep grooves were formed on the calvaria of sixteen rabbits by using a trephine bur 8 mm in diameter for the fixation of a titanium cap. The treatments were performed respectively as follows: without any graft for the control group (n=8), autogenous iliac bone graft for experimental group 1 (n=8), alloplastic bone graft ($SynthoGraft^{(R)}$, USA) for experimental group 2 (n=8), and xenogenic bone graft ($NuOss^{(R)}$, USA) for experimental group 3 (n=8). After the treatments, a titanium cap (8 mm in diameter, 4 mm high, and 0.2 mm thick) was fixed into the groove. At the third and sixth postoperative weeks, rabbits in each group were sacrificed for histological analysis. Results: 1. In gross examination, the surgical sites showed no signs of inflammation or wound dehiscence, and semicircular-shaped bone remodeling was shown both in the experimental and control groups. 2. In histological analysis, the control group at the third week showed bone remodeling along the inner surface of the cap and at the contact region of the calvarium without any specific infiltration of inflammation tissue. Also, there was no soft tissue infiltration. Bone remodeling was observed around the grafted bone and along the inner surface of the titanium cap in experimental group 1, 2, and 3. 3. Histologically, all groups at the sixth week showed the increased area of bone remodeling and maturation compared to those at the third week. In experimental group 2, the grafted bone was partially absorbed by multi nucleated giant cells and new bone was formed by osteoblasts. In group 3, however, resorption of the grafted bone was not observed. 4. Autogenous bone at the third and sixth week showed the most powerful ability of new bone formation. The size of newly formed bone was in decreasing order by autogenous, alloplastic, and heterogenous bone graft. There was no statistically significant difference among autogenous, alloplastic, and heterogenous bones(p>0.05). Summary: This result suggests that autogenous bone is the best choice for new bone formation, but when autogenous bone graft is in limited availability, alloplastic and xenogenic bone graft also can be an alternative bone graft material to use with a suitably guided membrane.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.20 no.4
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• pp.305-315
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• 1998

The fibrous layer of mandibular condyle of the neonatal, 1-, 7-, 14-, 27-, 55-days and 1 year old rats were examined in the electron microscope with particular attention to the ultrastructure and diameter of collagen fibrils. In the 1-day rats, most of the cells of the fibrous layer were undifferentiated mesenchymal cells and fibroblasts with rough a little developed rough endoplasmic reticulum(RER) and golgi apparatus(GA). In 7-, 17 and 27-days old rats, most of the fibroblast showed well developed GA and RER with widely distended cisternae containing granular materials. In many of these cells contained intracytoplasmic filaments among the cytoplamic organelle. In 55-day and 1-year old rats, three types of cells were observed, ie, cells containing well developed cytoplasmic organelle presumed to be involved in the collagen fibril synthesis, cells showing well developed lysosomes, golgi apparatus, mitochondria and short cytoplasmic process presumed to be involved in the active resorption of the injured collagen fibrils or cellular debris, cells containing many intracytoplasmic filaments and a little organelle presumed to be cells of inactive state. The average diameters of collagen fibrils were similar in 1- and 7-day old rats as $38.48{\pm}3.81nm$, $38.06{\pm}3.86nm$. That was thickest in 14 days old rats as $50.21{\pm}3.93nm$ among experimental groups. They were gradually thinner in 27-, 55-day rats as $40.05{\pm}2.52nm$, $43.63{\pm}1.20nm$ and thinnest in 1-year old rats as $37.38{\pm}2.17nm$. The distribution pattern of diameters of collagen fibrils were unimordal with peak of 30-60nm in rats from 1-day to 17-day old. With aging from 27-day to 1 year old rats, collagen fibril diameters showed wide distribution pattern and percentage of thin collagen fibrils increased. These results may show the functional adaptation of fibrous layer of mandibular condyle to the increased mechanical forces with aging.