Matrix metalloproteinase(MMP) sensitive hyaluronic acid hydrogel-nanoparticle complex와 rhBMP-2를 이용한 골재생

남정훈(Jeong Hun Nam),박종철(Jong Chul Park),유상배(Sang Bae Yu),정용일(Yong Il Chung),태기융(Gi Yoong Tae),김정주(Jung Ju Kim),박용두(Yong Doo Park),장정원(Jeong Won Jahng),이종호(Jong Ho Lee) 대한구강악안면외과학회 2009 대한구강악안면외과학회지 Vol.35 No.3

As an efficient controlled release system for rhBMP-2, a functional nanoparticle-hydrogel complex, incorporated with matrix metalloproteinase(MMP) sensitive peptide cross-linker, was developed and used as a bone transplant. In vivo bone formation was evaluated by soft x-ray, histology, alkaline phosphatase(ALP) activity and mineral contents analysis, based on the rat calvarial critical size defect(8mm in diameter) model. Significantly, effective bone regeneration was achieved with the rhBMP-2 loaded MMP sensitive hyaluronic acid(HA) based hydrogel-Nanoparticles(NP) complex, as compared to only MMP HA, the MMP HA-NP without rhBMP-2, or even with the rhBMP-2. These improvements included the formation pattern of bone and functional marrow, the degree of calcium quantification, and the ALP activity. These results indicate that the MMP sensitive HA with nano-particle complex can be a promising candidate for a new bone defect replacement matrix, and an enhanced rhBMP-2 scaffold.

인회석 박막 피복 도관과 Brain-derived neurotrophic factor(BDNF) 유전자 이입 슈반세포를 이용한 백서 좌골신경 재생에 관한 연구

최원재(Won-Jae Choi),안강민(Kang-Min Ahn),황순정(Soon-Jeong Hwang),정필훈(Pill-Hoon Choung),김명진(Myung-Jin Kim),김남열(Nam-Yeol Kim),유상배(Sang-Bae Yoo),장정원(Jeong-Won Jahng),김현만(Hyun-Man Kim),김중수(Joong-Soo Kim),김윤희(Yun 대한구강악안면외과학회 2005 대한구강악안면외과학회지 Vol.31 No.3

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with 􀝜-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x106) or BDNF-Ad infected Schwann cells(1x106) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54±4.0×106 and 9.66±9.6×106. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 μg/μl of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 μg/μl of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

칼슘포스페이트 나노-크리스탈이 코팅된 골이식재와 자가골을 병행 이용한 상악동 거상술

방강미,이보한,알라쉬단,유상배,성미애,김성민,장정원,김명진,고재승,이종호,Pang, Kang-Mi,Li, Bo-Han,Alrashidan, Mohamed,Yoo, Sang-Bae,Sung, Mi-Ae,Kim, Soung-Min,Jahng, Jeong-Won,Kim, Myung-Jin,Ko, Jea-Seung,Lee, Jong-Ho 대한악안면성형재건외과학회 2009 Maxillofacial Plastic Reconstructive Surgery Vol.31 No.3

Purpose: Rehabilitation of the edentulous posterior maxilla with dental implants often poses difficulty because of insufficient bone volume caused by pneumatization of the maxillary sinus and by crestal bone resorption. Sinus grafting technique was developed to increase the vertical height to overcome this problem. The present study was designed to evaluate the sinus floor augmentation with anorganic bovine bone (Bio-$cera^{TM}$) using histomorphometric and clinical measures. Patients and methods: Thirteen patients were involved in this study and underwent total 14 sinus lift procedures. Residual bone height was ${\geq}2mm$ and ${\leq}6mm$. Lateral window approach was used, with grafting using Bio-$cera^{TM}$ only(n=1) or mixed with autogenous bone from ramus and/or maxillary tuberosity(n=13). After 6 months of healing, implant sites were created with 3mm diameter trephine and biopsies taken for histomorphometric analysis. The parameters assessed were area fraction of new bone, graft material and connective tissue. Immediate and 6 months after grafting surgery, and 6 months after implantation, computed tomography (CT) was taken and the sinus graft was evaluated morphometric analysis. After implant installation at the grafted area, the clinical outcome was checked. Results: Histomorphometry was done in ten patients.Bio-$cera^{TM}$ particles were surrounded by newly formed bone. The graft particles and newly formed bone were surrounded by connective tissue including small capillaries in some fields. Imaging processing revealed $24.86{\pm}7.59%$ of new bone, $38.20{\pm}13.19%$ connective tissue, and $36.92{\pm}14.51%$ of remaining Bio-$cera^{TM}$ particles. All grafted sites received an implant, and in all cases sufficient bone height was achieved to install implants. The increase in ridge height was about $15.9{\pm}1.8mm$ immediately after operation (from 13mm to 19mm). After 6 months operation, ridge height was reduced about $11.5{\pm}13.5%$. After implant installation, average marginal bone loss after 6 months was $0.3{\pm}0.15mm$. Conclusion: Bio-$cera^{TM}$ showed new bone formation similar with Bio-$Oss^{(R)}$ histomorphometrically and appeared to be an effective bone substitute in maxillary sinus augmentation procedure with the residual bone height from 2 to 6mm.

Dehydrothermal Treatment로 제작한 흡수성 콜라겐 골유도재생술 차단막

방강미,정한울,김성포,양은경,김기호,김성민,김명진,장정원,이종호,Pang, Kang-Mi,Choung, Han-Wool,Kim, Sung-Po,Yang, Eun-Kyung,Kim, Ki-Ho,Kim, Soung-Min,Kim, Myung-Jin,Jahng, Jeong-Won,Lee, Jong-Ho 대한악안면성형재건외과학회 2011 Maxillofacial Plastic Reconstructive Surgery Vol.33 No.2

Purpose: Collagen membranes are used extensively as bioabsorbable barriers in guided bone regeneration. However, collagen has different effects on tissue restoration depending on the type, structure, degree of cross-linking and chemical treatment. The purpose of this study was to evaluate the inflammatory reaction, bone formation, and degradation of dehydrothermal treated porcine type I atelocollagen (CollaGuide$^{(R)}$) compared to of the non-crosslinked porcine type I, III collagen (BioGide$^{(R)}$) and the glutaldehyde cross-linked bovine type I collagen (BioMend$^{(R)}$) in surgically created bone defects in rat mandible. Methods: Bone defect model was based upon 3 mm sized full-thickness transcortical bone defects in the mandibular ramus of Sprague-Dawley rats. The defects were covered bucolingually with CollaGuide$^{(R)}$, BioMend$^{(R)}$, or BioGide$^{(R)}$ (n=12). For control, the defects were not covered by any membrane. Lymphocyte, multinucleated giant cell infiltration, bone formation over the defect area and membrane absorption were evaluated at 4 weeks postimplantation. For comparison of the membrane effect over the bone augmentation, rats received a bone graft plus different covering of membrane. A $3{\times}4$ mm sized block graft was harvested from the mandibular angle and was laid and stabilized with a microscrew on the naturally existing curvature of mandibular inferior border. After 10 weeks postimplantation, same histologic analysis were done. Results: In the defect model at 4 weeks post-implantation, the amount of new bone formed in defects was similar for all types of membrane. Bio-Gide$^{(R)}$ membranes induced significantly greater inflammatory response and membrane resorption than other two membranes; characterized by lymphocytes and multinucleated giant cells. At 10 weeks postoperatively, all membranes were completely resorbed. Conclusion: Dehydrotheramal treated cross-linked collagen was safe and effective in guiding bone regeneration in alveolar ridge defects and bone augmentation in rats, similar to BioGide$^{(R)}$ and BioMend$^{(R)}$, thus, could be clinically useful.

백서 설신경 압박손상모델에서 신경성장인자 유전자 주입이 신경재생에 미치는 영향

고은봉,정헌종,안강민,김성민,김윤희,장정원,이종호,Gao, En-Feng,Chung, Hun-Jong,Ahn, Kang-Min,Kim, Soung-Min,Kim, Yun-Hee,Jahng, Jeong-Won,Lee, Jong-Ho 대한악안면성형재건외과학회 2006 Maxillofacial Plastic Reconstructive Surgery Vol.28 No.5

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

백서에서 금식으로 인한 스트레스 대응축 활성화의 회복조절기전에서 구강인두로부터 입수되는 다양한 맛 자극의 효과

유상배(Sang-Bae Yoo),이종호(Jong-Ho Lee),류비탈리(Vitaly Ryu),장정원(Jeong-Won Jahng) 대한구강악안면외과학회 2011 대한구강악안면외과학회지 Vol.37 No.3

ntroduction: This study examined the regulatory mechanism underlying the meal-induced changes in the hypothalamic-pituitary-adrenal gland (HPA) axis activity. Materials and Methods: Male Sprague-Dawley rats (250-300 g) were hired for two different experiments as follows; 1) rats received either 8% sucrose or 0.2% saccharin ad libitum after 48 h of food deprivation with the gastric fistula closed (real feeding) or opened (sham feeding). 2). rats received 5 ml of intra-oral infusion with 0.2% saccharin or distilled water after 48 h of food deprivation. One hour after food access, all rats were sacrificed by a transcardiac perfusion with 4% paraformaldehyde. The brains were processed for c-Fos immunohistochemistry and the cardiac blood was collected for the plasma corticosterone assay. Results: Real feedings with sucrose or saccharin and sham feeding saccharin but not sucrose, following food deprivation decreased the plasma corticosterone level. c-Fos expression in the nucleus tractus of solitarius (NTS) of the fasted rats was increased by the consumption of sucrose but not saccharin, regardless of the feeding method. On the other hand, the consumption of sucrose or saccharin with real feeding but not the sham, induced cFos expression in the paraventricular nucleus (PVN) of the fasted rats. The intra-oral infusion with saccharin or water decreased the plasma corticosterone level of the fasted rats. Intra-oral water infusion increased c-Fos expression in both the PVN and NTS, but saccharin only in the NTS in the fasted rats. Conclusion: Neither restoration of the fasting-induced elevation of plasma corticosterone nor the activation of neurons in the PVN and NTS after refeeding requires the palatability of food or the post-ingestive satiety and caloric load. In addition, neuronal activation in the hypothalamic PVN may not be an implication in the restoration of the fasting-induced elevation of the plasma corticosterone by oropharyngeal stimuli of palatable food.

가토모델에서 배양 구강상피를 이용한 근-점막 피판의 형성에 관한 연구

신영민,정헌종,안강민,박희정,성미애,김성민,황순정,김명진,장정원,김성포,양은경,송계영,이종호,Shin, Young-Min,Chung, Hun-Jong,Ahn, Kang-Min,Park, Hee-Jung,Sung, Mi-Ae,Kim, Soung-Min,Hwang, Soon-Jung,Kim, Myung-Jin,Jahng, Jeong-Won,Kim, Sung-P 대한악안면성형재건외과학회 2005 Maxillofacial Plastic Reconstructive Surgery Vol.27 No.3

Purpose : Extensive defect of oral and maxillofacial area is usually reconstructed with composite flap including skin paddle. However, if the defects are lined with only skin components, the mucosa's role in mastication and texture are not restored. Furthermore, stiffness and hair-growing prevent denture rehabilitation and good oral hygiene. This study was performed to overcome the disadvantages of composite soft tissue flaps including the skin and to make a model for myo-mucosal flaps. Materials and methods : Buccal mucosa sized $0.5\times1.0\;cm^2$ from New Zealand rabbit (around 1.5kg) was harvested and cultivated by the modification of Rheinwald and Green's keratinocyte culture method. Cultured mucosa was grafted on the fascia of latismus dorsi as form of mucosal sheet. After 7, 10, 14 days, the myomucosal flap was excised and evaluated under light microscope with H & E and immunohistochemical staining. As control group, harvested buccal mucosa from rabbit was transplanted to gracilis muscle(n=6). Results : From 7 days after prelamination, the basal layer of the grafted mucosa resembled that of normal mucosa. As control group, transplanted mucosa had original shape but there's slight inflammatory reaction. Prelaminated mucosa has 19.8$\pm$4.59 cell layers and some samples have more than 20 layers. The expression rate of PCNA was relatively strong (42.9%$\pm$14.1) at the basal layer of grafted mucosa and the laminin was found at the basal layer. On the contrary, prelaminated mucosa at 10 days showed moderate expression rate of PCNA(32.4%$\pm$4.62). We found the mucosal layer was somehow disappeared and there is strong inflammatory reaction. After 14 days prelamination, the grafted oral keratinocytes were almost disappeared and expression of PCNA was not observed. Conclusion : We can make 75 fold large mucosal($3850mm^2$) sheet from small samples of mucosa $(50mm^2)$. Epithelial sheet that grafted on the fascia of muscle underwent differentiation and proliferation. But after 10, 14 days, there was strong inflammatory reaction and the grafted mucosa was destroyed from surface layer. In rabbit model, transfer of fascio-mucosal flap should be done from 7 to 10 days after prelamination.

신생 백서 척수후근절의 슈반세포 배양을 위한 Ara-C 분열억제제의 최적 효과에 대한 연구

김성민(Soung-Min Kim),이종호(Jong-Ho Lee),안강민(Kang-Min Ahn),김남열(Nam-Yeol Kim),성미애(Mi-Ae Sung),황순정(Soon-Jeong Hwang),김지혁(Ji-Hyuck Kim),장정원(Jeong-Won Jahng) 대한구강악안면외과학회 2004 대한구강악안면외과학회지 Vol.30 No.2

Schwann cell, one of important components of peripheral nervous system, interact with neurons to mutually support the growth and replication of embryonal nerves and to maintain the different functions of adult nerves. The Ara-C, known as an antimitotic agent, have been used to have high effectiveness in eliminating fibroblasts during Schwann cell culture period. This enrichment effect is also known to be cummulative with each successive pulse of Ara-C applied and is due to a progressive loss of fibroblasts. But the cytotoxicity by Ara-C is also cummulative and noticeable over the period. To determine the most effective application time and interval of Ara-C in the Schwann cell culture, we observed the Schwann cell purity and density with the Ara-C treatment in plain and three-dimensional culture from dorsal root ganglion of new born rat. By culturing dispersed dorsal root ganglia, we can repeatedly generate homogenous Schwann cells, and cellular morphology and cell count with mean percentages were evaluated in the plain culture dishes and in the immunostainings of S-100 and GFAP in the three-dimensional culture. The Ara-C treated cultures showed a higher Schwann cell percentage (31.0%±8.09% in P4 group to 65.5%±24.08% in P2 group), compared with that obtained in the abscence of Ara-C (17.6%±6.03%) in the plain culture after 2 weeks. And in the three-dimensional culture, S-100 positive cells increased to 56.22%±0.67% and GFAP positive cells to 66.46%±1.83% in G2 group (p<0.05), higher yield than other groups with Ara-C application. Therefore, we concluded that the Ara-C treatment is effective for the proliferation of Schwann cells contrast to the fibroblasts in vitro culture, and the first application after 24 hours from cell harvesting and subsequent 2 pulse treatment (P2 group in plain culture and G2 group in three-dimensional culture) was more effective than other application protocols.

백서 척수후근절로부터 슈반세포의 효과적인 체외 배양법

김성민,이종호,김남열,안강민,최원재,최시호,차미주,이주영,황순정,장정원,명훈,최진영,서병무,정필훈,김명진 大韓顎顔面成形再建外科學會 2003 Maxillofacial Plastic Reconstructive Surgery Vol.25 No.4

Schwann cells(SCs), an important component of the peripheral nervous system, intract with nerous to mutually support growth and replication for the peripheral nerve regentation. Recently, ading SCs to the lumen of guidance channel is widely tried to improve regeneration or to make regeneration possible over otherwise irreparable gaps. however, it is not easy to isolate and multiplicate SCs as much as enough to help the axonal regeneration. For the allogeneic SCs source for tubular nerve guidance, we developed a little bit improved technique of harvesting and multiplicating SCs. by culturing dispersed dorsal root ganglia in specially designed medium with growth factors and serial processing, we repeatedlly generate relatively homogenous SC cultures. Our technique was compared with other methods of literature using immunostaining methods such as GFAP, S100, BDNF and the total SC count assessment at different time interval after primary culture.