Scaffold상에 식립한 사람치주인대섬유모세포를 통한 치주조직공학

Human periodontal ligament fibroblasts (hPDLF) are very important for curing the periodontal tissue because they can be differentiated into various cells. A tissue engineering approach using a cell-scaffold is essential for comprehending today's periodontal tissue regeneration procedure. This study examined the possibility of using an acellular dermal matrix as a scaffold for human periodontalligament fibroblast (hPDLF). The hPDLF was isolated from the middle third of the root of periodontally healthy teeth extracted for orthodontic reasons. The cells were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at in humidified air with 5% . The acellular dermal matrix(ADM) was provided by the US tissue banks(USA). Second passage cells were used in this study. The hPDLF cells were cultured with the acellular dermal matrix for 2 days, and the dermal matrix cultured by the hPDLF was transferred to a new petri dish and used as the experimental group. The control group was cultured without the acellular dermal matrix, The control and experimental cells were cultured for six weeks. The hPDLF cultured on the acellular dermal matrix was observed by Transmission Electron microscopy (TEM). Electron micrography shows that the hPDLF was proliferated on the acellular dermal matrix. This study suggests that the acellular dermal matrix can be used as a scaffold for hPDLF.이 논문은 2005년 정부(교육인적자원부)의 재원으로 한국학술진흥재단의 지원을 받아 수행된 연구입니다

The effects of Acellular dermal matrix on the healing of 1 wall intrabony defects in dogs

Although the main purpose of periodontal treatment to regenerate is the complete regeneration of periodontal tissue due to periodontal disease, most of the treatment cannot meet such purpose because healing by long epithelial junction. Therefore, diverse materials of resorbable and non-resorbable have been used to regenerate the periodontal tissue. Due to high risk of exposure and necessity of secondary surgical procedure when using non-resorbable membrane, guided tissue regeneration using the resorbable membrane has gain popularity, recently. However, present resorbable membrane has the disadvantage of not having sufficient time to regenerate date to the difference of resorption rate according to surgical site. Meanwhile, other than the structure stability and facile manipulation, acellular dermal matrix has been reported to be a possible scaffold for cellular proliferation due to rapid revascularization and favorable physical properties for cellular attachment and proliferation. The purpose of this study is to estimate the influence of acellular dermal matrix on periodontal ligament, cementum and alveolar bone when acellular dermal matrix is implanted to 1-wall alveolar bone defect. 4 dogs of 12 to 16 month old irrelevant to sex , which below 15Kg of body weight, has been used in this study. ADM has been used for the material of guided tissue regeneration. The 3rd premolar of the lower jaw was extracted bilaterally and awaited for self-healing. subsequently buccal and lingual flap was elevated to form one wall intrabony defect with the depth and width of 4mm on the distal surface of 2nd premolar and the mesial surface of 4th premolar. After the removal of periodontal ligament by root planing. notch was formed on the basal position. Following the root surface treatment, while the control group had the flap sutured without any treatment on surgically induced intrabony defect. Following the root surface treatment, the flap of intrabony defect was sutured with the ADM inserted while the control group sutured without any insertion. The histologic specimen was observed after 4 and 8 weeks of treatment. The control group was partially regenerated by periodontal ligament, new cementum and new alveolar bone. the level of regeneration is not reached on the previous formed notch. but, experimental group was fully regenerated by functionally oriented periodontal ligament fiber. new cementum and new alveolar bone. In conclusion, we think that ADM seems to be used by scaffold for periodontal ligament cells and the matrix is expected to use on guided tissue regeneration.이 논문은 2005년도 조선대학교 학술연구비의 지원을 받아 연구되었음

가토의 두개골 결손부에서 골재생에 끼치는 골막의 역할

The role of the periosteum on osteointegration of (Geistlich, Wolhusen/Switzerland) was studied in rabbit calvarial defect. 12 New Zealand white male rabbits between 2.8 and 4 kg were included in this randomized, blinded, prospective study. Each rabbit was anesthetized with Ketamine HCl(5 mg/kg) and Xylazine HCl(1.5 ml/kg). An incision was made to the bony cranium and the periosteum was reflected. Using a 6-mm trephine bur(3i. USA), four 8-mm defects were created with copious irrigation. The defects were classified into barrier membrane(, Lifecore Biomedical. Inc, U.S.A.) only group as a control, with barrier membrane group, with periosteum covering group, and without periosteum covering group. There were 2 rabbits in each group. The wound was closed with resorbable suture materials. Rabbits were sacrificed using phentobarbital(100 mg/kg) intravenously at 1, 2, and 4 weeks after surgery. The samples were fixed in 4% paraformaldehyde, and decalcified in hydrochloric acid decalcifying solution(Fisher Scientific, Tustin, CA) at for 2-4 weeks. It was embedded in paraffin and cut into 6 thickness. The sections were stained with H & E and observed by optical microscope. The results were as follows; 1. The periosteum played an important role in osteointegration of in bone defects. 2. When the periosteum remained intact and was placed on the defect, with periosteum covering has been incorporated into the newly formed bone from 2-week postoperatively. 3. When the periosteum was removed at the surgical procedure, invasion of connective tissue took place among the granules, and new bone formation was delayed compared to periosteum covering group. Therefore, when the bone grafting was performed with periosteal incision procedure to achieve tension-free suture, the integrity of the overlying periosteum should be maintained to avoid fibrous tissue ingrowth.이 논문은 2004년도 조선대학교 학술연구비의 지원을 받았습니다

토끼의 두개골내에 형성된 골결손부에서 HA/ß-TCP composite powders의 골형성에 관한 조직학적 연구

The purpose of the present study was to evaluate the histologic results of bone cavities that were surgically created in the calvaria of rabbit and filled with HA/ß-TCP composite powders, which had been developed in Korea (Dentium, Korea). Ten young adult rabbits were used. Four defects were surgically produced in calvaria of each rabbit. Each rabbit was anesthetized with Ketamine-HCI (5 mg/kg, Yuhan Cor. Korea) and Xylazine-HCI (1.5 ml/kg, Yuhan Cor. Korea)). An incision was made to the bony cranium and the periosteum was reflected. Using a trephine bur (external diameter: 8 mm, 3i, USA), 4 'through-and-through' bone defects were created with copious irrigation, and classified into 4 groups: control group: no graft materials, experimental group I: normal saline + graft materials: experimental group II: venous blood + graft materials: experimental group III: graft materials only. The defects were randomly filled with graft materials. The defects were closed with resorbable suture material. At the end of the surgical procedure, all animals received a single intramuscular injection of antibiotics Gentamicin (0.1 mg/kg, Dae Sung Microb. Korea). Rabbits were sacrificed with phentobarbital (100 mg/kg) intravenously at 1-, 2-, 4-, 6- and 8-week after. Specimens were treated with hydrochloric acid decalcifying solution (Fisher Scientific, Tustin, CA) and sectioned by bisecting the 8 mm diameter defects. The histologic specimens were prepared in the general method with H & E staining at 6 in thickness. The results were as follows; 1. New bone formation showed from after 2-week of surgery in defect area. As time lapsed, lots of new bone formation and mature bones showed. 2. Histologically, degree of new bone formation could not be discerned among the experimental groups. But, for experimental group II, lots of cells gathered around graft materials after 1-week of surgery, new bone formed slightly faster and than the others at 1-week after. For experimental group I, a few inflammatory finding showed around graft material at after 1-week and after 2-week of surgery. 3. No bone formation did show for control group. Based on histologic results, the new HA/ß-TCP composite powders appeared to act as a scaffolding material for regeneration of osseous defects.This study was supported by a grant of the Korea Health 21 R & D Project. Ministry of Health & Welfare. Republic of Kore

분자생물학을 이용하여 복제노화된 사람치주인대섬유모세포의 세포학적 연구

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% . For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.이 논문은 2004년도 한국학술진흥재단의 지원에 의하여 연구되었습니다

상아모세포의 조건배지를 이용한 백악모세포의 분화와 석회화 조절

For the regeneration of periodontal tissues, the microenvironment for new attachment of connective tissue fibers should be provided, At this point of view, cementum formation in root surface plays a key role for this new attachment. This study was performed to figure out which factor promotes differentiation of cementoblast Considering anatomical structure of tooth, we selected the cells which may affect the differentiation of cementoblast - Ameloblast, OD11&MDPC23 for odontoblasts, NIH3T3 for fibroblsts and MG63 for osteoblasts. And OCCM30 was selected for cementoblast cell line. Then, the cell lines were cultured respectively and transferred the conditioned media to OCCM30. To evaluate the result, Alizarin red S stain was proceeded for evaluation of mineralization. The subjected mRNA genes are bone sialoprotein(BSP), alkaline phosphate(ALP) , osteocalcin(OC), type I collagen(Col I), osteonectin(SPARC ; secreted protein acidic and rich in cysteine). Expression of the gene were analysed by RT-PCR, The results were as follows: 1. For alizarin red S staining, control OCCM30 didn't show any mineralized red nodules until 14 days. But red nodules started to appear from about 4 days in MDPC-OCCM30 & OD11-OCCM30. 2. For results of RT-PCR, ESP mRNAs of control-OCCM30 and others were expressed from 14 days, but in MDPC23-OCCM30 & OD11-OCCM30 from 4 days. Like this, the gene expression of MDPC23-OCCM30 & OD11-OCCM30 were detected much earlier than others. 3. For confirmation of odontoblast effect on cementoblast, conditioned media of osteoblasts(MG63) which is mineralized by producing matrix vesicles didn't affect on the mineralized nodule formation of cementoblasts(OCCM30). This suggest the possibility that cementoblast mineralization is regulated by specific factor in dentin matrix protein rather than matrix vesicles. Therefore, we proved that the dentin/odontoblast promotes differentiation/mineralization of cementoblasts. This new approach might hole promise as diverse possibilities for the regeneration of tissues after periodontal disease.본 연구는 교육인적자원부 누리사업 국고보조금으로 수행한 과제입니다

Enamel Matrix Derivatives가 사람 치주인대 세포의 특이유전자인 PDLs17, PDLs22의 발현에 끼치는 효과

The enamel matrix derivative (EMD) has been recently used in the periodontal regenerative techniques. The present study was established to investigate the influence of EMD on human periodontal ligament cells using expression of mRNA of periodontal ligament specific gene (PDLs)17, PDLs22, type I collagen when EMD applied to periodontal ligament cells. Periodontal ligament cells were obtained from a healthy periodontium and cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum and with ascorbic acid. Test groups were two; One adds EMD in culture media and another added EMD and Dexamethasone (DEX) in culture media. Positive control group added DEX in culture media, and negative control group adds niether of EMD nor DEX. (Biora, Sweden, 30 mg/ml) was diluted by 75 concentration to culture media. For reverse transcription-polymerase chain reaction (RT-PCR), total RNA isolated on days 0, 7, 14 and 21. mRNA of PDLs17 was expressed on days 14 and 21 in EMD or DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group, the other side, expressed on days 21 in negative control group. mRNA of PDLs22 expressed on days 7, 14 and 21 in EMD group, and expressed on days 14 and 21 in DEX group, and expressed on days 7, 14 and 21 in EMD plus DEX group. Negative control group expressed on days 14 and 21. Type I collagen was expressed on all days and all groups. These results indicate that EMD promotes differentiation of periodontal ligament cells, and this is considered to offer basis that can apply EMD to periodontal tissue regeneration technique.This study was supported by a grant of the Korea Health 21 R & D Project. Ministry of Health & Welfare. Republic of Kore

치주인대섬유모세포의 분화과정에서 아미노산 수송계 L의 발현

The periodontium is a topographically complex organ consisting of epithelial tissue, soft and mineralized tissues. Structures comprising the periodontium include the gingiva, periodontal ligament (PDL) , cementum and the alveolar bone. The molecular mechanism of differentiation in PDL fibroblast cells remain unclear. Amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In this study, the expression pattern of amino acid transport system L was, therefore, investigated in the differentiation of PDL fibroblast cells. To determine the expression level of amino acid transport system L participating in intracellular transport of amino acids in the differentiation of PDL fibroblast cells, it was examined by RT-PCR, observation of cell morphology, Alizaline red-S staining and uptake analysis after inducing experimental differentiation in PDL fibroblast cells isolated from mouse molar teeth. The results are as follows. 1. The LAT1 mRNA was expressed in the early stage of PDL fibroblast cell differentiation. This expression level was gradually reduced by differentiation- inducing time and it was not observed after the late stage. 2. The expression level of LAT2 mRNA was increased in time-dependent manner during differentiation induction of PDL fibroblast cells. 3. There was no changes in. the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during differentiation of PDL fibroblast cells. 4. The expression level of ALP mRNA was gradually increased and the expression level of Col I mRNA was decreased during differentiation of PDL fibroblast cells. 5. The L-leucine transport was reduced by time from the early stage to the late stage in PDL fibroblast cell differentiation. As the results, it is considered that among neutral ammo acid transport system L in differentiation of PDL fibroblast cells, the LATl has a key role in cell proliferation in the early stage of cell differentiation and the LAT2 has an important role in the late stage of cell differentiation for providing cells with neutral amino acids including several essential amino acids.이 연구에 참여한 연구자(의 일부)는 [2단계 BK21 사업]의 지원비를 받았습니다