• Maxillofacial Plastic and Reconstructive Surgery
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• 제38권
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• pp.27.1-27.9
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• 2016

Background: The objective of this study was to place bone graft materials in cranial defects in a rabbit model and compare their bone regenerating ability according to the size and density of demineralized dentin matrix (DDM). Methods: We selected nine healthy male rabbits that were raised under the same conditions and that weighed about 3 kg. Two circular defects 8 mm in diameter were created in each side of the cranium. The defects were grafted with DDM using four different particle sizes and densities: 0.1 mL of 0.25- to 1.0-mm particles (group 1); 0. 2 mL of 0.25- to 1.0-mm particles (group 2); 0.1 mL of 1.0- to 2.0-mm particles (group 3); and 0.2 mL of 1.0- to 2. 0-mm particles (group 4). After 2, 4, and 8 weeks, the rabbits were sacrificed, and bone samples were evaluated by means of histologic, histomorphometric, and quantitative RT-PCR analysis. Results: In group 1, osteoblast activity and bone formation were greater than in the other three groups on histological examination. In groups 2, 3, and 4, dense connective tissue was seen around original bone even after 8 weeks. Histomorphometric analysis of representative sections in group 1 showed a higher rate of new bone formation, but the difference from the other groups was not statistically significant. RT-PCR analysis indicated a correlation between bone formation and protein (osteonectin and osteopontin) expression. Conclusions: DDM with a space between particles of $200{\mu}m$ was effective in bone formation, suggesting that materials with a small particle size could reasonably be used for bone grafting.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.744-757
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• 1995

Implantation of demineralized bone matrices was done into the amputated pulp in vivo and sequential reaction of the pulpal ectomesenchymal cells was observed. The bone matrices, obtained from cat long bone were crushed into below $700{\mu}m$, demineralized with 0.5N HCl and allografted into pulp of molar teeth. At seven days after implantation many undifferentiated mesenchymal cells aggregated near the matrices in the pulpal tissue. At fourteen days after implantation, the cells differentiated into preosteoblast-like cells which have secretory cell characteristics. At one or two months after implantation osteoid tissue was formed. The cells, which are located at the surface of the tissue, contained abundant dilated rough endoplasmic reticulum, Golgi apparatus and secretory granules in the cytoplasm. The matrix of the tissue has less collagen fibers than those in normal dentin. These results suggest that the interaction of pulpal mesenchymal cells with demineralized bone matrix can be a model which induces mineralization.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제42권2호
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• pp.90-98
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• 2016

Objectives: The aim of this study was to compare the osteogenic effects of demineralized dentin matrix (DDM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbit calvarial defects with DDM and anorganic bovine bone (ABB) combined with rhBMP-2. Materials and Methods: Four round defects with 8-mm diameters were created in each rabbit calvaria. Each defect was treated with one of the following: 1) DDM, 2) ABB/rhBMP-2, or 3) DDM/rhBMP-2. The rhBMP-2 was combined with DDM and ABB according to a stepwise dry and dip lyophilizing protocol. Histological and microcomputed tomography (${\mu}CT$) analyses were performed to measure the amount of bone formation and bone volume after 2- and 8-week healing intervals. Results: Upon histological observation at two weeks, the DDM and ABB/rhBMP-2 groups showed osteoconductive bone formation, while the DDM/rhBMP-2 group showed osteoconductive and osteoinductive bone formation. New bone formation was higher in DDM/rhBMP-2, DDM and ABB decreasing order. The amounts of bone formation were very similar at two weeks; however, at eight weeks, the DDM/rhBMP-2 group showed a twofold greater amount of bone formation compared to the DDM and ABB/rhBMP-2 groups. The ${\mu}CT$ analysis showed markedly increased bone volume in the DDM/rhBMP-2 group at eight weeks compared with that of the DDM group. Notably, there was a slight decrease in bone volume in the ABB/rhBMP-2 group at eight weeks. There were no significant differences among the DDM, ABB/rhBMP-2, and DDM/rhBMP-2 groups at two or eight weeks. Conclusion: Within the limitations of this study, DDM appears to be a suitable carrier for rhBMP-2 in orthotopic sites.

• Journal of Periodontal and Implant Science
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• 제26권4호
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• pp.873-887
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• 1996

Platelet-derived growth factor(PDGF) is one of the polypeptide growth fators. PDGF has been reported as a biological mediator which regulates activities of wound healing process including the cell proliferation, migration and metabolism. Recent studies indicated that demineralized root surface as the primary site for growth factor application has advantages over other application method, especially due to binding capacity of growth factor for exposed matrix component of deminera1ized dentin surface. The purpose of this study is to evaluate optimal application time of PDGF-BB on proliferation of human gingival fibroblasts using deminera1ized dentin surface as primary application site. Human gingival fibroblasts and dentin slabs were prepared from the first premolar tooth extracted for the orthodontic treatment, cells were cultured in DMEM/I0% FBS at the $37^{\circ}C$, 5% CO2 incubator. All of the dentin slabs were preconditioned with Tetracycline HCI(100mg/ml) solution and rinsed in PBS. In the cell proliferation experiment, experimental group was immersed in DMEM containing 10% FBS, 50ng/rnl PDGF-BB during different time(30sec, 1, 2, 4, 8 minutes) and dried. Cells at concentration of $1{\times}10^5$cells/ml were seeded in each culture well which contained dentin slabs and incubated for 6 hours. Then, all of the dentin slabs were moved into new 24 well culture dish and incubated for 24, 48, 72 hours. The cell counting was done by hemocytometer with inverted phase contrast microscope after trypsinization. The results were as follows : The application of PDGF-BB for 1, 2 min slightly increased the number of gingival fibroblasts, and the application of PDGF-BB for 4, 8 min prominently increased the number of gingival fibroblasts. The application of PDGF-BB for 4 min showed maximum proliferation rate of gingival fibroblasts at 24, 48, 72 hours, and the application of PDGF-BB for 8 min showed less proliferation rate of gingival fibroblasts compared to the application of PDGF-BB for 4 min at 24, 48, 72 hours. In conclusion, the application of PDGF-BB for 4 min appeared to be optimal to obtain maximum proliferation of gingival fibroblasts using demineralized dentin surface as primary applicaton site of PDGF-BB.

• International Journal of Oral Biology
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• 제42권4호
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• pp.203-211
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• 2017

The aim of this study was to evaluate the role of demineralized and particulate autogenous tooth, and interleukin-6 in bone regeneration. A demineralized and particulate autogenous tooth was prepared and human osteoblast-like cells (MG63) and human osteosarcoma cells were inoculated into the culture. The rate of cell adhesion, proliferation and mineralization were examined, and the appearance of cellular attachment was observed. An 8 mm critical size defect was created in the cranium of rabbits. Nine rabbits were divided into three groups including: An experimental group A (3 rabbits), in which a demineralised and particulate autogenous tooth was grafted; an experimental group B (3 rabbits), in which a demineralized, particulate autogenous tooth was grafted in addition to interleukin-6 (20 ng/mL); and a control group. The rabbits were sacrificed at 1, 2, 4 and 6 weeks for histopathological examination with H-E and Masson's Trichrome, and immunohistochemistry with osteocalcin. The cell-based assay showed a higher rate of cell adhesion, mineralization and cellular attachment in the experimental group A compared with the control group. The animal study revealed an increased number of osteoclasts, newly formed and mature bones in the experimental group A compared with the control group. Eventually, a higher number of osteoclasts were observed in the experimental group B. However, the emergence of newly formed and mature bone was lower than in the experimental group A. The current results suggest that treatment with demineralized and particulate autogenous tooth and interleukin-6 is not effective in stimulating bone regeneration during the bone grafting procedure.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제39권3호
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• pp.103-111
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• 2013

With successful extraction of growth factors and bone morphogenic proteins (BMPs) from mammalian teeth, many researchers have supported development of a bone substitute using tooth-derived substances. Some studies have also expanded the potential use of teeth as a carrier for growth factors and stem cells. A broad overview of the published findings with regard to tooth-derived regenerative tissue engineering technique is outlined. Considering more than 100 published papers, our team has developed the protocols and techniques for processing of bone graft material using extracted teeth. Based on current studies and studies that will be needed in the future, we can anticipate development of scaffolds, homogenous and xenogenous tooth bone grafts, and dental restorative materials using extracted teeth.

• 대한소아치과학회지
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• 제27권2호
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• pp.318-332
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• 2000

탈회냉동건조골과 교원질을 치수치료제로서 이용할 수 있는지의 여부를 규명하기 위하여, 성견의 치아를 대상으로 치수절단술을 시행하여 치수조직의 염증반응, 재생성 상아질의 형성 등 치수조직반응을 평가하기 위하여 시행되었다. 체중 10kg 내외의 4마리의 성견 치아에 소독된 round bur을 이용하여 5급 와동을 형성한 후 치수절단술을 시행하였다. 대조군으로는 수산화칼슘으로 치수 복조을 시행하고 IRM으로 밀봉하였으며, 실험군으로는 교원질과 탈회냉동건조골을 이용하여 복조한 후 대조군에서와 같은 방법으로 밀봉하였다. 실험동물은 3일, 1주일, 2주일, 4주일 경과 후 관류 고정으로 희생되었으며, 조직표본을 제작한 후 광학현미경과 투과전자현미경으로 치수 조직 반응을 관찰 평가한 결과 다음과 같은 결론을 얻었다. 1. 모든 군에서 치수괴사 소견은 보이지 않았으며, 대조군과 실험 2군에서는 1주일 후부터 염증이 소실되었으나, 실험 1군에서는 2주까지 염증소견을 보이며 절단된 치수 측벽의 조상아세포들도 배열이 불규칙하였다. 2. 대조군에서는 2주부터 불완전한 상아질교가, 4주 경과 후에는 골양상아질과 완전한 상아질교가 관찰되었으며, 1군에서는 섬유성피막은 형성되었으나 조상아세포와 상아질교의 형성소견은 관찰되지 않았다. 2군의 경우 2주 경과 후 탈회냉동건조골 주위에 분화된 조상아세포들에 의해 형성된 상아전질과 재생성 상아질이 관찰되었으나 상아질교의 형성소견은 관찰되지 않았다. 3. 대조군과 2주군에서 1주일 경과 후에 크고 호염기성 핵을 갖는 세포들이 치수절단부 하방과 탈회냉동건조골 주위에 모여드는 양상을 보였는데, 이와 같은 현상은 대조군에 비해 2군에서 더욱 현저하였으며 시간이 경과될수록 규칙적으로 배열한 조상아세포와 재생성 상아질을 관찰할 수 있었다. 4. 대조군과 2군에서 조상아세포는 극성을 띤 핵, 조면내형질세망, 골지체, 분비과립 등을 포함하는 세포질과 유기기질을 분비하는 소견 등, 석회화가 이루어지는 초미세구조를 보였다. 이상의 결과를 종합하면 탈회냉동건조골은 치수의 미분화 간엽세포로부터 조상아세포의 분화를 유도하여 치수절단부위에 재생 상아질을 형성할 수 있는 것으로 사료된다.

• Restorative Dentistry and Endodontics
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• 제21권1호
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• pp.35-53
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• 1996

The development and repair requires the formation of new tissues comprised of various extracellular matrix components. The present study investigated the formation and distribution of the major ECM components such as type I collagen, type III collagen, fibronection, bone sialoprotein, and osteonection during development and repair. For developing observation. Sprague-Dawley rats weighing $27{\pm}1gm$ were sacrificed. For repair observation, Sprague-Dawley rats weighing $110{\pm}5gm$ were used. The pulp perforation were prepared on mesial surface of the maxillary first molar by using 1/2round bur. At 5 days after perforation, rats were sacrificed by perfusion with 3 % paroformaldehyde. The maxillary first molar region were cut, demineralized, dehydrated and embedded in paraffin. Immunostaining the ECM components was achieved by the avidin-biotin complex method. The results as follows : 1. Bright immunoreaction for fibronectin was present in the basement membrane at the inner epithelial-mesenchymal interface, especially concentrated in the blood vessel walls, cell membrane of odontoblasts, and initial predentin. 2. Type I and III collagen was observed in the newly formed pulp tissue, predentin, and its intensity increased as more of these components during repair. 3. Strong immunostaining for bone sialoprotein and osteonectin was found in dentin while no or weaker staining was observed loose connective tissue of the pulp. 4. These results suggest that develpment and repair is achieved through a series of cell differentiation and attachment by the specific ECM components.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제35권6호
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• pp.353-359
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• 2013

Purpose: Extensive research is actively ongoing for development of an ideal bone substitute that meets the gold standard. Tooth was selected as a donor site for evaluation of potentials in bone substitutes based on its similar chemical compositions to alveolar bone. Previous studies have evaluated inorganic components of autogenous tooth bone graft material (AutoBT) and osteoconductivity. In continuation from the previous studies, the current study was conducted for analysis of organic components and evaluation of osteoinductivity of AutoBT. Methods: Forty-six extracted teeth were collected from actual patients (Korea Tooth Bank, R&D Institute). Extracted teeth were processed into AutoBT and implanted in dorsal subcutaneous muscular tissues of 15 athymic mice. Biopsy samples were harvested at two, five, and eight weeks. The Bradford assay, sodium dodecyl sulphate polyacrylamide gradient gel, and western blotting were performed for investigation of organic contents of AutoBT. Results: Histology analyses showed signs of new bone formation as early as two weeks. Results of the Bradford assay indicated the existence of noncollagenous proteins (NCP). 0.29% (2.89 mg/g) of proteins were extracted by weight in the root portion of AutoBT; 0.02% (0.029 mg/g) and 1.79% (17.93 mg/g) of proteins were measured by weight in crown and block-form of AutoBT, respectively. However, recombinant human bone morphogenetic protein-2 was not observed in AutoBT. Conclusion: Within the limitation of the current study, AutoBT induced new bone formation by NCP embedded in dentin.

• Restorative Dentistry and Endodontics
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• 제32권2호
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• pp.151-161
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• 2007

치아 우식증은 구강 영역에서 가장 흔한 질환이나 여러 가지 복잡한 요소가 작용하기 때문에 이에 대한 발생기전과 치료법에 대해서는 완전히 이해되지 않은 상태이다. 특히 초기우식의 재광화에 있어서 pH의 영향에 대하여 논란의 여지가 있다. 본 연구에서는 유산 완충용액을 이용하여 상아질에 인공 우식을 형성하고 이때의 무기질 소실을 측정하고, 탈회된 시편을 pH가 다른 세 가지 재광화 용액 (pH 4.3, 5.0, 5.5 군)으로 재광화시켰을 때 나타나는 무기질의 침착되는 양과 침착이 일어나는 부위를 Microradiograph를 이용하여 정량적으로 비교 분석하였다. 또한 주사전자현미경을 이용하여 수산화인회석 결정의 정상 상태 탈회된 상태, 그리고 재광화가 일어났을 때의 상태를 정성적으로 비교하여 다음과 같은 결과를 얻었다. 1. Microradiograph분석시 pH가 증가할수록 탈회된 상아질에서 재광화양이 유의차 있게 증가되는 경향을 보였고, pH가 낮아질수록 재광화가 일어나면서 더 깊게 탈회되는 양상을 보였으나, pH 5.5 군은 전반적으로 재광화가 일어나는 경 향을 보였다 (p<0.05). 2. 주사전자현미경 소견에서 상아질 우식의 재광화는, 유기기질 망을 둘러싸고 있는 수산화인회석 결정의 표면으로부터 진행되며 결국에는 탈회 시 파괴된 공간을 채워가는 양상으로 관찰되었다. 3. 재광화 5일 후 pH 4.3. 5.0군에서 무기질이 침착되어 수산화인회석결정이 정상보다 커졌으며, pH 5.5군에서 재광화된 부위의 결정은 정상으로 회복되는 양상을 보였다. 재광화 10일 후 pH 4.3, 5.0 군에서 재광화 5일 후에 정상보다 커졌던 결정들이 정상상아질의 결정 크기로 줄어들었으며 pH 5.5군에서는 결정의 크기가 2배정도 커진 경우도 관찰되었다. 본 실험의 결과에 의하면 상아질의 우식과 재광화 과정은 단순한 탈회와 재광화의 독립적인 과정이 아니고 동력학적으로 탈회와 재광화가 동시에 일어나는 과정이며, 이때 재광화는 기존의 수산화인회석 결정의 표면으로부터 진행되었다.