• International Journal of Oral Biology
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• 제33권3호
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• pp.91-95
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• 2008

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and $\beta$-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

• International Journal of Oral Biology
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• 제33권4호
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• pp.131-135
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• 2008

Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with $10^{-10}$ to $10^{-4}M$ SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

• KSBB Journal
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• 제23권4호
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• pp.291-296
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• 2008

양막은 다양한 생체재료로 이용되어 왔으며 생체적합성과 환부의 치료효능이 우수한 것으로 알려져 있다. 본 연구에서는 양막을 갈아 양막스폰지를 제조하고 중간엽줄기세포를 배양한 뒤 누드마우스이식을 통하여 콜라젠스폰지와 비교하여 골형성유도능에 대한 연구를 실시하였다. 연구결과 세포의 부착과 증식면에서는 두 시료가 유사하였으나, 체내에 이식한 결과 양막스포지군에서 좀 더 많은 콜라젠 분비와 칼슘이 침착되었음을 확인하였다. 그리고 면역화학염색 결과 골형성 시 필요한 오스테오칼신과 오스테오넥틴이 좀 더 발현된 것을 관찰할 수 있었다. 따라서 양막은 골형성유도를 향상시킬 수 있는 가능성이 있음을 알 수 있었다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제34권6호
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• pp.588-593
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• 2008

There are increasing reports regarding regeneration of the defected tissues using tissue engineering technique. In this technique, multipotential stem cells are essential. There are many potential sources of adult stem cells, such as bone marrow, umbilical cord blood, fat, muscle, dental tissues and skin. Among them, skin is highly accessible and easily obtained with a minimum of donor site complications. Moreover, skin is an abundant adult stem cell sources and has the potential for self-replication and immune privilege. In this study, we isolated skin-derived precursor cells (SKPs) from the ear of adult miniature pigs. In these SKPs, the expression of transcriptional factors, Oct-4, Sox-2, and Nanog were detected by RT-PCR. In vitro osteogenesis and adipogenesis were observed at 3 weeks after transdifferentiations as assayed by positive von Kossa and Oil-red O staining, respectively. In addition, expression of osteocalcin and osteonectin in the osteogenic differentiation medium and $PPAR{\gamma}2$ and aP2 in the adipogenic differentiation medium were detected by RT-PCR. In vitro neurogenesis of porcine SKPs was observed during 24 and 72 hours after treatment of neurogenic differentiation medium. The results of this study suggest that SKPs demonstrate the properties of pluripotence or multipotence and multi-lineage differentiation. This indicates that autogenous SKPs are a reliable and useful source of adult stem cells for regenerative medicine.

• 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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• 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.

• 치위생과학회지
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• 제14권2호
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• pp.101-106
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• 2014

본 연구는 저 출력 초음파 장치를 이용하여 osteoblastlike MG-63 세포에 1 MHz, 3 Mz 강도의 초음파 처리 후 조골세포의 변화를 관찰하였다. 3 MHz 강도의 주파수를 osteoblast-like MG-63 세포에 처리한 결과 7일 후 성장률이 대조군보다 증가하였다. 저 출력 초음파가 골의 치유에 영향을 미치는 인자를 확인하였을 때 ALP, osteocalcin, VEGF, colla 1A1의 증가를 확인하였으며, 1 MHz 주파수 처리시 integrin alpha 2의 발현이 대조군과 유사하게 발현되는 것을 확인하였다. 반면에, osteonectin, osteopontin, fibronectin, MMP 2 등은 변화가 없음을 확인하였다. 그러므로 본 연구는 저 출력 초음파 장치는 뼈 치유 촉진 단백질들의 발현을 증가시켜 구강 내 외과적 수술 시 골 치유 촉진 효과에 대한 가능성을 제시하였으며 임상에서 골 치유 촉진을 위한 장비로서의 사용 가능성을 제시하였다고 생각된다.

• 대한소아치과학회지
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• 제45권2호
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• pp.242-249
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• 2018

이 연구의 목적은 정량적 실시간 중합효소 연쇄 반응법을 이용하여 발거된 과잉치 치수 및 치주인대 줄기세포의 유전자 발현을 비교하는 것이다. 전신병력이 없는 만 6세 남아 2명의 상악 전치부에 매복된 과잉치를 발거하였다. 같은 날 치수 및 치주인대 세포를 채취하였고, 3계대까지 계대배양하였다. 분석을 위해 상아질모세포 특이 유전자인 Alkaline phosphatase (ALP), Dentin Matrix Protein 1 (DMP-1), Dentin sialophosphoprotein (DSPP), Osteocalcin (OCN) 그리고 Osteonectin (ONT)을 사용했고, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)을 대조군으로 설정했다. 과잉치 치수 세포에서는 ONT, OCN, ALP, DMP-1, DSPP 순서로 발현량이 많았고, 치주인대 세포에서는 DMP-1과 DSPP의 순서만 바뀌었다. 치주인대 세포보다 치수 세포에서 모든 유전자의 발현량이 많았다. 이러한 상아질모세포의 특성을 고려해 보았을 때, 다른 조직으로의 분화 가능성이 있는 과잉치 줄기세포는 유용한 공여부로서 그 잠재력이 있음을 알 수 있었다.

• Restorative Dentistry and Endodontics
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• 제29권4호
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• pp.399-408
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• 2004

Odontoblasts are responsible for the formation and maintenance of dentin. They are known to synthesize unique gene products including dentin sialophosphoprotein (DSPP). Another unique genes of the cells remain unclear. OD314 was isolated from the odontoblasts/pulp cells of rats and partially characterized as an odontoblast-enriched gene (Dey et al., 2001). This study aimed to elucidate the biological function of OD314, relating to odontoblast differentiation and dentinogenesis. After determining the open reading frame (ORP) of OD314 by transient transfection analysis using green fluorescent protein (GPP) expression vector, mRNA in-situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western analysis were performed. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were expressed in odontoblasts of developing coronal and root pulp. 2. OD314 was a novel protein encoding 154 amino acids, and the protein was mainly expressed in cytoplasm by transient transfection analysis. 3. Mineralized nodules were associated with multilayer cell nodules in the culture of human dental pulp cells and first detected from day 21 using alizarin-red S staining. 4. In RT-PCR analysis, OD314, osteocalcin (OC) and DSPP strongly expressed throughout 28 days of culture. Whereas, osteonectin (ON) mRNA expression stayed low up to day 14, and then gradually decreased from day 21. 5. Western blots showed an approximately 17 kDa band. OD314 protein was expressed from the start of culture and then increased greatly from day 21. In conclusion, OD314 is considered as an odontoblast-enriched gene and may play important roles in odontoblast differentiation and dentin mineralization.

• 한국수정란이식학회지
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• 제27권3호
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• pp.175-181
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• 2012

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% $CO_2$ atmosphere at $38.5^{\circ}C$. The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-${\gamma}2$, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.