• Restorative Dentistry and Endodontics
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• v.48 no.2
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• pp.18.1-18.10
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• 2023

Objectives: This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). Materials and Methods: The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 ㎍/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. Results: CTHRC1 doses of 5, 10, and 20 ㎍/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. Conclusions: CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.5
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• pp.453-456
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• 2001

Background: Autogenous alveolar bone cell transplantation may be suitable for tissue engineering for alveolar bone reconstruction. This study aimed to isolate human alveolar bone-derived cells (HABDCs) and to evaluate the ability of collagen gels to support HABDC proliferation and differentiation for human alveolar bone tissue engineering applications. Method: Cultures of primary HABDCs were established from alveolar bone chips obtained from 10 persons undergoing tooth extraction. These cells were expanded in vitro until passage 3 and used for the in vitro characterization of HABDCs and the in vitro analysis of collagen gels for alveolar bone tissue engineering. Results: Of the 10 attempts made to obtain HABDC cultures, eight were successful. HABDCs expressed the osteoblastic phenotype characterized by alkaline phosphatase activity, osteocalcin expression and the mineralization of the extracellular matrix in vitro. When seeded on collagen gels, HABDCs penetrated into the collagen gel matrices and proliferated inside the gels. Significantly, when HABDCs were embedded into the gels, collagen fibers and mineralization were produced within the gels. Conclusion: This study demonstrates the feasibility of using cultured HABDCs and collagen gels for human alveolar bone tissue engineering applications.

• Restorative Dentistry and Endodontics
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• v.20 no.2
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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.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.1
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• pp.1-8
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• 2005

This study was aimed to characterize osteogenic potential of rat bone marrow stromal cells (BMSC) isolated with standard flushing method and investigate the plasticity of transdifferentiation between osteoblastic and adipocytic lineage of cultured BMSC. Unlike aspiration method in human, rat bone marrow was extracted by means of irrigation with culture media that elevates the possibility of co-extraction of committed osteoprogenitor, or preosteoblast or other progenitor cells of several types present inside bone marrow. The cultured stromal cells showed high ALP activity which is representative marker of osteoblast without any treatment. Osteogenic inducers such as Dex and BMP-2 were examined for the evaluation of their effect on osteogenic and adipocytic differentiation of stromal cells, because they function as osteoinductive agent in stromal cells, but simultaneously induce adipogenic differentiation. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity or mRNA expression of osteoblast markers such as osteopontin, bone sialoprotein, collagen type I and CbfaI, and in vitro matrix mineralization by von Kossa staining. Oil red staining method was used to detect adipocyte and adipocytic marker, aP2 and $PPAR{\gamma}2$ expression was examined using RT-PCR. It can be supposed that irrigation procedure resulted in high portion of already differentiation-committed osteoprogenitor cell showing elevated ALP activity and strong mineralization only under the supplement of $100{\mu}M$ ascorbic 2-phosphate and 10mM ${\beta}$-glycerophosphate without any treatment of osteogenic inducers such as Dex and BMP-2. Dex and BMP-2 seemed to transdifferentiate osteoprogenitor cells having high ALP activity into adipocytes temporarily, but continuous treatment redifferentiated into osteoblast and developed in vitro matrix mineralization. This property must be considered either in tissue engineering for bone regeneration, or in research of characterization of osteogenic differentiation, with rat BMSC isolated by the standard irrigation method.

• International Journal of Oral Biology
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• v.34 no.2
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• pp.105-113
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• 2009

Dentin, a major component of teeth, is formed by odontoblasts which produce the dentin matrix beneath the dental epithelium and induce the mineralization of dentin. To date, the biochemical properties of dentin matrix proteins have been well characterized, but upstream regulators of these proteins are not yet well known. Recently in this regard, several transcription factors have been identified as potential regulators of matrix proteins. Most transcription factors are generally involved in diverse biological processes and it is essential to identify those that are odontoblast-specific transactivators to further understand the process of dentin formation. We thus analyzed the expression pattern of dentin matrix proteins and the activities of established transactivators containing a Cre-locus. Expression analyses using in situ hybridization showed that dentin matrix proteins are sequentially expressed in differentiating odontoblasts, including type-I collagen, Dmp-1 and Dspp. The activities of the transactivators were evaluated using ${\beta}$-galactosidase following the generation of double transgenic mice with each transactivator and the ROSA26R reporter line. The ${\beta}$-galactosidase activity of each transactivator paralled the expression of the matrix proteins. These results thus showed that these transactivators could be utilized for odontoblastspecific conditional gene targeting. In addition, time- and tissue-specific conditional gene targeting might also be achieved using a combination of these transactivators. Odontoblast-specific conditional gene targeting with these transactivators will likely also provide new insights into the molecular mechanisms underlying dentin formation.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.445-456
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• 2002

The purpose of this study were to determine that dexamethasone(Dex) induces differentiation of periodontal ligament(PDL) cells to osteoblastic cells and to investigate expression of matrix Gla protein(MGP), which is one of bone matrix protein. The isolated human PDL cells and gingival fibroblasts were prepared and cultured. The fourth or sixth sub-passage cells were used in this experiments. control group, ascorbic acid and ${\beta}$-glycerophosphate treated group, ascorbic acid, ${\beta}$-glycerophosphate and l00nM Dex treated group, ascorbic acid, ${\beta}$-glycerophosphate, and 5 ${\mu}M$ Dex treated group were made for study. The results were as follows: Cellular morphological change of PDL cells according to time was investigated. At first, the cells exhibited confluent monolayer of spindle or polygonal appearance. The multilayer of cells were seen after 7 days of treatment. After 14 days, the cells lost polarity and were densely packed. The mineralized nodule formation was seen at 21 days in the only Dex treated PDL cell groups. In the gingival fibroblast groups and no Dex treated PDL cell groups, the mineralized nodule was not seen. The mineralized nodule formation of 5 ${\mu}M$ Dex treated group was higher than 100 nM Dex treated group. Alkaline phosphatase(ALP) activity was higher in the Dex treated PDL cell groups of 14 and 21 days than 0 and 7 days. MGP was expressed in the control and all experimental groups and the expression was constant at 0,7,14,21 day. The above results confirm that Dex is affected to differentiation of the PDL cells to osteoblastic or cementoblastic cells and has dose-dependent effect for mineralization. And, MGP is expressed in the PDL cells and is not affected to mineralization of PDL cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.6
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• pp.511-519
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• 2006

Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Journal of Periodontal and Implant Science
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• v.43 no.6
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• pp.251-261
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• 2013

A paradigm shift is taking place in medicine and dentistry from using synthetic implants and tissue grafts to a tissue engineering approach that uses degradable porous three-dimensional (3D) material hydrogels integrated with cells and bioactive factors to regenerate tissues such as dental bone and other oral tissues. Hydrogels have been established as a biomaterial of choice for many years, as they offer diverse properties that make them ideal in regenerative medicine, including dental applications. Being highly biocompatible and similar to native extracellular matrix, hydrogels have emerged as ideal candidates in the design of 3D scaffolds for tissue regeneration and drug delivery applications. However, precise control over hydrogel properties, such as porosity, pore size, and pore interconnectivity, remains a challenge. Traditional techniques for creating conventional crosslinked polymers have demonstrated limited success in the formation of hydrogels with large pore size, thus limiting cellular infiltration, tissue ingrowth, vascularization, and matrix mineralization (in the case of bone) of tissue-engineered constructs. Emerging technologies have demonstrated the ability to control microarchitectural features in hydrogels such as the creation of large pore size, porosity, and pore interconnectivity, thus allowing the creation of engineered hydrogel scaffolds with a structure and function closely mimicking native tissues. In this review, we explore the various technologies available for the preparation of macroporous scaffolds and their potential applications.

• International Journal of Oral Biology
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• v.44 no.4
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• pp.125-129
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• 2019

Osteocalcin is the most abundant non-collagenous protein produced in bone. It has traditionally been regarded as a marker of bone turnover and is thought to act in the bone matrix to regulate mineralization. However, emerging knowledge regarding osteocalcin has expanded to include functions in energy metabolism, fertilization, and regulation of cognition. Fully carboxylated osteocalcin binds to hydroxyapatite, thereby modulating bone turnover, whereas undercarboxylated osteocalcin in the circulation binds to osteocalcin-sensing receptors and acts as a hormone that affects multiple physiological aspects. In this review, we summarize the current knowledge regarding the hormonal actions of osteocalcin in various organs and potential cellular downstream signaling pathway that may be involved.