• Journal of Food Hygiene and Safety
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• v.37 no.5
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• pp.364-371
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• 2022

The bones of the human body support the structures of the body and provide protection for a person's internal organs. Bone metabolic diseases are on the rise due to a significant increase in life expectancy over a short period of time. Therefore, we investigated the osteoblast differentiation promoting and osteoclastogenesis inhibitory activities of fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf). We evaluated the alkaline phosphatase (ALP) activity of MC3T3-E1 mouse calvarial-derived osteoblasts. We also evaluated expression of ALP, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2), which regulate osteoblast differentiation. To assess effects on osteoclast formation, tartrate-resistant acid phosphatase (TRAP) activity in RAW264.7 cells was analyzed. ALP activity increased by 121-136% and 140-156%, respectively in the presence of HR1901-BS and HR1901-BSaf. Expression of osteoblast differentiation factor also increased significantly. We also confirmed that HR1901-BS and HR1901-BSaf decreased TRAP activity in osteoclasts by 35-47% and 23-39%, respectively. Our results showed that fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) increase bone mineralization and osteoblast differentiation activity in MC3T3-E1 cells, and inhibit bone resorption activity in RAW264.7 cells. In conclusion, fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) can be used as an effective natural resource for preventing and treating bone-related diseases.

• Journal of Nutrition and Health
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• v.55 no.6
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• pp.617-629
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• 2022

Purpose: Osteoporosis is characterized by structural deterioration of the bone tissue because of the loss of osteoblastic activity or the increase in osteoclastic activity, resulting in bone fragility and an increased risk of fractures. Hederagenin (Hed) is a pentacyclic triterpenoid saponin isolated from Dipsaci Radix, the dried root of Dipsacus asper Wall. Dipsaci Radix has been used in Korean herbal medicine to treat bone fractures. In this study, we attempted to demonstrate the potential anti-osteoporotic effect of Hed by examining its effect on osteoblast differentiation in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured in 0, 1, and 10 ㎍/mL Hed for 3 and 7 days. The activity of alkaline phosphatase (ALP), bone nodule formation and level of expression of bone-related genes and proteins were measured in MC3T3-E1 cells exposed to Hed. The western blot test was used to detect the activation of the bone morphogenetic protein-2 (BMP2)/ Suppressor of Mothers against Decapentaplegic (SMAD)1 pathway. Results: Hed significantly increased the proliferation of MC3T3-E1 cells. Intracellular ALP activity was significantly increased in the 1 ㎍/mL Hed-treated group. Hed significantly increased the concentration of calcified nodules. Furthermore, Hed significantly upregulated the expression of genes and proteins associated with osteoblast proliferation and differentiation, such as Runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN), and type I procollagen (ProCOL1). Induction of osteoblast differentiation by Hed was associated with increased BMP2. In addition, Hed induced osteoblast differentiation by increasing the activity of SMAD1/5/8. These results suggest that Hed has the potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells via the modulation of the BMP2/SMAD1 pathway. Conclusion: The results presented in this study indicate that Hed isolated from Dipsaci Radix has the potential to be developed as a healthcare food and functional material possessing anti-osteoporosis effects.

• Journal of dental hygiene science
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• v.24 no.3
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• pp.181-189
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• 2024

Background: Focal adhesions (FAs) is the most important process in the first step of osseointegration between preosteoblasts and titanium (Ti). FAs improvement and pre-osteoblasts cell proliferation leads to successful Ti-based dental implants. This study aimed to confirm the applicability of rosmarinic acid (RA) as a functional substance for improving FAs and cell proliferation of MC3T3-E1 preosteoblasts on Ti surfaces during the first stage of osseointegration for successful Ti-based dental implants. Methods: We used MC3T3-E1 preosteoblasts on Ti discs incubated in a medium supplemented with or without 14 ㎍/ml to decipher the effects of RA on FAs and cell proliferation. FAs and proliferation of MC3T3-E1 cells on Ti discs were assessed via MTT assay. Actin-labeled cells and paxillin contacts were observed and imaged by fluorescent microscopy, and the associated signaling pathways were revealed through western blot analysis. Results: In RA-treated MC3T3-E1 cells on Ti discs, FAs between MC3T3-E1 preosteoblasts and Ti surfaces and the expression of focal adhesion kinase (FAK), phosphorylated FAK and paxillin proteins and filamentous-actin formation increased. RA increased the proliferation of MC3T3-E1 preosteoblasts on the Ti surface as well as the expression of Grab2, Ras, pERK1/2, and ERK1/2. In addition, the expression of secretory leukocyte protease inhibitor and thymosin b4, known as nanomolecules that enhance the interaction between implanted Ti materials and preosteoblasts in the RA-treated MC3T3-E1 preosteoblasts, increased. RA not only increased the FAs of MC3T3-E1 preosteoblasts on the Ti surface through the FAK/Paxillin signaling pathway, but also increased cell proliferation and mitosis through the FAK/Grab2/Ras/ERK1/2 signaling pathway. Conclusion: RA can be applied as an effective functional substrate to improve the FAs and proliferation of MC3T3-E1 preosteoblats on Ti surfaces, which are essential in the first step of osseointegration between implanted Ti and bone tissue for the clinical success of Ti based dental implants.

• The korean journal of orthodontics
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• v.31 no.2 s.85
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• pp.173-185
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• 2001

The orthodontic osseointegrated titanium implant, a kind of intraoral skeletal anchorage can be an alternative to tooth-borne anchorage, in case that the conventional tooth-borne anchorage is not available or the anchorage is critical. This study was conducted to elucidate the effect of early loading on the osseointegration of the orthodontic titanium implant and the healing process of the impaired bone at the site of implant after removing it. In two adult beagle dogs24 osseointegrated titanium implants were inserted into the alveolar bone, with 12 implants placed in each dog. In dog1, 6 out of 12 implants were loaded with 200-300gm of force immediately after placing, and the remaining 6 implants were not loaded for 4weeks. In dog2, all 12 implants had healing period of 4weeks, and then were loaded with 200-300gm of force for another 4weeks. Following an observation period of 4 and 8 weeks, the animals were sacrificed. Then the implants and the surrounding bone of dog1 and dog2 were removed, respectively. Undecalcified sections along the long axis of implant were made and the degree of osseointegration was examined under the light microscope. The results were as follows. 1. In the histologic features of tissues around implants anchored in dog1, there was no difference between immediately loaded implants and unloaded implants. Immature woven bone was ingrowing into the thread spaces from the original compacta and in direct contact with the implant surface in part. 2. The premature loading just after 4weeks healing period did not halt the progress of the osseointegration between bone and implant surface. The woven bone around the implants was maturing into the lamellar bone which resembled the structure of the original compacta at the end of 8weeks observation period. 3. Most implants with the inflammed surrounding mucosa were lost or mobile. The mobile implants were encapsulated by fibrous connective tissue which separated the implant surface from the bone. 4. The impaired bone at the site of the implant failed to anchor was showing recovery without inflammatory reaction 2weeks after removing, with the immaure woven bone lined by active osteoblasts and osteoid. Based on the results of this study, the integration of this orthodontic implant seemed to be impaired by the inflammation of the tissue surrounding the Implant rather than by early loading on implant, and increased with time lapsed after placing the implant. The use of implant described in this report can be recommended as an orthodontic anchorage unit immediately after insertion under the careful control of orthodontic force applied and plaque.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.3
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• pp.391-405
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• 2003

Craniosynostosis, known as a premature fusion of cranial sutures, is a developmental disorder characterized by precocious differentiation and mineralization of osteoblasts in the calvarial sutures. Recent genetic studies have demonstrated that mutation in the homeobox gene Msx2 causes Boston-type human craniosynostosis. Additionally, the phenotype of Dlx5 homozygote mutant mouse presents craniofacial abnormalities including a delayed ossification of calvarial bone. Furthermore transcription of osteocalcin, a mature osteoblast marker, is reciprocally regulated by the homeodomain proteins Msx2 and Dlx5. These facts suggest important roles of osteocalcin, Msx2 and Dlx5 genes in the calvarial bone growth and suture morphogenesis. To elucidate the function of these molecules in the early morphogenesis of mouse cranial sutures, we have first analyzed by in situ hybridization the expression of osteocalcin, Msx2 and Dlx5 genes in the developing parietal bone and sagittal suture of mouse calvaria during the embryonic (E15-E18) stage. Osteocalcin mRNA was found in the periosteum of parietal bones from E15, and gradually more highly expressed with aging. Msx2 mRNA was intensely expressed in the sutural mesenchyme, osteogenic fronts and mildly expressed in the dura mater during the embryonic stage. Dlx5 mRNA was intensely expressed osteogenic fronts and the periostem of parietal bones. To further examine the upstream signaling molecules of transcription factor Msx2 and Dlx5, we have done in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of BMP2-, BMP4-soaked beads onto the osteogenic fronts after 48 hours organ culture induced etopic expressions of Msx2 and Dlx5 genes. On the other hand, overexpression of $TGF{\beta}1$, GDF-6, -7, FGF-2, -4 and Shh did not induce the expression of Msx2 and Dlx5. Taken together. these data indicate that transcription factor Msx2 and Dlx5 play critical roles in the calvarial bone and suture development, and that BMP siganling is involved in the osteogenesis of calvarial bones and the maintenance of cranial sutures through regulating these two transcriotpn factors. Furthermore, different expression patterns between Msx2 and Dlx5 suggest their specific functions in the osteoblast differentiation.

• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.1-17
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• 1997

The purpose of this study was to investigate the aspects of proliferation and bone nodule formation of osteogenic precursor cells. To determine the effects of ascorbic acid and dexamethasone upon capacity of osteoblast proliferation and bone nodule formation, cells were maintained in the presence of one or some of these additives for up to 30 days. Group I culture was maintained in standard medium(DMEM plus 10% plus antibiotics), group II was maintained in supplemented medium containing dexamethasone, group III was maintained in supplemented medium containing ascorbic acid and sodium-${\beta}$-glycerophosphate, and group IV was maintained in supplemented containing ascorbic acid, sodium-${\beta}$-glycerophosphate and dexamethasone. Morphology of bone nodules was observed with light microscope and electron microscope. The results were as follows: ${\bullet}$ Proliferation capacity of osteoblasts was not affected by single use of dexamethasone, but it was chiefly affected by ascorbic acid. ${\bullet}$ Cellular morphology was fibroblastic appearance initially, but, it was gradually changed to polygonal shape accompanied by confluency stage. ${\bullet}$ Pluripotent mesenchymal cells existed during primary culture, they were differentiated to adipocyte, chondrocyte, osteocyte according to culture condition. ${\bullet}$ Dexamethasone increased bone nodule formation under the condition that the culture was maintained with supplemented medium ascorbic acid and sodium-${\beta}$-glycerophosphate. ${\bullet}$ when the cultures were stained with alizarin red, the group supplemented with dexamethasone, ascorbic acid and sodium-${\beta}$-glycerophosphate showed the marked increase of bone nodule formation, but the group supplemented with ascorbic acid and sodium-${\beta}$-glycerophosphate revealed only small amounts of bone nodules. And the groups cultured without ascorbic acid showed no observed any of bone-like mass independent of dexamethasone addition.

• The korean journal of orthodontics
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• v.28 no.5 s.70
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• pp.699-716
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• 1998

This study was designed to evaluate the expression of non-collagenous protein in periodontal tissue during the experimental movement of rat incisors, by LSAB(labelled streptavidine biotin) immunohistochemical staining for osteonectin and osteocalcin. Twenty seven Sprague-Dawley rats were divided into a control group(3 rats) and 6 experimental groups(24 rats) where 75g of force was applied from helical springs across the maxillary incisors. Rats of experimental groups were sacrificed at 12 hours, 1, 4, 7, 14 and 28 days after force application, respectively. And the tissues of a control group and experimental groups were studied immunohistochemically and histologically. The results were as follows : 1. Until 28 days after force application, periodontal fibers had been strectched on tension side and compressed in pressure side of all the experimental groups, and the arrangement of periodontal fibers had not been recovered yet. 2. The expression of osteonectin in control group was rare in dentin, cementum and osteocyte, and was mild in odontoblasts and matrix of alveolar bone. 3. The expression of osteocalcin in control group was negative in gingiva, osteoblasts, osteocyte and cementum, and was rare in predentin, capillaries in pulp and periodontal ligament and the matrix of alveolar bone. 4. There was no difference in the expression of osteocalcin or osteonectin in dentin, cementum, pulp, odontoblasts, between of control and of experimental groups. 5. The expression of osteonectin in intermaxillary suture got the peak in 7-day and was declined after 14-day. The expression of osteocalcin remained in a same degree since it became mild in 14-day. 6. The expression of osteonectin in pressure side of periodontal ligament of experimental group was rare, which was similar to control group. But in tension side, it was increased until 14-day aftrer which it was declined. 7. The expression of osteocalcin in periodntal ligament was rare in 12-hour to 14-day, but became severe in 28-day, which was greater in tension side than in pressure side, and in the periodontal fiber next to alveolar bone than to tooth surface. 8. The expression of osteocalcin in alveolar bone was rare until 14-day in pressure side, but became moderate in 28-day. The expression of osteonectin was increased from 7-day by time dependency, which was greater in tension side than in pressure side.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.11
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• pp.1532-1536
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• 2011

In this study, the antioxidative activity of Artemisia capillaris T. extract on the proliferation and differentiation of MC3T3-E1 cells under $H_2O_2$-induced oxidative stress was investigated in order to determine its protective effect against oxidative stress as well as its availability as an antioxidant material related to treatment of bone diseases. As a result, the total polyphenol content of A. capillaris extract was 90.10 mg/g, whereas the flavonoid content was 4.45 mg/g. A. capillaris extract increased proliferation of MC3T3-E1 cells under $H_2O_2$-induced oxidative stress, and also increased the proliferation of differentiated osteoblast cells under oxidative stress. In addition, two differentiation markers, alkaline phosphatase activity and mineralization level, in A. capillaris extract tended to increase. These results indicate that A. capillaris extract suppresses the damage to osteoblasts caused by oxidative stress, which demonstrates its availability as an antioxidant material for preventing bone diseases.

• The korean journal of orthodontics
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• v.31 no.6 s.89
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• pp.589-600
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• 2001

Insulin-like growth factor I (IGF-I) has the local tissue regulating actions. In bone, IGF-I increases the replication of osteoblastic lineage, probably preosteoblasts, and enhances osteoblastic collagen synthesis and matrix composition rates. The purpose of this study was to investigate the local regulatory effect of IGF-I on periodontium totally, both in an autocrine and paracrine manner. To examine the effect of IGF-I directly on osteoblast (OB) of test rats, and indirectlv on OB via periodontal ligament fibroblast (PDLF), and the effect of gingival fibroblast (GF) on OB via cellular paracrine manner for the understanding of humoral action of adjacent tissue, GF and PDLF were obtained from male Sprague-Dawley rats of six to eight weeks of age. OB was obtained iron frontal and parietal calvarial bone of Sprague-Dawley 21day-old-fetus. After each tell was Incubated 24 hours, for collecting conditioned medium, different concentrations of IGF-I (1,10,100 ng/ml,1ml/well) was adding in the GF, PDLF cells, and the supernatant from these cultures was put into the primary OB culture with $1{\times}10^4$cell/ml/well. The experimental group was divided into six groups control OB, IGF-I treated OB, OB culture with conditioned medium from PDLF, OB culture with conditioned medium from IGF-I treated PDLF, OB culture with conditioned medium from GF, OB culture with conditioned medium from IGF-I treated GF. After final IGF-I treatment, OB was Incubated for 24 hours, and alkaline phosphatase activity assay, BMP expression, cell proliferation measurement using MTT assay, total protein measurement, Collagen synthesis assay using western blot, and examination of bone nodule synthesis were done. Alkaline phosphatase expressions were increased in the group of PDLF-IGF-I supernatant treatment. Direct IGF-I treatment with concentrations of 100ng/m1 showed increased viable tell number measured by MTT assay. And IGF-I treatment did not increase total protein amount. The entire experimental group showed BMP2, 4 expression in western blot, and there was no significant difference between control and experimental groups. These results suggested that supernatant from PDLF effects on increasing cellular activities of OB regardless of IGF-I, and at high concentration, IGF-I increases OB tell proliferation.

• 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.