• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.37 no.3
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• pp.214-224
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• 2011

Objective: This study examined the potential of the in vitro osteogenesis of microtopographically modified surfaces, RBM (resorbable blasting media) surfaces, which generate hydroxyapatite grit-blasting. Methods: RBM surfaces were modified hydroxyapatite grit-blasting to produce microtopographically modified surfaces and the surface morphology, roughness or elements were examined. To investigate the potential of the in vitro osteogenesis, the osteoblastic cell adhesion, proliferation, and differentiation were examined using the human osteoblast-like cell line, MG-63 cells. Osteoblastic cell proliferation was examined as a function of time. In addition, osteoblastic cell differentiation was verified using four different methods of an ALP activity assay, a mineralization assay using alizarin red-s staining, and gene expression of osteoblastic differentiation marker using RT-PCR or ELISA. Results: Osteoblastic cell adhesion, proliferation and ALP activity was elevated on the RBM surfaces compared to the machined group. The cells exhibited a high level of gene expression of the osteoblastic differentiation makers (osteonectin, type I collagen, Runx-2, osterix). imilar data was represented in the ELISA produced similar results in that the RBM surface increased the level of osteocalcin, osteopontin, TGF-beta1 and PGE2 secretion, which was known to stimulate the osteogenesis. Moreover, alizarin red-s staining revealed significantly more mineralized nodules on the RBM surfaces than the machined discs. Conclusion: RBM surfaces modified with hydroxyapatite grit-blasting stimulate the in vitro osteogenesis of MG-63 cells and may accelerate bone formation and increase bone-implant contact.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.3
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• pp.216-225
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• 2002

Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in ${\alpha}-MEM$ medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.

• Journal of Periodontal and Implant Science
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• v.53 no.1
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• pp.54-68
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• 2023

Purpose: The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. Methods: BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 µM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 µM, ROS scavenger) group, (4) the drBMSCs + MF (200 µM) group, and (5) the drBMSCs + MF (200 µM) + H₂O₂ (50 µM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. Results: MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H₂O₂ inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. Conclusions: MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.198-206
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• 2009

Purpose: The aims of this study were to assess the in vitro co-culturing pattern of isolated skin-derived precursor cells (SKPs) with a mixed demineralized bone (DMB) and fibrin glue scaffold and to evaluate in vivo osteogenesis after transplantation of autogenous SKPs with a these mixed scaffold in the animal's mandibular defects. Materials and Methods: We isolated SKPs from the ears of adult 4 miniature pigs. The isolated SKPs were co-cultured with a mixed DMB and fibrin glue scaffold in a non-osteogenic medium for 1, 2, and 4 weeks. Histological characteristics of in vitro co-cultured cells and scaffold were evaluated. $1{\times}10^7\;cells/100\;{\mu}l$ of autogenous porcine SKPs were grafted into the mandibular defects with a DMB and fibrin glue scaffold. In the control sites, only a scaffold was grafted, without SKPs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: Homogeneously shaped skin-derived cells were isolated from porcine ear skin after 3 or 4 weeks of primary culture. In vitro osteogenic differentiation of SKPs was observed after co-culturing with a DMB and fibrin glue scaffold in a non-osteogenic medium. Von Kossa-positive bone minerals were also noted in the co-cultured medium at 4 weeks. As the culture time progressed, the number of observable cells increased. Trabecular new bone formation and osteocalcin expression were more pronounced in the SKP-grafted group compared to the control group. Conclusion: These findings suggest that autogenous SKP grafting with a DMB and fibrin glue scaffold can serve as a useful alternative to bone grafting technique.

• Archives of Plastic Surgery
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• v.34 no.2
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• pp.141-148
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• 2007

Purpose: The object of this study was to evaluate the development of continuous osteogenic differentiation and bone formation after the subcutaneous implantation of the tissue-engineered bone, in vitro. Methods: Human adipose-derived stem cells were obtained by proteolytic digestion of liposuction aspirates. Adipose-derived stem cells were seeded in PLGA scaffolds after being labeled with PKH26 and cultured in osteogenic differentiation media for 1 month. The PLGA scaffolds with osteogenic stimulated adipose-derived stem cells were implanted in subcutaneous layer of four nude mice. Osteogenesis was assessed by RT-PCR for mRNA of osteopontin and bone sialoprotein(BSP), and immunohistochemistry for osteocalcin, and von Kossa staining for calcification of extracellular matrix at 1 and 2 months. Results: Implanted PLGA scaffold with adipose-derived stem cells were well vascularized, and PLGA scaffolds degraded and were substituted by host tissues. The mRNA of osteopontin and BSP was detected by RT-PCR in both osteogenic stimulation group and also osteocalcin was detected by immunohistochemistry at osteogenic stimulation 1 and 2 months, but no calcified extracellular deposit in von Kossa stain was found in all groups. Conclusion: In vivo, it could also maintain the characteristics of osteogenic differentiation that adipose-derived stem cells within PLGA scaffold after stimulation of osteogenic differentiation in vitro, but there were not normal bone formation in subcutaneous area. Another important factor to consider is in vivo, heterologous environment would have negative effect on bone formation as.[p1]

• Proceedings of the Korean Society for Agricultural Machinery Conference
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• 2017.04a
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• pp.109-109
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• 2017

Cirsium setidens nakai belonging to cirsium has been reported to have various physiological activities including anticancer activity because it contains polyphenols, dietary fiber, minerals and vitamins. Despite these positive positive efficacies, however, no studies have studied cirsium setidens nakai products as biomaterials such as cellular metabolism and bone formation. Thus, the aim of this study was evaluate of osteogenesis differentiation a natural material extracted from cirsium setidens nakai. The natural materials in this studys in this studywere created by 40% ethanol extraction process and then dried. FabricatedFabricatedpowders were added to a medium at various concentrations (0.01, 0.05, 0.1, 0.2, and $0.25{\mu}g/mL$), and pure medium was used as a control. The natural material caused positive increases in cell metabolic activity and mineralized bone formation without cytotoxicity. In addition, we observed higher expression of genes such as ALP, BSP, Runx2 and COL1 in cirsium setidens nakai treatment cells. As a result, this study produced and investigated cirsium setidens nakai extracts and the natural materials showed potential biomaterials. In this research indicated that the cirsium setidens nakai extracts might have promising applications in areas of agricultural, biological and food engineering as a biomaterial.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.6
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• pp.474-484
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• 2007

Purpose: The effect of platelet-rich plasma(PRP) on osteogenesis of marrow-derived osteoblasts on histomorphometric analysis in the mandible of rabbit was assessed. Materials and Method: Bone marrow cells were obtained from iliac bone of rabbits and were cultured in a Dulbecco's Modified Eagle's Medium(DMEM) with Dexamethasone, L-Ascortic acid, ${\beta}$-Glycerophosphate to proliferate and differentiate into osteoblasts for $4{\sim}5$ weeks. The expression of osteogenic mar-kers was detected by reverse transcription-polymerase chain reaction(RT-PCR) and silver nitrate stain. Then we prepared bony defects in the mandible of rabbit, 10.0mm in diameter and 4.0mm deep, by trephine bur. In the control group, the defects were filled with autogenous bone and cultured osteoblasts. In the experimental group, the defects were filled with autogenous bone, cultured osteoblasts and PRP. 2 weeks, 4 weeks, 8 weeks later, each group was evaluated with histological and histomorphometric analyses. Results: In vitro, osteoblasts were identified on RT-PCR and silver nitrate stain. According to histological observation, at 2 weeks well-developed anasto-mosing newly-formed woven bone was observed, at 4 weeks anastomosing newly-formed woven bone having osteoblastic activation was observed, and at 8 weeks thick newly-formed woven bone was observed in both control and experimental groups. According to histomorphometric analysis, there were 1.5% more newly-formed bone volume in experimental group than control group at 2 weeks, 28.4% more at 4 weeks, 4.3% more at 8 weeks. Particularly there were significant differences in bone volume at 4 weeks and 8 weeks new bone. Conclusion: Our results demonstrated PRP may enhance osteogenesis of marrow-derived osteoblasts at 4 weeks, 8 weeks.

• Journal of Korean Medicine Rehabilitation
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• v.30 no.1
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• pp.13-29
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• 2020

Objectives This study was performed to decide the bone union effect of Hwaweo-jeon on tibia fractured mice. Methods In this study, laboratory experiments were implemented by the stage of in vitro and in vivo. In in vitro, MC3T3-E1 cells were treated with various concentration of Hwaweo-jeon extract (HWJ). To investigate effect of HWJ for osteoblast, relative mRNA expression of 5 substances (alkaline phosphatase [ALP], runt-related transcription factor 2 [Runx2], osteocalcin [OCN], osterix [OSX] and collagen type II alpha 1 chain [Col2a1]) was used as a marker of osteogenesis. In order to determine HWJ's effect for fracture healing, relative gene expression level of ALP, Runx2, OCN, OSX and Col2a1 were used to find out the influence to osteoblast. Furthermore, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin relative mRNA expression were used to estimate the impact to osteoclast. Also, X-ray was used for the purpose of identifying bone union in tibia-fracture mouse model. Results In in vitro experiment, most part of relative mRNA expression were increased compared to control group. In in vivo and in vitro experiment, HWJ induced osteoblast activitation by verifying relative mRNA expression of 5 substances. And in vivo experiment, we can also identify that HWJ triggered osteoclast activation during early stage of tibia fracture. Furthermore, X-ray pictures show noticeable recovery of tibia fracture. Conclusions HWJ extract promotes bone union by facilitating the osteoblast. But, HWJ may occur liver & kidney toxicity over specific concentration. Therefore, when HWJ is applied to human body, doctors have to follow up the liver function test & renal function test of patient.

• Molecules and Cells
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• v.41 no.5
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• pp.476-485
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• 2018

Although tectorigenin (TG), a major compound in the rhizome of Belamcanda chinensis, is conventionally used for the treatment of inflammatory diseases, its effects on osteogenesis and osteoclastogenesis have not been reported. The objective of this study was to investigate the effects and possible underlying mechanism of TG on in vitro osteoblastic differentiation and in vivo bone formation, as well as in vitro osteoclast differentiation and in vivo bone resorption. TG promoted the osteogenic differentiation of primary osteoblasts and periodontal ligament cells. Moreover, TG upregulated the expression of the BMP2, BMP4, and Smad-4 genes, and enhanced the expression of Runx2 and Osterix. In vivo studies involving mouse calvarial bone defects with ${\mu}CT$ and histologic analysis revealed that TG significantly increased new bone formation. Furthermore, TG treatment inhibited osteoclast differentiation and the mRNA levels of osteoclast markers. In vivo studies of mice demonstrated that TG caused the marked attenuation of bone resorption. These results collectively demonstrated that TG stimulated osteogenic differentiation in vitro, increased in vivo bone regeneration, inhibited osteoclast differentiation in vitro, and suppressed inflammatory bone loss in vivo. These novel findings suggest that TG may be useful for bone regeneration and treatment of bone diseases.

• Journal of Periodontal and Implant Science
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• v.50 no.6
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• pp.392-405
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• 2020

Purpose: Titanium implants are widely used in the treatment of dentition defects; however, due to problems such as osseointegration failure, peri-implant bone resorption, and periimplant inflammation, their application is subject to certain restrictions. The surface modification of titanium implants can improve the implant success rate and meet the needs of clinical applications. The goal of this study was to evaluate the effect of the use of porous titanium with a chitosan/hydroxyapatite coating on osseointegration. Methods: Titanium implants with a dense core and a porous outer structure were prepared using a computer-aided design model and selective laser sintering technology, with a fabricated chitosan/hydroxyapatite composite coating on their surfaces. In vivo and in vitro experiments were used to assess osteogenesis. Results: The quasi-elastic gradient and compressive strength of porous titanium implants were observed to decrease as the porosity increased. The in vitro experiments demonstrated that, the porous titanium implants had no biological toxicity; additionally, the porous structure was shown to be superior to dense titanium with regard to facilitating the adhesion and proliferation of osteoblast-like MC3T3-E1 cells. The in vivo experimental results also showed that the porous structure was beneficial, as bone tissue could grow into the pores, thereby exhibiting good osseointegration. Conclusions: Porous titanium with a chitosan/hydroxyapatite coating promoted MC3T3-E1 cell proliferation and differentiation, and also improved osseointegration in vitro. This study has meaningful implications for research into ways of improving the surface structures of implants and promoting implant osseointegration.