• Journal of Acupuncture Research
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• 제26권5호
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• pp.65-75
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• 2009

Objectives : Pyrite is one of the important prescriptions that has been used in oriental medicine for healing of fracture. It is reasonable, therefore, to postulate that native copper affects the process of bone metabolism and bone formation. The purpose of this study is to discover the effect of Pyrite on the healing of tibia fracture. Methods : 1. In vitro test : MG-63 cell in human body and the Pyritum in the ratio of 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml were incubated for 24 hours. After 24 hours, RNA was extracted via trizol reagent (Sigma, USA). In order to understand the activation of osteoblast, the level of OPN mRNA, osteopontin, was measured. 2. In vivo tesgroups normal group, control group and experimental group. Left tibia bones of mice in CON and JT groups were fractured by bone cutters. Pyrite was orally administered to the experimental group. After 14 days, each group's tibia specimen was constructed to observe changes in activation of proinflmmatory cytokines in relation to MIF and IL-6. Also, proliferation of osteoblast and osteopontin were measured via changes in levels of OPN and OPN mRNA. Results : In jn-Titro test, the level of OPN mRNA, osteopontin production was remarkably increased in Pyritum-treated MG-63 cells. In in-vitro test, fractured area in external tibia morphology was increased more in the JT group than that of the CON group. Osteogenesis, endochodrial ossification, and osteoid in fractured area were also increased more in the JT group than that of the CON group. Increase in OPN mRNA, osteopontin level and osteoblast's proliferation were observed. Activation of MIF and IL-6 was confirmed from the fracture region. Conclusions : From the result, development of a new stimulator in healing fracture via pyrite is expected.

• 대한치과보철학회지
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• 제43권6호
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• pp.751-763
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• 2005

Statement of problem. To improve a direct implant fixation to the bone, various strategies have been developed focusing on the surface of materials. The surface quality of the implant depends on the chemical, physical, mechanical and topographical properties of the surface. The different properties will interact with each other and a change in thickness of the oxide layer may also result in a change in surface energy, the surface topography and surface, chemical composition. However, there is limited the comprehensive study with regard to changed surface and biologic behavior of osteoblast by anodization. Purpose of study. The aim of this study was to analyze the characteristics of an oxide layer formed and to evaluate the cellular biologic behaviors on titanium by anodic oxidation (anodization) by cellular proliferation, differentiation, ECM formation and gene expression. And the phospholipase activity was measured on the anodized surface as preliminary study to understand how surface properties of Ti implant are transduced into downstream cellular events. Methods and Materials. The surface of a commercially pure titanium(Grade 2) was modified by anodic oxidation. The group 1 samples had a machined surface and other three experimental specimens were anodized under a constant voltage of 270 V(Group 2), 350 V(Group 3), and 450 V(Group 4). The specimen characteristics were inspected using the following five categories; the surface morphology, the surface roughness, the thickness of oxide layer, the crystallinity, and the chemical composition of the oxide layer. Cell numbers were taken as a marker for cell proliferation. While the expression of alkaline phosphatase and Runx2 (Cbfa1) was used as early differentiation marker for osteoblast. The type I collagen production was determined, which constitutes the main structural protein of the extracellular matrix. Phospholipase $A_2$ and D activity were detected. Results. (1) The anodized titanium had a porous oxide layer, and there was increase in both the size and number of pores with increasing anodizing voltage. (2) With increasing voltage, the surface roughness and thickness of the oxide film increased significantly (p<0.01), the $TiO_2$phase changed from anatase to rutile. During the anodic oxidization, Ca and P ions were more incorporated into the oxide layer. (3) The in vitro cell responses of the specimen were also dependant on the oxidation conditions. With increasing voltage, the ALP activity, type I collagen production, and Cbfa 1 gene expression increased significantly (p<0.01), while the cell proliferation decreased. (4) In preliminary study on the relation of surface property and phospholipase, PLD activity was increased but $PLA_2$ activity did not changed according to applied voltage. Conclusion. The anodized titanium shows improved surface characteristics than the machined titanium. The surface properties acquired by anodization appear to give rise more mature osteoblast characteristics and might result in increased bone growth, and contribute to the achievement of a tight fixation. The precise mechanism of surface property signaling is not known, may be related to phospholipase D.

• 한국식품영양과학회지
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• 제45권5호
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• pp.664-670
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• 2016

Polyglutamic acid(PGA) 함유량이 증대된 청국장이 조골세포에 미치는 영향을 세포 수준에서 관찰하고자 하였다. 조골세포에 미치는 영향을 관찰하기 위하여 세포 생존율, 염기성 인산분해효소(alkaline phosphatase, ALP) 활성, 골 석회화 형성능, 조골세포 분화 관련 유전자 발현능을 측정하였다. 세포 독성이 없는 농도에서 염기성 인산분해효소 활성을 측정한 결과 농도에 의존적으로 ALP 활성이 증가하였으며, 일반 청국장 A보다 PGA 함유량이 증가한 청국장 B가 더 높은 ALP 활성 증가 효과를 나타내었다. 청국장의 골 석회화 형성능을 측정한 결과 역시 청국장 A보다 B가 더 높은 석회화 형성능을 나타내었다. 또한 분화 관련 유전자인 OCN, OPN, Col1, ALP 역시 청국장 B가 A보다 유전자 발현이 더 높게 나타났다. 이러한 실험 결과를 바탕으로 PGA가 증대된 청국장이 기존 청국장보다 조골세포의 활성에 효과적일 것으로 생각한다.

• Archives of Plastic Surgery
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• 제36권3호
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• pp.247-253
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• 2009

Purpose: Hydroxyapatite(HA) has been widely used due to its chemical similarity to bone and good biocompatibility. HA is composed of macropores and micropores. Too much irregularities of the micropores are ineffective against the adhesion and proliferation of osteoblast. Many efforts have been tried to overcome these drawbacks. HA crystal coating on the irregular surface of HA scaffold, crystallized HA, is one of the method to improve cell adhesion. Meanwhile, the collagen has been incorporated with HA to create composite scaffold that chemically resembles the natural extracellular matrix components of bone. The authors proposed to examine the effect of collagen - coated crystallized HA on the adhesion and proliferation of osteoblast. Method: HA powder containing $10{\mu}m$ pore size was manufactured as 1 cm pellet size. For the making crystallized HA, 0.1 M EDTA solution was used to dissolve HA powder and heated $100^{\circ}C$ for 48 hours. Next, the crystallized HA pellets were coated with collagen (0.1, 0.5, and 1%). The osteoblasts were seeded into HA pellets and incubated for the various times (1, 5, and 9 days). After the indicating days, methylthiazol tetrazolium (MTT) assay was performed for cell proliferation and alkaline phosphatase (ALP) activty was measured for bone formation. Result: In SEM study, the surface of crystallized HA pellet was more regular than HA pellet. MTT assay showed that the proliferation of osteoblasts increased in a collagen dose - dependent and time - dependent manner and had a maximum effect at 1% collagen concentration. ALP activity also increased in a collagen dose - dependent manner and had a highest effect at 1% collagen concentration. Conclusion: These data showed that crystallization and collagen coating of HA was effective for osteoblast proliferation and ALP activity. Therefore, our results suggest that crystallized - HA scaffold with collagen coating is may be a good strategy for tissue engineering application for bone formation.

• Archives of Plastic Surgery
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• 제32권2호
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• pp.143-148
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• 2005

Chemotactic migration of bone forming cell, osteoblast, is an important event during bone formation, bone remodeling, and fracture healing. Migration of cells is mediated by adhesion receptors, such as integrins, that link the cell to extracellular matrix ligands, type I collagen, fibronectin, laminin and depend on interaction between integrin and extracellular ligand. Our study was designed to investigate the effect of extracellular matrix like fibronectin, laminin, type I collagen on migration of osteoblast. Migration distance and speed of MC3T3-E1 cell on extracellular matrix-coated glass were measured for 24 hours using 0.01% type I collagen, 0.01% fibronectin, 100 microliter/ml laminin. The migration distance and speed of MC3T3-E1 cell was compared using a video-microscopy system. To determine migration speed, cells were viewed with a 4 phase- contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. The migration distance on 0.01% type I collagen or 0.01% fibronectin was longer than that on $100{\mu}l/ml$ laminin-coated glass. The migration speed on fibronectin-coated glass was 68 micrometer/hour which was fastest. The migration speed on type I collagen-coated glass was similar with that on fibronectin-coated glass. The latter two migration speeds were faster than that on no-coated glass. On the other hand, the average migration speed on laminin-coated glass was 37micrometer/hour and not different from that of control group. In conclusion, the extracelluar matrix ligands such as type I collagen and fibronectin seem to play an important role in cell migration. The type I collagen or fibronectin coated scaffold is more effective for migration of osteoblast in tissue engineering process.

• 한국한의학연구원논문집
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• 제6권1호
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• pp.123-132
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• 2000

Bone is continuously remodeled during adult life with the resorption of old bone by osteoclasts and its subsequent replacement by osteoblast. Bone homeostasis is maintained by a balance between activities of osteoblasts and osteoclasts, but an imbalance between resorption and formation results in bone diseases including osteoporosis. Osteoblasts line up on the bone surface, especially regions of new bone formation, lay down bone matrix (osteoid) in orderly lamellae and induce its mineralization. Thus, the increased activity of osteoblasts is helpful to treat and prevent osteoporosis. In this study, we examined whether 80% EtOH extract of yukmijiwhang-whan is capable of affecting osteoblast proliferation using human osteoblast-like cell line, MG-63 and Saos-2. In an in vivo experiment, extract of yukmijiwhang-whan was administered for 9 weeks to ovariectomized (OVX) rats. At necropsy, uterus weights were measured, and trabecular bone areas (TBAS) of tibia and the sixth lumbar vertebra were measured by bone histomorphology. The maximum cell proliferation of MG-63 caused by extract of yukmijiwhang-whan at $5\;{\times}\;10^{-6}\;mg/ml$ was approximately 115% compared with control. In Saos-2, cell proliferation was approximately 145% of control at $5\;{\times}\;10^{-4}\;mg/ml$ and maximum alkaline phosphatase (ALP) activity was approximately 143% of control at $5\;{\times}\;10^{-5}\;mg/ml$. In animal study, however, the tibia and lumbar TBAS of the yukmijiwhang-whan group did not increased than the OVX control group. In conclusion, the 80% EtOH extract of yukmijiwhang-whan increased proliferation of osteoblasts but did not prevent bone loss in OVX rats.

• 폴리머
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• 제36권3호
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• pp.357-363
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• 2012

Poly(L-lactic acid)(PLLA) 고분자 필름 및 지지체의 세포 친화성을 향상시키기 위하여 산소 플라즈마 처리후 카복실기를 함유한 아크릴산(AA)을 $in$ $situ$ 그래프트시켰다. Stimulated body fluid(SBF) 용액에 15일간 담지시킨 후 hydroxyapatite(HA)를 형성시킨 시료와 phosphate-buffered saline(PBS), fetal bovine serum(FBS), 식염수 및 세포 배양용 배지에 담지시킨 다음 PLLA 시료 표면의 접촉각을 비교해 본 결과, HA 표면이 가장 낮은 접촉각을 나타내었다. 또한 연골세포와 조골세포는 HA 표면 위에서 높은 점착과 성장을 보였으며 연골세포가 HA에 많은 영향을 받는 것으로 확인되었다. 조골세포의 경우 HA 표면 이외에도 FBS나 세포 배양배지에 담지된 표면에서도 높은 세포 증식을 보였다. 더욱이 필름형태보다는 3차원 입체 구조의 다공성 지지체에서 연골세포와 조골세포의 점착과 세포 증식이 향상됨도 확인할 수 있었다. 이러한 표면개질된 PLLA는 조직공학적으로 연골이나 뼈 재생을 위한 유-무기 하이브리드 지지체로 응용될 수 있을 것으로 기대된다.

• International Journal of Oral Biology
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• 제33권3호
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• pp.105-111
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• 2008

Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권1호
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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.

• 생명과학회지
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• 제30권5호
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• pp.468-475
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• 2020

본 연구에서는 홍국색소의 항산화 활성 및 조골세포의 분화에 미치는 영향을 검토하였다. 홍국색소의 항산화 활성은 DPPH radical 소거능, ABTS radical 소거능 및 SOD 유사 활성을 측정하여 평가하였다. 홍국색소의 DPPH radical 및 ABTS radical 소거 활성은 농도 의존적으로 증가하였으며, 특히 1,000 ㎍/ml 농도에서 각각 94% 및 99%의 최대 활성을 나타내었다. 또한 SOD 유사 활성을 검토한 결과에서, 홍국색소 1,000 ㎍/ml 농도에서 62%의 최대 활성을 나타내었다. 우선 홍국색소 1~1,000 ㎍/ml의 농도에서 세포독성을 검토하기 위해 MTT assay를 실시한 결과, 1~100 ㎍/ml 농도의 범위에서 세포 독성은 나타나지 않았다. ALP 활성은 1~100 ㎍/ml 농도에서 농도의 존적으로 증가하였으며, 100 ㎍/ml의 농도에서 최대 124%의 활성을 나타내었다. 또한, 석회화 형성능 시험에서도 대조군 대비 모든 농도에서 유의적으로 증가하는 경향이 나타났으며, 100 ㎍/ml의 농도에서 대조군 대비 1.5배의 석회화 형성 결과가 나타났다. 이러한 결과를 통해 홍국색소는 항산화 활성이 우수하며, 조골세포 분화에 긍정적인 영향을 줌으로써 골다공증 예방 소재로서의 개발 가능성을 가진 부가가치가 높은 천연물 소재로 활용 가능할 것으로 생각된다.