Tissue engineering has been rapidly developed in oral and maxillofacial reconstruction. Biocompatible scaffold from chemically composites seeded with stem cells is essential and several growth factors for bone formation and angiogenesis are also required. To overcome limited activity of new bone formation with scaffolds, several biomechanical stimulation methods on cells have been made to grow cells in scaffold. Several bioreactors have been developed for real tissue growth in culture laboratory. In addition to biological stimulants like BMP, growth factors and exogenous drugs, biomechanical stimulation technique has also been known as an effective method in cell differentiation. We developed our own bioreactor with tensile mechanical strains. Then we tested with it for detection of suitable biomechanical effect on the cell differentiation and proliferation. And we also compared the results with the effect of low intensity pulsed ultrasound (LIPUS). Mechanical strain group showed more rapid reaction with cell differentiation and proliferation than non-mechanical strain group. Mechanical strain groups stimulated with 0.5∼0.7Hz for 6 hours and 8 hours showed more active cell differentiation than the group with 0.5∼0.7Hz for 2.5 hours tensile strain stimulation. Group of LIPUS also showed more rapid reaction in cell differentiation and proliferation. LIPUS with 3MHz showed more cell reaction than the LIPUS group with 1MHz. Our results showed the positive effect on differentiation and proliferation of cell with mechanical tensile strain, LIPUS both.

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

The attachment and adhesion of RAW 264.7 and MC3T3-E1 cells to titanium (Ti) discs with various degrees of roughness was investigated. The attachment, adhesion, and proliferation of these cells were evaluated after 4 hr, 24 hr and 7 day incubations. Both RAW 264.7 and MC3T3-E1 cells showed a time-dependant correlation between attachment and adhesion on the surface of the titanium discs. Both types of cells tended to have higher survival rate on these discs as the surface roughness increased. The percentage of adherent inflammatory RAW 264.7 cells was greater than MC3T3-E1 cells at 24 hr, but this was reversed at 7 days in culture. The morphology of osteoblastic MC3T3-E1 cells at 24 hr, determined using a surface emission microscope (SEM), appeared flattened and spread out while inflammatory RAW 264.7 cells were predominantly spherical in shape. The adhesion of both cell types on the titanium discs was dependant on the levels of fibronectin adsorbed on the disc surface, indicating that serum constituents modulate the efficient adhesion of these cells. Our data indicate that the cellular response to the titanium surface is dependent on the types of cells, surface roughness and serum constituents.

We have investigated the effects of cottonseed extract on the proliferation, differentiation and lipopolysaccharide (LPS)-induced production of local factors in murine clonal osteoblastic MC3T3-E1 cells. Ethanol extract of cotton seed (4~63 μg/mL) significantly increased the proliferatin of MC3T3-E1 cells (p <O.05). Moreover, cottonseed extract (10~50 μg/mL) caused a significant elevation of alkaline phosphatase (ALP) activity and collagen content in the cells. Lipopolysaccharide (LPS) is a potent stimulator of bone resorption in inflarnmatory diseases. We examined the effect of cottonseed extract on the LPS-induced production of tumor necrosis factor a (TNF-a) and nitric oxide (NO) in MC3T3-El cells. Treatment with cottonseed extract (10~50 μg/mL) decreased ilie 5 μg/mL LPS-induced production of TNF-α and NO in osteoblasts， suggesting that the antiresorptive action of cottonseed extract may be mediated by decrease in these local factors. This study suggests that cottenseed may contribute to antiresorptive action against osteoblastic cells, resulting in a beneficial effect in promoting the function of osteoblastic cells.

Previous ly we have s hown that fï brob last• growth factor-2 (FGF-2) and dexamethasone (Dex) in combination strongly stimulate both p l 이 i fe rati o n a nd differe nt iation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes, In the present s tudy we invesL igaLed whether inhibition 01' FGF-2 and Dex-induced adipogenic differentiation of bone marrow derived s Lem cells (BMSCs) by GW9662, an antagoni s t of proxisome proliferators-activated receptol γ (PPARy) which plays a key role in ad ipogenic differentiation , enhances proliferation and osteoblastic differentiation of BMSCs Proliferation 01' BMSCs t reated wi 네 FGF-2 a nd Dex was further increased by GW9662 up to 9,7, 10,6, and 7,2% at 3, 5, and 7 days of cul Lu re , Expansion of BMSCs with FGF-2, Dex and GW9662 followed by osteoblastic different iation showed that osteoblas tic differentiation 01' BMSCs was in creased by 37 % (p=O, 01) compared to those expanded with FGF-2 and Dex, ln contrast , ad i pogenic di fferenti a tion of FGF-2 and Dex-expanded BMSCs was substantially reduced to 14% (p=O, 036) by GW9662, Taken toget her , these resul ts demonstrate that FGF-2 and Dex in combination with GW9662 f ur t her stimu late proliferation 01' BMSCs and those cells expanded with these factors acquire enhanced potentiaIs to be dif ferentiated i n to osteoblas ts

Biosynthetic processing of fibrillar procollagens is essential for producing mature collagen monomers that polymerize into fibrils by a self-assembly process. The metalloproteinase ADAMTS-2 is the major enzyme that processes the N-propeptide of type I procollagen in the skin and also of type II and type III procollagens. Mutations in the ADAMTS-2 gene cause dermatospraxis in animals and Ehlers-Danlos syndrome VIIC in humans, both of which are characterized by the accumulation of type I pN-collagen and the formation of abnormal collagen fibrils in the skin. Despite its importance in procollagen processing, little is known about the regulation of ADAMTS-2 expression. Here, we demonstrate that ADAMTS-2 can be regulated by 1,25-dihydroxyvitamin D3, an inducer of type I procollagen synthesis. This steroid hormone induced ADAMTS-2 mRNA ~3-fold in MG-63 human osteosarcoma cells and MC3T3-E1 murine osteoblastic cells. This induction was dose- and time-dependent in MG-63 cells. In contrast, secreted ADAMTS-2 protein was increased only 1.4-fold with 1,25-dihydroxyvitamin D3. Finally, 1,25-dihydroxyvitamin D3 in the presence of ascorbate increased levels of secreted ADAMTS-2 1.9-fold over ascorbate treatment alone, which did not appreciably change ADAMTS-2 expression. These data indicate that the regulation of ADAMTS-2 is coupled with the synthesis of type I procollagen through 1,25-dihydroxyvitamin D3 signaling and may involve translational or posttranslational control.

Cell adhesion is used as a parameter to evaluate the biocompatibility of dental implant and also affected by the surface form of dental implant. Most study have showed different cell reaction by the composition and the surface morphology of implant. Therefore it is thought that the osteoblastic activity would be affected by the surface roughness and composition of implants. This study was performed to evaluate the biological activity and morphological change of normal human osteoblastic cells(NHost) depending on the variations of implant surfaces. We used grade 2 titanium disks which were being air-blasted with TiO2 50 ㎛, 110 ㎛, 250 ㎛ powder by 3psi compressed air and non-blasted as control. We evaluated and compared morphologic change, adhesion assay, and Ca, P, ALP concentration of NHost in vitro. The obtained results were as follows. 1. In the growth curve, although the growth of experimental groups were lower than that of the group of NHost only, there was no significant difference between each groups. 2. Inverted microscopic findings showed NHosts in early stage of each group were adherant perpendicular to the titanium disk and the multilayered NHosts were attached with various directions after 4 weeks. 3. Scanning electron microscopic(SEM) features showed that NHosts in all groups seemed to be attached multilayered and connected with each process after 2 weeks. 4. NHosts' processes were found by the SEM after one day culture. The cell adhesion of experiment group was higher than that of control group. 110 ㎛(the 3rd group) showed prominant process of NHost on the titanium disk surface. 5. Although the concentrations of Ca, P and ALP were gradually reduced, ANOVA analysis of each groups were partially different, and ANOVA analysis of 4th group were significantly different with others. From the aboving results, NHosts cultured on the titanium disks showed similar morphological change and cell proliferation. There were partially differences in each group except the 4th group, and the 4th group were significantly different with other's in biological activity. We thought that biological activity and adhesion of NHost cell on titanium had been affected by the variation of the titanium surface roughness.

고령사회에서 노년기 건강의 큰 문제로 대두되고 있는 골다공증은 특히 폐경 후 여성들에게서 가장 그 발생빈도가 높게 나타났으며, 현재 골다공증 예방 및 치료에 사용되고 있는 약제는 대부분 골흡수 억제제로써 진행된 골소실을 회복 시킬 수는 없기 때문에 골형성 증가를 통한 골다공증 예방과 치료에 관한 연구가 활발히 이루어지고 있다. 산양삼(cultivated wild Panax ginseng, CWP)에 대한 연구는 다수가 원기회복, 자양강장 및 면역증강 효과 등에 대한 것이나 골대사에 미치는 영향에 대한 연구는 거의 없는 실정이다. 이에 본 연구에서는 산양삼 추출물이 조골세포에서 골관련 유전자 발현에 미치는 영향을 확인함으로써 골다공증 예방 및 치료 효과를 갖는 천연 소재로의 활용 가능성을 검토하고자 하였다. 산양삼 추출물 처리가 조골 세포 의 증식에 미치는 영향을 알아보기 위해 MTT assay를 실시하였고, MC3T3-E1 세포생존률은 FBS가 첨가되지 않은 배양액만 처리한 대조군과 산양삼 추출물을 처리한 실험군 모두에서 동일한 수준으로 나타났으며 이로써 산양삼 추출물의 안전성을 확인할 수 있었다. 또한 산양삼 추출물을 처리한 실험군과 대조군과의 세포증식률을 비교하였을 때 산양삼 추출물 50 ㎍/mL 농도 처리군에서 유의적으로 세포증식이 촉진되었으며 25 ㎍ /mL과 100 ㎍/mL 농도 처리군에서도 대조군보다 높은 경향을 나타내었다. 산양삼 추출물이 조골 세포의 활성에 미치는 영향을 알아보기 위해 조골세포의 분화초기 표지인자인 ALP 활성을 측정하였으며 그 결과 모든 산양삼 추출물 처리군이 대조군과 비교하여 유의적으로 높은 활성을 나타내었으며 특히 산양삼 추출물 50 ㎍/mL 농도 처리군에서 가장 높은 활성을 나타내었다. 산양삼 추출물의 농도에 따른 석회화 형성도를 확인하기 위해 무기질화된 세포의 기질을 alizarin red로 염색하였고 산양삼 추출물을 처리한 실험군과 대조군과의 석회화 형성도를 비교하였을 때 산양삼 추출물 50 ㎍/mL 농도 처리군에서 유의적으로 석회화 형성이 촉진되었으며 25 ㎍/mL과 100 ㎍/mL 농도 처리군에서도 대조군보다 높은 경향을 나타내었다. 산양삼 추출물이 MC3T3-E1 조골세포에서 골 형성 관련 유전자 발현에 미치는 영향을 확인하기 위해 Runx2, ALP, OPN, OCN 등의 유전자를 정량 real-time PCR을 통해 분석하였으며 대조군과 비교하여 모든 산양삼 추출물 처리군에서 농도 의존적이고 유의적으로 골 형성 관련 유전자발현이 증가되었다. 따라서 산양삼 추출물이 골 형성 관련 유전자인 Runx2, ALP, OPN, OCN 발현을 증가시켜 MC3T3-E1 조골세포의 분화를 촉진하고, 골 석회화 형성 촉진에 기여하였을 것으로 사료된다. 그러나 산양삼 추출물이 골형성과 관련하여 어떠한 기전으로 유전자의 발현을 조절하였는지에 대한 유전자 및 단백질 수준의 추가적인 연구와 산양삼 추출물의 분화 촉진과 석회화 형성능이 산양삼의 사포닌계 진세노사이드 성분의 영향인지에 대한 후속 연구가 필 요할 것으로 사료된다.

본 연구는 복분자 미숙과 추출물과 황기 뿌리 추출물로 이루어진 혼합물의 다양한 혼합비율을 이용하여 조골세포의 증식과 활성변화를 확인하고, ALP 활성 및 osteocalcin의 분리량 측정을 통하여 복분자 추출물과 황기추출물의 최적혼합비율을 결정한 것이다. 구체적으로는 각 혼합비율(복분자:황기=1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1)에 따른 혼합물을 MG-63 조골세포에 처리하여 세포의 증식과, 분화마커 ALP의 활성 및 osteocalcin 분비량을 측정함으로써 골다공증 치료에 유용한 복분자 추출물과 황기추출물의 혼합비율을 7:3으로 최적화하였다. 혼합비율 0:0에서 조골세포증식이, 혼합비율 72:28이 세포 내 ALP 활성 증가를, 68:32이 세포 내 osteocalcin의 분비가 가장 높게 나타났다. 이러한 결과는 7:3의 혼합물이 항골다공증 효과에 우수한 결과가 있을 가능성을 나타내며, 특히 조골세포의 활성화 촉진으로 인해 골파괴 진행을 억제하는 효과, 즉 병의 진행을 지연시키는 작용기전이 아니라 골을 다시 형성해줄 수 있는 긍정적 효능에 대한 가능성을 제공한다. 이에 본 연구결과를 바탕으로 본 연구결과에 의해서 제공되는 최적혼합비율의 혼합물에 대한 골다공증 예방 및 치료에 대한 임상실험과 그 작용기전에 대한 연구가 더욱 진행되어야 할 것으로 보인다.