• 한국수정란이식학회지
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• 제28권3호
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Journal of Periodontal and Implant Science
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• 제23권3호
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• pp.535-563
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• 1993

치주질환 진행의 최종적인 결과는 하반 지지조직의 소실로 치아상실을 초래하는 것이다. 이러한 치주질환의 이상적인 치유형태는 부착상실 예방을 비롯 상실된 조직의 재상 즉 신생 치조골과 백악질이 형성되고 두 조직사이에 치주인대가 재형성되는 것이다. 최근까지의 치주조직재생을 위한 처치법으로 이환된 병소부의 단순제거, 치관변위판막술, 약제의 치근면처리법, 조직유도재생술, 골전도물질 삽입, 골유도 혹 골형성물질 사용등 다양한 방법이 제시되었으나 아직 이상적인 치료법은 밝혀지지 않고 있다. 치아지지의 가장 중요한 역할을 하는 치조골의 재생에 대해 많은 연구들과 아울러 조직화학적 연구에서 Polypeptide Growth Factor(PGF) 가 다양한 종류의 세포증식과 이주 및 기질합성에 촉진효과가 있다고 하여 조직재생에 사용 가능성이 보고된 바 있었다. 이 PGF중 혈소판유래성장인자가 섬유아세포와 골아세포의 유사분열 및 단백질합성에 촉진작용이 있으며 조직재생에 영향을 미친다는 보고도 있었다. 본 연구의 목적은 총 8 마리의 실험동물을 이용하여 치근이개부병변을 형성한 후 혈소판유래성장인자를 처리하고 기존 조직결손부에 사용하였던 Tricalcium Phosphate(TCP) 와 콜라젠을 성장인자 함유매개체로 하여 이개부병변에 병용삽입한 경우 치유과정에 미치는 효과를 규명하여 임상적용의 가능성을 규명하고 실제 임상적용방법을 개발할 목적으로 실시하였다. 실험동물 Pentobarbital Sodium으로 전신마취를 시킨후, 초음파치석제거기등을 이용하여 구강위생을 청결하게 한 다음, 치은열구절개를 이용하여 전층판막을 형성하였다. 피질골과 치조골을 삭제하고 형성된 이개부 결손부에 치근면활택술을 시행하였으며, 구연산으로 치면처리 후, 계획된 재료를 삽입하고, 치관변위판막술과 유사한 형태의 봉합을 시행하였다. 실험동물을 2, 4, 8, 12 주에 관류고정과 아울러 회생시켜 악골을 채취하고 통법에 의해 후고정, 탈회, 탈수과정을 거쳐 파라핀으로 포매한 후 $7{\mu}m$두께로 절편하고 H & E 염색후 광학현미경으로 관찰하여 다음과 같은 결과를 얻었다. PDGF-BB 만 처리한 군에서는 2주 소견에서부터 결손부 전체에 걸쳐 활성도가 높은 조골세포들이 균일하게 분포하면서 이들로부터 생성된 골양조직이 기초적인 골소주의 형태를 이루고 있음이 관찰되었으며 그후 매우 빠른 골형성이 계속되어 8주 소견에서는 결손부 정상에 이르기까지 성숙된 치밀골이 채워져 있었다. 신생골형성의 전반적 형태는 치근면의 외형에 따라 치근이개부상단부로 형성되는 양상이었다. PDGF-BB 군에서 신생백악질의 형성은 2주소견 에서는 미약하였으나 4주이후 치근면에 수직으로 배열된 교원섬유들과 함께 균일한 두께로 형성되기 시작하여, 8주에 이르러서는 비 손상부위에서보다 더욱 두터운 신생백악질이 형성되었고 전형적인 샤피스씨 섬유가 완성되어 있음이 관찰되었다. PDGF-BB와 TCP를 병용한 경우 및 PDGF-BB와 콜라젠을 병용한 경우에서는 PDGF-BB군에 비해 신생골 및 신생백악질의 형성, 치주인대강의 발달이 상대적으로 미약하였으며 이러한 현상은 수술후 초기단계에서 더욱 두드러져 함유매개체로서의 기능을 기대하였던 이들 이식재들이 도리어 급속하게 분화 증식되는 세포들의 이동에 장애물로서 작용하는 것으로 나타났다. 결론적으로 PDGF-BB의 치근면 처리가 치근이 개부병변 치료에 있어 전반적으로 조직재생의 속도가 빠르고 그 치유양상도 시간경과에 따라 치주조직 고유형태로 진행됨이 관찰되어 동일 병소치료에 응용가능성을 확인하였다. 또한 차후 결손부에 주입방법과 성장인자의 관리법 및 적용량 그리고 적응중의 규명을 위해 연이은 연구가 있어야 할것으로 사료된다.

• 대한치과교정학회지
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• 제21권2호
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• pp.259-272
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• 1991

Orthodontic traction has been suggested as the treatment of choice for intrusive luxation injuries. Prior research has shown orthodontic forces to be ineffective in the presence of ankylosis or in cases with zero mobility following the injury. If orthodontic traction is to be effective, it must be initiated prior to the onset of ankylosis. The purpose of this study was to describe the effects of intrusive luxation at various times following the injury, and to determine the time of the onset of ankylosis, and to examine what effect immediate partial luxation has on the onset of ankylosis. Eight young mongrel dogs were utilized for this study. Intrusive luxation was produced with an axial impact using a gravity hammer and a specially designed holding device on 4 teeth (2 max. and 2 man. first premolars) in each dog. The teeth were intruded approximately 3-4mm in an axial direction. One maxillary and one mandibular premolars were partially luxated with the other two teeth being untouched. Pre and posttrauma tooth position was documented with plaster models and radiographs taken with an individualized X-ray jig. Dogs were sacrificed immediately following the injury and at 1, 2, 4, 7, 10, 14 and 21 days respectively. Tetracycline was administered as a vital bone marker 24 hours before sacrifice. Block sections of the tooth and alveolus were prepared for decalcified and non decalcified histologic sections. The effects of traumatic intrusion were analyzed by means of model casts, radiographs, tetracycline bone marking and histologic preparations. The results obtained were as follows: 1. The animal sacrificed immediately following the injury displayed alveolar fractures, torn periodontal ligaments, and areas of direct tooth-bone contact. 2. The odontoblastic layer of the pulp was disorganized as early as 24 hours after the injury. 3. Bony remodeling was noted at 4 days along with active surface resorption. 4. Ankylosis was first seen 7 days after the injury. 5. Osteogenesis in the dentin (thick tetracycline bands) was observed 7 days after the injury. 6. There was no progressive root resorption and ankylosis where the periodontal ligament has been healed. 7. The Luxated group showed significantly more root resolution and ankylosis than the Nonluxated group with increased observation periods. The results suggest that ankylosis may occur within the first week following the injury, and hence orthodontic traction should be initiated as soon after the injury as possible.

• Journal of Periodontal and Implant Science
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• 제29권1호
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• pp.209-231
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• 1999

Even though titanium(Ti) and its alloys are the most used dental implant materials, there are some problems that Ti wears easily and interferes normal osteogenesis due to the metal ions. Ti coated with bioactive ceramics such as hydroxyapatite has also such problems as the exfoliation or resorption of the coated layer, Recent studies on implant materials have been proceeding to improve physical properties of the implant substrate and biocompatibility of the implant surfaces. The purpose of the present study was to examine the physical property and bone tissue compatibility of bioinert nitrides ion plated Ti, Button type specimens(14mm in diameter, 2.32rrun in height) for the abrasion test and cytotoxicity test and thread type implants(3.75mm in diameter, 6mm in length) for the animal experiments were made from Ti(grade 2) and 316LVM stainless steel. Ti specimens were ion plated with TiN, ZrN by the low temperature arc vapor deposition, and the depth profile of the TiN/Ti, ZrN/Ti ion plated surface was examined by Auger Electron Spectroscopy. Three kind of button type specimens .of TiN/Ti, ZrN/Ti and Ti were used for abrasion test, and HEPAlClC7 cells and CCD cells were cultivated for 4 days with the specimens for cytotoxicity test. Thread type implants of TiN/Ti, ZrN/Ti, Ti, 316LVM were implanted on the femur of 6 adult dogs weighing 10kg-13kg. Two dogs were sacrified for histological examination after 45 days and 90 days, and four dogs were sacrified for the removal torque test of the implant') after 90 days. The removal torque force was measured by Autograph (Shimadzu Co., AGS-1000D series, Japan). Abrasion resistance of TiN/Ti was the highest, and that of ZrN/Ti and Ti were followed. The bioinert nitride ion plated Ti had much better abrasion resistance, compared with Ti, In the cytotoxicity test, the number of both cells were increased in all specimens, and there were no significant difference in cytotoxic reaction among all groups (p>0.1), In histological examination, 316LVM showed the soft tissue engagement in interface between the implant and bone, but the other materials after 45 days noted immature new bone formation in the medullary portion along the implant surface, and those after 90 days showed implant support by new bone formation in both the cortical and the medullary portion, The removal torque force of Tilv/Ti showed significantly higher than that of Ti(p(O,05). The difference in removal torque force between TiN/Ti and ZrN/Ti was not significant(p>0.05), and that of 316LVM was lowest among all groups(p<0.05). These results suggest that bioinert nitrides ion plated Ti can resolve the existing problems of Ti and bioactive ceramics, and it may be clinically applicable to human.

• 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.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제20권2호
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• pp.139-147
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• 1998

동종 이식골 내의 탈회 정도에 따른 잔존 칼슘치가 이소성 골형성능에 미치는 영향을 파악하고자 Sprague-dowley계 백서에서 채취한 경골 및 대퇴골의 골간부를 0.5cm 크기로 절단하여 부착 연조직은 제거한 다음, 초음파 세척장치를 이용하여 $60^{\circ}C$ 0,6N HCl용액으로 5분, 10분, 15분, 20분, 25분, 30분, 35분, 40분간 각각 처리하여 탈회 동종 이식골을 준비하였다. 이때 탈회시간에 따른 시편의 무게를 측정하였고, 각 탈회용액으로부터 1cc를 취한 다음 Sigma사의 진단용 칼슘치 측정kit를 이용하여 spectrophotometer로 600nm 파장하에서 칼슘치를 측정하였다. 그리고 탈회 정도에 따른 이식골편의 골형성 유도능을 확인하기 위하여 24마리의 Sprague-dowley계 백서를 탈회시간별로 8군으로 나누어 배부에 0.5cm 크기로 4군데의 피하낭을 형성한후 각각의 처리된 탈회 이식골편을 이식하였다. 매식된 동종 이식골편들을 술후 1, 2, 3주째 채취하여 통법에 따라 H&E염색 표본을 제작하여 광학현미경으로 관찰하였다. 이상의 실험에서 다음과 같은 결과를 얻었다. 1. 탈회 30분까지의 용출되는 칼슘 농도 변화는 평균 15.91mg/ml로, 탈회 20분 이후에 평균 99.65%의 탈회정도를 보였다. 2. 동종골 무게 변화량은 탈회시작 25분까지 뚜렷한 무게변화를 보였으나, 그 이후에는 변화의 정도가 미약하였다. 3. 탈회된 이식 통종골에의한 이소성 골형성 유도능 비교에서 20분에서 30분간 탈회된 군에서 가장 양호하였고 그 외의 군에서는 그 정도가 열등하였다. 이상의 결과는 탈회된 동종골에 있어 골형성 유도능을 극대화하기 위해서는 골기질내 무기성분의 완전한 탈회가 선행되어야 하며, 탈회골 기질의 변성이 최소화되어야 함을 시사한다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제20권2호
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• pp.112-126
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• 1998

In dentistry, bony defects can be formed by cyst, tumor, inflammation, trauma and surgery in maxilla and mandible. If the overlying soft tissue invades and preoccupies the jaw bony defects, regenerated bony tissue same as adjacent bone can not replace whole space of the defects, thus preventing osteogenesis from occurring. Guided bone regeneration(GBR) is based on the prevention of overlying soft tissue from entering the bony defect during the initial healing periods. E-polytetrafluoroethylene(e-PTFE) is one of an effective and widely used barrier membrane for GBR, but it has the disadvantages such as surgical removal and high price. To overcome such disadvantages of e-PTFE, many investigators have proposed various absorbable barrier membranes. Inexpensive oxidized cellulose($Surgicel^{(R)}$) membrane was shown to have potential for use as an absorbable barrier membrane for regenerative procedure and it would not require surgical removal. The purpose of this study is to investigate the absorption periods of oxidized cellulose at the implant site and usefulness as a mechanical barrier, preventing the ingrowth of the overlying soft tissue into the bony defects. Two bony defects were made in each tibia of a dog using drill and one defect covered with oxidized cellulose and the other covered with periosteum directly as control. The experimental animals were sacrificed at 1st-7th, 10th, 14th, 21th, 28th day postoperatively, Inspection of the specimens was done to evaluate gross changes. Specimens were examined histopathologically by hematoxylin-eosin and Masson's trichrome staining under light microscope. The results were as follows : 1. There was no significant differences of inflammatory reaction between the experimental and the control group. 2. The resorption of oxidized cellulose was almost completed within 14th day. 3. Histologically, bone formation in the experimental group was somewhat more than that of the control group at 10th, 14th, 21th and 28th day postoperatively. The bone forming pattern of the experimental group was more regular than that of the control group. 4. There was no evidence of soft tissue invasion into the bony defect in the experimental group. In conclusion, oxidized cellulose membrane might be used as an alternative absorbable barrier membrane to prevent overlying soft tissue invasion into the bony defects.

• Journal of Periodontal and Implant Science
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• 제33권4호
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• pp.585-597
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• 2003

Porphyromonas gingivalis has been implicated as an important periodontophathic bacterium in the etiology and progression of periodontal diseases. It has been reported that P.gingivalis may mediate periodontal destruction not only directly through its virulence factors, but also indirectly by including complex host mediated inflammatory reponses. The purpose of this study was t o evaluate the effects of P.gingivalis on the bone formation and resorption by osteoblasts. For this purpose, after determining the concentration below which sonicated P.gingivalis extracts (SPEs) have no cytotoxicity on mouse calvarial primary osteoblastic (POB) cells, we investigated the effects of SPEs on the alkaline phosphatase (ALP) activity, matrix metalloproteinase (MMP) expression (MMP-2, -9, 13), and prostaglandin $E_2$ ($PGE_2$) release in POB cells by treatment with SPEs below that concentration. The results were as follows; 1. SPEs showed no cytotoxic effect on POB cells up to a concentration of 1 ${\mu}m$/ml. 2. The treatment with SPEs reduced ALP activity in a dose-dependent manner in POB cells, In addition, when we investigated the effect of SPEs (1 ${\mu}m$/ml) on ALP activity for different exposure periods, statistically significant inhibition of ALP activity was shown at 2 days of exposure, and further significant inhibition occurred by extending the periods of exposure. 3. The treatment with SPEs stimulated the gene expression of MMP-9 in POB cells. 4. The pre-treatment with SPEs increased the amount of $PGE_2$ released in POB cells. In summary, the present study shows that P.gingivalis could inhibit osteogenesis and stimulate bone resorption not only by reducing ALP activity but also by increasing MMP-9 mRNA expression in osteoblasts, possibly through an endogenous $PGE_2$ pathway. In addition, our results suggest that if P.gingivalis affects osteoblasts in early differentiation stage, such effects by P. gingivalis could be irreversible.

• Journal of Periodontal and Implant Science
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• 제40권3호
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• pp.132-138
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• 2010

Purpose: The purpose of this study was to compare the bone regeneration effects of cortical, cancellous, and cortico-cancellous human bone substitutes on calvarial defects of rabbits. Methods: Four 8-mm diameter calvarial defects were created in each of nine New Zealand white rabbits. Freeze-dried cortical bone, freeze-dried cortico-cancellous bone, and demineralized bone matrix with freeze-dried cancellous bone were inserted into the defects, while the non-grafted defect was regarded as the control. After 4, 8, and 12 weeks of healing, the experimental animals were euthanized for specimen preparation. Micro-computed tomography (micro-CT) was performed to calculate the percent bone volume. After histological evaluation, histomorphometric analysis was performed to quantify new bone formation. Results: In micro-CT evaluation, freeze-dried cortico-cancellous human bone showed the highest percent bone volume value among the experimental groups at week 4. At week 8 and week 12, freeze-dried cortical human bone showed the highest percent bone volume value among the experimental groups. In histologic evaluation, at week 4, freeze-dried cortico-cancellous human bone showed more prominent osteoid tissue than any other group. New bone formation was increased in all of the experimental groups at week 8 and 12. Histomorphometric data showed that freeze-dried cortico-cancellous human bone showed a significantly higher new bone formation percentile value than any other experimental group at week 4. At week 8, freeze-dried cortical human bone showed the highest value, of which a significant difference existed between freeze-dried cortical human bone and demineralized bone matrix with freeze-dried cancellous human bone. At week 12, there were no significant differences among the experimental groups. Conclusions: Freeze-dried cortico-cancellous human bone showed swift new bone formation at the 4-week healing phase, whereas there was less difference in new bone formation among the experimental groups in the following healing phases.

• BMB Reports
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• 제45권9호
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• pp.509-514
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• 2012

We have found that the previously uncharacterized bone morphogenetic protein-9 (BMP9) is one of the most osteogenic factors. However, it is unclear if BMP9 cross-talks with $TGF{\beta}1$ during osteogenic differentiation. Using the recombinant BMP9 adenovirus, we find that low concentration of rh$TGF{\beta}1$ synergistically induces alkaline phosphatase activity in BMP9-transduced C3H10T1/2 cells and produces more pronounced matrix mineralization. However, higher concentrations of $TGF{\beta}1$ inhibit BMP9-induced osteogenic activity. Real-time PCR and Western blotting indicate that BMP9 in combination with low dose of $TGF{\beta}1$ potentiates the expression of later osteogenic markers osteopontin, osteocalcin and collagen type 1 (COL1a2), while higher concentrations of $TGF{\beta}1$ decrease the expression of osteopontin and osteocalcin but not COL1a2. Cell cycle analysis reveals that $TGF{\beta}1$ inhibits C3H10T1/2 proliferation in BMP9-induced osteogenesis and restricts the cells in $G_0/G_1$ phase. Our findings strongly suggest that $TGF{\beta}1$ may exert a biphasic effect on BMP9-induced osteogenic differentiation of mesenchymal stem cells.