模拟微重力环境对人牙周膜干细胞增殖分化的影响及其机制的研究
|
摘要
长期空间飞行可引起身体多个系统的改变如血液—心血管系统、内分泌系统、骨骼肌肉系统、自主平衡系统、免疫系统、神经系统等。由于实验耗资巨大,具备空间实验能力的人力资源缺乏和实验设备重量严格受限等因素使得空间载体科学实验非常有限,因此被美国航空航天局认可的旋转细胞培养系统(rotary cell culture system,RCCS),成为地面模拟微重力环境进行细胞或组织实验的主要仪器。RCCS中培养体系对细胞产生的机械剪切力小,充氧不引起涡流,有利于细胞营养的及时补充,加速代谢产物的尽快排除,从而改善离体细胞的培养条件,有利于细胞培养和组织生长。
成体干细胞是指从机体某些组织中分离出来的具有自我更新和多向分化能力的一类细胞,它分布于不同的组织,还可以通过血液循环到达躯体各处,参与组织的修复和再生。牙周膜干细胞是一种典型的牙源性间充质干细胞,可以从新鲜拔除牙齿的牙周膜组织中获得。尽管在体外普通培养环境下这种牙周膜干细胞呈现出一种较低的成骨分化潜能,但是在矿化诱导培养条件下,牙周膜干细胞可以表达成牙骨质/成骨特性标记物,如碱性磷酸酶、细胞外基质磷酸化糖蛋白、骨涎蛋白、骨钙素和转移生长因子β1;牙周膜干细胞植入免疫缺陷鼠体内能够生成类似牙周膜样结构。这些特性表明牙周膜干细胞作为一种新的成体干细胞,具有其特殊性和潜在的临床应用价值。
TGF-β超家族细胞因子经过其各自的信号转导引起不同的生物学效应,而信号转导的基本过程相似:通过TGF-β族配体——膜受体——Smads蛋白——转录因子——基因表达等步骤。Smads家族是TGF-β超家族成员下游的一种细胞内信号的特异性转导分子,可将信号直接从细胞膜转导入细胞核内,具有转录活化的作用。组织学和细胞学研究证实牙周膜的发育过程中,TGF-β及其受体在成骨细胞、成牙骨质细胞和成纤维细胞内均有较强的表达,提示TGF-β能够调节牙周膜组织的发生和成熟。
本课题旨在:探讨地面静止及模拟微重力环境中牙周膜干细胞形态,内部结构,增殖、分化等生物学特性有何种变化;明确smads信号转导分子在两种不同培养环境下对细胞生物学行为变化是否起到关键性的调控作用;探明微重力环境下牙周膜细胞的生物力学反应,及牙周膜细胞在微重力环境下生物力学响应的主要信号转导途径和关键信号分子,为维护航天员的口腔健康提供有效的防护措施,并且为临床研究牙周组织再生和牙齿松动等牙周疾病提供新的治疗思路。
所取得的主要结果如下:
1.牙周膜干细胞的分离、培养、鉴定及多向分化能力检测
通过组织块联合酶消化法获得了牙周膜细胞,利用单克隆培养法在间充质干细胞培养基内成功分离出牙周膜干细胞;采用成骨诱导、成脂诱导方式验证了牙周膜干细胞具备多向分化能力;采用免疫组织化学染色和流式细胞仪定量分析细胞群体中间充质干细胞标志物Stro-1的阳性表达率。
2.微重力环境下牙周膜干细胞的生物学特性分析
利用旋转生物反应器(RCCS)和Cytodex-3微载体成功对牙周膜干细胞接种并悬浮培养。对牙周膜干细胞在悬浮模拟微重力和普通重力环境的细胞生物状态进行了比较,主要观察了细胞形态学改变、增殖潜能变化、细胞成骨/成牙骨质向分化能力改变。结果提示模拟微重力环境能够加强细胞增殖能力,促进细胞成骨/成牙骨质向分化潜能,同时细胞出现不同于普通重力下的形态。
3. Smads信号转导分子在微重力引起牙周膜干细胞成骨/成牙骨质向分化中的作用机制研究。
TGF-β1可以促进牙周膜干细胞成骨/成牙骨质向分化;Smads信号在此过程中充当了重要角色;微重力引起的牙周膜干细胞成骨/成牙骨质向分化过程中,Smads信号分子担当了重要作用,可能通过TGF-β族配体——膜受体——Smads蛋白——转录因子——基因表达等步骤来促进细胞成骨/成牙骨质向分化过程。
综上所述,牙周膜干细胞在微重力环境下其生物学行为发生了特定的改变, Smads信号转导分子在此过程中发挥了重要作用。研究结果为空间牙周生理病理学提供了研究基础;同时为牙周组织工程研究提供了新的研究思路。
Introduction
The periodontal ligament functions as a cushion to mitigate the mechanical forces of mastication and maintains homeostasis such as remodeling of the adjacent bone. Periodontal ligament retains regenerative capacity to some degrees throughout adulthood, which is attributed to stem cells maintaining their proliferation and differentiation potential in the area. Previous studies have determined that periodontal ligament stem cells (PDLSCs) play a crucial role in regeneration of periodontal defects, contributing to the formation of new cementum, alveolar bone, and periodontal ligament. In addition, several in vitro findings showed that PDLSCs can differentiate into osteoblast-like cells when challenged with dexamethasone,β-glycerophosphate and ascorbic acid. Recently, tissue engineering based on PDLSCs to enhance periodontal regeneration has been the focus of periodontal research. In situ engineered constructs containing in vitro expanded autologous cells have been used to regenerate periodontal defects. While how to rapidly gain plenty of functional seeded cells in vitro is the key technique for bioengineering tissue. A theoretical way to achieve this goal would be to provide a“stimulatory’’environment for PDLSCs to expedite tissue engineering of periodontium.
With the acceleration of human space exploration, aerospace medicine has been gradually developed recently. Due to payload constraints, flight cost, spaceflight experiments are limited; several ground-based systems have been invented to simulate microgravity. Rotary cell culture system (RCCS), recommended by National Aeronautics and Space Administration (NASA) as an effective tool for analysis of cells characteristics in conditions similar to microgravity in space, is simultaneously a kind of three dimensional (3D) dynamic culture system. Its simulated microgravity (SMG) function is due to the alteration of the cell's perception of a continuously changing gravitational direction as a result of rotational culture conditions. At the same time, the rotational motion of this system prevents sedimentation, creating a suspension culture environment, and seems to be ideal for overcoming some drawbacks associated with static culturing systems. Several researches indicated that RCCS benefit certain type of cellular aggregation, subsequent intercellular adhesion, and gradual formation of 3-dimensional (3D) cell clumps. So we wonder that whether RCCS is an effective tool in vitro for expansion of functional stem cells in 3D form, which can facilitate periodontal tissue engineering.
Although, SMG is known to affect the biological behavior of several kinds of cells, the biology of different cells cultured in SMG is different; even a few conflicting findings have recently been reported with respect to the effects of SMG on definite cell types such as osteogenic cells. Up to now, no data exist illustrating the behavior of periodontal ligament stem cells under SMG. Thus, the objective of current study was to clarify the effects of three-dimensional dynamic simulated microgravity created by RCCS on hPDLSCs proliferation, osteoblastic differentiation and morphological changes. For it is the first time to investigate the effects of simulated microgravity on periodontal ligament stem cells, the research may lend insight into variations of cell response in 3D environment, and contribute to achievement of desirable periodontal regeneration utilizing PDLSCs-based tissue engineering approaches.
Main results
1. Isolation of periodontal ligament stem cells and investigation of multiple differentiation ability.
HPDLSCs were isolated, cultured and expanded as previously described. Procedures were performed according to the approval of the institutional review board and the informed consent of the patients. Wisdom and premolar teeth intended for extraction due to orthodontic reasons were used as the cell source. Periodontal ligament tissue was cultured in MesenPRO RS medium HPDLSCs were enriched by collecting multiple colonies. HPDLSCs at passage three were used in experiments. Growth characteristics and multipotent differentiation of the cell were assessed.
2. Cell cultures under SMG and biological behavior analysis
Cells were co-incubated with microcarrier beads of Cytodex3, and were placed in the rotating bioreactor (55-mL rotating wall vessel, Synthecon, U.S.A.) Cells morphology observation was examined such as scanning electron microscopy, transmission electron microscopy, fluorescence staining of microfilaments. Cell proliferation assessment including cell counting, flow cytometry and BrdU incorporation were investigated. And also, osteogenic differentiation potential was assessed. The results showed that simulated microgravity can change cell morphology, enhance cell proliferation and cell osteogenic differentiation ability.
3. The mechanism of Smads in hPDLSCs ostegenic differentiation induced by simulated microgravity
TGF-β1 can enhance ostegenic differentiation ability of hPDLSCs, as indicated by increased ALP, OCN and COL1 levels. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity. The mechanism maybe activated by pathway of TGF-βligament—membrane receptor—Smads proteins—transcriptor—gene expression.
Conclussions
In this article, we have found that this simulated microgravity environment can enhance periodontal ligament stem cells proliferation, viability and osteogenic differentiation. This environment also stimulates morphological changes and tended to aggregate as compared to the static control. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity.We conclude that the 3D dynamic simulated microgravity culture system has the potential to be used for the bioengineering reconstruction of the periodontal tissues. And the observation can benefit physiopathology of periodontium in aerospace flight.
成体干细胞是指从机体某些组织中分离出来的具有自我更新和多向分化能力的一类细胞,它分布于不同的组织,还可以通过血液循环到达躯体各处,参与组织的修复和再生。牙周膜干细胞是一种典型的牙源性间充质干细胞,可以从新鲜拔除牙齿的牙周膜组织中获得。尽管在体外普通培养环境下这种牙周膜干细胞呈现出一种较低的成骨分化潜能,但是在矿化诱导培养条件下,牙周膜干细胞可以表达成牙骨质/成骨特性标记物,如碱性磷酸酶、细胞外基质磷酸化糖蛋白、骨涎蛋白、骨钙素和转移生长因子β1;牙周膜干细胞植入免疫缺陷鼠体内能够生成类似牙周膜样结构。这些特性表明牙周膜干细胞作为一种新的成体干细胞,具有其特殊性和潜在的临床应用价值。
TGF-β超家族细胞因子经过其各自的信号转导引起不同的生物学效应,而信号转导的基本过程相似:通过TGF-β族配体——膜受体——Smads蛋白——转录因子——基因表达等步骤。Smads家族是TGF-β超家族成员下游的一种细胞内信号的特异性转导分子,可将信号直接从细胞膜转导入细胞核内,具有转录活化的作用。组织学和细胞学研究证实牙周膜的发育过程中,TGF-β及其受体在成骨细胞、成牙骨质细胞和成纤维细胞内均有较强的表达,提示TGF-β能够调节牙周膜组织的发生和成熟。
本课题旨在:探讨地面静止及模拟微重力环境中牙周膜干细胞形态,内部结构,增殖、分化等生物学特性有何种变化;明确smads信号转导分子在两种不同培养环境下对细胞生物学行为变化是否起到关键性的调控作用;探明微重力环境下牙周膜细胞的生物力学反应,及牙周膜细胞在微重力环境下生物力学响应的主要信号转导途径和关键信号分子,为维护航天员的口腔健康提供有效的防护措施,并且为临床研究牙周组织再生和牙齿松动等牙周疾病提供新的治疗思路。
所取得的主要结果如下:
1.牙周膜干细胞的分离、培养、鉴定及多向分化能力检测
通过组织块联合酶消化法获得了牙周膜细胞,利用单克隆培养法在间充质干细胞培养基内成功分离出牙周膜干细胞;采用成骨诱导、成脂诱导方式验证了牙周膜干细胞具备多向分化能力;采用免疫组织化学染色和流式细胞仪定量分析细胞群体中间充质干细胞标志物Stro-1的阳性表达率。
2.微重力环境下牙周膜干细胞的生物学特性分析
利用旋转生物反应器(RCCS)和Cytodex-3微载体成功对牙周膜干细胞接种并悬浮培养。对牙周膜干细胞在悬浮模拟微重力和普通重力环境的细胞生物状态进行了比较,主要观察了细胞形态学改变、增殖潜能变化、细胞成骨/成牙骨质向分化能力改变。结果提示模拟微重力环境能够加强细胞增殖能力,促进细胞成骨/成牙骨质向分化潜能,同时细胞出现不同于普通重力下的形态。
3. Smads信号转导分子在微重力引起牙周膜干细胞成骨/成牙骨质向分化中的作用机制研究。
TGF-β1可以促进牙周膜干细胞成骨/成牙骨质向分化;Smads信号在此过程中充当了重要角色;微重力引起的牙周膜干细胞成骨/成牙骨质向分化过程中,Smads信号分子担当了重要作用,可能通过TGF-β族配体——膜受体——Smads蛋白——转录因子——基因表达等步骤来促进细胞成骨/成牙骨质向分化过程。
综上所述,牙周膜干细胞在微重力环境下其生物学行为发生了特定的改变, Smads信号转导分子在此过程中发挥了重要作用。研究结果为空间牙周生理病理学提供了研究基础;同时为牙周组织工程研究提供了新的研究思路。
Introduction
The periodontal ligament functions as a cushion to mitigate the mechanical forces of mastication and maintains homeostasis such as remodeling of the adjacent bone. Periodontal ligament retains regenerative capacity to some degrees throughout adulthood, which is attributed to stem cells maintaining their proliferation and differentiation potential in the area. Previous studies have determined that periodontal ligament stem cells (PDLSCs) play a crucial role in regeneration of periodontal defects, contributing to the formation of new cementum, alveolar bone, and periodontal ligament. In addition, several in vitro findings showed that PDLSCs can differentiate into osteoblast-like cells when challenged with dexamethasone,β-glycerophosphate and ascorbic acid. Recently, tissue engineering based on PDLSCs to enhance periodontal regeneration has been the focus of periodontal research. In situ engineered constructs containing in vitro expanded autologous cells have been used to regenerate periodontal defects. While how to rapidly gain plenty of functional seeded cells in vitro is the key technique for bioengineering tissue. A theoretical way to achieve this goal would be to provide a“stimulatory’’environment for PDLSCs to expedite tissue engineering of periodontium.
With the acceleration of human space exploration, aerospace medicine has been gradually developed recently. Due to payload constraints, flight cost, spaceflight experiments are limited; several ground-based systems have been invented to simulate microgravity. Rotary cell culture system (RCCS), recommended by National Aeronautics and Space Administration (NASA) as an effective tool for analysis of cells characteristics in conditions similar to microgravity in space, is simultaneously a kind of three dimensional (3D) dynamic culture system. Its simulated microgravity (SMG) function is due to the alteration of the cell's perception of a continuously changing gravitational direction as a result of rotational culture conditions. At the same time, the rotational motion of this system prevents sedimentation, creating a suspension culture environment, and seems to be ideal for overcoming some drawbacks associated with static culturing systems. Several researches indicated that RCCS benefit certain type of cellular aggregation, subsequent intercellular adhesion, and gradual formation of 3-dimensional (3D) cell clumps. So we wonder that whether RCCS is an effective tool in vitro for expansion of functional stem cells in 3D form, which can facilitate periodontal tissue engineering.
Although, SMG is known to affect the biological behavior of several kinds of cells, the biology of different cells cultured in SMG is different; even a few conflicting findings have recently been reported with respect to the effects of SMG on definite cell types such as osteogenic cells. Up to now, no data exist illustrating the behavior of periodontal ligament stem cells under SMG. Thus, the objective of current study was to clarify the effects of three-dimensional dynamic simulated microgravity created by RCCS on hPDLSCs proliferation, osteoblastic differentiation and morphological changes. For it is the first time to investigate the effects of simulated microgravity on periodontal ligament stem cells, the research may lend insight into variations of cell response in 3D environment, and contribute to achievement of desirable periodontal regeneration utilizing PDLSCs-based tissue engineering approaches.
Main results
1. Isolation of periodontal ligament stem cells and investigation of multiple differentiation ability.
HPDLSCs were isolated, cultured and expanded as previously described. Procedures were performed according to the approval of the institutional review board and the informed consent of the patients. Wisdom and premolar teeth intended for extraction due to orthodontic reasons were used as the cell source. Periodontal ligament tissue was cultured in MesenPRO RS medium HPDLSCs were enriched by collecting multiple colonies. HPDLSCs at passage three were used in experiments. Growth characteristics and multipotent differentiation of the cell were assessed.
2. Cell cultures under SMG and biological behavior analysis
Cells were co-incubated with microcarrier beads of Cytodex3, and were placed in the rotating bioreactor (55-mL rotating wall vessel, Synthecon, U.S.A.) Cells morphology observation was examined such as scanning electron microscopy, transmission electron microscopy, fluorescence staining of microfilaments. Cell proliferation assessment including cell counting, flow cytometry and BrdU incorporation were investigated. And also, osteogenic differentiation potential was assessed. The results showed that simulated microgravity can change cell morphology, enhance cell proliferation and cell osteogenic differentiation ability.
3. The mechanism of Smads in hPDLSCs ostegenic differentiation induced by simulated microgravity
TGF-β1 can enhance ostegenic differentiation ability of hPDLSCs, as indicated by increased ALP, OCN and COL1 levels. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity. The mechanism maybe activated by pathway of TGF-βligament—membrane receptor—Smads proteins—transcriptor—gene expression.
Conclussions
In this article, we have found that this simulated microgravity environment can enhance periodontal ligament stem cells proliferation, viability and osteogenic differentiation. This environment also stimulates morphological changes and tended to aggregate as compared to the static control. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity.We conclude that the 3D dynamic simulated microgravity culture system has the potential to be used for the bioengineering reconstruction of the periodontal tissues. And the observation can benefit physiopathology of periodontium in aerospace flight.
引文
1杨唐斌,曾昆.空间飞行和微重力对免疫应答的影响.中华航空航天医学杂志. 2006, 17(1):56-61
2姚宇华严洪熊江辉等.模拟微重力条件下心肌细胞骨架的灰度统计特征分析.中国生物医学工程学报. 2005,24(3):268-270.
3孙联文庄逢源.微重力导致航天员骨质疏松的研究进展.中华航空航天医学杂志.2004,15(1):54-58.
4 Zayzafoon M, VE Meyers and JM McDonald. Microgravity: the immune response and bone. Immunol Rev, 2005, 208: 267-280.
5 Hammond TG, FC Lewis, TJ Goodwin, RM Linnehan, DA Wolf, KP Hire, WC Campbell, E Benes, KC O’Reilly, RK Globus and JH Kaysen. Gene expression in space. Nat. Med, 1999, 5: 359.
6 Klement BJ, QM Young, BJ George and M Nokkaew. Skeletal tissue growth, differentiation and mineralization in the NASA rotating wall vessel. Bone 2004, 34, 487-498.
7 Carmeliet G, G Nys and I Stockmans. Gene expression related to the differentiation of osteoblastic cells is altered by microgravity. Bone, 1998, 22: 139S-143S
8 Basso N, Bellows CG, Heersche JN. Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats. Bone. 2005, 36(1):173-83.
9 Oganov VS, Rakhmanov AS, Novikov VE, et al. The state of human bone tissue during space flight. Acta Astronaut. 1991, 23:129-33.
10袁林天,文玲英,罗亚宁.尾悬吊大鼠牙体、牙髓、牙周组织的变化.航天医学与医学工程. 2003, 16(4): 248-252.
11 Hasegawa M, M Yamato, A Kikuchi, T Okano and I Ishikawa. Human Periodontal Ligament Cell Sheets Can Regenerate Periodontal LigamentTissue in an Athymic Rat Model. 2005, Tissue Eng 11: 469-478.
12 Inan? B, A Eser Elcin and YM Elcin. (2006). Osteogenic Induction of Human Periodontal Ligament Fibroblasts Under Two- and Three-Dimensional Culture Conditions. Tissue Eng 12: 257-266.
13 Inan? B, A Eser El?in, A Ko?, K Balo?, A Parlar and Y Murat El?in. (2007). Encapsulation and osteoinduction of human periodontal ligament fibroblasts in chitosan-hydroxyapatite microspheres. J Biomed Mater Res A 82, 917-926.
14 Garetto LP, Gonsalves MR, Morey ER,et al Preosteoblast production 55 hours after a 12.5-day spaceflight on Cosmos 1887. FASEB J. 1990 Jan;4(1):24-8.
15 Fielder PJ, Morey ER, Roberts WE. Osteoblast histogenesis in periodontal ligament and tibial metaphysis during simulated weightlessness. Aviat Space Environ Med. 1986 Dec;57(12 Pt 1):1125-30.
16涂小丽.生长因子与牙周组织工程,国外医学生物医学工程分册,2002, 25(2):70-72
17杨隽;司书毅;张月琴. Smad在TGF-β超家族信号通路中的调控作用.中国生物工程杂志,2003, 23(12):9-12.
18宋亚文,谢利民.骨发生和形成过程中TGF-β/BMPs的信号传导.中国中医骨伤科杂志. 2004,12(2):41-44.
19牙周病学,孟焕新主编,人民卫生出版社,2008年7月,第三版。
20 Desvarieux M, Demmer RT, Rundek T, et al. Relationship between periodontal disease , tooth los s , and carotid artery plaque:the Oral Infections and Vascular Disease Epidemiology Study (INVEST). Stroke 2003; 4(9):2120-2125
21唐旭,王素云,刘琪.工程化基质在牙周组织再生中的临床应用.中华老年口腔医学杂志,2007,5(3):185-188
22 Melcher AH. On the repair potential of periodontal tissues. J periodontol , 1976 ,47 (5) :256 - 260
23 Gay, I.C., Chen, S. and MacDougall, M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod Craniofacial Res 2007, 10, 149.
24 Seo BM, Miura M, Gronthos S, Bartold PM, et al . Investigation of multipotent postnatal stem cells from human periodontal ligament. Lanect ,2004 ,364(9429):149–155.
25 Christian M, Gottfried S, Torsten E, et al . Somatic stem cells for regenerative dentistry. Clin Oral Invest,2008, 12:113–118
26 Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C,Liu H, Gronthos S, Wang CY, Shi S, Wang S (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE 1:e79
27贺慧霞,金岩,史俊南,等.人牙髓干细胞的体外分离、培养及鉴定.临床口腔医学杂志, 2004, 20 (9) : 515
28 Jo, Y.Y., Lee, H.J., Kook, S.Y., Choung, H.W., Park, J.Y., Chung, J.H., Choung, Y.H., Kim, E.S., Yang, H.C. and Choung, P.H. Isolation and Characterization of Postnatal Stem Cells from Human Dental Tissues. Tissue Eng 2007(.13)767—776.
29刘怡,房殿吉,李静远,海波,张春梅,施松涛,王松灵.小型猪牙周膜细胞体外培养及与三维支架材料的生物相容性研究.北京口腔医学, 2006,14(1):20-22
30 Yi Liu, Ying Zheng, Gang Ding, Dianji Fang, Chunmei Zhang, Peter Mark Bartold, StanGronthos, Songtao Shi and Songlin Wang. Periodontal Ligament Stem Cell-mediated Treatment for Periodontitis in Miniature Swine. StemCells published online Jan 31, 2008.
31 Hasegawa, M., Yamato, M., Kikuchi, A., Okano, T. and Ishikawa, I. Human Periodontal Ligament Cell Sheets Can Regenerate Periodontal Ligament Tissue in an Athymic Rat Model. Tissue Eng. 11, 469, 2005.
32 Margarita, Zeichner-David. Regeneration of periodontal tissues: cementogenesis revisited. Periodontology 2000. 2006,41,196–217.
33 Duray PH, Hatfill SJ, Pellis NR. Tissue culture in microgravity [J ] . Sci Med (Phila), 1997, 4(3): 46 -55.
34 Begleycm, Kleis J. RWV bioreactor mass transport: earth - based and in microgravity[J ] . Biotechnol Bioeng, 2002 , 80(4) :465 - 476.
35 Klaus DM. Clinostats and bioreactors [J] . Gravit Space Biol Bull, 2001,14(2) :55 - 64.
36 Licatol L, Grimm EA. Multiple interleukin - 2 signaling pathways differentially regulated by microgravity [J] Immunopharmacology, 1999, 44(3):273 - 279.
37 Baldwin SP, Saltzman WM. Aggregation enhances catecholamine secretion in cultured cells [J ]. Tissue Eng, 2001, 7(2):179 - 190.
38 Qiu QQ, Ducheyne P, Ayyaswamy PS. 3D bone tissue engineered with bioactive microspheres in simulated microgravity. In Vitro Cell Dev Biol Anim, 2001, 37(3): 157 - 165.
39 Freed LE, Martin I, Vunjak-Novakovic G. Frontiers in tissue engineering. In vitro modulation of chondrogenesis. Clin Orthop, 1999, 367:S46 - S58.
40 Gorit GK, Lo J, Falsafi S. Cartilage tissue engineering using cryogenic chondrocytes. Arch Otolaryngol Head Neck Surg, 2003, 129(8):889 - 893.
41 Rutzkyl P, Bilinski S, Kloc M. Microgravity culture condition reducesimmunogenicity and improves function of pancreatic islets. Transplantation, 2002, 74(1): 13 - 21.
42 Song C, Duan XQ, Li X. Experimental study of rat beta islet cells cultured under simulated microgravity conditions. Shengwu Huaxue Yu Shengwu Wuli Xuebao, 2004, 36(1):47 - 50.
43 Dabos KJ, Nelson LJ, Bradnock TJ. The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes. Biochim Biophys Acta, 2001, 1526(2):119- 130.
44 Khaoustov VI, Darlington GJ, Soriano HE. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev Biol Anim, 1999, 35(9):501 - 509.
45 Seitzer U, Bodo M, Mller PK. Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts. Cell Tissue Res, 1995, 282(3) : 513 - 517.
46 Carrier RL, Papadakim, Rupnickm. Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. Biotechnol Bioeng, 1999, 64(5): 580 - 589.
47 Buravkova LB, Romanov YA. The role of cytoskeleton in cell changes under condition of simulated microgravity. Acta Astronaut, 2001, 48(5 - 12): 647 - 650.
48 Carlsson SI, Bertilacciom T, Ballabio. Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization. Biochim Biophys Acta, 2003, 1642(3): 173-179.
49 Plett PA, Frankovitz SM, Abonour R. Proliferation of human hematopoietic bone marrow cells in simulated microgravity. In Vitro Cell Dev Biol Anim,2001, 37(2): 73 - 78.
50 Dutt K, Harris- Hooker S, Ellerson D. Generation of 3D retina-like structures from a human retinal cell line in a NASA bioreactor. Cell Transplant, 2003, 12(7): 717 - 731.
51 Lappa M. Organic tissues in rotating bioreactors: fluid - mechanical aspects, dynamic growth models , and morphological evolution. Biotechnol Bioeng, 2003, 84(5): 518 - 532.
52 Infanger M, Kossmehl P, Shakibaei M l. Simulated weightlessness changes the cytoskeleton andextracellular matrix proteins in papillary thyroid carcinomacells. Cell Tis sue Res, 2006, 324(2): 267- 277.
53 Carlsson SI, Bertilaccio MT, Ascarii. Modulation of human endothelial cell behavior in simulated microgravity. J Gravit Physiol, 2002, 9(1): 273-274.
54 Grimm D, Bauer J, Kossmehl P. Simulated microgravity alters differentiation and increases apoptos is in human follicular thyroid carcinoma Ecells. FASEB J, 2002, 16(6): 604- 606.
55 Massague J.TGF-βsignal transduction.Annu Rev Biochem,1998,67:753~791
56 Gao J, Symons AL, Bartold PM. Exp ression of transforming growth factor beta recep tors typesⅡandⅢwithin various cells in the rat periodontium [J]. J periodontal Res, 1999, 34 (2): 113-122.
57 Narayanan AS, Bartold PM. Biochemistry of periodontal connective tissues and their regeneration: a current perspective [J]. Connect Tissueres, 1996, 34 (3) : 191-201.
58 Dennison DK,V allone DR, P inero GJ , et al. Differential effect of TGF-B1 and PDGF on proliferation of periodontal ligament cells and gingival fibroblasts [J ]. J Periodontal, 1994, 65: 641-648.
59 Sodek JO, Verall CM. Metalloproteinases in periodontal tissue remodeling [J]. M at rix2Supp l, 1992, 1: 352.
60 Oates TW, Rouse CA, Coch ran DL, et al. M itogenic effects of grow th facto rs on human periodontal ligaments cells in vit ro [J]. J Perodonto l, 1993, 64 (2): 142.
61 Bartold PM, McCulloch CA, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000. 2000, 24: 253–69.
62 Pitaru S, Pritzki A, Bar-Kana I, Grosskopf A, Savion N, Narayanan AS. Bone morphogenetic protein 2 induces the expression of cementum attachment protein in human periodontal ligament clones. Connect Tissue Res 2002; 43: 257–64.
63 Shimono M, Ishikawa T, Ishikawa H, et al. Regulatory mechanisms of periodontal regeneration. Microsc Res Tech 2003; 60: 491–502.
64 Cochran DL,Jones A,Heijl L,et al.Periodontal regeneration with a combination of enamel matrix proteins and autogenous bone grafting [J].J Periodontol,2003,74(9):1269-1281.
65 Gould TR, Melcher AH, Brunette DM. Migration and division of progenitor cell populations in periodontal ligament after wounding. J Periodontal Res 1980; 15: 20–42.
66 McCulloch CA, Melcher AH. Cell density and cell generation in the periodontal ligament of mice. Am J Anat 1983; 167: 43–58.
67 McCulloch CA, Bordin S. Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodontal Res 1991; 26: 144–54.
68 Isaka J, Ohazama A, Kobayashi M, et al. Participation of periodontal ligament cells with regeneration of alveolar bone. J Periodontol 2001; 72:314–23.
69 Beertsen W, McCulloch CA, Sodek J. The periodontal ligament: a unique, multifunctional connective tissue. Periodontol 2000, 1997; 13: 20–40.
70 Ma Z, Li S, Song Y, The biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Tissue Eng Part A. 2008;14(12): 2059-68.
71 Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003; 18: 696–704.
72 Shi S, Gehron RP, Gronthos S. Comparison of gene expression profiles for human, dental pulp and bone marrow stromal stemcells by cDNA microarray analysis. Bone 2001; 29: 532–39.
73 Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood, 1994; 84: 4164–73.
74 Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002; 105: 93–8.
75 Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci USA 2000; 97: 13625–30.
76 Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; 81: 531–5.
77 Gronthos S, CN, Gehron Robey P, Shi S. Human dental pulp stem cells: characterization and developmental potential. Adult Stem Cells, 2004. 67–82.
78 Nojima N, Kobayashi M, Shionome M, et al. Fibroblas tic cells derived from bovine periodontal ligaments have the phenotypes of os teoblasts. Jperiodontal Res 1990; 25(3):179-185
79韩劼,孟焕新,唐军民,等.人牙周膜细胞与BIO-OSS胶原体外三维复合体的构建[J ].实用口腔医学杂志, 2006, 22(3): 412-413
80 Cai Z, Zhang L, Shu J, et al. The application ofspace biotechnology in the s tudy of s tem cells and tis sueengineering [J].Chin J Rehabil Theory Practice, 2004, 10( 1): 9- 10.
81 Malda J, van Blitterswijk C A, GrojecM, et al. Expansion of bovine chondrocytes on microcarriers enhances redifferentiation [J]. Tissue Eng, 2003, 9 (5): 939 - 948.
82 Nam JH, Ermonval M, Sharfstein ST.. Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture Biotechnol Prog. 2007, 23: 652-60
83 Unsworth B R, Lelkes P I. Growing tissues in microgravity [J]. Nat Med, 1998, 4 (8): 901 - 907.
84 Liu T Q, Li X Q, Sun X Y, et al. Analysis on forces and movement of cultivated particles in a rotatingwall vessel bioreactor[J]. Biochemical Engineering Journal, 2004, 18 (2): 97 - 104.
85 Khaoustov V I, Risin D, Pellis N R, et al. Microarray analysis of genes differentially exp ressed in HepG2 cells cultured in simulated microgravity: p reliminary report [J]. In Vitro Cell Dev Biol Anim, 2001, 37(2): 84 - 88.
86 Chiu B, Wan J Z, Abley D, et al. Induction of vascular endothelial phenotype and cellular p roliferation from human cord blood stem cells cultured in simulated microgravity [J]. Acta Astronaut, 2005, 56 (9-12): 918 - 922.
87 Licato LL, Grimm EA. Multip le interleukin-2 signaling pathways differentially regulated by microgravity [J]. Immunopharmacology, 1999, 44(3): 273 - 279.
88 Wu J, F Jin, L Tang, J Yu, L Xu, Z Yang, G Wu, Y Duan and Y Jin. Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages. Biol Cell, 2008,100: 291-302.
89 Meyers VE, M Zayzafoon, JT Douglas and JM McDonald.. RhoA and Cytoskeletal Disruption Mediate Reduced Osteoblastogenesis and Enhanced Adipogenesis of Human Mesenchymal Stem Cells in Modeled Microgravity. J Bone Miner Res,2005, 20: 1858-1866.
90 Dai ZQ, R Wang, SK Ling, YM Wan and YH Li. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Prolif, 2007, 40: 671-684.
91 Matsuura T. Bioreactors for 3-dimensional high-density culture of human cells.. Human Cell. 2006, 19: 11–16.
92 Ingber D. How cells (might) sense microgravity. FASEB J. 1999, 13:S3-15.
93 Meloni MA, G Galleri, P Pippia, M Cogoli-Greuter.. Cytoskeleton changes and impaired motility of monocytes at modelled low gravity. Protoplasma. 2006, 229: 243-249.
94 McBeath R, DM Pirone, CM Nelson, K Bhadriraju and CS Chen. (2004). Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6: 483-495.
95 Ko YJ, RS Zaharias, DA Seabold, J Lafoon and GB Schneider. Osteoblast Differentiation Is Enhanced in Rotary Cell Culture Simulated Microgravity Environments. J Prosthodont, 2007,16: 431-8.
96罗飞,许建中,王序全等.模拟微重力环境促进人间充质干细胞体外高效增殖[J].第三军医大学学报,2005, 27(16): 1640-1643
97 Khaoustov VI, GJ Darlington, HE Soriano, B Krishnan, D Risin, NR Pellis,and B Yoffe.. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev. Biol. Anim, 1999, 35: 501-509.
98 Martin A, A Zhou, RE Gordon, SC Henderson, AE Schwartz, EW Friedman and TF Davies. Thyroid organoid formation in simulated microgravity: influence of keratinocyte growth factor. Thyroid, 2000, 10: 481-487.
99 Saxena R, G Pan and JM McDonald. Osteoblast and Osteoclast Differentiation in Modeled Microgravity. Ann N Y Acad Sci, 2007, 1116: 494-498.
100 Nagai N, K Mori, Y Satoh, N Takahashi, S Yunoki, K Tajima and M Munekata. In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel. J Biomed Mater Res A, 2007, 82: 395-402.
101 Wei F, C Wang, G Zhou, D Liu, X Zhang, Y Zhao, Y Zhang and Q Yang. The effect of centrifugal force on the mRNA and protein levels of ATF4 in cultured human periodontal ligament fibroblasts. Arch Oral Biol, 2008, 53: 35-43.
102 Choi CW, Kim BI, Joung KE,et al.Decreased Expression of Transforming Growth Factor-beta1 in Bronchoalveolar Lavage Cells of Preterm Infants with Maternal ChorioamnionitisJ Korean Med Sci. 2008 Aug;23(4):609-15.
103 Masatoshi Jinnin, Hironobu Ihn, Kunihiko Tamaki.Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on TGF-β1 Induced Extracellular Matrix Expression. Molecular Pharmacology, 2006,69, (2):597-607
2姚宇华严洪熊江辉等.模拟微重力条件下心肌细胞骨架的灰度统计特征分析.中国生物医学工程学报. 2005,24(3):268-270.
3孙联文庄逢源.微重力导致航天员骨质疏松的研究进展.中华航空航天医学杂志.2004,15(1):54-58.
4 Zayzafoon M, VE Meyers and JM McDonald. Microgravity: the immune response and bone. Immunol Rev, 2005, 208: 267-280.
5 Hammond TG, FC Lewis, TJ Goodwin, RM Linnehan, DA Wolf, KP Hire, WC Campbell, E Benes, KC O’Reilly, RK Globus and JH Kaysen. Gene expression in space. Nat. Med, 1999, 5: 359.
6 Klement BJ, QM Young, BJ George and M Nokkaew. Skeletal tissue growth, differentiation and mineralization in the NASA rotating wall vessel. Bone 2004, 34, 487-498.
7 Carmeliet G, G Nys and I Stockmans. Gene expression related to the differentiation of osteoblastic cells is altered by microgravity. Bone, 1998, 22: 139S-143S
8 Basso N, Bellows CG, Heersche JN. Effect of simulated weightlessness on osteoprogenitor cell number and proliferation in young and adult rats. Bone. 2005, 36(1):173-83.
9 Oganov VS, Rakhmanov AS, Novikov VE, et al. The state of human bone tissue during space flight. Acta Astronaut. 1991, 23:129-33.
10袁林天,文玲英,罗亚宁.尾悬吊大鼠牙体、牙髓、牙周组织的变化.航天医学与医学工程. 2003, 16(4): 248-252.
11 Hasegawa M, M Yamato, A Kikuchi, T Okano and I Ishikawa. Human Periodontal Ligament Cell Sheets Can Regenerate Periodontal LigamentTissue in an Athymic Rat Model. 2005, Tissue Eng 11: 469-478.
12 Inan? B, A Eser Elcin and YM Elcin. (2006). Osteogenic Induction of Human Periodontal Ligament Fibroblasts Under Two- and Three-Dimensional Culture Conditions. Tissue Eng 12: 257-266.
13 Inan? B, A Eser El?in, A Ko?, K Balo?, A Parlar and Y Murat El?in. (2007). Encapsulation and osteoinduction of human periodontal ligament fibroblasts in chitosan-hydroxyapatite microspheres. J Biomed Mater Res A 82, 917-926.
14 Garetto LP, Gonsalves MR, Morey ER,et al Preosteoblast production 55 hours after a 12.5-day spaceflight on Cosmos 1887. FASEB J. 1990 Jan;4(1):24-8.
15 Fielder PJ, Morey ER, Roberts WE. Osteoblast histogenesis in periodontal ligament and tibial metaphysis during simulated weightlessness. Aviat Space Environ Med. 1986 Dec;57(12 Pt 1):1125-30.
16涂小丽.生长因子与牙周组织工程,国外医学生物医学工程分册,2002, 25(2):70-72
17杨隽;司书毅;张月琴. Smad在TGF-β超家族信号通路中的调控作用.中国生物工程杂志,2003, 23(12):9-12.
18宋亚文,谢利民.骨发生和形成过程中TGF-β/BMPs的信号传导.中国中医骨伤科杂志. 2004,12(2):41-44.
19牙周病学,孟焕新主编,人民卫生出版社,2008年7月,第三版。
20 Desvarieux M, Demmer RT, Rundek T, et al. Relationship between periodontal disease , tooth los s , and carotid artery plaque:the Oral Infections and Vascular Disease Epidemiology Study (INVEST). Stroke 2003; 4(9):2120-2125
21唐旭,王素云,刘琪.工程化基质在牙周组织再生中的临床应用.中华老年口腔医学杂志,2007,5(3):185-188
22 Melcher AH. On the repair potential of periodontal tissues. J periodontol , 1976 ,47 (5) :256 - 260
23 Gay, I.C., Chen, S. and MacDougall, M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod Craniofacial Res 2007, 10, 149.
24 Seo BM, Miura M, Gronthos S, Bartold PM, et al . Investigation of multipotent postnatal stem cells from human periodontal ligament. Lanect ,2004 ,364(9429):149–155.
25 Christian M, Gottfried S, Torsten E, et al . Somatic stem cells for regenerative dentistry. Clin Oral Invest,2008, 12:113–118
26 Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C,Liu H, Gronthos S, Wang CY, Shi S, Wang S (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE 1:e79
27贺慧霞,金岩,史俊南,等.人牙髓干细胞的体外分离、培养及鉴定.临床口腔医学杂志, 2004, 20 (9) : 515
28 Jo, Y.Y., Lee, H.J., Kook, S.Y., Choung, H.W., Park, J.Y., Chung, J.H., Choung, Y.H., Kim, E.S., Yang, H.C. and Choung, P.H. Isolation and Characterization of Postnatal Stem Cells from Human Dental Tissues. Tissue Eng 2007(.13)767—776.
29刘怡,房殿吉,李静远,海波,张春梅,施松涛,王松灵.小型猪牙周膜细胞体外培养及与三维支架材料的生物相容性研究.北京口腔医学, 2006,14(1):20-22
30 Yi Liu, Ying Zheng, Gang Ding, Dianji Fang, Chunmei Zhang, Peter Mark Bartold, StanGronthos, Songtao Shi and Songlin Wang. Periodontal Ligament Stem Cell-mediated Treatment for Periodontitis in Miniature Swine. StemCells published online Jan 31, 2008.
31 Hasegawa, M., Yamato, M., Kikuchi, A., Okano, T. and Ishikawa, I. Human Periodontal Ligament Cell Sheets Can Regenerate Periodontal Ligament Tissue in an Athymic Rat Model. Tissue Eng. 11, 469, 2005.
32 Margarita, Zeichner-David. Regeneration of periodontal tissues: cementogenesis revisited. Periodontology 2000. 2006,41,196–217.
33 Duray PH, Hatfill SJ, Pellis NR. Tissue culture in microgravity [J ] . Sci Med (Phila), 1997, 4(3): 46 -55.
34 Begleycm, Kleis J. RWV bioreactor mass transport: earth - based and in microgravity[J ] . Biotechnol Bioeng, 2002 , 80(4) :465 - 476.
35 Klaus DM. Clinostats and bioreactors [J] . Gravit Space Biol Bull, 2001,14(2) :55 - 64.
36 Licatol L, Grimm EA. Multiple interleukin - 2 signaling pathways differentially regulated by microgravity [J] Immunopharmacology, 1999, 44(3):273 - 279.
37 Baldwin SP, Saltzman WM. Aggregation enhances catecholamine secretion in cultured cells [J ]. Tissue Eng, 2001, 7(2):179 - 190.
38 Qiu QQ, Ducheyne P, Ayyaswamy PS. 3D bone tissue engineered with bioactive microspheres in simulated microgravity. In Vitro Cell Dev Biol Anim, 2001, 37(3): 157 - 165.
39 Freed LE, Martin I, Vunjak-Novakovic G. Frontiers in tissue engineering. In vitro modulation of chondrogenesis. Clin Orthop, 1999, 367:S46 - S58.
40 Gorit GK, Lo J, Falsafi S. Cartilage tissue engineering using cryogenic chondrocytes. Arch Otolaryngol Head Neck Surg, 2003, 129(8):889 - 893.
41 Rutzkyl P, Bilinski S, Kloc M. Microgravity culture condition reducesimmunogenicity and improves function of pancreatic islets. Transplantation, 2002, 74(1): 13 - 21.
42 Song C, Duan XQ, Li X. Experimental study of rat beta islet cells cultured under simulated microgravity conditions. Shengwu Huaxue Yu Shengwu Wuli Xuebao, 2004, 36(1):47 - 50.
43 Dabos KJ, Nelson LJ, Bradnock TJ. The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes. Biochim Biophys Acta, 2001, 1526(2):119- 130.
44 Khaoustov VI, Darlington GJ, Soriano HE. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev Biol Anim, 1999, 35(9):501 - 509.
45 Seitzer U, Bodo M, Mller PK. Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts. Cell Tissue Res, 1995, 282(3) : 513 - 517.
46 Carrier RL, Papadakim, Rupnickm. Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. Biotechnol Bioeng, 1999, 64(5): 580 - 589.
47 Buravkova LB, Romanov YA. The role of cytoskeleton in cell changes under condition of simulated microgravity. Acta Astronaut, 2001, 48(5 - 12): 647 - 650.
48 Carlsson SI, Bertilacciom T, Ballabio. Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization. Biochim Biophys Acta, 2003, 1642(3): 173-179.
49 Plett PA, Frankovitz SM, Abonour R. Proliferation of human hematopoietic bone marrow cells in simulated microgravity. In Vitro Cell Dev Biol Anim,2001, 37(2): 73 - 78.
50 Dutt K, Harris- Hooker S, Ellerson D. Generation of 3D retina-like structures from a human retinal cell line in a NASA bioreactor. Cell Transplant, 2003, 12(7): 717 - 731.
51 Lappa M. Organic tissues in rotating bioreactors: fluid - mechanical aspects, dynamic growth models , and morphological evolution. Biotechnol Bioeng, 2003, 84(5): 518 - 532.
52 Infanger M, Kossmehl P, Shakibaei M l. Simulated weightlessness changes the cytoskeleton andextracellular matrix proteins in papillary thyroid carcinomacells. Cell Tis sue Res, 2006, 324(2): 267- 277.
53 Carlsson SI, Bertilaccio MT, Ascarii. Modulation of human endothelial cell behavior in simulated microgravity. J Gravit Physiol, 2002, 9(1): 273-274.
54 Grimm D, Bauer J, Kossmehl P. Simulated microgravity alters differentiation and increases apoptos is in human follicular thyroid carcinoma Ecells. FASEB J, 2002, 16(6): 604- 606.
55 Massague J.TGF-βsignal transduction.Annu Rev Biochem,1998,67:753~791
56 Gao J, Symons AL, Bartold PM. Exp ression of transforming growth factor beta recep tors typesⅡandⅢwithin various cells in the rat periodontium [J]. J periodontal Res, 1999, 34 (2): 113-122.
57 Narayanan AS, Bartold PM. Biochemistry of periodontal connective tissues and their regeneration: a current perspective [J]. Connect Tissueres, 1996, 34 (3) : 191-201.
58 Dennison DK,V allone DR, P inero GJ , et al. Differential effect of TGF-B1 and PDGF on proliferation of periodontal ligament cells and gingival fibroblasts [J ]. J Periodontal, 1994, 65: 641-648.
59 Sodek JO, Verall CM. Metalloproteinases in periodontal tissue remodeling [J]. M at rix2Supp l, 1992, 1: 352.
60 Oates TW, Rouse CA, Coch ran DL, et al. M itogenic effects of grow th facto rs on human periodontal ligaments cells in vit ro [J]. J Perodonto l, 1993, 64 (2): 142.
61 Bartold PM, McCulloch CA, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000. 2000, 24: 253–69.
62 Pitaru S, Pritzki A, Bar-Kana I, Grosskopf A, Savion N, Narayanan AS. Bone morphogenetic protein 2 induces the expression of cementum attachment protein in human periodontal ligament clones. Connect Tissue Res 2002; 43: 257–64.
63 Shimono M, Ishikawa T, Ishikawa H, et al. Regulatory mechanisms of periodontal regeneration. Microsc Res Tech 2003; 60: 491–502.
64 Cochran DL,Jones A,Heijl L,et al.Periodontal regeneration with a combination of enamel matrix proteins and autogenous bone grafting [J].J Periodontol,2003,74(9):1269-1281.
65 Gould TR, Melcher AH, Brunette DM. Migration and division of progenitor cell populations in periodontal ligament after wounding. J Periodontal Res 1980; 15: 20–42.
66 McCulloch CA, Melcher AH. Cell density and cell generation in the periodontal ligament of mice. Am J Anat 1983; 167: 43–58.
67 McCulloch CA, Bordin S. Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodontal Res 1991; 26: 144–54.
68 Isaka J, Ohazama A, Kobayashi M, et al. Participation of periodontal ligament cells with regeneration of alveolar bone. J Periodontol 2001; 72:314–23.
69 Beertsen W, McCulloch CA, Sodek J. The periodontal ligament: a unique, multifunctional connective tissue. Periodontol 2000, 1997; 13: 20–40.
70 Ma Z, Li S, Song Y, The biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Tissue Eng Part A. 2008;14(12): 2059-68.
71 Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003; 18: 696–704.
72 Shi S, Gehron RP, Gronthos S. Comparison of gene expression profiles for human, dental pulp and bone marrow stromal stemcells by cDNA microarray analysis. Bone 2001; 29: 532–39.
73 Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood, 1994; 84: 4164–73.
74 Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002; 105: 93–8.
75 Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci USA 2000; 97: 13625–30.
76 Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; 81: 531–5.
77 Gronthos S, CN, Gehron Robey P, Shi S. Human dental pulp stem cells: characterization and developmental potential. Adult Stem Cells, 2004. 67–82.
78 Nojima N, Kobayashi M, Shionome M, et al. Fibroblas tic cells derived from bovine periodontal ligaments have the phenotypes of os teoblasts. Jperiodontal Res 1990; 25(3):179-185
79韩劼,孟焕新,唐军民,等.人牙周膜细胞与BIO-OSS胶原体外三维复合体的构建[J ].实用口腔医学杂志, 2006, 22(3): 412-413
80 Cai Z, Zhang L, Shu J, et al. The application ofspace biotechnology in the s tudy of s tem cells and tis sueengineering [J].Chin J Rehabil Theory Practice, 2004, 10( 1): 9- 10.
81 Malda J, van Blitterswijk C A, GrojecM, et al. Expansion of bovine chondrocytes on microcarriers enhances redifferentiation [J]. Tissue Eng, 2003, 9 (5): 939 - 948.
82 Nam JH, Ermonval M, Sharfstein ST.. Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture Biotechnol Prog. 2007, 23: 652-60
83 Unsworth B R, Lelkes P I. Growing tissues in microgravity [J]. Nat Med, 1998, 4 (8): 901 - 907.
84 Liu T Q, Li X Q, Sun X Y, et al. Analysis on forces and movement of cultivated particles in a rotatingwall vessel bioreactor[J]. Biochemical Engineering Journal, 2004, 18 (2): 97 - 104.
85 Khaoustov V I, Risin D, Pellis N R, et al. Microarray analysis of genes differentially exp ressed in HepG2 cells cultured in simulated microgravity: p reliminary report [J]. In Vitro Cell Dev Biol Anim, 2001, 37(2): 84 - 88.
86 Chiu B, Wan J Z, Abley D, et al. Induction of vascular endothelial phenotype and cellular p roliferation from human cord blood stem cells cultured in simulated microgravity [J]. Acta Astronaut, 2005, 56 (9-12): 918 - 922.
87 Licato LL, Grimm EA. Multip le interleukin-2 signaling pathways differentially regulated by microgravity [J]. Immunopharmacology, 1999, 44(3): 273 - 279.
88 Wu J, F Jin, L Tang, J Yu, L Xu, Z Yang, G Wu, Y Duan and Y Jin. Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages. Biol Cell, 2008,100: 291-302.
89 Meyers VE, M Zayzafoon, JT Douglas and JM McDonald.. RhoA and Cytoskeletal Disruption Mediate Reduced Osteoblastogenesis and Enhanced Adipogenesis of Human Mesenchymal Stem Cells in Modeled Microgravity. J Bone Miner Res,2005, 20: 1858-1866.
90 Dai ZQ, R Wang, SK Ling, YM Wan and YH Li. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Prolif, 2007, 40: 671-684.
91 Matsuura T. Bioreactors for 3-dimensional high-density culture of human cells.. Human Cell. 2006, 19: 11–16.
92 Ingber D. How cells (might) sense microgravity. FASEB J. 1999, 13:S3-15.
93 Meloni MA, G Galleri, P Pippia, M Cogoli-Greuter.. Cytoskeleton changes and impaired motility of monocytes at modelled low gravity. Protoplasma. 2006, 229: 243-249.
94 McBeath R, DM Pirone, CM Nelson, K Bhadriraju and CS Chen. (2004). Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6: 483-495.
95 Ko YJ, RS Zaharias, DA Seabold, J Lafoon and GB Schneider. Osteoblast Differentiation Is Enhanced in Rotary Cell Culture Simulated Microgravity Environments. J Prosthodont, 2007,16: 431-8.
96罗飞,许建中,王序全等.模拟微重力环境促进人间充质干细胞体外高效增殖[J].第三军医大学学报,2005, 27(16): 1640-1643
97 Khaoustov VI, GJ Darlington, HE Soriano, B Krishnan, D Risin, NR Pellis,and B Yoffe.. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev. Biol. Anim, 1999, 35: 501-509.
98 Martin A, A Zhou, RE Gordon, SC Henderson, AE Schwartz, EW Friedman and TF Davies. Thyroid organoid formation in simulated microgravity: influence of keratinocyte growth factor. Thyroid, 2000, 10: 481-487.
99 Saxena R, G Pan and JM McDonald. Osteoblast and Osteoclast Differentiation in Modeled Microgravity. Ann N Y Acad Sci, 2007, 1116: 494-498.
100 Nagai N, K Mori, Y Satoh, N Takahashi, S Yunoki, K Tajima and M Munekata. In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel. J Biomed Mater Res A, 2007, 82: 395-402.
101 Wei F, C Wang, G Zhou, D Liu, X Zhang, Y Zhao, Y Zhang and Q Yang. The effect of centrifugal force on the mRNA and protein levels of ATF4 in cultured human periodontal ligament fibroblasts. Arch Oral Biol, 2008, 53: 35-43.
102 Choi CW, Kim BI, Joung KE,et al.Decreased Expression of Transforming Growth Factor-beta1 in Bronchoalveolar Lavage Cells of Preterm Infants with Maternal ChorioamnionitisJ Korean Med Sci. 2008 Aug;23(4):609-15.
103 Masatoshi Jinnin, Hironobu Ihn, Kunihiko Tamaki.Characterization of SIS3, a Novel Specific Inhibitor of Smad3, and Its Effect on TGF-β1 Induced Extracellular Matrix Expression. Molecular Pharmacology, 2006,69, (2):597-607