摘要
细胞在材料表面的粘附是贴壁依赖型细胞生长的前提，细胞只有在表面以一定的粘附力发生粘附并铺展后，细胞才能生长。作为骨组织工程细胞外基质替代物的人工合成的可降解高分子聚合物材料绝大多数亲水性差，细胞吸附力弱，需要进行必要的表面改性和表面修饰，以提高表面生物活性，利于细胞在材料上的粘附，进而影响细胞的增殖和分化。研究表明，细胞-材料粘附主要由细胞膜上的一个特殊受体整合素介导。而大多数蛋白如纤维连接蛋白、骨桥蛋白、Ⅰ型胶原、骨涎蛋白、玻基结合素等分子结构上包含精-甘-天冬氨酸(Arg-Gly-Asp，RGD)序列，后者可以特异性结合整合素，从而介导细胞粘附。因此将这些蛋白包被、固定到材料表面，观察骨组织工程种子细胞MSCs细胞的粘附、生长特性是本研究的中心环节，并从以下方面进行探讨：(1)采用不同原代细胞分离方法，研究其对MSCs细胞的生物学特性影响。(2)检测基因胜肽胶对MSCs细胞粘附、增殖及分化的影响。(3)分别采用Ⅰ型胶原及纤维粘连蛋白(fibronectin，FN)包被聚乙醇酸-乳酸共聚物(Poly(1actide-co-glycolide)，PLGA)膜及多孔块型PLGA材料，观察细胞在单层或三维培养状态下，Ⅰ型胶原及FN对MSCs细胞粘附、增殖及向成骨细胞分化效应及能力。(4)检测Ⅰ型胶原对MSCs细胞骨架肌动蛋白及细胞内游离钙离子的影响，了解Ⅰ型胶原促进MSCs细胞粘附的内在机制。(5)观察经Ⅰ型胶原包被的多孔块型PLGA材料体内成骨效果，尤其是材料中心部位的成骨情况。
第四军医大学博士学位论文
一
为研制具有细胞粘附能力、高效诱骨效应的新型活化人工骨提供实验依据和新
的解决途径。
研究结果显示：（1）密度梯度高心法接种后24小时可见少数细胞贴壁，
3天后，细胞增殖活跃。红细胞裂解法较密度梯度离心法细胞贴壁较慢，36
小时才可见零星细胞贴壁，两种方法细胞形态相似。活细胞百分比，红细胞
裂解法为（gi士 4）％，密度梯度离心治（83士 5）％，有明显差异。传代培
养：2～6代范围内，红细胞裂解法较梯度离心法细胞增殖更旺盛（分别是按
种时细胞数的4．67／4．10倍人其他细胞生物学行为如细胞贴壁率，克隆形成
率两者无明显差异。（2）电镜下 PLGA膜有均匀一致的文理结构，表面结构均
～，无明显突起及裂痕；多孔块型PLGA材料可见相互贯通的大孔结构，大孔
的形貌较圆滑，较少尖锐的棱角，孔隙平均尺寸约为400 v你在大孔的周围
布满了相互贯通的微孔结构，孔隙形貌多为近似的圆形，孔隙平均尺寸约为
5 u m。同样结构均一，符合实验要求。u）基因胜肽胶可促进 MSCs细胞的早
期粘附：细胞接种后3小时，实验组（1．5士0．18）X m’，与对照组（0．47
士0．22）X10’有显著性差异（P＜0．01），与空白对照组（1．04士0．21）X10’
有差异O钥．05人 接种6小时及12小时，实验组与空白对照组均无差异，
不能继续促进粘附：其对细胞增殖、细胞碱性磷酸酶活性也均无明显促进作
用。N）1型胶原、纤维连接蛋白均能促进细胞粘附：单层培养状态下，纤
维粘连蛋白于细胞接种6小时后、I型胶原于细胞接种后12小时开始促进细
胞粘附；三维培养体系，I型胶原、纤维粘连蛋白于细胞接种6小时即促进
细胞粘附，纤维粘连蛋自作用更明显。电镜照片显示，经1型胶原、纤维粘
连蛋白修饰后，细胞分布均匀，铺展良好。G）I型胶原、纤维粘连蛋白促
进细胞增殖，细胞接种后 3、6、gd三个检测时间点，实验组细胞均明显高于
对照组。与 1型胶原相比，纤维粘连蛋白刺激作用更强。①）I型胶原、纤
维粘连蛋白尚能诱导MSCS细胞向成骨细胞分化，不仅表达成骨细胞标志物
OCN、ALP、OPN mRNA，而且碱性磷酸酶活性明显增高，碱性磷酸酶及钙结节
7
第四军医大学博士学位论文
一
染色均强阳性，I型胶原组MSCS细胞碱性磷酸酶活性较FN组更高，有显著
性差异；同时，兔疫组化染色表明，经纤维粘连蛋白作用的MSCs1型胶原表
达阳性。（7）参照EIAmin方法，细胞骨架肌动蛋白（actinj的结构可根据
不同细胞类型加以描述。根据细胞形态，将细胞分为1、11两种类型。1型
细胞为多角形或长校形，actin表达较少并相对聚集；11型细胞细胞圆形，
铺展良好，。cdn形成丝状结构，表达丰富。I型胶原包被后细胞接种6小
时，实验组1型细胞占56％，11型细胞为44％，而对照组1型细胞则占79
％，11型细胞为21％；接种12小时，实验组I型细胞占门％，大部分为11
型细胞，为而对照组 1型细胞只占 45％。提示，材料经 1型胶原修饰后，细
胞多数为圆形，铺展良好，细胞骨架肌动蛋白构建活跃。（8）在1型胶原存
在的条件下生长的细胞内荧光较强，而对照组细胞内仅可见少许荧光；在细
胞内钙离子浓度曲线图上同样实验组单个细胞内钙离子浓度较高，说明1型
胶原可增加细胞内游离钙离于浓度。（8）通过样本连续切片，图像分析系统
分析各组动物新骨形成‘情况，显示应用多孔PLGA块状材料经1型胶原修饰后
体内成骨明显优于对照组，表现在：1．早期成?
Cell adhesion to surface of the substrate is essential to development of the anchorage-dependent cells.Only after adhering to surface followed by spreading can cells develop and proliferate.Most synthetic polymers used as orthopaedic matrix substitute present hydrophobicity,which may correlates to the low degree of cell attachment.Modification with cell adhesion protein/peptides can be benificial to the cell adhesion on polymers and then affect the cell proliferation and differentiation.Cell attachment to substrate is primarily mediated by integrins,a widely expressed family of heterodimeric surface receptors.Most extrcellular matrix proteins such as fibronectin,osteopontin,collagen type I , bone sialoprotein and vitronectin contain an Arg-Gly-Asp (RGD) sequence which is specific to the fixation of cell membrane receptors like integrin.The main aim of this research is to measure,assess adhesion,proliferation of rabbit marrow stromal cells(MSCs) on the polymers coated by fibronectin, collagen type I or biotie
gen,which includes:(1)Biologic characteristics of rabbit MSCs were observed by two types of separating method in primary culture.(2)Adhesion,proliferation and differentiation of MSCs cultured on polymers coated with biotiegen were assessed.(3)also, adhesion,proliferation and differentiation of MSCs were assessed on PLGA film or
porous PLGA substrates coated with fibronectin, or collagen type I respectively.(4)Bone formation was observed on the porous PLGA substrates coated with collagen type I in vivo.This research aims to give new way to make novel synthetic bone with cell adhesion and high bone induction capabilities.
Results obtained included:
(1) MSCs began to attach to culture flask after 24h cell inoculation by centrifugation method.Cell proliferation was active after 3d.and MSCs began to attach to culture flask only after 36h cell inoculation by erythrocyte splitting method. Cell morphology was similar no matter what method was used.Percentage of live cells was significantly higher in erythrocyte splitting method that was (91 ±4)%than in centrifugation method that was (83±5) % .Proliferative ability of MSCs seperated by erythrocyte splitting method at 2 to 6 generation was much higher than by another method.The maximal cell number was 4.67 vs 4.10 times that of the initial cell number at cell inoculation. There was no difference in other biologic characteristic of MSCs between the two separation method,such as cell anchorage ratio and clone formation ratio.(2)PLGA film presented uniformity frame with no protuberance and fissure under scanning electron microscopy(SEM).Big aperture with smooth wall and average 400 μ m i
n size running-through each other was observed in porous PLGA substrate,around the big aperture there were many round micropores about 5μm size.All of the structure were equal and uniform,which satisfied the further research work.(3)MSCs adhesion at earlier time was promoted by biotiegenrAfter 3h ,cell number was (1.5 ±0.18) × 105 in the PLGA film coated with biotiegen group,which was significantly higher than that in PLGA film group (P<0.01) and higher than that in coverslip group (P<0.05) ,which cell number was (1.04± 0.21) × 105 .After 6h and 12h biotiegen could not promote cell adhesion,and cell proliferation and alkaline phosphatase(ALP) activity were not promoted
dramatically during 9 days.(4) Cell adhesion was promoted by fibronectin or collagen type I . Cell adhesion dramatically increased after 6h by fibronectin and after 12h by collagen type I in monolayer culture. Fibronectin or collagen type I promoted cell adhesion after 6h under three dimension condition. Fibronectin had more ability to promote cell adhsion than collagen type I under such circumstances,SEM micrographs showed that MSCs all had a spread-out appearance with uniform distribution on polymer substrates precoated with fibronectin or collagen type I .(5) Fibronectin or collagen type I also promoted cell proliferation within 9 days culture under three dimension condi
引文
1. Menko AS and Boettiger D. Occupation of the extracellular matrix receptor integrin is a control point for myogenic differentiation. Cell, 1987, 51: 51-57
2. Adams JC, Watt FM. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes α5β1 integrin loss from the cell surface. cell, 1990, 63: 425-435
3. MacDonald DE, Deo N, Markovic B, et al. Adsorption and dissolution behavior of human plasma fibronectin on thermally and chemically modified titanium dioxide particles. Biomaterials 2002 Feb; 23(4): 1269-79
4. Wang YC, Kao SH, Hsieh HJ. A chemical surface modification of chitosan by glycoconjugates to enhance the cell-biomaterial interaction. Biomacromolecules 2003 Mar-Apr;4(2):224-31
5．杨健 贝建中。改进高分子材料细胞亲和性的研究—聚(D,L-乳酸)的等离子体处理改性。中国修复重建外科杂志，2001，15(5)：269-272
6. Cai K, Liu W, Li F, et al. Modulation of osteoblast function using poly(D, L-lactic acid) surfaces modified with alkylation derivative of ch itosan. J Biomater Sci Polym Ed 2002; 13(1): 53-66
7. Hosh D, Sengupta J. Molecular mechanism of embryo endometrial adhesion in primates:a future task for implantation biologists.Mol Hum Reprod, 1998, 4(8):733-795
8. Cowles EA, Brailey LL, Gronowicz GA. Integrin-mediated signaling regulatesAP-1 transcription factors and proliferation in osteoblasts. J Biomed Mater Res, 2000, 52(4): 725-737
9. Bloom L, Ingham KC, Hynes RO. Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat Ⅲ13.Mol BioI Cell, 1999, 10:1521-1536
10. Stanford CM, Solursh M, Keller JC. Significant role of adhesion properties
of primary osteoblast-like cells in early adhesion events for chondroitin sulfate and dermatan sulfate surface molecules. J Biomed Mater Res, 1999, 47(3): 345-352
11．王前，郑磊。电刺激促进成骨细胞粘附特性的实验研究。暨南大学学报：自然科学与医学版，2000，21(1)：46-49
12. Bennett JH, Moffatt S, Horton M. Cell adhesion molecules in human osteoblasts: structure and function. Histol Histopathol. 2001 Apr; 16(2): 603-11. Review.
13. Gronthos S, Simmons PJ, Graves SE, Robey PG. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone. 2001 Feb; 28(2): 174-81.
14. Wozniak M, Fausto A, Carron CP, Meyer DM, Hruska KA. Mechanically strained cells of the osteoblast lineage organize their extracellular matrix through unique sites of alphavbeta3-integrin expression. J Bone Miner Res. 2000 Sep; 15(9): 1731-45
15. Webster TJ, Schadler LS, Siegel RW, Bizios R. Mechanisms of enhanced osteoblast adhesion on nanophase alumina involve vitronectin. Tissue Eng. 2001 Jun;7(3):291-301
16. El-Amin SF, Lu HH, Khan Y, Burems J, Mitchell J, Tuan RS, Laurencin CT. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials. 2003 Mar; 24(7): 1213-21
17. Gronthos S, Stewart K, Grave SE, et al. Integrin expression and function on human osteoblast-like cells. J Bone Miner Res, 1997,12:1189-1197
18. Grzesik WJ, Robey PG. Bone matrix RGD glycoproteins-immunolocalization and interaction with human primary osteoblastic bone cells in vitro.J Bone Miner Res, 1994, 9: 487-496
19. Lacouture ME, Schaffer JL, Klickstein LB. A comparison of type I collagen, fibronectin, and vitronectin in supporting adhesion of mechanically strained osteoblasts. J Bone Miner Res. 2002
Mar; 17(3): 481-92.
20. Malaval L, Chenu C, Delmas PD, et a.l.Maladies metaboliques osseuse de l'adulte. in: Kuntz D, ed. Flammarion Medecine Sciences, Paris, 1996: 17-35
21. Puleo DA, Bizios R. RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin mediated adhesion. Bone, 1991, 12: 271-276
22. Gronowicz G, McCarthy MB. Response of human osteoblasts to implant materials: Integrin-mediated adhesion. J Orthopaed Res, 1996, 14: 878-887
23. Woolley DE, Tetlow LC, Adlam DJ, Gearey D, Eden RD. Electrochemical monitoring of cell behaviour in vitro: a new technology. Biotechnol Bioeng 2002 Mar 30; 77(7): 725-33
24. Owali K, Goto M, Ikariyama Y, et al. Optical immuno-sensing for IgG. Sensors Actuators B, 1993, 13-14: 7233-7234
25. MacDonald DE, Deo N, Markovic B, Stranick M, Somasundaran P. Adsorption and dissolution behavior of human plasma fibronectin on thermally and chemically modified titanium dioxide particles. Biomaterials 2002 Feb; 23(4): 1269-79
26．周红星。骨组织工程中种子细胞和材料的粘附。第三军医大学学报，2003，25(2)．-173-175
27. Goessl A, Golledge SL, Hoffman AS. Plasma lithography--thin-film patterning of polymers by RF plasma polymerization Ⅱ: Study of differential binding using adsorption probes. J Biomater Sci Polym Ed, 2001; 12(7):739-53
28. Sorribas H, Padeste C, Tiefenauer L. Photolithographic generation of protein micropatterns for neuron culture applications. Biomaterials. 2002 Feb; 23(3): 893-900
29. Sigrist H. Surface immobilization of biomolecules by light. Optic Eng, 1995, 34(8): 2339-2348
30. Allara DL. Critical issues in applications of self assembled monolayers. Biosensors Bioelectron, 1995, 10: 771-783
31. Hodneland CD, Lee YS, Min DH, Mrksich M. Selective immobilization
of proteins to self-assembled monolayers presenting active site-directed capture ligands. Proc Natl Acad Sci U S A. 2002 Apr 16; 99(8): 5048-52.
32. Boltovets PN, Diachenko NS, Snopok BA, Shirshov IuM. Method of orienting immobilized proteins on gold surface sensory modified with rhodanide. Ukr Biokhim Zh, 2002 Mar-Apr; 74(2): 51-5
33. Sorribas H, Padeste C, Tiefenauer L. Photolithographic generation of protein micropatterns for neuron culture applications. Biomaterials. 2002 Feb; 23(3): 893-900
34. Sumper M. A phase separation model for the nanopatterning of diatom biosilica. Science. 2002 Mar 29; 295(5564): 2430-3.
35. Blawas AS, Reichert WM. Protein patterning. Biomaterials. 1998 Apr-May; 19(7-9): 595-609.
36. Nann T, Kielmann U, Dietrich C. Electrochemical metallization of self-assembled porphyrin monolayers. Anal Bioanal Chem, 2002 Apr; 373(8):749-53
37. El-Amin SF, Lu HH, Khan Y, et al. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials, 2003 Mar, 24(7): 1213-21
38. Saadeh PB, Khosla RK, Mehrara BJ et al. Repair of a critical size defect in the rat mandible using allogenic type Ⅰ collagen. J Craniofac Surg, 2001, 12(6): 573-9
39. Vilamitjana-Amedee J, Bareille R, Rouais F, Caplan AI. Harmand MF. Human bone marrow stromal cells express an osteoblastic phenotype in culture. In Vitro Cell Dev Biol Anim, 1993, 29A(9): 699-707
40. Roehlecke C, Witt M, Kasper M, et al. Synergistic effect of titanium alloy and collagen type Ⅰ on cell adhesion, proliferation and differentiation of osteoblast-like cells. Cells Tissues Organs, 2001; 168(3): 178-87
41. Geissler U, Hempel U, Wolf C, Scharnweber D, et al. Collagen type I-coating of Ti6A14V promotes adhesion of osteoblasts. J Biomed Mater Res, 2000, 15; 51(4): 752-60
42. Tatsumi J, Kurihara N, Kanai A, et al. Clinical application of sintered bone (Ⅱ). Effects of collagen coating on implant materials in vitro Nippon Shishubyo Gakkai Kaishi 1989 Mar; 31(1): 200-12
43. Nagai M, Hayakawa T, Fukatsu A, et al. In vitro study of collagen coating of titanium implants for initial cell attachment. Dent Mater J 2002 Sep; 21(3): 250-60
44. Anselme K. Osteoblast adhesion on biomaterials. Biomaterials, 2000, 21: 667-681
45. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol, 1999, 181: 67-73
46. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol, 1999, 181(1): 67-73
47. Mizuno M, Kuboki Y. Osteoblast-Related Gene Expression of Bone Marrow Cells during the Osteoblastic Differentiation Induced by Type I Collagen. J Biochem (Tokyo), 2001, 129: 133-138
48. Moursi AM, Damsky CH, Lull J, Zimmerman D, Doty SB, Aota S, Globus RK. Fibronectin regulates calvarial osteoblast differentiation. J Cell Sci. 1996 Jun; 109(Pt 6): 1369-80
49. Winnard RG, Gerstenfeld LC, Toma CD, Franceschi RT. Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts.J Bone Miner Res. 1995 Dec; 10(12): 1969-77
50. Nordahl J, Mengarelli-Widholm S, Hultenby K, Reinholt FP. Ultrastructural immunolocalization of fibronectin in epiphyseal and metaphyseal bone of young rats. Calcif Tissue Int. 1995 Dec; 57(6): 442-9.
51. Garcia AJ, Ducheyne P, Boettiger D.Effect of surface reaction stage on fibronectin-mediated adhesion of osteoblast-like cells to bioactive glass. J Biomed Mater Res, 1998, 40(1): 48-56
52. Toesca A, Pagnotta A, Specchia N. Characterization of human bone cells
in culture. Ital J Anat Embryol 2001, 106(1): 13-26
53. C. H. Damsky, Extracellular matrix-integrin interactions in osteoblast function and tissue remodeling. Bone, 1999(25), 95-96
54. Liu S. Heparin/heparan sulfate(HP/HS)interacting peotein (HIP) supports cell attachment and selective, high affinity binding of HP/HS.J Biol Chem, 1997, 272: 25856-25862
55. Massia SP, Hubbell JA. Immobilized amines and basic aminoacide as mimetic heparin-binding domains for cell surface proteoglycan-mediated adhesion. J Biol Chem, 1992, 267: 10133-10141
56. Helfrich MH, Nesbitt SA, Lakkakorpi PT, et al. Beta 1 integrins and osteoclast function: involvement in collagen recognition and bone resorption. Bone, 1996, 19(4): 317-28
57. Shibata Y, Hosaka M, Kawai H, Miyazaki T. Glow discharge plasma treatment of titanium plates enhances adhesion of osteoblast-like cells to the plates through the integrin-mediated mechanism. Int J Oral Maxillofac Implants, 2002, 17(6): 771-7
58. Wong JY, Weng Z, Moll S, Kim S, Brown CT. Identification and validation of a novel cell-recognition site (KNEED) on the 8th type Ⅲ domain of fibronectin. Biomaterials 2002 Sep; 23(18): 3865-70
59. Dettin M, Conconi MT, Gambaretto R, Pasquato A, Folin M, Di Bello C, Parnigotto PP. Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials. J Biomed Mater Res, 2002, 60(3): 466-71
60. Olbrich KC, Andersen TT, Blumenstock FA, et al. Surfaces modified with covalently-immobilized adhesive peptides affect fibroblast population motility. Biomaterials, 1996, 17(8): 759-64
61. LebaronRG, Athanasiou KA. Extracellular matrix cell adhesion peptide: functional applications in orthopedic materials. Tiss Eng, 2000, 6(2): 85-103
62. Massia SP, Hubbell JA. Covalently attached GRGD on polymer surfaces promotes biospecific adhesion of mammalian cells. Ann N Y Acad Sci
1990;589:261-70
63. Chinn JA, Sauter JA, Phillips RE, et al. Blood and tissue compatibility of modified polyester:thrombosis, inflammation and healing. J Biomed Mater Res, 1998, 39: 130-140
64. Fujisawa R, Mizuno M, Nodasaka Y, Kuboki Y. Attachment of osteoblastic cells to hydroxyapatite crystals by a synthetic peptide (Glu7-Pro-Arg-Gly-Asp-Thr) containing two functional sequences of bone sialoprotein. Matrix Biol, 1997,16(1):21-8
65. Kouvroukoglou S, Dee KC, Bizios R, et al. Endothelial cell migration on surfaces modified with immobilized adhesive peptides. Biomaterials, 2000, 21: 1725-1733
66. Maheshwari G, Brown G, Douglas A, et al. Cell adhesion and motility depend on manoscale RGD clustering. J Cell Science, 2000, 113: 1677-1686
67. Lebaron RG, Athanasiou KA. Extracellular matrix cell adhesion peptides: functional applications in orthopedic materials. Tissue engineering, 2000, 6: 85-103
68. Shah AK, Lazatin J, Sinha RK, et al. Mechanism of BMP-2 stimulated adhesion of osteoblastic cells to titanium alloy. Biol Cell 1999(91):131-142
69. Zheng J, Ito Y, Imanishi Y. Cell growth on immobilized cell-growth factor. 10. Insulin and polyallylamine co-immobilized materials. Biomaterials 1994 Oct; 15(12): 963-8
70. Ito Y, Inoue M, Liu SQ, Imanishi Y. Cell growth on immobilized cell growth factor. 6. Enhancement of fibroblast cell growth by immobilized insulin and/or fibronectin. J Biomed Mater Res. 1993, 27(7):901-7.
71. Liu SQ, Ito Y, Imanishi Y. Cell growth on immobilized cell growth factor. 9. Covalent immobilization of insulin, transferrin, and collagen to enhance growth of bovine endothelial cells. J Biomed Mater Res. 1993 Jul; 27(7): 909-15.
72．郑磊 颜晓慧。碱性成纤维细胞生长因子对成骨细胞粘附特性影响的
实验研究。中国修复重建外科杂志，2000，14(5)：305-307
73. Molitoris BA. Putting the actin cytoskeleton into perspective:pathophysiology of ischemic alteration. Am J Physiol, 1997, 272(4Pt2): 430-433
74. Burridge K, Fath K. Focal contacts-transmembrane links between the extracellular matrix and the cytoskeleton. Bioessays, 1989, 10: 104-108
75. Burridge K, Fath K. Focal contacts-transmembrane links between the extracellular matrix and the cytoskeleton. Bioessays, 1989,10:104-108
76. Yamada KM and Geiger B. Molecular interactions in cell adhesion complexes. Curr Opin Cell Bio1, 1997(9), 76-85
77. Sutherland JD, Witke W. Molecular genetic approaches to undersdanding the actin cytoskeleton. Curr Opin Cell Biol, 1999, 11: 142
78. Janmey PA. The cytoskeleton and cell signaling-component localization and mechanical coupling. Physiol Rev, 1998, 78: 763
79. Alberts B, Bray D, Lewis J et al. Molecular Biology of the Cell, chapter 16. 3rd edition. New York and London: Garland publishing 1994: 787
80. Lombardi T, Samson J, Muhlhauser J et al. Expression of intermediate filaments and actins in human dental pulp and embryonic dental papilla. Anat Rec, 1992, 234: 587
81. Pollard TD, Cooper JA. Actin and actin-binding proteins:a critical eveluation of mechanisms and functions. Annu Rev Biochem, 1986, 55: 987
82. Garcia AJ and Boettiger D. Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling. Biomaterials, 1999(20): 2427-2433
83. El-Amina SF, Lua HH, Khan Y, et al. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering. Biomaterials, 2003(24): 1213-1221
84. Gullberg-D, Gehlsen-KR, Turner-DC, et al. Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta l integrins in cell--collagen interactions:
identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I. EMBO-J, 1992; 11(11): 3865-3873
85. Malek-Hedayal S, Rome LH. Cloning and sequence of the cDNA encoding the rat oligodendrocyte β_1 integrin subunit. Gene, 1995; 158:287-290
86. Bosman F. Integrins: cell adhesives and modulators of cell function. Histochem J, 1993; 25: 469-477
87. Miyamoto S, Teramoto H, Goso O, et al. Integrins function: molecular hierarchies of cytoskeletal and signal molecules. J Cell Biol, 1995; 131: 791-805
88. Shibata Y, Hosaka M, Kawai H, Miyazaki T. Glow discharge plasma treatment of titanium plates enhances adhesion of osteoblast-like cells to the plates through the integrin-mediated mechanism. Int J Oral Maxillofac Implants, 2002, 17(6): 771-7
89. Kornberg LJ, Earp TS, Turner CE, et al. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of β_1 integrins. Proc Natl Acad Sci, 1991; 88: 8392-8396
90. Novella A, Bergamaschi G, Canale C, et al. Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM-CSF and role of the Bcr gene. Br J Haematol, 1997; 98(3): 621-626
91. Carvalho RS, Scott JE, Suga DM and et al. Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone. J Bone Miner Res, 1994(9):999-1011
92. Saadiq F. El-Amina, d, Mohamed Attawiaa, et al. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.Journal of Orthopaedic Research, 2002(20):20-28
93. Lacouture ME, Schaffer JL, Klickstein LB.A comparison of type I collagen, fibronectin, and vitronectin in supporting adhesion of mechanically strained osteoblasts. J Bone Miner Res 2002 Mar; 17(3): 481-92
94. Shibata Y, Hosaka M, Kawai H, Miyazaki T. Glow discharge plasma treatment of titanium plates enhances adhesion of osteoblast-like cells to the plates through the integrin-mediated mechanism. Int J Oral Maxillofac Implants, 2002, 17(6): 771-7
95. Cowles EA, Brailey LL, Gronowicz GA. Integrin-mediated signaling regulates AP-1 transcription factors and proliferation in osteoblasts. J Biomed Mater Res 2000 Dec 15;52(4):725-37
96. Xie L, Clunn GF, Lymn JS, Hughes AD. Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen. Cardiovasc Res 1998 Aug; 39(2): 475-84
97. Rowin ME, Whatley RE, Yednick T, et al. Intracellular calcium requirements for β1 integrin activation. J Cell Physio, 1998(175) 193-202
98. Schwartz MA. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J cell Bio, 1993(120): 1003-1010
99. Takeichi M. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol, 1995(7): 619-627
100. Yamada KM and Geiger B. Molecular interactions in cell adhesion complexes. Curr Opin Cell Biol, 1997(9): 76-85
101. Okasaki M, Takeshita S, Kawai S, and et al. Molecular cloning and characterization of OB-cadherin, a new member of cadherin family expressed in osteoblasts. J Biol Chem, 1994 (269): 12092-12098
102. Cheng SL,Lecanda F, Davidson MK and et al. Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation. J Bone Miner Res 1998 (13): 633-644
103. Hay E, Lemonnier J, Modrowski D, N-and E-cadherin mediate early human calvaria osteoblast differentiation promoted by bone morphogenetic protein-2. J Cell Physiol 2000 Apr; 183(1): 117-28
104. Lecanda F, Cheng SL, Shin CS, Differential regulation of cadherins
by dexamethasone in human osteoblastic cells J Cell Biochem 2000 Apr; 77(3): 499-506
105. Cheng SL, Lecanda F, Davidson MK, et al. Human Osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation. J Bone Miner Res, 1998, 13: 633-644
106. Delannoy P, Lemonnier J, Hay E, et al. Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts. Exp Cell Res, 2001, 269(1): 154-61
107. Cheng SL, Lecanda F, Davidson M, et al. Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation. J Bone Miner Res, 1998, 13(4): 633-644
108. Stanford CM, Solursh M, Keller JC. Significant role of adhesion properties of primary osteoblast-like cells in early adhesion events for chondroitin sulfate and dermatan sulfate surface molecules. J reed Mater Res, 1999, 47(3): 345-352
109. Talts U, Kuhn U, Roos G, et al. Modulation of extracellular matrix adhesiveness by neurocan and identification of its molecular basis. Exp Cell Res, 2000, 259(2): 378-368
110. Cardin AD, Weintraub HJ. Molecular modeling of protein glycosaminoglycan interactions. Arteriosclerosis, 1989, 9: 21-32
111. Dee KC, Andersen TT, Bizios R. Design and function of novel osteoblast adhesive peptides for chemical modification of biomaterials. J Biomed Mater Res, 1998, 40: 371-373
112. Craig SW, Johnson RP. Assembly Of focal adhesions:progress, paradigms, and portents. Curr Opin Cell Biol, 1996, 8: 74-85
113. Delannoy P, Lemonnier J, Hay E, Modrowski D, Marie PJ. Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts. Exp Cell Res. 2001 Sep 10;269(1):154-61
114. Brunette DM, Kenner GS, Gould TR. Grooved titanium surfaces orient growth and migration of cells from human gingival explants. J Dent Res, 1983, 62(10):1045-8
115. Toader O, John S. Square spiral photonic crystals: robust architecture for microfabrication of materials with large three-dimensional photonic band gaps. Phys Rev E Stat Nonlin Soft Matter Phys. 2002, 66(1 Pt 2):016610.
116. Toader O, John S. Proposed square spiral microfabrication architecture for large three-dimensional photonic band gap crystals. Science, 2001, 292(5519):1133-1135
117. Lange R, Luthen F, Beck U, Rychly J, et al. Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material. Biomol Eng, 2002, 19(2-6): 255-61
118. Linez-Bataillon P, Monchau F, Bigerelle M, et al. In vitro MC3T3 osteoblast adhesion with respect to surface roughness of Ti6A14V substrates. Biomol Eng, 2002 Aug; 19(2-6): 133-41
119. Shelton RM, Rasmussen AC, Davies JE. Protein adsorption at the interface between charged polymer substrata and migrating osteoblasts. Biomaterials, 1988, 9(1): 24-9
120. M. K(?)n(?)nen, M. Hormia, J. Kivilahti, J. Hautaniemi and I. Thesleff, Effect of surface processing on the attachment orientation, and proliferation of human gingival fibroblasts on titanium. J Biomed Mater Res 26(1992), pp. 1325-1341
121. Steele JG, Johnson G, McLean KM, et al. Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. J Biomed Mater Res, 2000, 15; 50(4): 475-82
122. Mikos AG, Lyman MD, Freed LE, et al. Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials, 1994, 15(1): 55-8
123. Hsu SH, Tsai CL, Tang CM. Evaluation of cellular affinity and compatibility to biodegradable polyesters and Type-Ⅱ collagen-modified scaffolds using immortalized rat chondrocytes. Artif Organs, 2002, 26(7): 647-58
124. Ying P, Jin G, Tao Z. MC3T3-El osteoblasts adhesion to micropatterned surfaces. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2002, 19(3): 370-3
125. Hasenbein ME, Andersen TT, Bizios R. Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts. Biomaterials, 2002, 23(19): 3937-42
126. Matsuda T, Sugawara T. Development of surface photochemical modification method for micropatterning of cultured cells. J Biomed Mater Res, 1995, 29(6): 749-56
127. DeFife KM, Colton E, Nakayama Y, et al. Spatial regulation and surface chemistry control of moncyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces. J Biomed Mater Res, 1999, 45: 148-154
128. Fortier LA, Nixon AJ, Williams J, et al. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res, 1998, 59(9): 1182-1187
129. Friedenstein AJ. Precursor cells of mechanocytes. Int Rev Cytol, 1976, 47: 327
130. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997, 276: 71-75
131. Kitano Y, Radu A, Shabban A, et al. Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. Exp Hematol, 2000, 28(12): 1460-1469
132. Qiu QQ, Ducheyne P, Ayyaswamy PS. Bioactive, degradable composite microspheres: effect of filler material on surface reactivity. Ann
N Y Acad Sci, 2002, 974: 556-564
133. Saito N, Okada T, Horiuchi H, Murakami N, Takahashi J, Nawata M, Ota H, Nozaki K, Takaoka K. A biodegradable polymer as a cytokine delivery system for inducing bone formation. Nat Biotechnol, 2001, 19(4): 332-335
134．熊卓，孙磊。骨组织工程聚左旋乳酸多孔框架快速成形研究。清华大学学报：自然科学版，2002，42(2)：157-160
135. Wake MC, Patrik CW, Mikos AG. Pore morphology affects on the fibrovascular tissue growth in porous polymer substrates. Cell Transplant, 1994, 3(4): 339-343
136．龚海鹏，钟映辉，公衍道，等。多聚赖氨酸改性壳聚糖对神经细胞的作用。生物物理学报，2000，16(3)：553-561
137. Petite H, Viateau V, Bensaid W, et al. Tissue-engineered bone regeneration. Nat Biotechnol, 2000, 18(9): 959-963
138. Pri Chen S, Pitaru S, Lokiec F, et al. Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture. Bone, 1998, 23: 111-121
139. Mizuno M, Kuboki Y. Osteoblast-Related Gene Expression of Bone Marrow Cells during the Osteoblastic Differentiation Induced by Type I Collagen. J Biochem (Tokyo), 2001, 129: 133-138
140. Gronthos S, Simmons PJ, Graves SE, Robey PG. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone 2001 Feb; 28(2): 174-181
141. Tjia JS, Aneskievich BJ, Moghe PV. Substrate-adsorbed collagen and cell secreted fibronectin concertedly induce cell migration on poly(lactide-glycolide) substrates. Biomaterials 1999 Dec; 20(23-24): 2223-33
142．韩君，陈槐卿。骨组织工程的种子细胞—骨髓基质细胞的研究进展。国外医学生物医学工程分册，2001，24(1)：12-15
143. Goshima J, Goldberg VM, Caplan AA. The osteogenic potential of culture expanded rat marrow phosphate ceramic block..Clin Orthop, 1991, 262(2): 298
144. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1. J Cell Physiol, 1996, 166(3): 585
145. Pri-Chen S, Pitaru S, Lokiec f, et al. Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone treated human bone marrow derived bone-like cells in culture. Bone, 1998, 23(2); 111
146. Gronthos S, Stewart K, Graves SE, et al. Integrin expression and function on human osteoblast-like cells. J Bone Miner Res, 1997, 12(8): 1189-1197
147．李桂臣 刘永锋。低温保存人肝细胞Na~∧+／H~∧+交换与细胞内钙和细胞活力的关系。中华实验外科杂志，2001，18(1)：80-81
148．王春梅，黄晓峰，臧益民。钙荧光探针及其在医学研究中的应用。第四军医大学学报，1997，18(12)：64-66
149. Goldstein SA, Patil PV, Moalli MR. Perspectives on tissue engineering of bone. Clin Orthop 1999 Oct; (367 Suppl): S419-23
150. Vacanti CA, Bonassar LJ. An overview of tissue engineered bone. Clin Orthop 1999 Oct; (367 Suppl): S375-81
151. Frost HM, et al. Tetracycline staining of newly forming bone and mineraling cartilage in vivo. Stain Technol, 1960(35):135
152．徐小良，王坤正，吕荣。四环素荧光标记不脱钙骨甲酯包埋法。临床与实验病理学杂志，2001，17(2)：173-174