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研究生：	謝明佑 Shie, Ming-You
論文名稱：	鈣矽材料對細胞行為之影響及其機制研究 Effect and mechanisms of calcium silicate materials on cell behavior
指導教授：	張憲彰 Chang, Hsien-Chang
共同指導教授:	丁信智 Ding, Shinn-Jyh
學位類別：	博士 Doctor
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	89
中文關鍵詞：	矽、生物活性、細胞生長、整合素、有絲分裂活化蛋白質激酶
外文關鍵詞：	Silicon (Si), bioactivity, proliferation, integrin, MAPK
相關次數：	點閱：131 下載：1
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矽是一種健康骨組織與結締組織必須的微量元素。目前，已發展出多種矽基底材料，如生物活性玻璃、矽取代氫氧基磷灰石、矽取代α–三鈣磷酸鹽、與其他多相型系統。然而，矽誘導細胞功能之最佳濃度並未被明確定義。因此，本研究主要目的為進行矽濃度對於細胞行為影響之探討。研究結果顯示，當MG63骨母細胞培養在含有2 mM與4 mM 矽之培養液中，細胞增生量會隨著培養時間增加而逐漸提升。當細胞培養於含有6 mM矽培養液之細胞量，則隨著培養時間的增加而顯著地減少（P < 0.05）。且細胞於此異常高濃度之矽含量下培養12小時後，其sub-G1相於細胞週期內所佔比例由原本的3.60%增加為43.01%（P < 0.05）。相較於培養於控制組培養液之細胞，培養於含有4 mM矽之培養液後，其第一型膠原蛋白（collagen type I, COL I）及細胞外訊息調節激酶（extracellular signal-regulated kinase, ERK）之基因表現量顯著提升（P < 0.05），並發現培養液中的矽會刺激MG63內ERK的活化。此外，培養於含有4 mM矽之細胞，其鈣化基質量為培養於控制組培養液之細胞的15倍。此研究結果也許有助於設計出具優異生物性質之鈣矽酸鹽基底材料。細胞於材料上的貼附、增生、以及分化行為，會因材料表面特性的差異而有所影響。因此，對於矽誘導提升細胞貼附及增生現象之機制進行探討，有利於拓展二氧化矽基底材料之應用。本研究之目的為藉由將人類間葉幹細胞（human mesenchymal stem cells, MSCs）與人類牙髓細胞（human dental pulp cells, DPCs）貼附於不同矽鈣莫耳比例之鈣矽酸鹽水泥上，對於其整合蛋白（integrin）次單位、磷酸化黏着斑激酶（phosphor-focal adhesion kinase, pFAK）、蛋白質分泌、與有絲分裂活化蛋白質激酶（mitogen-activated protein kinases, MAPKs）之表現進行評估。實驗結果顯示，細胞貼附量、pFAK與全integrin之表現量會隨著水泥內矽含量的增加而提升。且高矽含量之水泥有利於COL I的吸附與α2 integrin的表現；反之，高鈣含量之水泥則有助於纖連蛋白（fibronectin, FN）的吸附，與α5、αv integrins的表現。矽主要是藉由活化MAPK/ERK與p38訊號途徑刺激細胞貼附行為的進行。然而此並未對c-Jun胺基末端激酶（c-Jun NH2-terminal kinase, JNK）造成影響。此研究結果證實細胞貼附於生醫材料表面之機制為，材料成分的差異會影響integrin的表現，進而調節細胞行為。

Silicon (Si), an essential trace element required for healthy bone and connective tissues. The silicon-base material such as bioglass, Si substituted HA (Si-HA), Si substituted α-TCP (Si-α-TCP), and other multiphase systems were developed. The optimal concentration at which Si induces cell functions has not been fully elucidated. In the present study the effects of Si concentration on the biological. Cell proliferation in the presence of 2 mM Si- and 4 mM Si-containing media progressively increased with culture time, whereas that of 6 mM Si treated MG63 cell was significantly (P < 0.05) reduced. The unusually high Si concentration (6 mM) induced a significant (P < 0.05) increase in the sub-G1 phase of cells from the original 3.60% up to 43.01% after 12 h. 4 mM Si treated MG63 cells, but not 6 mM Si treated MG63 cells, showed remarkably enhanced collagen type I (COL I) gene expression and extracellular signal-regulated kinase (ERK) secretion, which were significantly (P < 0.05) higher than those in the control medium. The activation of ERK was also stimulated in MG63 cells by 4 mM Si. Cells cultured in the presence of 4 mM Si were found to have calcium matrix formation on day 7 that was 15-fold greater than that in the control medium. The results obtained in this study may be useful in designing calcium silicate-based materials with optimal biological properties. Therefore, cell attachment, proliferation, and differentiation behaviors on different materials depend on the surface properties of the material. Unraveling the mechanism of Si-induced cell attachment and proliferation enhancement is important to expand the applications of silica-based materials. The purpose of this study was to investigate the responses of three cell types (human mesenchymal stem cells (MSCs) and human dental pulp cells (DPCs)) to calcium silicate cements with different Si/Ca molar ratios. The study evaluated integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels, protein production, and MAPK signaling pathway activity at the cell attachment stage. Increased pFAK and total integrin levels and increased cell attachment were observed upon an increase in cement Si content. Cement with a higher Si content was beneficial for COL I adsorption and α2 integrin expression, whereas cement with a higher Ca content increased fibronectin (FN) adsorption and enhanced α5 and αv integrins. Si stimulated cell adhesion via activation of MAPK/ERK and p38 signaling pathways more effectively than did extracellular Ca, but it did not affect c-Jun NH2-terminal kinase (JNK) activity. These results establish composition-dependent differences in integrin binding as a mechanism regulating cellular responses to biomaterial surfaces.

Contents

Abstract 	i
Chinese Abstract 	ii
Contents 	iii
List of Figures 	vii
List of Tables	xii

Chapter 1   Introduction 	1
1.1 Bone cement	1
1.2 Si effect	1
1.3Silicate-based materials 	3
   1.3.1 Bioglass	4
   1.3.2 Mineral trioxide aggregate	5
   1.3.3 Calcium silicate cement	6
1.4 Cell-material interaction	6

Chapter 2   Materials and methods 	8
2.1 Specimen preparation	8
2.2 Physiccochemical property 	8 
   2.2.1 Roughness and topography 	8
   2.2.2 Zeta potential measurement	9
   2.2.3 Preparation of extract medium	9
   2.2.4 Osmolality measurement	10
   2.2.5 Protein adsorption	10
2.3 In vitro test	10
   2.3.1 Cell culture	10
   2.3.2 Cell attachment and proliferation assays	11
   2.3.3 Cell cycle analysis	12
   2.3.4 Cell death analysis	13
   2.3.5 Cell morphology	14
   2.3.6 Gene expression	14
   2.3.7 Western blot analysis	15
   2.3.8 Bone matrix formation	16
   2.3.9 Fluorescent staining	16
   2.3.10 pH value	17
   2.3.11 Transfection experiments	17
   2.3.12 Collagen and fibronectin synthesis	18
   2.3.13 Anti-adhesion assay	19
   2.3.14 Effect of MAPK inhibitors	19
   2.3.15 ALP	19
2.4 Statistical analysis	20

Chapter 3   Results 	21
3.1 The role of silicon 	21
   3.1.1 Ion concentration 	21
   3.1.2 Osmolality of medium	23
   3.1.3 Cell proliferation	23
   3.1.4 Cell cycle	25
   3.1.5 Cell death analysis	25
   3.1.6 Cell morphology	30
   3.1.7 COL I and ERK expression	30
   3.1.8 Bone matrix formation	35
3.2 Effects of Si/Ca molar ratio	37
   3.2.1 Cell attachment	37
   3.2.2 Actin staining observation	38
   3.2.3 Ion concentration	38
   3.2.4 Cell proliferation	41
   3.2.5 Osteogenic differentiation	42
   3.2.6 Alkaline phosphatase	42
3.3 Unraveling mechanism of Si-induced cell functions	45
   3.3.1 Roughness and Morphology	45
   3.3.2 Protein adsorption	45
   3.3.3 Osmolality measurement	45
   3.3.4 Cell Attachment and Proliferation	50
   3.3.5 pH value 	50
   3.3.6 COL I and FN Synthesis	53
   3.3.7 Integrin expression	53
   3.3.8 Anti-adhesion assay	57
   3.3.9 pFAK expression	57
   3.3.10 MAPK signaling	57
   3.3.11 MAPK inhibitor effect	58

Chapter 4   Discussion 	67

Chapter 5   Conclusions	80

Reference 	81

Curriculum vitae	87





 
List of Figures

Figure 1. Proliferation of MG63 cells treated with culture media containing (A) various Ca ion concentrations and (B) Si ion concentrations at a constant 0.35 mM Ca ion concentration	24
Figure 2. Phase percentage of MG63 cell cycle after culture with various test media for different time points determined using flow cytometry. 	27
Figure 3. (A) Representative diagrams of annexin V binding and propidium iodide uptake of MG63 cells undergoing apoptosis versus necrosis after culture for 6 h. (B) The corresponding quantitative number of cells cultured in various media for 6 h, 12 h, and 24 h. Data are mean ± standard derivation of three independent determinations	28
Figure 4. (A) Phase and (B) immunofluoresence images of MG63 cells cultured in the control, Ex4, or Ex6 medium for 24 h followed by staining with both Ann V (green) and PI (red).	29
Figure 5. In-situ and real-time photographs of MG63 cells in the presence of the control (a-c), Ex4 (d-f), or Ex6 (g-i) medium after culture for 0 h (a, d, g), 12 h (b, e, h), and 24 h (c, f, i). The arrows indicate vacuole formation	32
Figure 6. Col I (A) and ERK (B) gene expression levels of MG63 cells in the presence of the control, Ex4, or Ex6 medium at various culture time points	33
Figure 7. Col I (A), ERK (B) and phosphor-ERK (C) protein expression levels of MG63 cells in the presence of the control, Ex4, or Ex6 medium at various culture time points		34
Figure 8. (A) Alizarin Red S staining of MG63 cells in the presence of the control, Ex4, or Ex6 medium after culture for 7 and 14 days. (B) The corresponding calcium deposit quantification obtained using ImageJ software. 	36
Figure 9. Alamar Blue assay for MG63 cell attachment to various specimens at various time-points. *Statistically significant difference (P < 0.05) from S50C50 as a positive control. 	37
Figure 10. Immunofluorescent staining of MG63 cells cultured on (a) the control, (b) S40C60, (c) S50C50 and (d) S60C40 after 3, 6 and 12 h of seeding. Cells were stained for nuclei (blue) and actin cytoskeleton (red). Original magnification: 200X	39
Figure 11. Variations in (a) Si and (b) Ca ion concentrations of cell culture medium at various time-points. *Statistically significant difference (P < 0.05) between the cement groups	40
Figure 12. Alamar Blue assay for MG63 cell proliferation cultured on the specimens at various time-points. *Statistically significant difference (P < 0.05) from S50C50 as a positive control. 	41
Figure 13. RT-PCR analysis for the bone- related gene expression of different markers (a) COL I, (b) ALP, (c) BSP and (d) OC on various specimens. *Statistically significant difference (P < 0.05) from S50C50 as a positive control. 	43
Figure 14. Alkaline phosphatase (ALP) assay on the culture medium presented as optical density for differentiation of MG63 cultured on various test groups after 3 and 7 days. *Statistically significant difference (P < 0.05) from S50C50 as a positive control		44
Figure 15. Effect of the applied pressures on the roughness and microstructure of the specimen surfaces. (A) Surface roughness (Rz and Ra) of the cement specimens. (B) Surface SEM micrographs of S50C50 subjected to 0.7 MPa or 300 MPa. *, significant difference from S40C60 (P < 0.05). 	47
Figure 16. FN and COL I adsorption on the substrate surfaces for 3 h. *, significant difference from S40C60 (P < 0.05). 	48
Figure 17. Variations in osmolality of cement-immersed medium at different soaking time-points. *, statistically significant difference (P < 0.05) between the cements		49
Figure 18. Total DNA content for attachment and proliferation of (A) MSCs and (B-D) DPCs cultured on the TCP control and various cement surfaces subjected to applied pressures of 0.7, 100, and 300 MPa. Roughness does not significantly alter (P > 0.05) cell attachment and proliferation, but these parameters are affected by surface chemistry.		51
Figure 19. Variations in pH of medium after (A) MSCs and (B-D) DPCs cultured on the TCP control and various cement surfaces for different time-points. *, statistically significant difference (P < 0.05) between the cements. 	52
Figure 20. FN and COL I secreted by (A) MSCs, (B-D) DPCs cultured on various samples for 3 h. Protein expression level values were normalized to β-actin. *, statistically significant difference (P < 0.05) between S40C60. 	55
Figure 21. Analysis of integrin protein expression in (A) MSCs and (B-D) DPCs cultured on CSCs for 3 h. Western blot analyses show integrin expression. Integrin expression was quantified and normalized to actin expression. *, statistically significant difference (P < 0.05) between S40C60. 	56
Figure 22. Effects of anti-integrin α and β antibodies on the attachment of (A) MSCs and (B-D) DPCs on different CSCs and the TCP control for 3 h. Adhesion of cells on the test groups in the medium without antibodies (untreated) was used as the 100% reference level. *, statistically significant difference (P < 0.05) between S40C60	59
Figure 23. FAK phosphorylation by (A) MSCs, (B-D) DPCs cultured on the TCP control and CSCs at different timepoints. The values were normalized to FAK expression. The data show increased pFAK levels on the cement with increased Si content. *, statistically significant difference (P < 0.05) between the cements		60
Figure 24. Analyses of phosphorylated ERK, p38, and JNK expression in MSCs cultured on the TCP control and CSCs for different times before (A) and after (B) the addition of inhibitors. The quantification of protein was normalized to actin. *, statistically significant difference (P < 0.05) between S40C60. 	61
Figure 25. Analyses of phosphorylated ERK, p38, and JNK expression in DPC-1 cultured on the TCP control and CSCs for different times before (A) and after (B) the addition of inhibitors. The quantification of protein was normalized to actin. *, statistically significant difference (P < 0.05) between S40C60. 	62
Figure 26. Analyses of phosphorylated ERK, p38, and JNK expression in DPC-2 cultured on the TCP control and CSCs for different times before (A) and after (B) the addition of inhibitors. The quantification of protein was normalized to actin. *, statistically significant difference (P < 0.05) between S40C60. 	63
Figure 27. Analyses of phosphorylated ERK, p38, and JNK expression in DPC-3 cultured on the TCP control and CSCs for different times before (A) and after (B) the addition of inhibitors. The quantification of protein was normalized to actin. *, statistically significant difference (P < 0.05) between S40C60. 	64
Figure 28. (A) MSCs and (B-D) DPCs attachment and proliferation on various cement surfaces after treatment of PD98059, SB203580, and SP600125 inhibitors. The untreated groups was used as the 100% reference level. *, significant difference from S40C60 (P < 0.05) at the same culture time-point. PD98059 or SB20358 resulted in an decreased cell attachment and proliferation, but not for SP600125. 	65
Figure 29. ALP assay on (A) MSCs and (B-D) DPCs on various cement surfaces after treatment of PD98059, SB203580, and SP600125 inhibitors. The untreated groups was used as the 100% reference level. *, significant difference from S40C60 (P < 0.05) at the same culture time-point. 	66


 
List of Tables


Table 1. Gene-specific primers used in this study.	15
Table 2. Sequences of FN and COL I siRNAs.	18
Table 3. Ion concentrations of Si, Ca, and P elements in various test solutions measured by ICP-AES.	22

                                    

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校內：2018-01-01公開
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