Preparation of Cell-Specific Scaffolds by Peptide-Grafting for Bone Regeneration

本研究採用冷凍交換法 （freeze-extraction） 與冷凍膠化法（freeze-gelation）來製備具有高孔隙度的組織工程基材。以冷凍法製備組織工程基材時，孔洞結構在高分子溶液冷凍後即出現， 但須採用適當的方法來避免多孔結構在溶劑移除的階段中被破壞，方能保留此結構於基材中，本研究所使用的方式是在高分子溶液的冷凍環境下，溶劑與非溶劑進行交換（extraction）或是使高分子產生膠化（gelation），於是，基材便可以在室溫下進行乾燥而不至於使多孔結構塌陷。 與傳統用的冷凍乾燥法相比較，本研究所開發的方法在能源與時間上的花費較經濟、較不易有溶劑殘留、也較有利於大規模生產，此外，高沸點溶劑也可應用這個方法製備多孔基材。在本研究中，幾丁聚醣 (chitosan)、海藻酸鈉 (alginate)、聚乳酸 (PLA) 和聚乳酸-甘醇酸 (PLGA) 皆可藉由冷凍交換法或冷凍膠化法，成功地製備出可用於組織工程的多孔基材。 經由醯胺鍵形成反應，可將數種胺基酸序列 (RGDS、KRSR和FHRRIKA) 接枝於幾丁聚醣基材上。 研究中以傅立葉轉換紅外線光譜 (FTIR) 來確定接枝程序的完成，並以胺基酸分析儀得知所接上的胺基酸濃度約為10-12。細胞對改質後幾丁聚醣的貼附性較未改質前有顯著提升，將RGDS接枝的幾丁聚醣基材用來培養老鼠骨瘤細胞 (ROS，rat osteosarcoma)時，基材中的細胞密度較未改質的幾丁聚醣基材為高，也由於細胞數量的提升， RGDS接枝的幾丁聚醣基材中，細胞更容易發展出骨化的組織。KRSR與FHRRIKA的接枝則使幾丁聚醣基材對類似骨母細胞的細胞 (osteoblastic cell) 具有專一性，對老鼠骨瘤細胞的貼附具有促進的效果，但對人類皮膚纖維母細胞 (human fetal skin fibroblast) 則是無效的；其對牙周纖維母細胞 (periodontal fibroblast)，貼附之促進效果雖然並不及RGDS接枝基材。 但培養於KRSR與FHRRIKA改質基材上的牙周纖維母細胞，卻比培養在RGDS改質基材上的牙周纖維母細胞，在成骨作用的指標上有著更顯著的表現。上述結果顯示KRSR與FHRRIKA的接枝會使幾丁聚醣基材具有對骨類細胞的選擇性，更適於用來促進骨骼組織的再生。 由於聚乳酸 (PLLA) 缺乏可直接進行化學改質的官能基，故不易將氨基酸序列接枝於其上，本研究利用電漿將聚乳酸活化，再將胺基酸序列固定於聚乳酸基材上。研究中使用傅立葉轉換紅外線光譜與表面分析電子能譜儀 (ESCA) 來證實接枝反應的發生， 並以胺基酸分析儀得知所接上的胺基酸濃度在有效範圍內。在聚乳酸基材上接枝的胺基酸序列所產生之效用與前述幾丁聚醣基材相仿：RGDS的固定使聚乳酸基材與細胞間的親合性大幅增加，而KRSR與FHRRIKA的固定則使其具有對骨類細胞的專一性。 本研究也提出一數學模式，嘗試解釋老鼠骨瘤細胞在幾丁聚醣及聚乳酸基材中的貼附與生長情形。以此模式分析實驗結果，可推論出在幾丁聚醣基材中，RGDS接枝對於細胞在材料上貼附的促進性是改質基材具有較高細胞密度的主要原因，而細胞成長速度並未改變。在聚乳酸基材中，RGDS接枝則同時促進了細胞貼附與成長。此外，此模式可描述培養時間較長時，細胞密度趨於定值的趨勢，造成此現象的原因，可能是基材內空間的限制，或細胞阻擋孔洞造成養分供給不足。Freeze-extraction and freeze-gelation methods are presented in this thesis which can be used to prepare highly porous scaffolds. The porous structure was generated after freeze of a polymer solution, following that either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the traditional freeze-drying method, the presented methods are time and energy saving, with less residual solvent, and easier to scale-up. Besides, by the methods presented, the limitation is lifted so only solvent with low boiling point can be used for scaffold preparation. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated. Chitosan scaffolds were modified with peptides, RGDS (Arg-Gly-Asp-Ser), KRSR (Lysine-Arginine-Serine-Arginine) and FHRRIKA (Phenylalanine- Histidine-Arginine-Arginine-Isoleucine-Lysine-Alanine), via an amide-bond forming reaction between amino groups in chitosan and carboxyl groups in peptides. Successful immobilization was verified with FTIR spectroscopy, and the immobilized amount was determined with an amino acid analyzer to be in the order. The RGDS immobilization can enhance the attachment of cells onto the chitosan, resulting in cells with higher density attached to the RGDS-modified scaffold than to the unmodified scaffold. Consequently, when being applied to culture of ROS (rat osteosarcoma cells), more cells were on the RGDS-modified scaffold than on the unmodified scaffold, which tended to form bone-like tissues. The immobilizations of KRSR and FHRRIKA made the chitosan scaffolds specific to osteoblastic cells, promoting attachment of ROS cells but ineffective on human fetal skin fibroblasts. For PF (periodontal fibroblast) cells, the graft of KRSR and FHRRIKA also increased the initial density of attached cells, although less effectively than the graft of RGDS did. However, the PF cells cultured on the KRSR and FHRRIKA immobilized chitosan expressed more significant markers in osteoconduction. The grafted KRSR and FHRRIKA might induce the attachment of the osteoblastic subgroup in the PF cells or make the non-osteoblastic subgroup in PF cells transform to osteoblastic cells. The results suggested that the immobilization of KRSR and FHRRIKA could make chitosan scaffolds osteoblastic cell specific and more suitable for regeneration of bones. The peptides were also grafted on the PLLA scaffolds with the plasma grafting technique. The successful graft was confirmed with FTIR spectroscopy and ESCA, and the graft amount was determined with an amino acid analyzer. The grafted RGDS enhanced cell attachment and the grafted KRSR and FHRRIKA had specific effects on osteoblastic cells, just like what was observed for chitosan scaffolds. In the last part, a mathematical model was proposed to describe the attachment and growth of ROS cells in scaffolds. For chitosan scaffolds, it revealed that the enhancement on the initial cell attachment by the grafted RGDS should be the major reason for the observed effect and the cell doubling time remains changeless with different modification. Besides, the model can well describe the occurrence of a plateau in cell density at long culture time, which might be caused by the space limitation in the scaffold. For PLLA scaffolds, the graft of RGDS can enhance not only the initial attached cell number but also the cell growth rate.CONTENTS ABSTRACT I ABSTRACT (in Chinese) V ACKNOWLEDGEMENT (in Chinese) VII CONTENTS IX FUGURE LIST XV TABLE LIST XXVII EQUATION LIST XXIX Chapter I Introduction 1 Chapter II Literature Survey 5 2.1 Tissue Engineering 5 2.1.1 The Role of Tissue Engineering 5 2.1.2 Triangle of Tissue Engineering 7 2.2 Scaffolds 10 2.2.1 Biocompatibility of Scaffolds 10 2.2.2 Biodegradable Polymer 12 2.2.3 Scaffold Preparation 20 2.2.4 Phase Separation 20 2.3 Signals 21 2.3.1 RGD 25 2.3.2 KRSR 27 2.3.3 FHRRIKA 28 2.4 Cells 29 2.4.1 Rat Osteosarcoma Cells (ROS 17/2.8 Cells) 29 2.4.2 Periodontal Fibroblast Cells (PF Cells) 29 2.5 Surface Modification of Scaffolds 30 2.6 Tissue Engineering for Bones 33 2.6.1 Bone Engineering 33 2.6.2 Bone Loss Caused by Periodontal Diseases 35 2.6.3 Biochemical Markers of Osteoconduction 36 Chapter III Materials and Experimental Procedures 41 3.1 Preparation of Scaffolds by Freeze-Gelation 41 3.1.1 Preparation of Chitosan Scaffolds 41 3.1.2 Preparation of Alginate Scaffolds 41 3.2 Preparation of Scaffolds by Freeze-Extraction 42 3.2.1 Preparation of PLLA Scaffolds 42 3.2.2 Preparation of PLGA Scaffolds 43 3.3 Freeze-Drying 43 3.4 Characterization of Scaffolds 44 3.4.1 Determination of the Porosity of Scaffolds 44 3.4.2 Pore Size Distribution 45 3.4.3 SEM Analysis 45 3.5 Preparation of Chitosan and PLLA Films 46 3.5.1 Preparation of Chitosan Films 46 3.5.2 Preparation of PLLA Films 46 3.6 Peptide Grafting 46 3.6.1 Peptide Grafting on Chitosan 46 3.6.2 Peptide Grafting on PLLA 47 3.7 Characterization of Peptide Grafted Scaffolds 47 3.7.1 FTIR-ATR Spectroscopy 47 3.7.2 Amino Acid Analysis 48 3.7.3 Electron Spectroscopy for Chemical Analysis (ESCA) 48 3.8 Cell Culture and In Vitro Mineralization 48 3.8.1 Cell Attachment to the Polymer Films 48 3.8.2 Cell Culture with Scaffolds 49 3.8.3 Determination of the Number of Cells Attached to Scaffolds 50 3.8.4 SEM Analysis for Samples with Cells 50 3.8.5 In Vitro Mineralization 51 3.8.6 ESCA for Samples with Cells 51 3.9 Histochemistry and Immunochemistry Staining 51 3.9.1 Frozen Section 51 3.9.2 ALPase Staining 52 3.9.3 OPN (Osteopontin) and BSP (Bone Sailoprotein) Staining 52 3.9.4 Von Kossa Staining 54 Chapter IV Preparation of Porous Scaffolds 57 4.1 Scaffolds Prepared by Freeze-Extraction and Freeze-Gelation Methods 59 4.2 Formation of Porous Structure 67 4.3 Advantages of Freeze-Extraction and Freeze-Gelation 69 Chapter V RGDS Immobilization on chitosan Scaffolds 73 5.1 Creation of Biomimetic Materials by Attaching Cell Adhesive Peptides 73 5.2 Immobilization of Peptides on Chitosan Scaffolds by Amide Bond Formation Reaction 75 5.3 Effect of RGD Immobilization on Cell Culture 81 5.4 The Mineralization Behavior of ROS Cells in RGDS Modified Chitosan Scaffolds 87 Chapter VI RGDS Immobilization on PLLA Scaffolds 93 6.1 Modification of PLA 93 6.2 Fabrication of Porous Scaffolds 94 6.3 Plasma Grafting of Peptides on PLLA 95 6.4 Effect of RGD Grafting on Cell Culture 100 6.5 Mineralization of Cultured ROS Cells 105 Chapter VII KRSR and FHRRIKA Immobilization on Scaffolds 109 7.1 Preparation of Functional Scaffolds 109 7.2 Characterization of KRSR and FHRRIKA Immobilized Porous Scaffolds 110 7.3 Effects of KRSR and FHRRIKA Immobilization on Cell Culture 114 7.4 Subpopulations of PF Cells in KRSR and FHRRIKA Immobilized Porous Scaffolds 132 7.5 Histochemistry and Immunochemistry Stainings on the PF Cells Cultured on the Peptide Grafted Films 137 7.6 Histochemistry and Immunochemistry Stainings on the PF Cells Cultured with the Peptide Grafted Scaffold 149 Chapter VIII Analysis of Cell Growth in Scaffolds 157 8.1 Development of the Mathematical Model 157 8.2 Verification and Application of the Mathematical Model 160 8.3 Analysis of the Growth of ROS Cells in PLLA Scaffolds 167 8.4 Mechanism of the Enhancement of Cell Density by Peptide Grafting 170 Chapter IX Conclusions and Recommendations 171 9.1 Conclusions 171 9.2 Recommendations 173 REFERENCE 177 APPENDIX 205 Appendix A Notations 205 Appendix B Calculations of the Surface Area in Scaffolds 210 Appendix C The efficiency of peptide grafting 211 AUTOBIOGRAPHY (in Chinese) XXXI RESUME XXXIII RESUME (in Chinese) XXXI