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研究生：	吳孟倩 Wu, Meng-Chian
論文名稱：	評估聚異丙基丙烯醯胺接枝幾丁聚醣之溫感水膠結合間質幹細胞與高濃度血小板血漿對於軟骨再生之成效 The Chondrogenic Effect of Thermoresponsive Poly(N-isopropylacrylamide)-g-chitosan Hydrogel Combined Mesenchymal Stem Cells and Platelet-Rich Plasma for Cartilage Regeneration
指導教授：	葉明龍 Yeh, Ming-Long
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2015
畢業學年度：	103
語文別：	中文
論文頁數：	71
中文關鍵詞：	軟骨再生、溫感水膠、間質幹細胞、高濃度血小板血漿
外文關鍵詞：	Cartilage regeneration, Thermoresponsive hydrogel, Mesenchymal stem cells, Platelet-rich plasma
相關次數：	點閱：201 下載：6
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由於透明軟骨的再生能力有限，因此關節軟骨一旦受損便難以自行修復，時至今日關節軟骨的修復仍面臨很大的挑戰。如果不及時治療，受損處在外力反覆的摩擦之下將會生成纖維軟骨，然後發展成關節炎。雖然學者們已經研發出多種修復關節軟骨的方法，但是至今仍然沒有一個明確且有效的治療方式。而軟骨組織工程結合了三維的支架、軟骨化的細胞以及促進軟骨化的信號這三大要素，希望可以提供一種用於修復軟骨組織的方案。
本研究第一部分利用自由基聚合法製備出三種不同比例的聚異丙基丙烯醯胺接枝幾丁聚醣之溫感水膠，希望能利用溫感水膠的特性，在室溫下與間質幹細胞混合之後，藉由手術注射至受損處來修復軟骨。經由傅立葉轉換紅外光譜、親水程度、機械性質、生物相容性以及掃描式電子顯微鏡等測試的初步結果中，得知CSPN05 (幾丁聚醣：聚異丙基丙烯醯胺=1：5) 的性質最適合用於膝關節軟骨的修復。
本研究第二部分是將高濃度血小板血漿與聚異丙基丙烯醯胺接枝幾丁聚醣混合成濃度為50%的溫感水膠，藉由生化的分析來探討高濃度血小板血漿對髕下脂肪墊間質幹細胞的影響。由螢光染色結果發現，高濃度血小板血漿在共培養的第一天就有促使細胞聚集的效果。在細胞活性分析中發現，添加高濃度血小板血漿之後，細胞的活性都遠大於其他兩組。另外，經由DMMB分析與染色結果證實，添加高濃度血小板血漿的組別，其醣胺多醣的表現量明顯高於其他兩組。
本研究第三部分主要是將混合高濃度血小板血漿的溫感水膠注射至兔子的內側股骨髁缺陷處(直徑與深度皆為3 mm)，在術後四週犧牲。藉由巨觀觀察、微電腦斷層掃描進行評估，在添加高濃度血小板血漿的組別中，組織的表面有似透明軟骨的再生同時也有促進骨頭的修復。結果顯示，在骨組織的比值(BV/TV)以及骨小樑的厚度(Tb. Th)方面，都比控制組的表現還要好。
整體而言，聚異丙基丙烯醯胺之溫感水膠與幾丁聚醣接枝後，有促進間質幹細胞聚集的效果。而溫感水膠與高濃度血小板血漿結合之後，對於細胞增生以及骨軟骨的再生有正向且顯著的影響。

Articular cartilage repair has become a significant challenge due to its limited regenerative capability of hyaline cartilage. Once articular cartilage was damaged, the progress of damaged cartilage would turn into osteoarthritis (OA). So far, several treatments are available for OA treatments but no defined conclusions are reported. Cartilage tissue engineering becomes emerging approach for cartilage repairing in recent years. It is the use of a combination of 3-dimensional scaffolds, chondrogenic cells and chondrogenic signals. Among many synthetic polymers, thermoresponsive poly(N-isopropylacrylamide)-g-chitosan hydrogel is exploited for the treatment of articular cartilage defects and has shown that can enhance cell viability, chondrogenic genes expression of MSCs and eventually generate good cartilage. Infrapatellar fat pad derived mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into chondrocytes under suitable culture conditions. In addition, platelet-rich plasma is rich in growth factors which stimulate chondrocytes activity and enhance extracellular matrix biosynthesis and chondrogenic differentiation of MSCs. Therefore, this study examines the chondrogenic effect of combining thermoresponsive hydrogel, MSCs and platelet-rich plasma for cartilage regeneration.
Part I of this study, NIPAAM was grafted to chitosan (CS) using free radical polymerization prepared from different ratio of reactants. This thermoresponsive hydrogel could mix with the MSCs at room temperature then was injected into human body through minimal invasive surgeries. The results from fourier transform infrared (FT-IR) spectroscopy, swelling ratio (SR), dynamic mechanical analysis (DMA), scanning electron microscope (SEM), and MTS assay found that the composition of CSPN05 group (the ratio of NIPAAM to CS was 5 to 1) was most suitable for knee joint cartilage repair.
Part II of this study, PRP and PNIPAAM-g-CS was mixed at a concentration of 50% and then investigated its proliferation and differentiation ability on IFPSCs. The results of live/dead assay showed that PRP had the effect to promote cells aggregated at the first day of co-culture. In the existence of PRP, the cell viability was significantly greater than other groups. In addition, the synthesis of GAG in PRP group was also significantly higher than other groups through Alcian blue staining and dimethylmethyleneblue (DMMB) assay.
Part III of this study, the thermoresponsive hydrogel was injected into the rabbits’ medial femoral condyle defects and sacrificed at 4 weeks after surgery. Based on macroscopic evaluation and micro-CT scanning, PRP could contribute to the regeneration of neo-cartilage and bone repair. The micro-CT results showed the bone volume/tissue volume (BV/TV) ratio and trabecular thickness (Tb. Th) in PRP group was better than control group.
In summary, the PNIPAAM-g-CS hydrogel had a slightly effect on cell aggregation because the presence of CS. When combined with PRP, the PNIPAAM-g-CS hydrogel could promote cell proliferation and osteochondral defect regeneration.

Table of Contents
中文摘要 ..............I
Abstract ............. III
誌謝 .............. V
Table of Contents ............ VII
List of Tables ............ XI
List of Figures ............. XII
Chapter 1: Introduction ........... 1
1.1 Biology and Composition of Articular cartilage ....... 1
1.2 Poly (N-isopropylacrylamide)-g-chitosan hydrogel ...... 4
1.3 Platelet-rich plasma ........... 6
1.4 Infrapatellar fat pad derived mesenchymal stem cells .... 11
1.5 Motivation and Aims ........... 12
1.5.1 Part I: Characterization of PNIPAAM and PNIPAAM-g-CS hydrogel . 12
1.5.2 Part II: In vitro analysis of PNIPAAM-g-CS hydrogel .... 13
1.5.3 Part III: Hydrogel implants for osteochondral defect repair in rabbit model.... 13
Chapter 2: Materials and Methods .......... 14
2.1 Experiment flow chart ........... 14
2.1.1 Characterization of PNIPAAM and PNIPAAM-g-CS hydrogel .. 14
2.1.2 In vitro analysis of PNIPAAM-g-CS hydrogel ..... 14
2.1.3 Hydrogel implants for osteochondral defect repair in rabbit model . 14
2.2 Instruments ............ 16
2.3 Materials ............ 17
2.4 Fabrication of PNIPAAM and PNIPAAM-g-CS hydrogel ..... 19
2.5 Isolation and culture of infrapatellar fat pad derived mesenchymal stem cells
............... 21
2.6 Preparation of platelet-rich plasma ........ 22
2.7 Characterization properties of PNIPAAM and PNIPAAM-g-CS hydrogel.. 23
2.7.1 Surface morphology of the hydrogels ....... 23
2.7.2 LCST measurement ........... 24
2.7.3 Swelling ratio ............ 24
2.7.4 Shrinking ratio ........... 24
2.7.5 Mechanical stiffness .......... 25
2.7.6 Fourier transform infrared (FT-IR) spectroscopy ..... 25
2.7.7 Cell proliferation ........... 26
2.8 In vitro analysis of PNIPAAM-g-CS hydrogel ....... 26
2.8.1 Cell proliferation ........... 27
2.8.2 Cell viability ............ 27
2.8.3 Quantification of GAG content ......... 28
2.8.4 Histological analysis ......... 28
2.9 Hydrogel implants for osteochondral defect repair in rabbit model ... 29
2.9.1 Surgical procedure .......... 29
2.9.2 Macroscopic evaluations ......... 31
2.9.3 Micro-CT evaluations ......... 31
2.10 Statistical analysis ........... 33
Chapter 3: Results ............. 34
3.1 Part I: Characterization of PNIPAAM and PNIPAAM-g-CS hydrogel .. 34
3.1.1 Fabrication of PNIPAAM and PNIPAAM-g-CS hydrogels ... 34
3.1.2 LCST measurement ........... 35
3.1.3 Swelling ratio ............ 35
3.1.4 Shrinking ratio ........... 37
3.1.5 Mechanical stiffness .......... 39
3.1.6 FT-IR spectroscopy ........... 39
3.1.7 Cell proliferation ........... 41
3.2 Part II: In vitro analysis of PNIPAAM-g-CS hydrogel ..... 42
3.2.1 Cell viability ............ 42
3.2.2 Cell proliferation ........... 43
3.2.3 Histological analysis ......... 44
3.2.4 Quantification of GAG content ......... 45
3.3 Part III: Hydrogel implants for osteochondral defect repair in rabbit model .. 46
3.3.1 Macroscopic evaluations ......... 46
3.3.2 Micro-CT evaluations ......... 49
Chapter 4: Discussion ........... 52
4.1 Part I: Characterization of PNIPAAM and PNIPAAM-g-CS hydrogel .. 52
4.2 Part II: In vitro analysis of PNIPAAM-g-CS hydrogel ..... 54
4.3 Part III: Hydrogel implants for osteochondral defect repair in rabbit model .. 56
Chapter 5: Conclusion........... 59
Chapter 6: Limitations and Future works ......... 60
References ............. 61
List of Tables
Table 1. Recent researches of PRP in animal model. ...... 9
Table 2. Composition of the reactants of PNIPAAM-g-CS hydrogel. .. 20
Table 3. Composition of control group and experimental group. ... 27
Table 4. The modified Wayne’s grading scheme. ....... 32
Table 5. The SR results. .......... 36
Table 6. Mann-Whitney U test analysis of SR. ....... 37
Table 7. The SHR results. .......... 38
Table 8. Mann-Whitney U test analysis of SHR. ...... 38
Table 9. Storage modulus of prepared hydrogels. ....... 39
Table 10. The results of the relative GAG content/DNA content. .... 45
Table 11. Mann-Whitney U test analysis of the relative GAG content/DNA content...... 45
List of Figures
Figure 1.Schematic representation of normal articular cartilage. ... 3
Figure 2. Schematic of mechanism of PNIPAAM-gelatin gelation ... 6
Figure 3. Technologic concept of PRP processing....7
Figure 4. Experiment flow chart. .......... 15
Figure 5. Structures of PNIPAAM-g-CS....... 20
Figure 6. Synthetic route for PNIPAAM and PNIPAAM-g-CS by the free radical
polymerization. .......... 21
Figure 7. Procedure of FT-IR analysis. ......... 25
Figure 8. The surgical procedure for knee joint of New Zealand white rabbits. .. 30
Figure 9. Scanning electron micrographs of the hydrogels. ..... 34
Figure 10. The phase transition of thermoresponsive hydrogel. ... 35
Figure 11. Results of the SR analyzed by Mann-Whitney U test. .... 36
Figure 12. Results of the SHR analyzed by Mann-Whitney U test. ... 38
Figure 13. Macroscopic image of shrinking hydrogels at 7th day. .... 39
Figure 14. FT-IR spectra of various hydrogels. ...... 40
Figure 15. MTS assay results of PN group and PNIPAAM-g-CS groups. ... 41
Figure 16. The results of cell viability for PN group, CSPN05 group and CSPN05 with PRP group..... 42
Figure 17. MTS assay results of the hydrogels. ...... 43
Figure 18. The results of Alcian blue stain for the hydrogels. .... 44
Figure 19. Results of the relative GAG content/DNA content analyzed by
Mann-Whitney U test. ......... 46
Figure 20. The images of the gross appearances in PN group and CSPN05 with PRP group at 4 weeks after operation... 47
Figure 21. The scores of gross appearance in in PN group and CSPN05 with
PRP group at 4 weeks after operation. ...... 48
Figure 22. The micro-CT images for bone evaluation in PN group and CSPN05
with PRP group at 4 weeks after operation. ...... 49
Figure 23. Quantification scores of BV/TV of PN group and CSPN05 with PRP
group at 4 weeks after operation. ........ 50
Figure 24. Quantification scores of Tb. Th of PN group and CSPN05 with PRP
group at 4 weeks after operation. ........ 51
Figure 25. The schema illustrated the procedures of MSCs chondrogenesis. .. 55
Figure 26. The schema illustrated the procedures of MSCs chondrogenesis in the scaffold..... 56
Figure 27. A cross-section of articular cartilage stained with toluidine blue.. . 58
                                    

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