幾丁聚醣、硫酸軟骨素與麩胺酸複合生醫基材之製程探討、性質改良與應用

Chitosan/Chondroitin Sulfate/Glutamic Acid Composite Scaffolds – Investigation of Fabrication Process, Enhancement of Properties and Applications

苑乃義(Nai-Yi Yuan)

指導教授 ： 謝學真

國立臺灣大學/工學院/化學工程學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.00437

摘要

在組織工程研究中，所使用的基材通常須符合基本的要求例如高孔隙度、適當的孔洞大小分佈、適合的機械強度與良好的細胞相容性等等。而新型生醫材料的發展則是針對特殊的功能和需求進行設計和製造，其趨勢乃是結合不同的材料製成複合材料 (composite)，以改善的單一材料的不足。因此，本研究著眼於多孔性的基材製備方法改進與新型複合材料的發展，並進行複合基材的性質測定與細胞的相容性測詴及功能分析。
在第一部分的研究中，比較了快速降溫 (FC) 或緩慢降溫 (SC) 的模式對於以冷凍凝膠法製備多孔狀基材所產生的影響，共製備幾丁聚醣 (CH)、褐藻膠 (AL) 與羧甲基纖維素 (CMC) 等多醣分子的基材。在FC或SC降溫過程中，先測量了溶液的溫度變化，之後測定基材機械性質及利用掃描式電子顯微鏡 (SEM) 與光學顯微鏡觀察基材孔洞的分佈。結果發現，過冷現象存在於冷凍過程中，而且採FC模式會誘導較大的溫度梯度而使多醣溶液中的水產生非同步成核，造成冰晶方向性排列，進而讓基材形成層狀的孔洞結構，其孔洞大小較緩慢降溫程序為大。改採緩慢降溫的SC模式 (0.83 ºC/min) 則可於相同過冷溫度下，誘導冰晶同步成核，進而形成等向性及大小較為均勻的孔洞形狀與結構分佈，孔洞的大小約為60-100 μm。而在基材機械性質的影響方面，以FC模式所製備的基材，其拉伸強度約為20 N/g，改採SC模式所製備之基材的拉伸強度可提高二至三倍 (達到約60 N/g)。這顯示出不同降溫模式不只會誘導基材生成不同的孔洞結構，還會進一步影響基材的機械性質，並且以SC模式製作的基材整體效能較佳。
第二部分的研究的目的在於幾丁聚醣、硫酸軟骨素 (CS) 及麩胺酸 (Glu) 三成分複合基材的製備與特性之探討。在配製溶液的過程中以EDC/NHS作為交聯接合劑，並測定反應之後溶液的黏度、pH值等。待以冷凍凝膠法將溶液製備成基材後，測量了基材表面的接觸角、內部的孔隙
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度、吸水能力與機械性質等，並以SEM觀察基材的微結構。研究過程中發現溶液中的CH與CS分子之間離子作用力極強，於高濃度條件下極易發生沉澱。因此加入第三成分—麩胺酸 (Glu)，利用其分子上的羧酸基先行與CH進行反應，減少CH上質子化胺基的存在，以降低CH與CS之間的離子作用力，再以緩慢加入醋酸的方式減少其沉澱產生。由基材的拉伸強度與抗壓縮強度之結果顯示，少量添加CS增加了CH基材的拉伸強度。然而當CS含量較高時 (CH/CS單體莫耳比小於94/6)，反而不利於CH基材的機械強度。在加入Glu後，CH/CS基材 (莫耳比96/4) 的拉伸強度則會隨所加入Glu的含量增加 (CH/CS/Glu莫耳比由96/4/0上升至96/4/16) 而上升 (由150 N/g增加至250 N/g)，抗壓強度亦可由143.39 N增加至160.50 N。在接觸角與吸水能力的測定結果顯示，加入CS與Glu後，CH基材的接觸角可由95º降低至50º (92/8/8) 並且吸水能力由15.32倍增加至21.34倍，顯示基材的親水性增加。
此外本研究利用化學分析電子能譜儀 (ESCA) 對基材中的元素C、N、O與S進行X光光電子能譜測定，結果中顯示純幾丁聚醣的帄均去乙醯化程度約為92%，而經過EDC/NHS的接合作用後，在CH/CS複合基材，會產生C=N共價鍵結 (N1S能譜)；且其比例在96/4的基材 (29.06%) 高於92/8組 (20.92%)，這說明少量的CS存在可增加CH/CS複合基材的共價交聯比例與交聯程度，進而增加基材的機械強度。此外，當CS含量較高，基材的磺酸基 (sulfonate，S2P能譜，可與水結合) 的絕對含量較高，使其親水性較好，與DSC測定結果中束縛水 (bound water) 的波峰隨CS含量增加向高溫處帄移，以及基材的親水性隨CS含量增加而增加相符。在三成分複合基材 (CH/CS/Glu) 的N1S能譜測定結果中，C=N鍵結 (398.9 eV) 的比例均高於兩成分系統 (CH/CS)，並且其S2P能譜測定結果顯示，-SO4-的比例下降，顯示加入Glu可有效的增加共價交聯及減少CH與CS兩成分之間的離子作用，這解釋了為何在加入Glu後，基材機械強度高於兩成分系統。此外CH/CS/Glu基材中的磺酸基部分亦大量增加，顯示加
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入Glu後可增加複合基材的親水性，此結果與基材的親水性測定相符合。而由基材之細胞相容性測定的結果中發現，CS的添加對不同形態細胞的貼附與增生具有不同的影響。少量的CS有助於類骨母細胞的增生。在添加Glu後，除了增進細胞增生外，根據osteocalcin和type I collagen的基因表現結果與鈣離子含量的測定，發現亦可增進類骨母細胞的分化與礦化。
綜而言之，本研究以不同降溫模式應用於冷凍凝膠法，可製備CH、AL和CMC多醣類為主體之多孔狀基材，並改變其機械性質。再將CH與CS兩種天然高分子，配合Glu作為交聯媒介物，製備新型CH/CS/Glu複合基材。此複合基材具有較高機械強度、較佳親水性等優點，再藉由類骨母細胞在基材表面培養，發現CH/CS/Glu複合基材具有良好的細胞相容性與促進骨細胞功能表現的效果，預期能作為骨組織工程等相關領域的應用。
關鍵字:幾丁聚醣、硫酸軟骨素、麩胺酸、冷凍凝膠法、複合材料、組織工程、類骨母細胞

關鍵字

幾丁聚醣 ； 硫酸軟骨素 ； 麩胺酸 ； 冷凍凝膠法 ； 複合材料 ； 組織工程 ； 類骨母細胞

並列摘要

In the tissue engineering research, scaffolds usually have to fulfill basic requirements such as high porosity, favorable pore size distribution, satisfactory mechanical strength and cytompatibility, and so on. Furthermore, the developments of novel biological materials are focusing on the design and fabrication of materials for the particular function and need. The trend of new biomaterial development includes the use of several materials to fabricate composite scaffolds for improving the performance of the scaffolds. Therefore, this research focused on the improvement in the fabrication method of porous scaffold, and the development of a novel composite material.
In the first part of research, using fast-cooling (FC) and slow-cooling (SC) modes in freeze-gelation method, polysaccharide scaffolds made of chitosan (CH), alginate (AL), and carboxymethyl cellulose (CMC) were fabricated. The temperature profiles with respect to time in cooling process were measured, and mechanical properties of the scaffolds were determined. In addition, the pore structure of the scaffolds was observed by scanning electron microscope (SEM) and optical microscope. The results showed that SC mode (0.83 ºC/min) induced simultaneous ice nucleation and formed the isotropic pore distribution of the scaffolds, with the mean pore size about 60-100 μm. In contrast, the use of FC mode for preparing the scaffolds would induce non-simultaneous ice nucleation and formed stratiform pore structure due to greater temperature gradient, and the mean pore size of the scaffolds was larger than that of SC mode. Moreover, for all cooling mode, the supercooling phenomenon occurred during the cooling processes. Regarding the effect on mechanical properties of the scaffolds, the normalized tensile strength of the scaffolds prepared by using SC mode (60 N/g) was two to three-folds higher than that of using FC mode (20 N/g). It demonstrates that cooling mode was able to affect the pore structure, and then induce the difference in mechanical properties of the scaffolds.
In the second part of research, the preparation of the chitosan/chondroitin sulfate (CH/CS) and CH/CS/glutamic acid (Glu) composite scaffolds were carried out by freeze-gelation method. During the preparation of scaffold solution,
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EDC/NHS was used as the crossing-linking reagent, and the viscosities, pH value of solution, CS binding efficiency were determined. After the fabrication of scaffolds, water contact angle, internal porosity, water uptake, and mechanical properties of the scaffolds were measured. The microstructures of scaffolds were observed by SEM. In the CH/CS solution, strong ionic interactions lead to the precipitation especially under the highly CH/CS concentrations. Therefore, the third component-glutamic acid (Glu) was added to the solution to decrease the amount of protonated amines and reduce the ionic interactions between CH and CS because carboxyl groups of Glu could interact with amino groups of CH, then acetic acid was added slowly to the solution to reduce precipitation. The results of tensile strength and compressive test of scaffolds showed that the addition of a small amount of CS to the scaffold improved mechanical strength of CH scaffolds. However, a large amount of CS in the scaffold was unfavorable to the mechanical strength of CH scaffolds. For the addition of Glu to the CH/CS scaffolds, the tensile strength increased with increasing the content of Glu in CH/CS/Glu scaffold (molar ratio of CH/CS/Glu increasing from 96/4/0 to 96/4/16, and the tensile strength increase in tensile strength: from 150 N/g to 250 N/g), the compressive strength may also increased from 143.39 N (96/4 scaffold) to 160.50 N (96/4/16 scaffold). In hydrophilicity test and water uptake measurement, the addition of CS and Glu caused the contact angle to decrease from 95º to 50º (92/8/8 scaffold surface), and the water uptake increased from 15.32 to 21.34, demonstrating that hydrophilicity of scaffolds increases.
In addition, we used Electron Spectroscopy for Chemical Analysis (ESCA) to determine the elements C, N, O and S of the scaffolds. The result showed that the chitosan had an average degree of deacetylation of 92%. For the N1S spectrum of CH/CS scaffolds showed that after reacted with EDC/NHS coupling agent, new covalent bonds of C=N was formed. Moreover, the C=N proportion of 92/8 group (29.06%) was higher than 96/4 group (20.92%). That is to say, the addition of small amount of CS to the scaffold increased tensile strength of the scaffold due to the covalent crosslinkages was increased. The S2P spectrum of CH/CS showed that the sulfonate groups (having affinity to water) increased in the scaffolds that led to the peak of bound water in the DSC graph shifted to the high
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temperature region as the CS content increased. From N1S spectrum analyses, the C=N (398.9 eV) proportion of the three component scaffolds (CH/CS/Glu) were higher than two component scaffolds (CH/CS), and the S2P spectrum of the scaffolds showed that the sulfate groups of the scaffolds was decreased. It demonstrated that the addition of Glu increased covalent crosslinkage again and reduced the ionic interaction between CH and CS, thus improving the tensile strength of the scaffolds. Besides, the amount of sulfonate groups increased in CH/CS/Glu to enhance the hydrophilicity of the scaffolds. As the result of the cell compatibility test using murine osteoblast-like cells (7F2 cells) revealed that the addition of a small amount of CS to CH is beneficial to the proliferation of 7F2 cells. And the addition of Glu, according to osteocalcin and the type I collagen gene expression analysis and the calcium ion measurement, CH/CS/Glu composite material was not only suitable for the 7F2 cell proliferation, but also beneficial to osteogenic the differentiation and the matrix mineralization.
In conclusion, this research used different cooling modes in the freeze-gelation method for fabricating polysaccharide scaffolds with different pore structure and thus different mechanical strength. And then a novel composite scaffold made of CH/CS/Glu was successfully fabricated. This scaffold possessed higher hydrophilicity and mechanical strength than other CH-based scaffolds. Besides, the proliferation, maintenance of osteogenic phenotype, and mineralization behavior of the osteoblast-like (7F2) cells were also improved. Therefore, we suggest that this novel composite biomaterial consisting of CH, CS, and Glu has great application potential in the field of bone-related tissue engineering.
Keywords: chitosan, chondroitin sulfate, glutamic acid, freeze-gelation method, composite, tissue engineering, osteoblast-like cells.

並列關鍵字

chitosan ； chondroitin sulfate ； glutamic acid ； freeze-gelation method ； composite ； tissue engineering ； osteoblast-like cells

參考文獻

262. 謝承軒，以乳化/冷凍凝膠法製備幾丁聚醣微粒及其特性分析與應用，國立台灣大學化學工程學研究所碩士論文，2006。

1. R. Langer, J. P. Vacant, Tissue engineering. Science 1993, 260, 920-926.

2. B. D. MacArthur, R. O. C. Oreffo, Bridging the gap. Nature 2005, 433, 19.

4. H. G. Machens, A. C. Berger, P. Mailaender, Bioartificial Skin. Cells Tissues Organs 2000, 167, 88-94.

5. K. H. Lee, Tissue-engineered human living skin substitutes: Development and clinical application. Yonsei Med. J. 2000, 40, 744-779.