Effect of allantoin addition and crosslinking treatment on the properties of plant polysaccharide/chitosan bioscaffolds

Abstract

尿囊素（Allantoin）廣泛存在於紫草科（Symphytum officinale, Comfrey）的葉及根中，因其具有抗刺激、刺激纖維母細胞增生及幫助傷口癒合等保護皮膚的功效，而常使用在化妝品、治療皮膚的藥物及個人保養品中。先前研究結果指出添加刺槐豆膠、蒟蒻膠之幾丁聚醣支架，能夠改善幾丁聚醣支架之物理性質，因此，本研究欲添加不同濃度（0、0.25、0.5%）的尿囊素於刺槐豆膠/幾丁聚醣及蒟蒻膠/幾丁聚醣支架中，再以低細胞毒性的Genipin（GP）及EDAC+NHS（EDAC）進行交聯，探討尿囊素的添加及交聯處理對刺槐豆膠/幾丁聚醣及蒟蒻膠/幾丁聚醣生物支架的微結構、機械性質、溶解率等物理性質及纖維母細胞（NIH-3T3）生長狀態的影響。 從掃描式電子顯微鏡結果可得，支架表面皆散佈著大小不一的孔洞，刺槐豆膠/幾丁聚醣支架的平均孔洞直徑約介於63~84 μm，蒟蒻膠/幾丁聚醣支架的的平均孔洞直徑約介於61~107 μm。孔洞率結果顯示，所有支架的孔洞率皆大於92%。 由膨潤率曲線可發現，支架的膨潤率皆隨時間上升達最大膨潤率後趨緩，且具有極高的反應速率常數。尿囊素的添加會使大部分支架的最大膨潤率下降，交聯處理可提升大部分支架的最大膨潤率。在溶解率試驗中，添加尿囊素支架的溶解率皆因尿囊素的釋出而顯著增加，而經GP交聯後可有效降低支架的溶解率。由交聯密度結果可發現，大部分支架的交聯密度隨著尿囊素添加濃度增加而下降，而經GP交聯後可有效提升支架的交聯密度。 在機械性質分析中，所有支架的彈性模量及壓縮應力皆隨尿囊素添加濃度的增加而增加，因交聯處理而下降，推測與支架孔洞大小的變化有關。應力緩和試驗中，尿囊素的添加濃度、多醣種類對應力緩和時間（λ）並無顯著影響，而蒟蒻膠/幾丁聚醣支架經GP交聯後，λ會增加；在刺槐豆膠/幾丁聚醣支架中，未經交聯支架的應力緩和係數（Y）會因尿囊素的添加而下降，經交聯後則隨尿囊素添加而上升，說明尿囊素的添加會增加未經交聯支架的彈性，經交聯後則會使支架具有較高的黏性性質，而蒟蒻膠/幾丁聚醣支架則具相反的趨勢。 細胞存活率的結果顯示，添加0%及0.5%尿囊素且經GP交聯的支架皆不具有細胞毒性，且添加尿囊素的支架確實具有促進NIH-3T3增生的效果。 綜合以上結果，說明添加0.5%尿囊素且經GP交聯的支架具有作為皮膚組織工程支架或藥物載體的潛力。

Allantoin exists in lots of plants, especially in leaves and roots of Comfrey. It has many effective effects on skin protective properties, such as anti-irritation, stimulates proliferation of fibroblast and help wound healing. Therefore, it's used in cosmetics, medicine that healing skin disease and skin care products extensively. According to previous study pointing out that adds locust bean gum and konjac can improve physical properties of pure chitosan scaffold. Hence, the purpose of this study is evaluating the effect of the addition of allantoin and different crosslinking treatments on the physical properties, such as micro-structure, mechanical properties and dissolution rate, and cell viability of NIH-3T3 cell of locust bean gum/chitosan and konjac/chitosan scaffolds. Results of SEM showed that there are lots of pores with various size distributing on the surface of all scaffolds. The mean pore size of locust/chitosan scaffolds ranged from 63~84 μm, and 61~107 μm for konjac/chitosan scaffolds. The porosity of all scaffolds is higher than 92%. As the swelling curve showed, the swelling ratio of scaffolds increased with time to a certain level then levels off. All scaffolds had high reaction rate constant. The addition of allantoin decreased the maximum swelling ratio of most scaffolds. After crosslinking, the maximum swelling ratio of most of scaffold increased. In dissolution test, the dissolution rate of scaffolds with allantoin increased significantly because of the release of allantoin while crosslinked with genipin could decrease the dissolution rate. The crosslinking density of scaffolds increased with the concentration of allanoin. Moreover, scaffolds crosslinked with genipin increased the crosslinking density of scaffolds effectively. The elastic modulus and compressive strength of the scaffolds increased with the concentration of allantoin but decreased with crosslinking treatment, which might related to the change of mean pore size of scaffolds. In relaxation test, there is no significant effect of the concentration of allantoin and different kinds of polysaccharides on relaxation time (λ) of scaffolds while an increase λ of konjac/chitosan scaffold after crosslinking by GP. Relaxation coefficient of locust bean gum/chitosan scaffold without crosslinking decreased with the addition of allantoin but increased after crosslinking that demonstrated the addition of allantoin could increase the elasticity of locust bean gum/chitosan scaffold without crosslinking while crosslinking treatment could increase its viscosity. However, there's a different tendency of konjac/chitosan scaffolds. Cell viability results revealed that GP-cross-linked scaffolds with 0% and 0.5% allantoin didn't have cytotoxicity and the addition of allantoin significantly promoted the proliferation of NIH-3T3. In conclusion, GP-cross-linked scaffolds with 0% and 0.5% allantoin had the potential for skin tissue engineering and drug delivery.