明膠與聚己內酯與聚麩胺酸團聯共聚物複合支架於小腸組織工程之研究

The Study of the Composite Scaffold of Gelatin and Poly(ε-caprolactone)-Poly(γ-glutamic acid) (PCL-PGA) Block Copolymer for Small Intestine Tissue Engineering

邱鉅樺(Chu-Hua Chiu)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/碩士(2011年)

https://doi.org/10.6840/CYCU.2011.00142

摘要

短腸症病患因為先天以及後天的因素導致小腸被大量切除，因而發生營養以及吸收不良等症狀，目前治療短腸症的方式有全靜脈營養治療、藥物治療以及外科手術治療，但是上述幾種方法均會造成某種程度的副作用，利用組織工程的方式再生小腸是現今亟需研究的目標。本研究首先合成聚己內酯與聚麩胺酸團聯共聚物，並且利用黏度法、傅立葉轉換紅外線光譜儀、液態核磁共振儀與凝膠滲透層析儀鑑定化學結構與分子量。支架製作方面以明膠當作主要材料，混合聚己內酯與聚麩胺酸團聯共聚物，再經由1-(3-二甲基胺丙基)-3-乙基碳二亞胺交聯，最後使用冷凍乾燥法製備出不同濃度比例的多孔性支架。在結果中，合成聚己內酯與聚麩胺酸團聯共聚物以掃描式電子顯微鏡觀察支架型態，表面及內面大致為光滑表面，截面可看到100-300 μm的孔洞，並利用多孔性材料孔洞分析儀分析具有通透性的孔洞大小，通透性孔洞大小隨著明膠濃度從10 wt%上升至20 wt%，孔洞大小分別為150-230 μm，70-140 μm，40-70 μm。浸泡二次去離子水後發現支架在前12小時吸水膨潤，隨後保持在穩定的狀態，膨潤後，利用流變儀測量支架機械強度，儲存模數介於70-140 Pa，體外降解實驗的結果，明膠濃度從10 wt%上升至20 wt%，其14天降解率從80 %下降至50 %。體外種植與增生評估方面，分別用靜態種植、靜態培養(SS)以及離心種植、靜態培養(CS)將平滑肌細胞植入支架中進行共培養，培養7天後，平滑肌細胞開始攤開於支架中生長，14天後，細胞大量貼附且增生於支架中，形成細胞網絡，在共軛焦顯微鏡影像中可以清楚看見，平滑肌細胞呈現三維分佈於支架中。在細胞增生測試中，利用PicoGreen® assay定量平滑肌細胞，SS組培養28天後，細胞密度約8×105 cells/cm3，CS組支架的細胞密度為1×106 cells/cm3，顯示利用離心法種植細胞可以得到較高的細胞密度，在SS組，正規化後發現培養28天後，細胞有7-16倍成長，CS組方面，也有13-15倍的成長，表示本研究製備的支架對平滑肌細胞為生物相容性的支架，未來適合應用於小腸組織工程再生研究。

關鍵字

明膠 ； 聚己內酯與聚麩胺酸團聯共聚物 ； 小腸組織工程

並列摘要

Short bowel syndrome (SBS) is an intestinal dysfunction resulting from massive intestinal resectioning, which may be brought upon by tumor, trauma, Crohn’s disease, radiation enteritis, etc. Patients with SBS require therapy, such as lifelong total parenteral nutrition support, medication, or surgical intervention. To date, SBS treatment remains a clinical challenge. Intestinal tissue engineering is an alternative treatment which has been documented in several animal studies of SBS. In this study, poly(ε-caprolactone)-poly(γ-glutamic acid) block copolymer was synthesized and characterized by solution viscometry, Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spcrtroscopy, and gel permeation chromatography. Composite tubular scaffolds of gelatin and poly(ε-caprolactone)-poly(γ-glutamic acid) block copolymer (G/CG) were fabricated and cross-linked using a carbodiimide method, frozen at -80 oC, and lyophilized, giving porous tissue-engineered scaffolds. SEM micrographs showed that the outer and inner surfaces of the G/CG scaffolds were smooth, but the cross section was porous with pore sizes in the range of 100-300 μm. Open pores were measured by a capillary flow porometer. Size distribution of open pores decreased with an increase in gelatin concentration, e.g., 150-230 μm, 70-140 μm, 40-70 μm for 10-20 wt%, respectively. G/CG scaffolds could swell in ddH2O in 12 hours, but its storage moduli was around 70-140 Pa, depending on gelatin concentration. The in vitro degradation of G/CG scaffolds decreased from 80 % to 50 % with an increase in gelatin concentration. To optimize cell seeding in the scaffolds, smooth muscle cells (SMCs) were seeded using static seeding (SS) and centrifugal cell immobilization (CS). Confocal microscopic images show the adherence of SMCs on G/CG scaffolds at Day 7. After a co-culture of 14 days, SMCs were observed to have spread extensively and formed of 3D cellular networks. Quantified by PicoGreen® reagents, the cell density of CS groups (1×106 cells/cm3) was higher than that of SS groups (8×105 cells/cm3) after 28 days of incubation. In summary, the composite scaffolds of gelatin and poly(ε-caprolactone)- poly(γ-glutamic acid) block copolymer prepared in this study showed good biocompatibility for the application to small intestine tissue engineering.

並列關鍵字

poly(ε-caprolactone)-poly(γ-glutamic acid) block copolymer ； gelatin ； small intestine tissue engineering

參考文獻

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