明膠/軟骨素/透明質酸三重共聚物應用於脊椎椎間盤本核再生之研究

Regeneration of Nucleus Pulposus by Using Gelatin/Chondroitin-6-Sulfate/Hyaluronan Tri-copolymer Scaffold

楊曙華(Shu-Hua Yang)

指導教授 ： 林峰輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00140

摘要

【研究設計】首先發展一種包含明膠、軟骨素及透明質酸的生醫活性材料，再於此三重聚合物支架中體外培養人類脊椎椎間盤本核細胞，並藉由ELISA檢測法、組織切片的免疫化學染色及即時定量反轉錄酶－聚合酶鏈反應(real-time RT-PCR)檢驗細胞增生及製造細胞外基質之能力。
【研究目的】評估明膠/軟骨素/透明質酸三重聚合物作為脊椎椎間盤本核再生之生醫活性支架的合適性。
【背景簡介】脊椎椎間盤退化始於本核部位，且會導致多種相關之脊椎病變，而有臨床症狀之脊椎椎間盤退化會造成疼痛並增加健康照護的支出。目前現有之治療方式或可減輕臨床症狀，但無法停止脊椎椎間盤的退化進程，甚至有時會加重其退化速度。針對治療脊椎椎間盤退化之組織工程研究目的在增進組織再生而使其退化進程得以減慢或甚至逆轉，結合細胞及生醫活性支架的方法是其中一種深具潛力的組織工程方法。
【材料與方法】將明膠/軟骨素/透明質酸三重聚合物製成圓盤狀之支架，並以戊二醛進行交聯反應。本核細胞先自人體組織分離，以單層培養方式增加其細胞數量，再將一百萬個細胞種植到一個盤型支架中後，於體外環境下培養四週。針對培養細胞之實驗組支架及對照組支架進行一系列的檢驗，檢驗項目包括細胞活性及增生、硫化葡萄醣胺聚醣含量、特定基因之訊息核糖核酸(mRNA)表現、組織學及免疫組織化學研究。
【實驗結果】實驗組支架在四週的體外培養後，可持續呈現出明顯的細胞活性及細胞增生，且硫化醣葡萄醣胺聚醣淨增量也持續地增加。實驗組支架中的細胞在與單層培養細胞的比較中可發現其第二型膠原蛋白、aggrecan、Sox9、TGF-β1及TIMP-1的mRNA表現明顯較高，而第一型膠原蛋白、第十型膠原蛋白、IL-1、Fas-associated death domain protein的mRNA表現則明顯較低。組織學檢驗可發現有包含醣胺多醣及第二型膠原蛋白的新生成細胞外基質。
【研究結論】本研究顯示明膠/軟骨素/透明質酸三重聚合物生醫支架對於人類脊椎椎間盤本核細胞在細胞增生及細胞外基質製造上有正面的影響，此三重聚合物對脊椎椎間盤本核組織工程研究而言是一種深具潛力的材料，但仍須進一步實驗證實其臨床運用於椎間盤本核再生的可行性。
【未來研究】在運用明膠/軟骨素/透明質酸三重聚合物生醫支架至人類身上之前，動物實驗是絕對必須的步驟。尋求健康具活性的椎間盤本核細胞以增進細胞增生及基質製造也極為重要，結合間質幹細胞及生醫活性支架的研究也是一個值得研究的方向。

關鍵字

明膠 ； 軟骨素 ； 透明質酸 ； 脊椎椎間盤 ； 本核 ； 組織工程

並列摘要

【Study Design】A bioactive material was developed with the composition of gelatin, chondoitin-6-sulfate, and hyaluronan. Human nucleus pulposus cells were 3- dimensionally cultured in this tri-copolymer scaffold in vitro, and then their cell proliferation and matrix productivity were measured by ELISA assays, immuno- histochemical staining, and real-time reverse-transcriptase polymerase chain reaction (RT-PCR).
【Objective】To evaluate the feasibility of gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer serving as a bioactive scaffold for regeneration of human nucleus pulposus.
【Summary of Background Data】Degeneration of the intervertebral disc starts from the nucleus pulposus and finally leads to various associated spinal disorders. Symptomatic intervertebral disc degeneration results in substantial pain and increased health cost. Current treatment options for degenerative disc disease may alleviate clinical symptoms but are not able to stop and sometimes even would accelerate the degeneration in the intervertebral disc. Tissue engineering approaches for treating degenerative intervertebral discs aim to promote tissue regeneration then retard or even reverse degenerative process. Combination of cells and bioactive scaffold is one of the promising tissue engineering methods for regeneration of the intervertebral disc including the nucleus pulposus.
【Materials and Methods】The gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer was fabricated into scaffold discs and was cross-linked by glutaraldehyde. Nucleus pulposus cells were isolated from human nucleus pulposus tissues and expanded in monolayer culture. Each scaffold disc was seeded with 1x106 cells and then cultured in vitro for 4 weeks. The cell-scaffold hybrids as well as the control scaffolds were then analyzed on their cell viability/proliferation, content of sulfated glycos- aminoglycans, mRNA expression of selected genes, histological and immuno- histochemical studies.
【Results】The cell-scaffold hybrids demonstrated active cell viability/proliferation and their net increases of sulfated glycosaminoglycans were progressively higher during a 4-week cultivation. In comparison to monolayer cells, scaffold-cultured cells showed significantly higher mRNA expression in type II collagen, aggrecan, Sox9, TGF-β1, and TIMP-1. Expressions of mRNA were significantly suppressed in type I collagen, type X collagen, IL-1 and Fas-associating death domain protein. Histological studies showed newly synthesized matrices containing glycos- aminoglycans deposits and type II collagen in the cell-scaffold hybrids while the scaffold substrate lost its originally entrapped glycosaminoglycans during the in vitro cultivation.
【Conclusions】The gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold has positive effects on cell proliferation and matrix production to human nucleus pulposus cells. The tri-copolymer scaffold is promising bioactive material that warrants further investigation into their application in regeneration of nucleus pulposus.
【Future Works】After a successful in vitro experiment on culturing human nucleus pulposus cells in the tri-copolymer scaffold, an animal study is a mandatory step before proceeding to clinical application. In addition, searching for healthy and viable nucleus pulposus cells is also important for promoting cell proliferation and matrix productivity. Combination of mesenchymal stem cells and current/future bioactive scaffolds is also worthwhile being investigated.

並列關鍵字

gelatin ； chondroitin-6-sulfate ； hyaluronan ； intervertebral disc ； nucleus pulposus ； tissue engineering

參考文獻

151. Salem AK, Stevens R, Pearson RG, Davies MC, Tendler SJ, Roberts CJ, Williams PM, Shakesheff KM. Interaction of 3T3 fibroblasts and endothelial cells with defined pore features. Journal of Biomedical Materials Research 2002, 61:212-7.

4. Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. Journal of Clinical Investigation 1996, 98:996-1003.

6. Eyre DR, Muir H. Quantitative analysis of type I and II collagens in human intervertebral discs at various ages. Biochimica et Biophysica Acta 1977, 492:29-42.

7. Eyre DR, Matsui Y, Wu JJ. Collagen polymorphisms of the intervertebral disc. Biochemical Society Transactions 2002, 30:844-8.

8. Roberts S, Ayad S, Menage PJ. Immunolocalisation of type VI collagen in the intervertebral disc. Annals of the Rheumatic Diseases 1991, 50:787-91.