以新微滴技術製作三維高規則排列細胞支架進行軟骨組織工程之研究

Abstract

【研究設計】首先以新的微滴技術研發出一細胞支架，此支架為具有三維高規則排列孔洞結構的支架，再於此支架上培養豬的軟骨細胞，並藉由細胞活性測試，硫化葡萄胺聚醣 (sGAG) 定量分析及即時定量反轉錄酶-聚合酶鏈反應 (real-time PCR) 來檢驗軟骨細胞在此新型支架上的增生與分泌細胞外基質的能力。第二階段則是將豬的軟骨細胞種入此新型支架後，再植入免疫缺陷鼠的背部內，數週後取出以觀察軟骨組織再生的情形。 【研究目的】評估以新微滴技術研發的細胞支架作為軟骨組織工程再生的可行性與適切性。 【背景簡介】老化所造成的關節退化是一普遍的疾病，以美國為例，每年僅就治療退化性關節炎所支出的醫療給付金額高達一千多億美元之譜。過去治療軟骨缺損或退化包括軟骨鑽洞，軟骨摩擦整形，軟骨移植手術等，但其軟骨再生的成效仍不一致，效果存疑，故現在多以組織工程研究為主,組織工程是近來很熱門的一個學問，軟骨再生的研究即是其中一項。若能成功以組織工程達到軟骨再生的目的，對人類將是莫大的福祉。 【材料與方法】先以新的微滴技術來製作褐藻膠立體支架，測試其機械強度，量測支架孔隙度與膨脹率，並以共軛焦顯微鏡觀察支架的型態。再來將單層細胞培養的豬軟骨細胞種植到支架中，在體外環境下培養一、二、三週，進行一系列細胞接種後的分析，包括細胞存活測試，支架毒性測試，細胞外基質硫化葡萄胺聚醣 (sGAG) 分析，去氧核醣核酸 (DNA) 定量，即時定量反轉錄酶-聚合酶鏈反應(real-time PCR) 測試基因表現等研究。動物體內實驗部分則是將細胞接種於支架後，培養三天，再植入免疫缺陷小鼠背部皮下內，再分別於接種2週、4週、6週時，將此移植物取出作組織切片染色，免疫化學染色，即時定量反轉錄酶-聚合酶鏈反應 (real-time PCR) 測試基因表現等的分析。 【實驗結果】此支架的特色為孔洞大小與排列非常規則，其構造有如蜂巢，機械強度不輸冷凍乾燥法製造的支架，孔隙度與膨脹率則更佳。體外培養的結果顯示軟骨細胞在支架內存活良好，發現隨著時間增加，支架內的軟骨細胞量也增加，硫化葡萄胺聚醣 (sGAG) 淨增量持續增加，基因表現出強烈的第二型膠原蛋白和聚蛋白多糖 (aggrecan)，符合軟骨應有的特性且能複製許多細胞，並維持軟骨細胞應有的表徵 (phenotype)，以電子顯微鏡及共軛焦顯微鏡觀察可發現細胞可沿著支架壁生長良好，甚至一個月後仍有許多細胞存活於支架內，證明此新製程的支架確實可行。在免疫缺陷小鼠體內培養植入種植有豬軟骨細胞的支架實驗，取出時可見支架已從透明變成白色, 與軟骨極為類似, 發現隨著時間增加，支架內的軟骨細胞量也增加，且支架強度也變硬，免疫化學染色呈現出明顯S100蛋白與第二型膠原蛋白大量存在的組織，且基因表現出分泌大量的第二型膠原蛋白與聚蛋白多糖 (aggrecan)，顯示出培養產生的是透明軟骨。 【研究結論】本研究顯示此一新的微滴技術製作的褐藻膠立體支架對於豬的軟骨細胞培養與細胞外基質製造皆有正向的影響，不管是體外或是體內培養都有維持透明軟骨該有的表徵。此支架的特色為孔洞大小一致與排列非常規則，非常類似軟骨細胞生活的軟骨凹穴 (lacuna)，對軟骨組織工程研究相信是一深具潛力與值得進一步研究的材料。 【未來研究】目前此研究雖已進行至免疫缺陷小鼠體內培養軟骨的動物實驗，也獲致不錯的成果，但在應用至人類身上之前，仍應考慮做大型動物軟骨缺陷移植實驗，以了解實際上軟骨再生的能力。另外目前也已用此微滴技術製作其他材料的支架，也獲致初步的結果，再結合幹細胞研究以促進軟骨再生，相信是一值得研究的方向。

【Study Design】A novel highly organized alginate scaffold was developed by microfluidic technology. Porcine chondrocytes were cultured in this three-dimensional scaffold in vitro. The cell proliferation and extracellular matrix secretion were measured by cell viability test, 1,9-dimethylmethylene blue (DMMB) assay, and real-time reverse-transcriptase polymerase chain reaction (real-time PCR). The 2nd stage is transplanted porcine cells/scaffold constructs into the back of SCID mice. Mice were sacrificed later and analyzed the results of cartilage regeneration. 【Objective】 To evaluate the effectiveness and feasibility of the new highly organized alginate scaffold for cartilage tissue engineering. 【Background】Osteoarthritis is a degenerative disease and frequently involved knee and hip joint. The annual medical care expenditures in the U.S. are more than 150 billion. Current treatment includes multiple drilling and abrasion arthroplasty etc. for small cartilage defect. Mosaicplasty and autogenous chondrocyte transplantation are new options but gap existence and fibrocartilage formation are still problems. Combination of cells, scaffold and growth factor are considering a promising tissue engineering method for cartilage regeneration. 【Materials and Methods】Initially we developed a new method to produce a highly organized alginate scaffold by microfluidic device. The swelling ratio, porosity and mechanical strength of the new alginate scaffold were analyzed. The microstructure of the scaffold was examined by confocal laser scanning microscope. Porcine chondrocytes were seeded into the alginate scaffold and being cultured for 1, 2 and 3 weeks, respectively. The cells/scaffold constructs were analyzed with cell viability, cell toxicity, extracellular matrix, DNA quantification, gene expression (real- time PCR), SEM and confocal microscopy. In the in vivo study, cells/scaffold constructs were transplanted into the subcutaneous portion of the back of the SCID mice. The mice were sacrificed at 2, 4, and 6 weeks. The constructs were examined with histological studies, immunohistochemical staining and gene expression (real- time PCR). 【Results】The alginate scaffold revealed a highly organized porous structure with interconnection like a honeycomb. Comparing with the scaffold fabricated by freeze drying methods, the results revealed similar mechanical strength, higher porosity and swelling ratio. In vitro study, the chondrocytes can keep phenotype, highly expressed of aggrecan and collagen type II and secret much extracellular matrix (sGAG). SEM and confocal laser scanning microscope showed the chondrocytes were attached firmly on the scaffold. The results demonstrated the new scaffold is effective in chondrocyte culture. In vivo study, the morphology of harvested cells/scaffolds from the mice became harder and turned to white color like cartilage. Cells produced glycosaminoglycans can be proved by alcian blue stain. Immunohistochemical staining revealed cells secreted type II collagen and expressed S-100 protein. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated. The results demonstrated the regenerated cartilage tissue kept the phenotype of hyaline cartilage. 【Conclusions】The new highly organized alginate scaffold fabricated by microfluidic technology has positive effects on cell proliferation and extracellular matrix production. The in vitro and in vivo studies both revealed that the alginate scaffold can maintain normal phenotypes of chondrocytes. The pores mimic the shape and environment of the lacuna so that the chondrocytes will stay and proliferate. We believe the alginate scaffold may provide new possibilities for cartilage tissue engineering in the near future. 【Future works】In spite of the fact that this in vivo animal experiment, in combination with the previous in vitro study, revealed this alginate scaffold is a good candidate for cartilage tissue engineering, the chondrogenesis of this scaffold requires more large animal study to demonstrate for chondral defect in articular cartilage before application to human body. Currently, we also try other biomaterials to fabricate microbubble scaffolds and have the positive results. We suggest addressing mesenchymal stem cell or other source stem cell cultured in this scaffold to increase the chondrogenic properties and is a theoretically valuable research aim.