細胞層片用於複合型組織工程人工韌帶之韌帶化體外模型

Abstract

背景與目的：前十字韌帶傷害是常見的下肢運動傷害，尤其韌帶斷裂往往對於運動選手的運動生涯，或一般民眾的生活功能造成顯著的影響。治療方式除了保守治療外，另一種較常採用治療方式是十字韌帶重建手術1, 2。現行以自體韌帶移植為主的手術方式，仍存在著自體供給處功能受損、肌力下降與復原期長等副作用，然而人工韌帶易產生因植體長期使用發生的斷裂，因此使用並不普及。如何解決過往人工韌帶在生物相容性與使用年限不足等缺點，是人工韌帶設計上很重要的課題。本實驗目的是開發新型細胞複合型人工韌帶，合併使用間葉幹細胞實驗模型，探討其韌帶化之表現。研究方法:培養8周大SD大鼠的骨髓間葉幹細胞，種植於經過異型斷面纖維和膠原蛋白鍍層改質的PET材料，觀察其韌帶化結構的整合與成熟。進行的實驗包含: 以Alamar Blue 分析其細胞增生；電子顯微鏡、細胞活死染色和組織切片染色觀察細胞生長型態；qRT-PCR分析肌腱韌帶相關基因，包括轉錄因子 Egr1、Scx與基質分子Col1、Col3、Tnc、Tnmd的基因表現。細胞層片實驗部分，使用電紡纖維層片和市售熱感應培養盤，製作出細胞層片後再種植於PET材料，進行下列實驗:細胞增生實驗；qRT-PCR 分析肌腱、韌帶化的相關基因表現；組織切片染色。結果:第7、14、21天發現不同異型斷面纖維材料，不論是在細胞增生、基因表現和影像觀察，不會出現顯著差異。膠原纖維鍍層改質的部分，發現有鍍層的組別和未鍍層組相比，細胞增生在第21天有顯著增加。韌帶化相關的轉錄因子基因表現中，發現有鍍層組的Egr1在第7天表現最為明顯，Scx則是在第21天表現最明顯。基質分子Col1在7、14、21天表現明顯；Col3在第21天表現最明顯；Tnc在第7天表現最明顯；Tnmd在第21天表現最明顯。顯示膠原纖維鍍層可以增加幹細胞韌帶化的基因表現。影像觀察上，第7天電子顯微影像顯示膠原纖維鍍層組有較多的細胞貼附；組織切片染色顯示膠原纖維鍍層組在第2、3個月有更多的細胞貼附與胞外基質浸潤。在細胞層片實驗發現，加入了細胞層片後會使材料上的細胞增生增加，在第21天時出現顯著差異。觀察第1、2個月加入在細胞層片的材料染色切片後，發現加入了細胞層片的組別的細胞數目與胞外基質增加，此表現在第2個月更加明顯。另外也發現使用電紡細胞層片會影響細胞於材料上貼附的分布。然而在分析加入細胞層片組別的韌帶化相關的基因表現時，並未發現韌帶化相關基因表現出現顯著變化。結論:人工韌帶膠原蛋白鍍層可以增加細胞的貼附與韌帶化整合的表現，加入細胞層片後可增加細胞貼附數量，有助於臨床應用。

Background:Anterior cruciate ligament injury is a common lower extremity sports injury, especially ligament rupture often has a significant impact on the sports career of athletes or the people's physical function. In addition to conservative treatment, another commonly used treatment is cruciate ligament reconstruction surgery. The current surgical procedure is mainly based on autologous ligament transplantation, which has the advantages of good self-acceptance, low rejection and low inflammation. However, there are still side effects such as impaired function of the donor site, decreased muscle strength and long recovery period. Artificial ligaments are prone to ruptures caused by long-term use of ligament implants, and sequelae of knee joint synovitis caused by wearing debris. Therefore, the development of artificial ligaments is a very important issue, which solves the shortcomings such as the lack of biocompatibility of the artificial ligaments, as well as the problem of short product life span, while retaining the advantages of stable source of autologous mesenchymal stem cells, low rejection and low inflammation. The purpose of this study is to develop a new type of cell-composite artificial ligament, and use mesenchymal stem cells to conduct a cell experiment model. Methods: Mesenchymal stem cells (MSCs) harvested from bone marrow of Sprague-Dawley rats (8 weeks old) were seeded on PET material which was treated by profiled fiber and collagen coating. To measure MSCs proliferation, Alarma blue was assayed at 7, 14, 21 day. The gene expression level of ligamentization transcription factors Egr1 and Scx, and matrix proteins Col1, Col3, Tnc, Tnmd were assayed by qRT-PCR. MSCs growth pattern images were assayed by SEM, Live and Death assay kit and H E histology. Cell sheets used in this study include electrospinning nanofiber membrane and thermos responsive polymer cultured dish. Cell proliferation, gene expression and histology were assayed by above methods. Results: There is no significant differences in cell proliferation, gene expression and histology among 3 different profiled fibre groups. Collagen coated PET material has higher MSCs proliferation, ligamentization gene expression and more cell matrix on H E staining. Cell sheet groups had more cell attachment and distribution than control group. However, there is no difference on gene expression between cell sheet groups and control group. Conclusion: Collagen coating on PET material can increase ligamentization, cell proliferation and cell infiltration. Cell sheet technique can increase the number of cells attached, which is helpful for clinical application.