2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644151摘要：臨床上肌腱韌帶軟組織傷害十分常見，而關於肌腱韌帶細胞的研究，國內國外卻相當少見。本計畫主持人於臨床上專門從事韌帶重建和肌腱修補手術，經驗豐富，基於解決困難的臨床問題，及應用組織工程的技術以創新治療方法。故提出此三年計畫以研究肌腱韌帶組織工程中，細胞和基材之交互作用及其分子機轉，以期應用組織工程的技術以創新治療方法。第一年計畫子題為「韌帶組織工程之幾丁聚醣/聚己內脂混摻材料對前十字韌帶細胞及骨髓間葉細胞之細胞反應---轉化生長因子角色及分子機轉」，在國科會資助下，於2008年我們初步發現將前十字韌帶細胞培養於幾丁聚醣(chitosan)及聚己內脂(poly ε-caprolactone (PCL) )上，其前十字韌帶細胞在chitosan上會表現出大量的轉化生長因子(TGF-β1)，但卻無法貼附及生長良好；相反的，前十字韌帶細胞在PCL上會貼附良好並且生長，但其TGF-β1的表現不會出現異常的增加。此現象激發我們進一步創新研究，亦即將chitosan/PCL混摻，我們期望此混摻材料不僅可以促進細胞生長貼附並且可以分泌TGF-β1來促進本身產生更多的細胞外基質。此外，將前十字韌帶細胞或骨髓間葉細胞培養在chitosan/PCL混摻材料上，我們將利用西方點墨法(western-blot)先初步篩選，細胞內部參與MAPK 和 PI3K-Akt 訊號傳遞路徑中，具有磷酸化表現的蛋白質，如p38, JNK, ERK, 和PI3K/Akt。另外，為了進一步確定訊號傳遞的路徑，我們將利用SB203580 (p38 inhibitor), sp600125 (JNK inhibitor), U0126 (Erk inhibitor) and LY294002 (PI3K inhibitor)抑制訊號傳遞路徑，做反向驗證。因此，於第一年的研究，我們將建立細胞與chitosan/PCL混摻材料體外培養之分子機轉模型，提供肌腱韌帶組織工程的另一材料選擇。隨後，第二年計畫的子題為「韌帶組織工程之幾丁聚醣/聚己內脂混摻材料及體外共培養系統調控骨髓間葉細胞分化之細胞訊號傳遞機制探討」完全分化的前十字韌帶及膕旁肌腱纖維母細胞的增生及分化能力不足，因此，具有良好增生及分化能力之多功能性骨髓間葉細胞，提供了韌帶組織工程細胞來源的絕佳選擇。基於此，瞭解幹細胞於韌帶組織工程複雜的分化訊號傳遞機制，對將來的臨床治療應用而言是非常重要的。許多文獻指出，TGF-β1對於多功能性骨髓間葉細胞分化成軟骨有關，所以第二年我們以chitosan/PCL混摻材料的體外培養模型為基材，並將BMSCs種入此種混合材料，我們期望此混合材料會使BMSCs製造TGF-β1，並自我誘導成軟骨細胞；另外，我們應用體外共培養前十字韌帶細胞系統，期望BMSCs細胞會被誘導成前十字韌帶細胞，並因建構在chitosan/PCL混摻材料為基材，所以BMSCs將擁有既可分化又可表現細胞功能的雙向能力，因此導入韌帶細胞分化和韌帶-骨交界處界面軟骨的創新組織工程研究。第三年計畫的子題為「韌帶組織工程之物理調控骨髓間葉細胞於生醫材料之細胞訊號傳遞機制探討」。由於物理力量常用於臨床軟組織治療，可見物理因子或多或少會參與細胞訊號傳遞，進而影響細胞生理反應。然而，並無文獻探討低強度超音波和低功率雷射對骨髓間葉細胞-生醫材料的作用，而對其中細胞訊號傳遞分子機制更不清楚。因此，我們提出低強度超音波和低功率雷射是良好物理因子，可加速組織修復。我們假設低強度超音波和低功率雷射可促進骨髓間葉細胞增生及分化成骨母細胞，並研究物理刺激對骨髓間葉細胞培養在生醫材料之分子訊號傳遞機制。經由此三年計畫，我們將深入分子機轉證實我們所提供的創新混摻材料可運用於前十字韌帶組織工程， 並且提供有利BMSCs分化成骨母細胞，軟骨細胞及韌帶纖維母細胞的方法。<br> Abstract: Injury to tendons and ligaments are among the most common injuries to the body. In clinical practice, massive tendon tears and ruptured cruciate ligments require free tissue graft for reconstruction. However, there remain significant drawbacks in our current treatments. A novel approach in tissue engineering involves an optimal biodegradable scaffold loaded with specific living cells and/or tissue-inducing factors to facilitate tissue repair or regeneration.In the first year project-“The cellular response of ACL fibroblasts and BMSCs cultured on PCL/Chitosan blended membrane ---possible role of TGF β1and its mechanism”, The results of our last NSC project indicated that human ACL cells seeded on chitosan expressed lots of TGF-β1 than that of human ACL cells did on PCL. We also observed that ACL cells could not attach well on the chitosan, but attach and proliferate on the poly ε-caprolactone (PCL) substrate. Since the effects of the blended chitosan/PCL substrate on the BMSCs and ligament fibroblasts are poorly understood and have never been published in the literature, we will study the cellular responses and differentiation of the human BMSCs and ACL cells cultured on polymeric scaffolds, and further investigate the relationship among focal adhesion kinase (FAK), cytoskeleton, extracellular signal-regulated kinase (ERK) and TGF β1 signal pathways to establish the molecular mechanisms that up-regulate the expression of TGF β1 in human ACL cells.For the study of the signal mechanisms involved in our novel model, we will use Western blotting to screen the phosphorylated protein expression(s) of p38, JNK, ERK, and PI3K/Akt signaling transducers which are involved in MAPK and PI3K-Akt pathways, while human BMSCs and ACL cells are seeded on PCL/chitosan blended substrate. In order to further clarify the signal pathways involved, we will also use SB203580 (p38 inhibitor), sp600125 (JNK inhibitor), U0126 (Erk inhibitor) and LY294002 (PI3K inhibitor) to block the individual signal pathway.Thereafter, in the second year project-“Differentiation of human BMSCs co-cultured with ACL fibroblasts for ligament tissue engineering”, pluripotent BMSCs capable of proliferation and differentiation toward a variety of cell phenotypes, represent a promising alternative as cell sources in functional tissue engineering of ligament. Understanding the complex signal regulation of stem cell development in ligament tissue engineering is crucial for future therapeutic applications. In our second year study, the aims are to investigate the feasibility of using our in-vitro cell-biomaterials model in the first-year study to induce the differention of BMSCs into cartilage, and using co-culture system to induce the differentiation of BMSCs toward ligament. We hypothesize that co-culture has the ability to promote cell communication, and specific regulatory signals released from fibroblasts in co-culture system can enhance the differentiation of BMSCs, and our chitosan/PCL blended substrate would induce the differention of BMSCs into chondrocyte. In order to test this hypothesis, this study is designed (1) to co-culture BMSCs/scaffold with ACL fibroblasts, and (2) to observe the differentiation of BMSCs on the scaffold.We will demonstrate, for the first time that manipulation of chitosan/PCL blends can modulate the in vitro level of cell differentiation. The data acquired from this study will help develop and optimize chitosan/PCL-based scaffolds used in ligament regenerative therapies aimed at restoring the ligament proper and the ligament-bone junction. This work represents the first step in a unique optimization approach to solve a complex and challenging clinical problem-the need for readily available autologous ligament tissue for use in ACL surgical repair and reconstruction.In the third year project-“Physical regulation and the molecular mechanisms of the cellular response and differentiation of BMSC cultured on polymeric scaffold for ligament tissue engineering”. All mechanical forces must interact with one or more cellular pathways to elicit a biological effect. However, no study has been conducted to examine theeffects of low-intensity ultrasound or low power laser on the novel model of BMSC-scaffold-growth factors interaction, and the molecular pathways of their underlying mechanisms are virtually unknown.We propose that low-intensity ultrasound or low power laser applied in therapeutic medicine would be a useful physical energy to accelerate tissue repair process. We hypothesize that low-intensity ultrasound and low power laser could facilitate in vitro BMSC growth and differentiation toward osteoblast or fibroblat linage on the chitosan/PCL blended biomaterials. Also, the effects of low-intensity ultrasound and low power laser on the BMSCs plated on selected polymeric scaffolds will be compared and their underlying mechanisms explored in this study.