Abstract
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,基於探討創新臨床治療,首先須研發應用細胞與組織工程的新技術,以期創新組織工程的技術,並應用於臨床治療。不同型態細胞間的交互作用在組織癒合和再生醫學中扮演重要的角色,許多研究著重於研發最適宜的培養方法,以維持間葉細胞和相關的幹細胞的生長和分化。適當的細胞共培養系統能夠深入理解細胞的行為,包括細胞分佈型態和組織結構。本計畫的目的在探討三種不同的共培養系統對前十字韌帶細胞(ACLFs)和滑膜間葉幹細胞(SDMSCs)間行為的影響。此外除了傳統的間接共培養和直接共培養系統之外,我們的先驅研究發現,將前十字韌帶細胞和滑膜間葉幹細胞共同培養於幾丁聚醣(chitosan)基質上,會形成異質細胞交互混合的三度空間球形懸浮聚集,細胞間密切接觸,此與一般直接共培養系統中細胞間僅形成二維單層重疊不同,細胞間距雖小,但並不一定會完全接觸。同時,我們將進一步研究,此一創新的懸浮共培養系統中形成的異質圓球體,其不同的細胞顯微環境如何促進滑膜間葉幹細胞分化,如何調控前十字韌帶細胞功能,其機制為何。本研究的假說是最適宜的懸浮共培養系統藉由控制細胞間的距離、分佈和懸浮聚集的結構,可以產生最佳的細胞交互作用,此一結果將有助於探討間葉細胞和幹細胞間的反應,和研究調控細胞移行、增生、分化和功能的可能訊息傳遞路徑。本計畫為三年之研究,各年特定目標如下:第一年計畫在探討三種不同的共培養系統對前十字韌帶細胞和滑膜間葉幹細胞行為的影響。研究之特定目標在探討不同的共培養系統對前十字韌帶細胞和滑膜間葉幹細胞之細胞活性、增生、移動、相互分佈、功能和分化行為的影響;特別創新的是,兩者共培養於幾丁聚醣上會形成異質細胞混合之球形聚集懸浮共培養系統。第二年計畫將進一步探討此一懸浮共培養系統影響細胞與細胞間交互作用的內在機制。我們將研究懸浮共培養系統中細胞的基因表現變化,和特定具有磷酸化表現的蛋白質之訊息傳遞路徑。第三年計畫係在韌帶組織癒合研究模式下,研發適當條件的懸浮共培養系統,以促進滑膜間葉幹細胞成為具有功能性的前十字韌帶細胞。研究之特定目標在試驗球形聚集懸浮共培養系統對韌帶組織癒合的效用,我們將懸浮共培養圓球體種植於塗磨上第一型膠原蛋白的表面,以仿效韌帶組織癒合模式,研究將顯示在生長因子/無血清狀況下存活而聚集的細胞會分開,恢復為一定的生理形態,促進植體成形與韌帶組織癒合。
Abstract: The interaction between different types of cells plays an important role in the woundhealing and regenerative medicine. Many studies have been made on the optimization ofculture methods for the maintenance and differentiation of stroma and related stem cells,respectively. A proper co-culture system can lead to an increased understanding of cellbehaviors such as cell distribution pattern and tissue organization. The objective of the presentproject is to examine the effect of different co-culture systems on the interactionbetween anterior cruciate ligament fibroblasts (ACLFs) and synovium-derived mesenchymalstem cells (SDMSCs). In addition to traditional direct and indirect co-culture systems,suspension co-culture of ACLFs and SDMSCs on chitosan substrates will be developed andexamined. The so-called suspension co-culture system means that ACLFs and SDMSCs canintermix to form three-dimensional heterogeneous spheroids with intimate cell contact abovechitosan substrates, which is different from traditional direct co-culture system just formingtwo-dimensional monolayer without complete cell-cell contact on commercial substratesurface. Thus, how suspension co-culture creates different cellular microenvironments withinheterogeneous spheroids for enhancing SDMSC differentiation, regulating ACLF function,and the mechanisms involved will be studied. It is hypothesized that suspension co-culturesystem with an appropriate protocol can optimize cell-cell interaction by controlling cell-celldistance, distribution and organization within the suspension aggregates. This informationmay be helpful to investigate the stroma-stem cell response and its possible signaling pathwayinvolved in regulating cell migration, proliferation, differentiation, and function. The outcomein this study will allow us to work towards more tissue-engineered acceptable technology tofacilitate improving ACL repair and regeneration. At the end of this project, the outcome willbe published in famous journals, and the entire people join this project will gain good training.The objectives in this project will be achieved through the following specific aims.Specific Aim 1: To investigate the effect of different co-culture systems on behaviors ofACLFs and SDMSCs (Year 1). This specific aim will test the effectiveness of differentco-culture systems on behaviors of ACLFs and SDMSCs such as cell survival, proliferation,migration, distribution, function, and differentiation Especially, ACLFs and SDMSCs will beco-cultured on chitosan to form a suspension co-culture system.Specific Aim 2: The underlying mechanism of suspension co-culture effects on cell-cellinteraction for ligament tissue engineering (Year 2). The intrinsic mechanisms whichaccount for cell behaviors when they are co-cultured in a suspension system will be furtherexplored. Toward this aim, the change of gene expression and the specific proteins-dependentsignaling pathways of both in the suspension co-culture system will be examined.Specific Aim 3: To develop the proper suspension co-culture condition that is able tofacilitate SDMSCs into functional ACLFs in wound healing model (Year 3). The aim ofthe 3rd year is to evaluate the effectiveness of suspension co-culture spheroids on the ligamentwound healing. In this work, suspension co-cultured spheroids will be reinoculated oncollagen I-coated surface to mimic wound healing model. We will demonstrate thatsuspension co-cultured spheroids with an optimal protocol can show an ideal survival rate ingrowth factor/serum-deprived condition and disintegrate to return to a physiologicalmorphology.
Keyword(s)
懸浮共培養系統
前十字韌帶細胞
滑膜類間葉幹細胞
韌帶組織工程