機械應力刺激人眼角膜角質細胞之遷移模擬與傷口癒合研究

Abstract

全球視障主因之一為角膜受損，在損傷癒合後細胞排列雜亂導致折射率改變而影響視覺。鑒於捐贈數量不足無法手術，且人工角膜和體外細胞培養尚存不確定性，我們針對角膜基質細胞進行力學行為研究，期望對角膜細胞之生長更加了解。首先為細胞遷移模擬，我們改良Odde學者之遷移數學模型，並加入Gillespie隨機模擬演算法和Keren學者之膜力理論，建構新細胞遷移模型以剖析勻向機械力對其刺激與影響。模擬結果表示應變參數極其關鍵，同時機械力作用亦具細胞遷移導向能力。而機械應力為細胞型態與生理學中不容忽視之環境條件，常見除壓、張和剪切力外，週期性拉伸更是細胞研究廣泛使用之機械要素。為求體外擬真，本研究參考實際眼壓變化量及角膜硬度，提出一種氣動純張力拉伸系統，透過馬達精準調控應變及頻率，且適用表面處理之聚丙烯醯胺水膠作為細胞培養基材。實驗結果顯示動態與靜態角膜基質細胞培養相比，週期拉伸應變對其分化增殖具正向作用。更進一步我們分析角膜損傷癒合後混濁議題，利用聚焦型雷射燒熔，使單層人眼角膜基質細胞產生小面積之平行及垂直方形傷口。配合拉伸系統提供勻向應變動態環境，並置於相同位置透過活細胞相位差顯微平台進行即時紀錄。實驗結果顯示角膜基質細胞傷口癒合至二十小時後形狀逐漸轉化為橢圓環狀，超過四十小時接近完全癒合。整體而言，過程中機械刺激細胞易影響隨機之排列方式，而結果趨勢顯示垂直傷口較適合於靜態環境培養癒合，平行傷口則於動態拉伸條件下培養癒合效益較高。本論文之貢獻在於提出一種模擬人眼角膜機械環境之系統，並核實角膜基質細胞於動態刺激培養之適宜性。

One of the leading causes of global visual impairment is corneal damage. After healing of corneal injuries, disorganized cell arrangements may lead to changes of the refractive index. However, the quantity of corneal donations for the surgeries is insufficient, and there are still uncertainties surrounding artificial corneas and in vitro cell cultivation. Therefore, our research focuses on the mechanical behavior of corneal keratocytes and hopes to deeply understand the growth of corneal cells. Firstly, we analyzed the cell migration using a cell migration model, which is based on the migration mathematical model by Odde, and incorporated with the Gillespie stochastic algorithm and Keren's membrane force theory. Simulation results demonstrated that strain parameter played a critical role, and mechanical force transmission also possessed cell migration-guiding capabilities. To achieve in vitro authenticity, we developed a pneumatically controlled pure tension stretching system, which the strain and frequency were regulated, and the surface-treated polyacrylamide hydrogel was used as the cell culture substrate. The results exhibited that compared to the static cultivation, periodic stretching strain had a positive effect on the differentiation and proliferation of corneal keratocytes. Moreover, we examined the opacification of the cornea after wound healing. We created tiny wounds of human keratocytes and subjected to a dynamic environment with uniform strain, then recorded the healing process in real-time at the same location. Our results observed that the shapes of the keratocytes wounds gradually transformed into an elliptical ring after twenty hours, then approached complete healing and arranged regularly by stresses after about forty hours. Furthermore, the results indicated that vertical wounds were more suitable for healing under static conditions, while parallel wound healing benefited from cultivation that with dynamic stretching conditions. To conclude, this study proposes a system to simulate the mechanical environment of human corneal, and our results support that suitability of cultivating human keratocytes under dynamic stimulation.