超音波刺激對運動中鼠尾肌腱纖維束黏彈性質與椎間盤纖維環細胞培養的力學性質之影響

Abstract

目的：研究超音波刺激下之運動中鼠尾肌腱纖維束的黏彈性質變化以及牛尾椎間盤纖維環細胞之力學性質變化。背景簡介：本研究分為兩部分。第一部份：治療性超音波之效應可分為熱效應及非熱效應，廣泛在臨床使用與研究，相關研究多為針對超音波對軟組織修復之影響。本研究探討超音波的非熱效應如何造成軟組織機械性質的立即性改變。第二部份：椎間盤為位於脊椎椎體間的軟骨組織，讓脊椎擁有活動度，而椎間盤的突出與退化可能會造成嚴重的疼痛。更接近真實組織的培養的椎間盤組織，可能是解決此問題的新契機。但是，椎間盤組織的培養具相當難度，因為其中纖維環細胞結構具高度方向性，且以特定形式排列。微構形纖連蛋白塗層可控制細胞的形狀、使之排列有方向性。微構形纖連蛋白塗層可以控制纖維環細胞的形狀，使其生長更具方向性，而低強度脈衝超音波已被證實可使纖維環細胞分泌之第一型膠原蛋白增加。本研究探討超音波刺激下是否影響纖維環細胞之力學性質變化以及超音波在不同微構形的纖連蛋白塗層上，對細胞型態之影響。材料與方法：第一部份：此研究使用年齡四個月之SD品系大鼠鼠尾肌腱纖維束。肌腱纖維束以夾具固定在拉伸測試機上，進行循環應力鬆弛測試與循環潛變測試，以此模擬並評估鼠尾的運動狀態，並同時接受超音波刺激。循環測試後，會在試樣上進行拉伸測試。蒐集拉伸測試中的破壞應力與應變、循環應力鬆弛測試的應力鬆弛量、循環潛變測試潛變量等數據進行分析。以標準線性固體模型之麥克斯威爾模型分別擬合動態與靜態之鬆弛與潛變測試，得到楊式係數（E1、E2）與其他各項係數，黏滯係數（η）和時間常數（τ）。實驗使用之超音波為強度 Ispta200mW/cm2， 頻率3MHz。第二部份：纖維環細胞從牛尾椎間盤中取出，培養在上有無微構形或直線微構形纖連蛋白塗層的聚二甲基矽氧烷（PDMS）薄膜上。纖維環細胞培養皿放在 MIGO II 載台內進行超音波刺激，載台連接超音波系統。此研究使用之超音波為頻率1MHz，工作週期20%，強度依不同實驗分為Ispta3.25mW/cm2與Ispta0.79mW/cm2兩種。在無微構形塗層實驗，培養後會進行力學測試或螢光染色攝影，分析拉伸試驗中力與位移變化的線性區間，以線性回歸計算出試樣的楊氏模數；在直線微構形塗層實驗，培養後只進行螢光染色攝影。 結果與結論：第一部份：超音波刺激可能造成大鼠尾巴肌腱束黏彈性質的些微變化，黏彈性質的變化造成應力鬆弛量的降低及循環潛變後的破壞應力與應變上升；麥克斯威爾模型模擬出的係數，組間無顯著差異。可能是因為超音波造成微纖維的相互之間的位移影響蛋白多醣的連結，進而影響黏彈性反應，但並沒有顯著的影響其 材料性質。循環負載狀態中的肌腱鬆弛量較低，可能擁有較高的剛性進而有較高的動作效率。第二部份：（一）使用無微構形纖連蛋白塗層培養纖維環細胞，超音波刺激可刺激第一型膠原蛋白的產量顯著增加，高亮度之影像面積差異顯著 （p=0.01*），但第一型膠原蛋白所佔面積比例顯著較控制組少（p=0.0001*），顯示其分佈並不平均，在本實驗中尚不足以影響纖維環細胞的力學性質。（二）使用直線微構形纖連蛋白塗層培養纖維環細胞，超音波刺激可讓纖維環細胞顯著產生更多的第一型膠原蛋白，高亮度之影像面積差異顯著（p=0.0003*），所佔面積比 例亦顯著增加（p=0.0003*）；肌動蛋白所佔面積比例亦顯著增加（p=0.039*）；細胞之間以膠原蛋白與肌動蛋白互相連結的狀態增加。超音波使得纖維環細胞分泌膠原蛋白與肌動蛋白等基質之速度顯著增加，也讓細胞之間更早以基質相互連結。

Objective: To investigate the effect of ultrasound stimulation on viscoelastic properties of exercising rat-tail tendon and mechanical property of disc annulus fibrosus cell culture. Summary of background data: This research includes two different parts. Part 1. There are thermal effects and non-thermal effects in therapeutic ultrasound, which are widely used and studied in clinical therapy. Most of the research focus on the effect of ultrasound on soft tissue recovery. This study aims to verify how ultrasound non-thermal effects instantly affect the mechanical properties of soft tissues. Part 2. Intervertebral disc is a cartilaginous structure between two spinal vertebral bodies, plays an important role in mobility, disc herniation and degeneration may cause intense pain. Engineered disc tissue is more similar to real tissue, seemed to be a new possible solution. It is difficult to culture disc tissue because in which annulus fibrosus cell (AF cell) is highly directional in a specific pattern. Micro-patterned fibronectin coating can control the shape of AF cell, making it more directional, and low-intensity pulsed ultrasound stimulation (LIPUS) has been confirmed to increase collagen secretion in AF cell culture. This study aims to investigate the effect of ultrasound stimulation on mechanical property of AF cell culture and the ultrasound effect on cell morphology on different micro-patterned coating. Methods: Part 1. 4-month-old SD rats tail tendon fascicles were prepared for testing. The tendon fascicles were fixed with clamps on tensile test machine. Cyclic stress relaxation test and cyclic creep test were conducted to simulate and evaluate the exercising status of tendon fascicles which was simultaneously stimulated by ultrasound. After the tests, tensile tests were conducted on samples. The data from mechanical tests, failure stress and failure strain in tensile test, stress relaxation in cyclic stress relaxation test and strain change in cyclic creep test were collected and analyzed. Standard linear solid models in Maxwell representation were used to find viscoelastic parameters of tendon fascicles, which include Young’s modulus (E1, E2), viscosity (η) and time constant (τ). Ultrasound used in this study was ISPTA 200 mW/cm^2 and 3 MHz. Part 2. Annulus fibrosus cells (AF cell) were extracted from ox tail disc tissue and cultured on PDMS membrane, which was coated with non-patterned or straight micro-patterned fibronectin. AF cells were stimulated by LIPUS in MIGO II chamber which connected to the ultrasound system. Ultrasound used in this study was 1MHz, duty cycle 20%, Ispta 3.25mW/cm2 and Ispta 0.79 mW/cm2. In non-patterned coating experiments, after culture, tensile test or fluorescent confocal microscopy were conducted. Regression was used in linear region of force-strain data during tensile test to find out the Young’s modulus of samples. In straight micro-patterned coating experiments, only fluorescent confocal microscopy were conducted. Result and Conclusion: Part 1: Ultrasound stimulation may slightly change the viscoelastic behavior of tendon fascicles. The change of viscoelastic properties reduced the relaxed stress, and increase failure stress and failure strain after cyclic creep test. It is probably because ultrasound makes microfibers mutually move, thus affecting proteoglycan bridge between microfibers in turn impact on viscoelasticity, but does not affect material property. Tendon fascicle in cyclic loading with less stress relaxation may have higher stiffness, which improve the efficiency of movement. Part 2: (1) On non-patterned coating, ultrasound stimulates AF cell to secrete significantly more collagen I. Collagen I area with high red value is significantly more in experimental group than in control group (p=0.01*), but area portion is significantly lower (p=0.0001*), which shows it is unevenly distributed. The effect was not enough to change mechanical property. (2) On straight micro-patterned coating, ultrasound stimulates AF cell to secrete significantly more collagen I and actin. Collagen I area with high red value is significantly more in experimental group than in control group (p=0.0003*), area portion is also significantly higher (p=0.0003*). Actin area portion is significantly higher in experimental group than in control group (p=0.039*). Ultrasound makes AF cells secrete more extracellular matrix such as collagen I and actin at faster speed, also connected with each other with extracellular matrix earlier. Not only makes cultured AF cell growing faster, but being closer to real tissue.