使用超音波刺激之全椎間盤培養系統之開發

Abstract

目的：開發含有超音波刺激的全椎間盤培養系統，包含生物反應器之設計與特性探討、超音波刺激系統之建置與校正以及豬隻椎間盤的培養測試。 背景簡介：椎間盤退化是常見的脊椎病變，臨床上除了在退化末期使用侵入手術方法治療以外，目前尚未有有效的預防方法。低能量脈衝式超音波(Low-intensity pulsed ultrasound，LIPUS)是個安全且非侵入式的治療方法，已被證明可以用來促進骨折癒合與骨質增生。LIPUS已被證明於細胞層級可以促進髓核細胞的增生與細胞外基質之合成，但就組織與器官層級而言尚未見到相關的研究。本研究目的為開發一全椎間盤培養系統，此一系統可用於日後椎間盤生物與生化性質、分子擴散量、流變學及動力學、組織結構是否會因LIPUS刺激而改善的研究。 材料與方法：(一)全椎間盤培養系統之設計：系統包含生物反應器、培養液循環系統、氣動負載系統與超音波系統，生物反應器提供裝有培養液的營養環境；培養液循環系統供給椎間盤養份循環(流速200μl/min)；氣動負載系統模擬椎間盤於人體內所受到的負載(16小時動態負載0.2-0.8MPa 0.2Hz；8小時靜態負載0.2MPa)；超音波系統則提供給椎間盤超音波刺激。(二)超音波探頭校正：使用聲功率計與超音波機台將探頭的輸出能量調整與額定能量(0.2W, 1W, 2W, 3W)相符，先調整探頭的最大響應頻率，再調整放大倍率。(三)生物反應器內能量場分布量測：使用1MHz沉水式探頭和熱電偶分別從量測能量與升溫來觀察生物反應器內的能量場分布，施打超音波頻率為1MHz、能量為3W。沉水式探頭量測生物反應器內7個空間點的能量，並使用MatLab中的3D繪圖表示；熱電偶量測施打15分鐘超音波後，置入椎間盤前後周圍環境的升溫差異，以及椎間盤內部的升溫現象。(四)豬隻椎間盤培養測試：為了驗證本系統在培養豬隻椎間盤時能達到一定的存活率，將豬隻椎間盤分為新鮮試樣組(0 天)與培養7天試樣組，新鮮試樣組在試樣處理完後，進行細胞存活測試；培養7天試樣組則先進行培養，7天後取出進行細胞存活測試，並比較兩組的存活率。 結果：(一)超音波探頭校正結果中，額定能量與輸出能量的線性斜率接近1，可確定此探頭為準確的輸出源。(二)量測生物反應器內的能量場，沉水式探頭量測結果顯示超音波發射源正前方的能量不易衰退，但往上下左右偏離時量測到的能量變小許多；熱電偶量測結果顯示升溫無法判斷能量於生物反應器內的分布情形，但從中得知能量有傳遞至椎間盤內並轉化成熱量被吸收。(三)培養的豬隻椎間盤試樣目前無法存活至7天，為了解培養其間細胞存活率的變化，改為拍攝3天與6天之 Live/Dead 影像，0天與3天存活率較接近，6天則有明顯的下降。培養時感染的情形嚴重，推測可能原因為試樣取得來源、操作手法以及設備問題。 結論：使用超音波刺激之全椎間盤培養系統的硬體建置已完成，包含確定探頭打出能量、基本的能量分布狀況，另外，也透過升溫現象確認超音波可打入組織內並使其吸收。目前本研究中感染為此設備尚未解決的問題，因無法順利培養豬隻椎間盤至7天，仍需持續改善。 關鍵字：全椎間盤培養系統、低能量脈衝式超音波、超音波校正

Objective: To develop a whole disc culture system with ultrasound stimulation. Summary of background data: Intervertebral disc degeneration is one of the most common spinal disorders. Nevertheless, no effective treatment or preventative strategies are available in the management of degenerative disc disease. Low-intensity pulsed ultrasound (LIPUS), which has been proved to be affective in assisting bone healing and stimulating bone growth, is a safe and non-invasive treatment modality. Bench top studies conducted at the cellular level have shown the evidence of LIPUS stimulation in nucleus pulposus proliferation and extracellular matrix regeneration. Nevertheless, the potential effect of LIPUS on the intervertebral disc at tissue or organ level are yet to be explored. Therefore, the goal of the current project is to develop an ultrasound stimulated disc culture system used for the study of effectiveness of LIPUS on the biomechanical, biological and biochemical properties of the degenerative disc. Methods: (1) Development of the disc culture system: The system includes bioreactor chamber, media circulation system, pressure loading system and ultrasound system. The bioreactor chamber served as a culture environment for disc. Media circulation system supplied disc nutrient cycling with flow rate 200μl/min. The loading system mimics the daily disc loading in human body, which provided the dynamic loading at 0.2-0.8 MPa for 16 hours and static loading at 0.2 MPa for 8 hour. The ultrasound system was able to stimulate the disc at different power, frequency, and duty cycle. (2) Calibration of ultrasound probe: Acoustic power meter and ultrasound machine were used to calibrate the output power of probe. First, the frequency was tuned up to its best driving frequency, then the magnification was adjusted to match the output power and the nominal power in 0.2W, 1W, 2W, 2W. (3) The energy field inside the bioreactor: 1 MHz transducer and thermocouples were used to measure the energy field. The input frequency was 1 MHz, and the power was 3 W. This experiment is divided into two parts. In the first part, we measured ultrasound energy in seven points inside bioreactor. In the second part, we recorded temperature in 15 mins with and without disc, and calculated the temperature rise and heating rate. (4) Organ culture test using porcine discs: Live/Dead assay were used for the assessment of disc viability. To verify the survival rate, discs were assigned to two groups, the intact group and the cultured group. The cell viability rate of the intact group was measured at day 0, and the cultured group was measured after 7 days of incubation. Result: (1) The calibration rate of ultrasound probes are closed to one, the probes are therefore determined as accurate output sources. (2) From the measurement of energy field inside the bioreactor, we knew that output energy did not attenuate in front of ultrasound probe, but it reduced when transducer moved to other two directions. In addition, the results of thermocouple have shown that the energy passed through the disc and was absorbed. (3) Specimens did not survive over 7 days at present; thus, two groups were changed to three groups for better understanding of the cell viability in the culture process, which were intact group, 3 days group, and 6 days group. The cell viability rates of intact group and 3 days group were close, but the 6 days group shown a significant decrease. The potential infection risks may be the source of specimen, such as the endophyte generated inside the disc, human contamination, and liquid leakage from the connections of bioreactor. Conclusion: The construction of whole culture system has been established, but the infection is an issue that needs to be solved. Keywords: whole disc culture system, low-intensity pulsed ultrasound, ultrasound calibration