胜肽交聯劑、富含血小板之血漿與牽引治療回復退化性椎間盤生物力學功能之可行性分析

Feasibility Analysis of Crosslinker, Platelet-rich Plasma and Traction Treatment on the Recovery of Biomechanical Functions in Simulated Degenerative Intervertebral Disc

徐于鈞(Yu-Chun Hsu)

指導教授 ： 王兆麟

國立臺灣大學/工學院/醫學工程學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00195

摘要

椎間盤退化為造成下背痛的原因之一，適當的治療方式將有助於緩解椎間盤退化或減緩其退化速度。椎間盤退化起源於椎間盤內發炎物質及降解性酵素活性增加，使椎間環結構被破壞、椎間盤細胞外基質化學組成改變及水分含量降低。目前臨床上並沒有治療或預防退化椎間盤的有效方法與建議，因此退化椎間盤的治療或預防之研發有其臨床重要性。本研究認為椎間盤退化的治療策略應著重於維持細胞外間質的完整性以及細胞的存活率，因此天然交聯劑、富含血小板之血漿療法有可能為潛在的治療策略方式之一。天然交聯劑可強化椎間盤組織強度，但對於椎間盤生化性質上的影響則不得而知。富含血小板之血漿含有豐富的生長因子，已被證明可促進細胞生長與椎間盤基質合成，但目前是否能達到回復椎間盤力學功能則仍乏肯定。牽引治療則是復健醫學上普遍使用緩解下背痛的技術，可回復縮小的椎間孔，但長期的治療效果以及治療機制則尚未明朗。
本研究第一部分為利用體外全椎間盤培養系統以胰蛋白酶建立椎間盤退化模型，並評估胜肽交聯劑(梔子素)對回復不同退化程度的椎間盤細部結構及整體力學性質效益。椎間盤細部結構包含組織水含量以及椎間環結構完整性，椎間盤整體力學性質包括椎間盤受靜態負載時之聚合模數與液體滲透性，以及椎間盤受到衝擊式動態負載時之整體勁度及緩衝能力。結果顯示注射胰蛋白酶以及額外施加高強度疲勞負載(平均力量420N，負載頻率2.5Hz，負載時間4小時)可分別模擬早期退化以及晚期退化之椎間盤退化模型。早期退化組之靜態及動態性顯著下降、水分含量減少以及纖維環結構鬆散；晚期退化組之聚合模數以及整體勁度上升，但緩衝能力下降，椎間核水分含量減少且椎間環結構破壞嚴重。胜肽交聯劑治療則可回復早期退化椎間盤整體靜態及動態性質；然而，由於內部整體結構破壞嚴重，胜肽交聯劑治療雖可回復部分晚期退化椎間盤組織缺損部分，使椎間核內水分含量增加，但液體滲透性及緩衝能力仍顯著降低，這些組織破壞可能導致椎間盤退化持續惡化。
本研究第二部分為利用變性水解椎間盤退化模型，探討胜肽交聯劑以及富含血小板之血漿療法對於椎間盤力學性質以及生化性質的效益分析，生化性質包含組織間水分含量及醣胺聚醣含量。結果顯示利用胜肽交聯劑可回復椎間盤之聚合模數及整體勁度，椎間核內水含量上升；富含血小板之血漿治療則可同時回復聚合模數、液體滲透性以及動態特性中之椎間盤整體勁度、緩衝能力、椎間核水分含量以及醣胺聚醣含量，但無法回復至健康椎間盤狀態。由結果可知富含血小板之血漿之治療與回復退化椎間盤之力學及生化性質較胜肽交聯劑為佳。
本研究第三部分以全椎間盤培養系統探討牽引治療對於退化性椎間盤中纖維環微結構、分子傳輸以及細胞存活率的影響。實驗中纖維環微結構使用掃描式電子顯微攝影分析，而分子傳輸則利用螢光染劑並佐以螢光攝影平台進行觀測。其結果可觀測到退化性椎間盤中，纖維環遭受變性水解，使膠原蛋白結構產生不規則性排列甚至剝離分解，阻塞膠原蛋白間養分傳輸孔洞，進而導致養分傳輸減少，細胞存活率下降。而牽引治療則可將不規則性排列之膠原纖維結構伸直，使得被阻塞的孔隙再次打開，因此可有效的促進養分在組織間的傳輸效率，進而增加細胞存活率。
綜合以上結論，酵素降解作用會造成力學以及生化性質改變，並使椎間環內部養分傳輸孔徑阻塞，產生椎間盤退化。胜肽交聯劑、富含血小板之血漿以及牽引治療皆有助於回復部分椎間盤力學及生化性質，因此未來的研究中仍可探尋其他具潛力之生物性或機械性療法，供未來椎間盤退化治療選擇。

關鍵字

全椎間盤培養系統 ； 椎間盤退化模型 ； 天然交聯劑 ； 牽引治療 ； 富含血小板之血漿 ； 動態性質 ； 流變學性質 ； 水含量 ； 醣胺聚醣含量

並列摘要

Disc degeneration is one of the most common causes of low back pain and the development of appropriate treatment strategies to prevent or slow the degeneration process has gained enormous interest. It has been shown recently that the disc degeneration process may be initiated from inflammatory factors and the associated enzyme activation leading to a chain of reaction including the destruction of anular structural integrity, a change of disc biochemical compositions and significant reduction of water content. Subsequently, maintaining the integrity of extracellular matrix and cell viability by the injection of natural cross-linker and platelet-rich plasma (PRP) has been shown to be a treatment strategy with some great potential. Natural cross-linker has been proven to enhance the resistant strength of intervertebral disc; however, exact effects on disc biochemical properties remain unknown. Furthermore, although PRP is rich in growth factors and have been shown to stimulate the cell proliferation and matrix synthesis process, its ability to return the disc back to pre-injury status is yet to be investigated. Moreover, traction therapy is a physical therapy technique that is commonly prescribed for the treatment of low back pain. Biomechanically, traction is able to increase the foramen space and thus easing the symptoms of back pain, however, the long-term outcome and the associated mechanisms in the prevention of disc degeneration remain unclear.
The first part of this dissertation was to develop an appropriate disc degeneration model based on the injection of trypsin in a whole organ culture system, and to evaluate the treatment efficacy of natural cross-linker (genipin) on microstructure and biomechanical properties of degenerated disc. The results showed that the moderately degenerated disc and severely degenerated disc were successfully simulated by trypsin injection and high magnitude fatigue loading (Frms: 420N, Frequency: 2.5, Loading period: 4 Hours). The rheological, dynamic properties and water content significantly decreased and anular structure was loosen in moderately degenerated discs. In severely degenerated disc, the aggregate modulus and stiffness increased, while the reduction of water content in nucleus pulposus and broken anular structures were observed. Cross-linker therapy restored the rheological and dynamic properties of moderately degenerated disc. It also recovered the water content in nucleus pulposus, however, the hydraulic permeability and damping coefficient significantly decreased due to the unrecoverable anulus structure defects.
In the second part of the dissertation, the recovery efficacy of natural cross-linker and PRP therapy on trypsin-treated degenerated discs were determined by studying the change of disc water content, glycosaminoglycan content and disc biomechanics. The results showed that natural cross-linker has the capacity to recover the aggregate modulus, stiffness and water content in nucleus pulposus. The PRP therapy restored not only the disc biomechanics such as aggregate modulus, hydraulic permeability, stiffness, damping coefficient, but also recovered the disc water content and glycosaminoglycan content. However, both treatments failed to fully recover the disc functions back to its intact level. Overall, the PRP were found to have better treatment efficacy on the recovery of disc biomechanics and biology functions.
In the third part of the dissertation, the efficacy of the traction therapy on microstructure, molecular convection and cell viability of degenerated disc was determined. The results found that post-traction, the anular collagen fibers were delaminated and irregular. The pores in collagen fibrils were occluded which is likely to lower nutrient transportation and decrease cell viability. Traction therapy was found to straighten the collagen fibers and subsequently the annulus pores were found to be less occulded. Overall, the molecular convection and cell viability improved but not to the intact level.
In conclusion, the dissertation provided evidence that the enzymatic digestion on intervertebral disc has the capacity to alter disc biomechanics and biology to successfully simulate disc degeneration with the most profound effect being the occultation of the pores in anular fibrous. The natural cross-linker, PRP therapy, and traction therapy were all effective in the recovery of disc biomechanics and biology; however, all failed to return the degenerated disc back to pre-injury levels. Therefore, future studies should develop treatment strategies to enhance the recovery of degenerated disc that is applicable clinical practice.

並列關鍵字

Whole organ culture system ； Intervertebral disc degeneration ； Natural crosslinker ； Traction treatment ； Platelet-rich plasma ； Dynamic properties ； Rheological properties ； Water content ； Glycosaminoglycan content

參考文獻

1. Andersson GB. What are the age-related changes in the spine? Bailliere's clinical rheumatology 1998;12:161-73.

2. Cote P, Cassidy JD, Carroll L. The Saskatchewan Health and Back Pain Survey. The prevalence of neck pain and related disability in Saskatchewan adults. Spine (Phila Pa 1976) 1998;23:1689-98.

3. Walker BF. The prevalence of low back pain: a systematic review of the literature from 1966 to 1998. Journal of spinal disorders 2000;13:205-17.

4. Barrero LH, Hsu YH, Terwedow H, et al. Prevalence and physical determinants of low back pain in a rural Chinese population. Spine (Phila Pa 1976) 2006;31:2728-34.

5. Chou YC, Shih CC, Lin JG, et al. Low back pain associated with sociodemographic factors, lifestyle and osteoporosis: a population-based study. Journal of rehabilitation medicine : official journal of the UEMS European Board of Physical and Rehabilitation Medicine 2013;45:76-80.