使用明膠混合伽瑪-聚穀氨酸合成水凝膠進行椎間盤纖維環修補

Abstract

在目前椎間盤修補與再生的研究中，絕大部分都著重於髓核的治療，使用注射方式補充各種細胞或生長因子以期達到恢復椎間盤內壓的目的，但此種採用經由纖維環注射的方法就算使用細小孔徑的細針仍會 造成纖維環損傷，進而導致椎間盤退化。因此我們認為在椎間盤退化的治療上，修補纖維環的缺損是目前常被忽略，但卻是椎間盤修補與再生能得到長期效果的重要步驟。 目前發展中的纖維環修補技術常以縫線或缺損填塞的方式進行，但這些技術都尚未取得成功的臨床成效。發展組織膠則是另一種修補方法，其優點除了可提供力學穩定度之外，若在其中混入細胞與生長因子 將可提高組織再生的效果。本項研究使用明膠與伽瑪-聚穀氨酸並使用碳醯二亞胺當交聯劑製成水凝膠，並分析其力學強度、細胞毒性與分解速度以評估將其當作注射式組織膠用於椎間盤纖維環的可行性。 第一個實驗先在牛尾椎間盤纖維環上製造針刺缺損後，接著使用水凝膠填塞缺損，然後進行椎間盤內注水測試分析椎間盤內壓力的狀況，發現將濃度 10%的明膠與濃度 3%的伽瑪-聚穀氨酸溶液與濃度 2%的碳醯二亞胺以 10:1 的比例混合後所配置出來的水凝膠可提供足夠的填塞效果，即填塞缺損後可維持與正常椎間盤相當的椎間盤內壓力。第二個實驗再將牛尾椎間盤纖維環細胞種植於水凝膠表面進行細胞培養以評估細胞相容性，當碳醯二亞胺的濃度越高則細胞增殖速度越低，同時製造細胞外基質與去氧核糖核酸的總量越少，但是卻並不影響細胞存活率，當碳醯二亞胺濃度超過 40mM 時細胞無法生長；第三個實驗分析該水凝膠的分解速率，發現分解速度與碳醯二亞胺的濃度呈反比。綜合結論是此一水凝膠有足夠潛力用於修補椎間環纖維環的缺損，而權衡足夠的力學強度以維持填塞效果以及可接受的細胞毒性和組織膠分解速度，碳醯二亞胺的濃度需藉於 20-40mM。

Most recent disc regeneration studies have focused on the restoration of intradiscal pressure through percutaneous intradiscal injections which inevitably leaves a puncture hole on the annulus fibrosus (AF) that was previously thought to be related to minor and remote secondary degenerative changes, owing to the small needle diameters. Some concerns have recently been raised by the finding that even subtle AF impairments not only have an immediate effect on axial mechanics and disc height because of altered nucleus pulposus (NP) pressurization but also may subsequently compromise biologic homeostasis including altered cell viability and metabolism. Therefore, AF repair, with or without the inclusion of NP supplements, is an often ignored but essential procedure for long-term success in IVD regeneration. None of the developing AF repair strategies such as suturing or defect blocking devices has shown clinical success. Defect filling with tissue-engineered materials is an appealing alternative for AF repair because it provides both temporary mechanical stability and biologic benefit by the inclusion of cells or growth factors to enhance regeneration. Gelatin–poly (γ-glutamic acid) (gelatin/γ-PGA) hydrogel utilizing 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) for cross-linking has promising mechanical properties and biocompatibility. We evaluated the suitability of this material as a new injectable hydrogel for AF repair with mechanical strength validation, cytocompatibility evaluation and degradation behavior analysis. The capacity of this hydrogel to restore IVD integrity to filling a needle puncture defect in IVDs, in a model of a needle puncture for a diagnostic or therapeutic purpose, was determined through the quantitative discomanometry (QD) test. The hydrogel, which was constructed with 10% gelatin and 3% γ-PGA solution and crosslinked with EDC (2%) at a 10:1 ratio, can restore needle punctured discs with comparable intradiscal pressure. The cytotoxicity of the hydrogel crosslinking agent with different EDC concentration was assessed with AF cell hydrogel monolayer surface culture. EDC concentrations from 10 mM to 40 mM resulted in significant decreases in AF cell proliferation without obvious influence on cell viability. Higher EDC concentrations resulted in a decreased percentage of Alamar blue reduction and GAG and DNA concentration but did not affect GAG/DsDNA and live-dead ratios. The degradation behavior of the hydrogel was analyzed because the longevity of implanted hydrogel is a crucial factor in evaluating the sealing effect for intradiscal pressure maintenance. It revealed that higher EDC concentrations decreased the hydrogel degradation rate. The EDC concentration of 40 mM can maintain almost 90% of residual weight after two months, meaning that most of the filling effect remained. Based on the observations of our studies about intradiscal pressure restoration, cytocompatibility and degradation, the tradeoff EDC concentration should ideally be more than 20 mM to achieve adequate sealing effect and below 40 mM for acceptable cytotoxicity. In conclusion, the developed gelatin–poly (γ-PGA) hydrogel gel provides an effective gap-filling biomaterial with good cytocompatibility. Performance of this gel shows substantial promise for use as a sealant for small AF defects and suggests that it may be useful as an adhesive to augment other biomaterials for larger AF repairs.