以溫感性甲殼素水膠搭載洛伐他汀的奈米粒子持續釋放系統治療椎間盤髓核組織之研究

Abstract

腰椎椎間盤退行性病變（intervertebral disc degenerations; IVDD）的發病機制是多因素共同作用的結果，主要由基因易感性、衰老、慢性發作的炎症、氧化壓力以及機械應力引起的損傷等因素所驅動。雖然許多人並無臨床症狀，但仍有一部份患者會進展為有症狀的椎間盤病變，造成嚴重的下背痛或神經功能障礙。當保守治療無法有效緩解症狀時，往往必須訴諸外科手術干預。相反地，隨著對椎間盤退化背後分子機制更深入的理解，加上新型生物材料的近期進展，以生物醫學工程策略的治療方式也蓬勃發展。這些治療方法藉由椎間盤內引入生長因子、小分子藥物或治療性基因，來促進細胞外基質（extracellular matrix; ECM）的合成或抑制分解代謝降解，從而延緩或逆轉早期退變；或甚至是導入髓核細胞、幹細胞或提供物理性的支撐。在此框架下，水膠可根據其特定的結構設計，扮演多功能的結構性或藥物釋放的角色。 在這些策略中，相較於單次注射，理論上持續釋放洛伐他汀（lovastatin）等治療因子，能為髓核細胞與組織提供更具效益的合成代謝刺激。本研究旨在開發一種負載洛伐他汀的奈米顆粒（LVS-NPs），並將其整合至溫感性的甲殼素/明膠/甘油磷酸鹽（C/G/GP）的水膠中，用以治療椎間盤的退化。本研究採用油水乳化溶劑蒸發法製備 LVS-NPs，隨後進行分子外貌表徵與藥物封包率的分析。接著將 LVS-NPs 引入 C/G/GP 水膠中以達到藥物控釋，並進一步進行體外（in vitro）與體內（in vivo）評估。 結果顯示，LVS-NPs 呈圓形球體，具備中等範圍的聚分散指數，這有助於洛伐他汀的釋放。流變學研究表明，將 LVS-NPs 整合至水膠中並不會改變其溫感成膠的特性。藥物釋放測試證實，洛伐他汀能在7天之中持續從含有 LVS-NPs 的水膠中穩定釋放。在體外實驗（包括 WST-8 檢測、LDH 檢測、TUNEL 染色和蛋白質免疫印跡[Western blot]）中，含有 LVS-NPs 的水膠展現出良好的生物相容性與極低的細胞毒性。對培養在含有 LVS-NPs 水膠中的人類髓核細胞進行分析發現，基質聚醣（aggrecan）和第II型膠原蛋白的產量有所提升，且未引發細胞凋亡；然而，若要達到與 5μM游離洛伐他汀相當的效果，可能需要更高的藥物濃度或更長的作用時間。在動物實驗中，本研究將不同的治療方案應用於模擬早期椎間盤退變的大鼠尾椎模型。然而，無論水膠中是否含有游離洛伐他汀或 LVS-NPs，所有接受水膠治療的組別中，其椎間盤高度指數（disc height index; DHI）均顯著下降。透過核磁共振（MRI）與組織學染色觀察到的廣泛退化，顯示出髓核組織的完全喪失；這很可能是由於在纖維環限制的微小椎間盤空間內，注射的水膠產生了嚴重的機械性壓迫所致，此現象仍需進一步研究。 總結來說，LVS-NPs 在體外實驗中證實能持續釋放洛伐他汀，並對人類髓核細胞產生延伸的刺激效益。然而，未來仍需要更深入的研究來優化注射水膠的體積及其與椎間盤內空間的比例，並提高 LVS-NPs 內洛伐他汀的載藥濃度，以便在最大程度減少對周圍組織造成機械壓迫損傷的前提下，達到最佳的治療效果。

The pathogenesis of lumbar intervertebral disc degeneration (IVDD) is multifactorial, driven by factors such as genetic predisposition, aging, chronic inflammation, oxidative stress, and mechanical stress-induced injury. Although many individuals remain asymptomatic, a subset of patients develops symptomatic disc disease, presenting with debilitating lower back pain or neurological dysfunction. When conservative management fails to provide clinical relief, surgical intervention often becomes necessary. Conversely, a deeper understanding of the underlying molecular mechanisms of disc degeneration, combined with recent advancements in novel biomaterials, has fueled the development of biomedical engineering strategies. These therapeutic approaches aim to retard or reverse early-stage degeneration through the intradiscal injection of growth factors, small-molecule medications, or therapeutic genes designed to stimulate extracellular matrix (ECM) synthesis or inhibit catabolic degradation. Besides, we could also introduce NP cells, stem cells, or even provide mechanical support when needed. Within this framework, hydrogels can serve versatile structural or delivery roles depending on their specific macromolecular design. Among the strategies, sustained release of therapeutic factors, such as lovastatin, is theoretically more beneficial than a single injection in promoting anabolic stimulation to nucleus pulposus cells/tissues. This study aimed to develop lovastatin-loaded nanoparticles (LVS-NPs) incorporated into thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel for treatment of intervertebral disc (IVD) degeneration. LVS-NPs were prepared using an oil-in-water emulsion evaporation method, followed by characterization and drug encapsulation efficiency. LVS-NPs were then incorporated into C/G/GP hydrogel for controlled drug release, with subsequent in vitro and in vivo evaluations. The LVS-NPs showed a round shape with a mid-range polydispersity index, which shall benefit lovastatin release. Rheological study showed incorporation of LVS-NPs into hydrogel does not change thermal gelation property. Drug release tests demonstrated that lovastatin was released steadily from the hydrogel containing LVS-NPs over 7 days. The LVS-NPs-containing hydrogel demonstrated good biocompatibility and low cytotoxicity in the in vitro studies including WST-8 assay, LDH assay, TUNEL test and Western blot. Analysis of hydrogels containing LVS-NPs on human nucleus pulposus cells revealed enhanced production of aggrecan and type II collagen without inducing apoptosis, though higher concentrations or longer treatment durations may be required to achieve effects comparable to those of 5μM lovastatin. In animal study, various treatment regimens were applied to a rat caudal disc model mimicking early-stage IVD degeneration. Despite of lovastatin or LVS-NPs in hydrogel, significant decreases in disc height index (DHI) and extensive degeneration observed through MRI and histological staining indicated a complete loss of nucleus pulposus tissue in all hydrogel-treated groups, likely due to mechanical compression from the injected hydrogel within the confined space of the annulus fibrosus, which needs further investigation. LVS-NPs demonstrated sustained release of lovastatin and extended stimulatory effects on human nucleus pulposus cells in vitro. However, further research is needed to optimize the hydrogel volume and its ratio to intradiscal space and increase lovastatin concentration within LVS-NPs to achieve optimal therapeutic outcomes while minimizing mechanical compression to surrounding tissues.