2018-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/699727摘要：脊椎椎間盤結構包括外圍多層狀的纖維環(Annulus Fibrosus)、內層膠狀之本核(Nucleus Pulposus)、以及位於上下兩端的終板(Endplates)，其退化進程頗為複雜，可能起始於上述三組織之任一結構中。椎間盤退化過程中可能會導致纖維環承擔過大的受力，因而引起纖維環產生裂痕或破損，而本核中之膠狀物質就可能經由纖維環之缺損中外流以致壓迫脊髓或脊椎神經根而導致疼痛症狀，即為所謂之「椎間盤突出」。若臨床症狀未能借由非手術治療緩解時，則須以手術治療（椎間盤切除術）移除外溢之本核碎片以解除症狀。然而目前的臨床處置，均未能將因椎間盤突出或手術過程中形成的纖維環缺損加以修復，而未加以治療處理的纖維環缺損就是造成椎間盤突出復發的位置。纖維環修補對於重建本核空間完整性與提供椎間盤組織再生的有利環境實屬重要步驟，而長期有效的纖維環癒合需要細胞移入缺損部位及細胞外基質的合成與重塑，因此需要宿主免疫反應、細胞、活性材料的共同作用以達成修補及避免進一步退化之目的。纖維環修補的研究有兩大方向，包括機械性及生物性修補。機械性修補是以鈍性物質如縫線或合成聚合物進行縫合或圍堵，但於生物體內實驗顯示其對促進組織重整與長期癒合的效果不佳。生物性修復的常用方法為運用生物活性材料模擬原始纖維環組織結構，配合纖維環細胞或幹細胞，以達組織修復之目的。雖然目前有一些研究材料顯示此方法俱有相當的潛力，但仍未有特定材料及製備方式足以於臨床上使用。此三年期計劃將發展以蠶絲蛋白(Fibroin)為基底、模擬纖維環纖維排列之多層結構材料，配合蠶絲蛋白的強度與角蛋白(Keratin)或明膠(Gelatin)之親水性以利細胞貼附。使用兩種材料製成方式（電紡絲及3D列印），比較其材料特性、生物相容性、細胞行為、基質合成等效果。並以迷你豬進行動物實驗以驗證其對纖維環缺損之修補效果，驗證方法將包括X光片檢查椎間盤高度之變化、磁核共振檢驗椎間盤組織退化程度、椎間盤攝影及椎間盤內壓力測量以檢驗纖維環組織密合度、組織及免疫染色以檢驗纖維環組織修復及重整之成效。期待經此三年期研究後，能獲得足以於臨床使用之纖維環修復材料，以避免或減少椎間盤手術後椎間盤突出復發之機率。<br> Abstract: The intervertebral disc (IVD) is comprised of a multilaminar annulus fibrosus (AF) enclosing a gelatinous nucleus pulposus (NP) core, and adjacent cartilaginous endplates. IVD degeneration is a complicated process with various changes that may start at endplates, NP or AF. Degenerative processes of IVD may cause excessive loading to AF then subsequently lead to fissures or tears. NP materials may herniate through AF lesions then cause compression to spinal roots or cord. When clinical symptoms are not responded to nonsurgical managements, surgical intervention (microdiscectomy) is required to remove extruded NP fragments for alleviating symptoms. However, current clinical practice does not seal AF defects caused by processes of disc herniation or during procedure of microdiscectomy. The untreated defects in AF are potential sites for recurrent herniation. Repair of AF is needed to restore the containment to NP and to provide an environment for regeneration of IVD tissues. Long-term healing of AF demands infiltration of cells, synthesis and remodeling of extracellular matrix (ECM). A combination of host immune response, cells and biomaterials is warranted for achieving persistent repair and prevention of further degeneration.Two major approaches have been utilized in pre-clinical investigations of AF repair. Mechanical methods apply biologically inert materials, sutures or synthetic polymers, to close or barricade AF lesions. When tested in vivo, mechanical repairs fail to promote tissue integration, long-term healing/regeneration, or restore native biomechanical function to the spine. Common approaches for biologic repair of AF comprise biomaterials attempting to mimic native fibrous structure of AF with or without AF or stem cells. Although a number of biomaterials have been developed and investigated showing the biologic strategy is promising, none have reached clinical application in humans. This 3-year project will start from the development of fibroin-based membranes with aligned fibers mimicking the structure of a lamella in the AF. Silk fibroin has a very good mechanical strength but is hydrophobic not favoring cell seeding. Keratin is more hydrophilic and possesses numerous amounts of Arg-Gly-Asp (RGD) sequence for better cell adhesion. Gelatin is denatured Type I collagen with preservation of many signal domains in collagen. Gelatin-based scaffolds had been used in tissue engineering studies for IVDs. Two methods of fabrication, electro-spinning and 3-D bioprinting, will be tried and compared. Characteristics of the fabricated membranes including microstructures and tensile strength will be examined. Biocompatibility, adhesion, migration and proliferation of AF cells, and synthesis of ECM will be evaluation by cell culture on a single layer of fabricated membranes. After determining optimal composition and preparation of the fabricated membranes, plug-type scaffolds using multi-lamellae of fabricated membranes will be designed and manufactured. This plug scaffold will be implanted into created defects of porcine AFs for investigating the in vivo effects on AF repair. Evaluations will include changes of disc height by radiography, IVD tissue degeneration by MR imaging, containment of AF by discography and measurement of intradiscal pressure, AF tissue healing and remodeling by histologic/immunohistochemical studies.椎間盤纖維環修補蠶絲蛋白角蛋白明膠薄膜多層性支架細胞培養動物實驗椎間盤突出復發之預防Intervertebral DiscAnnulus FibrosusBiologic RepairFibroinKeratinGelatinMembraneMulti-Laminated ScaffoldCell CultureAnimal StudyPrevention of Re-Herniation