快速成型之膠原蛋白微纖維支架設計以幫助牙周韌帶再生

Abstract

牙周炎是牙周組織破壞性疾病，牙周組織包括齒槽骨、牙齦、牙周韌帶及牙骨質，牙周韌帶連接齒槽骨及牙骨質並在緩衝牙齒咬合時產生的負荷扮演重要角色且擁有其他如組織修復、運送養分、傳導神經感覺等的重要功能。然而牙周韌帶的再生及功能性恢復對現今的牙科技術仍是一大挑戰，本研究目標為開發出能承受剪力負荷之組織工程膠原蛋白微纖維支架，以引導牙周韌帶再生並幫助實現牙周組織的功能性重建。

本研究設計直線型及波浪型之膠原蛋白微纖維，以使用微量擠出技術(microextrusion)之三維生物列印機(bioprinter)印製，使用核黃素及紫外光交聯(crosslink)後執行後續的剪力實驗，實驗以平行板流體流動腔(parallel-plate fluid flow chamber)建構生物反應器(bioreactor)以形成流體剪力刺激並培養牙周韌帶細胞。將牙周韌帶細胞種植於直線型及波浪型之膠原蛋白微纖維後以0、6、12 dynes/cm2 之流體剪力培養與刺激1、4、8小時，並以DAPI/F-actin/Vinculin之免疫螢光染色、Live/Dead assay、RT-qPCR觀測細胞之形態、存活率及基因表現。 經過膠原蛋白列印最佳化並設定最佳列印參數後，膠原蛋白微纖維以250千帕(kPa)之列印壓力、2 mm/s之列印速度及34G針頭成功印製。直線型微纖維之寬度為189.9 ± 11.44 μm，波浪型微纖維之寬度為228.7 ± 13.06 μm，波浪型微纖維之振幅及波長分別為238.2 ± 17.12 μm及750.2 ± 13.80 μm。實驗結果發現牙周韌帶細胞及膠原蛋白微纖維成功承受6-12 dynes/cm2 之流體剪力並順利貼附。受剪力刺激後，貼附於波浪型膠原蛋白微纖維之細胞骨架有較明顯的伸展且細胞存活率較佳，細胞週期素D(cyclin D)、E-鈣粘蛋白(E-cadherin)及骨膜素(periostin)之表現量亦較高。

本研究的結論是經過列印最佳化後，模擬牙周韌帶之膠原蛋白微纖維能順利以三維生物列印機製成並以核黃素及紫外光交聯。與直線型膠原蛋白微纖維相比，波浪型微纖維更能承載剪力負荷且維持住細胞的存活率，細胞貼附於波浪型膠原蛋白微纖維之基因表現亦顯示出有較強的再生潛能。本實驗結果表明波浪型支架設計有望幫助牙周韌帶再生並實現牙周組織的功能性重建，因此評估波浪型膠原蛋白微纖維支架對牙周韌帶之功能性重建的臨床前試驗是必需且令人期待的。

Objective: The periodontal ligament (PDL) plays a pivotal role in occlusal load adaptation, in addition to other important functions. However, the functional reconstruction of the PDL remains a challenge. This study therefore sought to develop a PDL guiding microfiber scaffold which was capable of withstanding shear stress from the occlusal loads. Materials Methods: Collagen-based straight and wavy microfibers were designed to serve as substrates for PDL cell growth. The microfibers were prepared with a bioprinter using microextrusion technology coupled with riboflavin/UV 365 nm crosslinking. The capability of the scaffold to withstand occlusal load was assessed with a parallel-plate fluid flow chamber to generate shear stress levels within a 0–12 dynes/cm2 range, and the cell morphology, viability, and gene expression of PDL cells on the microfibers was evaluated via DAPI/F-actin/vinculin staining, the live/dead assay, and quantitative reverse transcription polymerase chain reaction (RT-qPCR), respectively. Results: Upon collagen bioprinting optimization (i.e., adjustment of the printing parameters), collagen-based microfibers were successfully fabricated under a 250 kPa pressure with a 34G needle at a 2 mm/s printing speed. The width of each straight microfiber was 189.9 ± 11.44 μm, whereas the width of the wavy microfibers was 228.7 ± 13.06 μm. Moreover, the amplitude and wavelength of wavy microfibers were 238.2 ± 17.12 μm and 750.2 ± 13.80 μm, respectively. Under shear stress stimulation, PDL cells were successfully seeded on both straight and wavy microfibers and cytoskeleton stretching was more evident on wavy microfibers. Additionally, PDL cell viability was higher on wavy microfibers and cyclin D, E-cadherin, and periostin gene expression was up-regulated. Conclusion: Collagen-based microfibers mimicking PDL fiber structure were rapidly prototyped using a bioprinter with riboflavin/UV 365 nm crosslinking. Notably, wavy microfibers were capable of withstanding shear load stimulation, preserved PDL cell viability and gene expression, and exhibited an enhanced tendency to promote healing and regeneration. Therefore, further preclinical research is required to confirm the fiber-guiding potential and function of this novel PDL cell/microfiber scaffold material.