生物支架設計於氣管重建之研究

Scaffold development for trachea reconstruction

洪士涵(Shih-Han Hung)

指導教授 ： 曾厚

臺北醫學大學/醫學院/醫學科學研究所/博士(2020年)

摘要

上呼吸道由於先天性疾病, 外傷, 癌症治療等等因素而受損時, 往往會造成患者極大的危害. 喉氣管的重建是一個非常複雜的議題. 本研究初期的設計是以製造生物支架, 即所謂的scaffold爲基礎 (Chapter I). 利用冷凍乾燥以及外加超音波能量的方式試圖製備喉部生物支架. 我們用新鮮的豬喉組織進行反復冷凍乾燥與超音波能量處理. 研究結果顯示單次進行冷凍乾燥處理的去細胞效率欠佳, 然而再加入超音波能量之後效果可以大幅度提升. 然而我們亦注意到這樣製備的生物支架結構強力有相當程度的下降, 顯微鏡下亦有相當程度結構的破壞. 基於這樣的問題, 在第二個階段(Chapter II) 我們嘗試只把黏膜的部分取下並進行去細胞, 至於提供支撐所需要的管狀強力就用可降解生物相容性材料來複合. 我們利用PLLA材料製造成螺旋環狀的氣管骨架, 外面包覆以電氣紡絲所製成的薄膜, 作為複合氣管生物支架的骨幹. 我們額外也對這個骨幹進行了抗壓縮強力的測試. 研究結果顯示以3mm 與 1.5mm所製成的環狀骨架所提供的支撐強度皆遠超過原生天然豬氣管的抗壓縮強力. 然而在嘗試植入以後卻發現無法此生物支架無法與host組織整合並逐漸壞死. 因此我們再度改變策略, 在第三個階段中 (chapter III), 為避免傷害過大, 採用1% SDS與超音波並用. 由於全結構去細胞涉及軟骨骨架的去細胞, 我們額外加入了SDS的這個去細胞要素. 在紐西蘭大白兔的動物實驗模式之下, 成功的利用冷凍乾燥, 超音波能量, 與SDS反覆週期法很有效率的製備了全結構去細胞氣管生物支架. 強力測試結果顯示經此處理的兔隻氣管仍然保有接近於原生氣管的強力特性. 進一步進行氣管移植實驗, 我們發現雖然呼吸上皮的再生良好, 然而氣管的管狀結構在經過一段時間之後仍然會逐漸崩塌, 兔隻仍在約平均兩周後因植入的氣管崩塌壞死而死亡, 雖然不會引起免疫排斥反應, 整段去細胞的氣管支架植入後無法引發有效血管增生與細胞移入. 我們再度修正策略 (chapter IV), 試圖進行保留軟骨細胞的部分去細胞氣管製程. 藉由成功保留一部分存活軟骨細胞的整段氣管生物支架原位植入, 可以達到兔隻的長期存活, 然而經內視鏡檢查與病理檢查仍可見相當程度之氣管狹窄. 此問題肇因於黏膜部分需依賴呼吸上皮逐漸移入, 難免有疤痕產生, 接下來考慮以片狀細胞技術克服.

關鍵字

生物支架 ； 氣管重建

並列摘要

The trachea is critical for respiration and airway protection in humans. Ideal methods for the reconstruction of the tracheal structure and restoration of tracheal function have not yet been developed once the trachea has been damaged or removed.
In Chapter I, the decellularization protocol is evaluated in the development of the laryngotracheal scaffold. We have found that combining freeze-drying and sonication can effectively decellularize the porcine laryngeal tissue. However, significant damage is notable. Therefore in Chapter II, only mucosa decellularization was done and the tubular framework of the trachea was made by biocompatible materials. While these two components can be hybridized to a transplantable segment, tissue necrosis with subsequent structure decomposition was found quickly after transplantation, and thus the cartilage supporting structure is mandatory. In Chapter III, the whole segment decellularization of the trachea was used on the rabbit model with a modified protocol using our previously developed freeze-dry-sonication-SDS (FDSS) decellularization process and transplanted orthotopically into segmental tracheal defects. We found that the FDSS decellularization process is effective in creating whole-segment, sub-totally decellularized tracheal scaffolds. However, although the respiratory epithelium regeneration on the inner surface appeared to be satisfactory, the tubular structures were not able to be maintained after transplantation, which ultimately led to the death of the animals. Therefore, it seems that the cartilage component appears to be very critical and it seems that host cell migrations and angiogenesis are difficult to happen once a complete decellularized tracheal cartilage scaffold is transplanted. Based on these results, in Chapter IV, a new protocol was developed with quick, partial decellularization of the harvested trachea. The idea is based on the low immunogenicity of the cartilage tissue and it is possible to implant the scaffold without serious rejection responses even with some donor cartilage cells remaining alive. By leaving at least a portion of live cartilage cells in the transplant, it is possible that these live cells continue to function and contribute to a higher structural strength after transplantation which is critical for maintaining the tracheal tubular structure. The result showed cartilage cells remain alive under vital stain through our new decellularization protocol. Long-term after implantation the implanted tracheal scaffold remained intact, especially the cartilage portion, without signs of rejection. However, we do find that at the transplanted segment, notable non-lethal stenosis or stricture was present, which might be the direct result of primary healing of the inner lining of the transplant. It is likely that the cell-sheet technique might be a potential solution if the cell sheet can be placed on the inner lining of the transplanted scaffold. Further investigations are still ongoing as scheduled.

並列關鍵字

Scaffold ； trachea reconstruction

參考文獻

References

CHAPTER II

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