幾丁聚醣傷口敷料止血性能的力學特性

Abstract

幾丁聚醣所製成的紗布，其止血性能優於一般傳統使用的紗布，一般認為是因為幾丁聚醣帶正電荷，吸引帶負電荷的紅血球黏附而形成黏膜屏蔽，阻擋了血液由傷口流出，達到止血的目的，但詳細的原因並未被解釋，本研究的第一個目標，便是從力學的角度去探討相關的止血機制。幾丁聚醣紗布在具有凝血功能障礙的動物實驗中，仍能產生止血的功能，但動物實驗耗費的資源龐大，因此本研究的第二個目標為：在生物體外的環境中，提供能定量量測止血性能的環境與裝置。 目前量測不同紗布敷料的止血性能多為靜態測試，若能在流動的環境中進行止血測量，其量測結果會更貼近於真實的情形；Jesty等人(2009)提出一套流體裝置(此稱為CPG裝置)，是利用恆定的壓力梯度，讓流體通過受測紗布後，依照所收集到的流體質量來評估紗布的止血性能，此裝置能以動態的方式進行實驗，但需要大量的測試液體，本研究的第三個目標：提出一個可使用較少測試液的替代實驗裝置。 本研究完成了三項工作： 1. 利用定流量(CFR)裝置量測各種紗布敷料的止血性能，以恆定的流動速率推動測試液體使其通過紗布敷料，觀察與記錄紗布兩側的液體壓力降，壓力降越大則表示紗布敷料的止血性能越佳，實驗結果與CPG裝置測量的結果相符合，但是CFR裝置消耗較少的測試液，而CPG裝置模擬出血的情況更為真實。 2. 利用定流量裝置以全血與洗滌紅血球液(即除去血小板與凝血因子)進行實驗，結果顯示幾丁聚醣的紗布在兩種血液中皆有優異的止血效果，由此可知，紅血球黏附於幾丁聚醣而達到止血的方式，是獨立於典型的人體凝血機制，即使沒有血小板與凝血因子的參與作用，仍能達到止血的 功效。 3. 架設一套光鉗系統，量測紅血球細胞在各種紗布纖維上的黏附力，量測到的黏附力約為3.82 pN，因為紅血球黏附於紗布纖維的力量太小，所以此黏附現象並不是造成止血的唯一原因。 然而，無論在靜態或是流動的環境下，可以觀察到紅血球細胞聚集於幾丁聚醣紗布纖維旁，並且堆疊排列成層狀結構，但並未發生於其它的紗布纖維上，此層狀結構是形成黏膜屏蔽的初始跡象，而後達到止血的目的。紅血球細胞聚集的原因，可透過量測血液與幾丁聚醣粉末的混合液體的界達電位(zeta potential)與pH值來解釋，由DLVO (Derjaguin-Landau-Verwey-Overbeek)理論得知，幾丁聚醣去質子化所釋出的H+離子與紅血球細胞膜上的COO¯離子相結合，使得紅血球彼此間相斥的電雙層力減弱而形成多層排列結構，又因幾丁聚醣的良好吸水性，增加了傷口附近的血液黏度，進而使血液的流量減少，再加上按壓於傷口的反向壓力，各個因素造就出優異的止血效果。本研究的結果有利於設計出提升止血效果的幾丁聚醣紗布，並可應用於一般生物醫學。

Chitosan-based wound dressings are superior to traditional dressings for hemostatsis, and can arrest bleeding with clotting dysfunction. It is generally claimed, but without detailed reasoning, that “the chitosan cross-links red blood cells (RBCs) to form mucoadhesive barrier” to block the bleeding. The first goal of this study is to understand the related mechanism from a mechanical view point. Hemostatsis succeeds using chitosan under clotting dysfunction was supported qualitatively by animal tests, but quantitative measurement was still lacking. The second goal of this study is provide such a quantitative result in an in vitro environment. Static tests were employed mostly for the hemostatic performance tests of various dressings, but it would be more realistic if the test was performed in a flowing environment. A flow-through device (called the CPG device here) was proposed by Jesty et al. (2009) under a constant pressure gradient, and the performance was assessed via measuring the amount of fluid masses through the device. However, a substantial amount of test fluid is required for the testing using the CPG device. The third goal of this study is to propose an alternative device using less test fluid. Three works were accomplished in this study. First, a flow-through device under a constant flow rate (called the CFR device) was proposed and demonstrated successfully for assessing dynamically the hemostatic performance of various wound dressings via comparing the testing using the CPG device. The pressure drops across the dressings are measured for assessing the performance in the CFR device. The CFR device consumes less test fluid, but the CPG device models more realistic the scenario of bleeding. Secondly, detailed experiments were performed using the CFR device with both the human whole blood and washed blood (with clotting factors and platelets removed) as test fluids, and the results agree with each other within experimental errors. Such quantitative findings show definitely that the hemostatic enhancement due to chitosan is independent of classical clotting pathways. Thirdly, the adhesion forces of red blood cells on various yarns were measured using an optical tweezers. The adhesion force is small, around 3.83 pN, such that the direct adhesion cannot be the sole cause for hemostasis. However, it was observed that layer structures of aggregated RBCs were formed next to chitosan objects in both static and flowing environments, but not formed next to other yarns. Such a layer structure is the clue for the initiation of the mucoadhesive barrier, and thus hemostatsis. Through the supporting measurements of zeta potentials of RBCs and pH’s using blood-chitosan mixtures, it is proposed here that the formation of the RBC layer structures next to chitosan objects is due to the reduction of repulsive electric double layer force between RBCs, because of the association of H+ deprotonated from chitosan with COO on RBC membrane, under the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory. The results are beneficial for designing effective chitosan-based wound dressings, and also for general biomedical applications.