201229339 六、發明說明： 【發明所屬之技術領域】 本發明是有關於-種止血材料與其製備方法且特別 是有關於一種微米級纖維狀止血材料及其製備方法。 【先前技術】 失血是人類死亡最重要的原因之一，據估計，約有5〇% 的人死於大量失a。人體的血液量約相當於體重的1/13, 當人體中血液-次失去朗15%就會發生休克現象；超過 30減會^命危險。因此，必須能夠及軌有效地止血， 才能夠穩定傷情’進而替後續治療創造有利的。 為了能夠有效地止血，目前的止血程序中經常會使用 止血敷料。幾丁聚畴是當前止錢料中主要的成分之-， 帶正電的ΐ丁聚醣材料本身沒有黏著能力，但遇到血液時 就會與帶著負電的紅血球、血小板、白血球等結合，形成 細胞栓子（或贿血拾），將&血處封住並且形成屏障，不 但能夠^血’還能夠降低細菌等感染原人侵人體的機率。 目刖已有多種市售的止血敷料如CELOX™ ( SAM Medical Products 生產）與 HemC〇,（ HEMC〇N，心生產） 止血粉等。然而，這些止血粉的抗溶解性並不理想，在與 血液接觸後’粉末會彳艮快地溶解而形成水凝膠。當使用在 大量出血的傷口時’此種水凝膠很容易被血液沖走；因此 通常需要使用大量的止血粉，才能達到止血的目的。為了 解決此一問題’可以外加支撐基材來避免凝膠被血液帶 走，或者是可將製得的止血材料進一步壓縮，以提高材料 201229339 的密度進而延緩材料的溶解速率。然而，上述方法需要使 用額外的材料或步驟，因而提高了製備止血材料的金錢和/ 或時間成本。 因而，如何利用簡便的方法提升止血敷料的抗溶解性 與降低凝血時間，仍是相關領域的研發重心之一。 【發明内容】 發明内容旨在提供本揭示内容的簡化摘要，以使閱讀 Φ 者對本揭示内容具備基本的理解。此發明内容並非本揭示 内容的完整概述，且其用意並非在指出本發明實施例的重 要/關鍵元件或界定本發明的範圍。 本發明之一態樣係有關於一種止血材料的製備方法， 此方法可運用任何適當方法製成的幾丁聚醣纖維，再將纖 維經酸化即可製成纖維狀的止血材料，其製程步驟較為簡 單。 根據本發明一實施例，上述製備方法包含以下步驟。 • 將纖維直徑約10-100 μιη的幾丁聚醣纖維浸泡於約 3-60 wt%的酸之乙醇溶液（或稱酸化溶液）中，以酸化該 幾丁聚醣纖維的表面。將酸化後之幾丁聚醣纖維粉末化， 以得到幾丁聚糖的纖維狀粉末，其長度小於約1 mm。 本發明之另一態樣係有關於一種微米級纖維狀止血材 料，其不具備顯著的生物毒性且具有理想的凝血效能與抗 溶解性，適用於處理各種傷口，且特別適用於大量出血的 傷口。 根據本發明一實施例，上述微米級纖維狀止血材料包 201229339 含表面經部分酸化的幾丁聚醣的纖維狀粉末，其纖維直徑 為約10-100 μηι且長度小於約1 mm。 在參閱下文實施方式後，本發明所屬技術領域中具有 通常知識者當可輕易瞭解本發明之基本精神及其他發明目 的’以及本發明所採用之技術手段與實施態樣。 【實施方式】 為了使本揭示内容的敘述更加詳盡與完備，下文針對 了本發明的實施態樣與具體實施例提出了說明性的描述； 但這並非實施或運用本發明具體實施例的唯一形式。實施 方式中涵蓋了多個具體實施例的特徵以及用以建構盥操作 這些具體實施例的方法步驟與其順序。然而，亦可利用直 他具體實施例來達成相同或均等的功能與步驟順序。 : ^施方式’用以製備微米級纖維狀止血 將適當的幾丁聚糖纖維經酸化再經粉末 化以付到微未級纖維狀止血材料。 試驗結果顯示 的止血效能。當纖維直徑 的，…止血材枓 液溶解性都非常不㈣目.㈣帅•’止血效能與抗血 過大，施用於傷D處，舍心:疋幾丁㈣纖維的直徑 發明-實施例，應先取:纖：趕到不適。因此，根據本 _維，較佳的纖維直二約：約I· _的幾丁聚 維的直徑可為約1〇、2〇:3〇為：10、20㈣。具體而言，纖 1〇0μιηβ 4〇、50、60、70、80、90或 根據本發明的原理與精神，可利用任何習用的技術來 201229339 製備適用於本方法的幾丁聚醣纖維；舉例來說，可利用濕 式紡絲技術來製備上述幾丁聚醣纖維。 根據任選的實施例，利用濕式紡絲來製備此幾丁聚醣 纖維包含以下步驟： (1) 將幾丁聚醣溶於醋酸水溶液中，以製備一纺絲液； (2) 將氫氧化鈉水溶液溶於體積比約1 : 1的親水性 溶劑與水中，以製備一成型液；以及 ,利用濕式紡絲設備’使紡絲液通過成型液，以製 _得纖維直徑約10-100 μηι的幾丁聚醣纖維。 在纺絲液中’幾丁聚醣的濃度為約3-8 wt% ;具體來 說’此一濃度可為約 3、3.5、4、4.5、5、5.5、6、6.5、7、 7·5或8 Wt%。此外，紡絲液中醋酸的濃度為約3-10 wt% ; 如 3 、 3.5 、 4 、 4.5 、 5 、 5.5 、 6 、 6.5 、 7 、 7.5 、 8 、 8.5 、 9 、 9.5 或 1〇 wt〇/o。 一般來說，幾丁聚醣係由幾丁質經去乙醯反應 /deaCetylation)所得’根據本發明任選的實施例，所用之 戍丁聚_的去乙醯度為約50-99% ;如，50、51、52、53、 54、55、56、57、58、59、60、6卜 62、63、64、65、66、 67、68、69、70、7卜 72、73、74、75、76、77、78、79、 80、81 ' 82、83、84、85、86、87、88、89、90、9卜 92、 93、94、95、96、97、98 或 99%。 在成型液中’氫氧化鈉的濃度為約3-10 wt%，如3、 3.5 、 4 、 4.5 、 5 、 5.5 、 6 、 6.5 、 7 、 7.5 、 8 、 8.5 、 9 、 9.5 或 10 wt%。 另外，成型液中所用的親水性溶劑可以是甲醇、乙醇、 201229339 水、丙酮或其中至少兩種溶劑所組成的混合溶劑。 作為例示而非限制，在一任選的實施例中，紡絲液中 醋酸的濃度為約5 wt%、幾丁聚醣的乙醯度約90%且濃度 為約5 wt% ;而成型液中使用了體積比約1 : 1的甲醇與水， 且氩氧化鈉的濃度約5wt°/〇。 在較佳的實施例中，於步驟（3)之後可利用去離子水 沖洗所得到的幾丁聚醣纖維，以洗除殘留的鹼。 在取得了適當的幾丁聚醣纖維（纖維直徑約10-100 φ μιη)之後，將其浸泡於酸之乙醇溶液中，以酸化該幾丁聚 醣纖維的表面。 在本發明實施例中，可使用有機酸或無機酸來製備上 述酸之乙醇溶液。有機酸的實施例包括，但不限於：醋酸、 乳酸、檸檬酸、琥珀酸、蘋果酸、馬來酸與丙烯酸；而無 機酸的實施例包括但不限於：鹽酸與硫酸。 在上述酸的乙醇溶液中，酸的重量百分比為約3-60 wt% ;較佳為約 5-20 wt% ’ 如約 3、4、5、6、7、8、9、 φ 10、1卜 12、13、14、15、16、17、18、19、20、2卜 22、 23、24、25、26、27、28、29、30、3卜 32、33、34、35、 36、37、38、39、40、4卜 42、43、44、45、46、47、48、 49、50、5卜 52、53、54、55、56、57、58、59 或 60 wt%。 一般來說，浸泡的時間係取決於所用的酸的種類、濃 度以及酸化的程度而定。然而，材料酸化的程度不易量化 表示；因此，在本發明中是以製程條件以及產物所展現的 效果來說明材料酸化之情形。 作為例示而非限制，當將幾丁聚醣浸泡於濃度約3-60 201229339 wt%的醋酸中時，約1〇一18〇分鐘即可得到兼顧理想抗溶解 性與凝血效能的止血材料（抗溶解性與凝jk效能之分析詳 見後文）。具體來說，上述浸泡時間可為約1〇、15、2〇、 25、30、35、40、45、50、55、60、65、70、75、80、85、 90、95、1〇〇、1〇5、110、115、120、125、130、135、140、 145、150、155、160、165、170、175 或 180 分鐘。 相較之下’在其他製程條件相同的前提下，若是將傳 統的的幾丁聚醣粉末浸泡於約酸化溶液中，則無法有效地 提升此材料的抗溶解性與凝血性能。 在浸泡完成後，可取出幾丁聚醣纖維，並加以乾燥。 例如，可將幾丁聚醣纖維放置於烘箱中，在約40-7〇。(：的 溫度下予以乾燥。 其後’將酸化後之幾丁聚醣纖維粉末化，以得到長度 小於約1 mm幾丁聚糖的纖維狀粉末。一般來說，若是纖 維狀粉末的長度過長，使用於傷口上的時候，會使得病患 感到不舒服，還可能會卡在傷口上不易清除；因此，在本 發明實施方式中，將纖維狀粉末的長度限定為 1 mm以下。 然而’在試驗過程中發現，當纖維狀粉末的長度過小時， 可能會大幅減損纖維狀粉末的抗溶解性與凝血效能；因 此’在本發明的替代性實施方式中，將纖維狀粉末的長度 限定為約ΙΟριη至約1mm，較佳的纖維狀粉末的長度為約 100—500 μιη 〇 可以利用任何適當的設備，來進行此一粉末化步驟。 舉例來說，可將幾丁聚醣纖維置於高速均質機中，並藉由 高速均質機的攪拌、混合、分散與剪切作用，以得到纖雉 201229339 狀的粉末。 試驗結=示，根據以上步驟即可得到不具顯著細胞 毒性的幾丁聚糖的纖維狀粉末；換句話說可將此材料直 接施用於動物身上作為止血材料，㈣需要經過 理步驟。 根據本發明具體實施例，利用上述方法可以得到一種 微米級纖=血材料’其主要包含表面經部分酸化的幾 丁㈣的_狀粉末’其纖維直經為約ι〇_謂哗且長度 小於約1賴。試驗結果顯示，此種微米級纖維狀止血材料 適用於處理f種傷Π;Μ於其具有較佳的抗溶解性 別適用於大量出血的傷口。 具體來說’在止血過財，當此種微米級纖維狀止血 材料在與血液接觸後，其卜部份的纖維狀止血材料會盘 血液中的各種分子仙產生塊狀_;然而，其㈣ 纖維狀止血材料仍_持原本的纖輪結構，因 = 一種纖維-凝塊結構。 …取 此種纖維-凝塊結構大致上是—種網狀結構，藉 將原本的纖維狀材料連結在一起。 硬膠 Π ’試驗結果證明，經吸收的 血液會被封包於凝膠之中，而不易再者，2 統止血材料遇到血液後產生的凝膠蛀 :、‘傳 纖維-凝塊結構的抗溶解性較佳.11 車下，、周狀的 凝塊結構較不容易被血液溶解（或沖散），因而可以提^維太 發明之止血材料在傷Π處的停滯時間，進而提升凝灰效果。 由於此處提出的微米級纖維狀止血材料在與灰液接觸 201229339 後，會先形成網狀的纖維-凝塊結構，此種纖維狀止血材料 在使用時，可以不需使用額外的支撐基材，其在傷口處停 留的時間就足以有效地止住傷口的出血情形。201229339 VI. Description of the Invention: [Technical Field] The present invention relates to a hemostatic material and a preparation method thereof, and more particularly to a micron-sized fibrous hemostatic material and a preparation method thereof. [Prior Art] Blood loss is one of the most important causes of human death. It is estimated that about 5% of people die from a large number of lost a. The amount of blood in the human body is about 1/13 of that of body weight. When the blood in the human body loses 15%, it will cause shock. If it exceeds 30, it will be dangerous. Therefore, it is necessary to be able to effectively stop bleeding in order to stabilize the injury and thus create favorable treatment for subsequent treatment. In order to effectively stop bleeding, hemostatic dressings are often used in current hemostasis procedures. Chitin polydomain is the main component of the current stop money - the positively charged guerto-glycan material itself has no adhesion ability, but when it encounters blood, it will combine with negatively charged red blood cells, platelets, white blood cells, etc. to form cells. The embolus (or bribe blood) will seal the blood and form a barrier, which will not only reduce the blood's ability to reduce the risk of bacteria invading the human body. Various commercially available hemostatic dressings such as CELOXTM (manufactured by SAM Medical Products) and HemC(R), (HEMC〇N, heart-produced) hemostatic powder have been observed. However, the anti-solubility of these hemostatic powders is not ideal, and after contact with blood, the powder dissolves rapidly to form a hydrogel. When used in a large number of bleeding wounds, this hydrogel is easily washed away by the blood; therefore, it is usually necessary to use a large amount of hemostatic powder to achieve hemostasis. In order to solve this problem, the support substrate may be additionally applied to prevent the gel from being carried away by the blood, or the prepared hemostatic material may be further compressed to increase the density of the material 201229339 and thereby retard the dissolution rate of the material. However, the above methods require the use of additional materials or steps, thereby increasing the cost and/or time cost of preparing the hemostatic material. Therefore, how to improve the anti-solubility of the hemostatic dressing and reduce the clotting time by using a simple method is still one of the research and development centers in related fields. SUMMARY OF THE INVENTION The Summary of the Invention is intended to provide a simplified summary of the present disclosure in order to provide a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is intended to be illustrative of the scope of the invention. One aspect of the present invention relates to a method for preparing a hemostatic material, which can be prepared by using any suitable method of chitosan fiber, and then the fiber is acidified to form a fibrous hemostatic material. It's simpler. According to an embodiment of the invention, the above preparation method comprises the following steps. • A chitosan fiber having a fiber diameter of about 10 to 100 μm is immersed in an acid solution (or acidified solution) of about 3-60 wt% to acidify the surface of the chitosan fiber. The acidified chitosan fiber is powdered to obtain a fibrous powder of chitosan having a length of less than about 1 mm. Another aspect of the present invention relates to a micron-sized fibrous hemostatic material which does not have significant biological toxicity and has desirable coagulation efficacy and solubility resistance, is suitable for treating various wounds, and is particularly suitable for wounds with massive bleeding. . According to an embodiment of the present invention, the above micron-sized fibrous hemostatic material package 201229339 comprises a fibrous powder having a partially acidified chitosan having a fiber diameter of about 10 to 100 μm and a length of less than about 1 mm. The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, can be readily understood by those of ordinary skill in the art. The present invention has been described in detail with reference to the embodiments of the present invention. . The features of the various embodiments are described in the embodiments and the method steps and sequences thereof used to construct the specific embodiments. However, the specific embodiments may be utilized to achieve the same or equivalent functions and sequence of steps. : ^ Application mode to prepare micron-sized fibrous hemostasis The appropriate chitosan fibers are acidified and then powdered to pay micro-grade fibrous hemostatic material. The test results show the hemostatic effect. When the fiber diameter, ... hemostatic material sputum solubility is very not (four) mesh. (four) handsome • 'hemostasis efficacy and anti-blood is too large, applied to the injury D, heart: 疋 丁 (4) fiber diameter invention - examples, Should be taken first: Fiber: rushed to discomfort. Therefore, according to the present invention, the preferred fiber diameter is about 1 〇, 2 〇: 3 〇 is 10, 20 (4). In particular, fiber 1 〇 0 μιη β 4 〇, 50, 60, 70, 80, 90 or according to the principles and spirit of the present invention, any conventional technique can be used to prepare a chitosan fiber suitable for use in the method 201229339; For example, the above-mentioned chitosan fibers can be prepared by a wet spinning technique. According to an optional embodiment, the preparation of the chitosan fiber by wet spinning comprises the steps of: (1) dissolving chitosan in an aqueous solution of acetic acid to prepare a spinning solution; (2) introducing hydrogen An aqueous solution of sodium oxide is dissolved in a hydrophilic solvent having a volume ratio of about 1:1 and water to prepare a molding liquid; and, by using a wet spinning apparatus, the spinning solution is passed through a molding liquid to obtain a fiber diameter of about 10 100 μηι of chitosan fiber. The concentration of chitosan in the spinning solution is about 3-8 wt%; specifically, the concentration can be about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7· 5 or 8 Wt%. In addition, the concentration of acetic acid in the spinning solution is about 3-10 wt%; such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 1 〇 wt〇 /o. In general, chitosan is obtained from chitin by deacetylation/deaCetylation. According to an optional embodiment of the present invention, the degree of de-acetylation of the ruthenium poly- _ is about 50-99%; For example, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 Bu 62, 63, 64, 65, 66, 67, 68, 69, 70, 7 Bu 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 '82, 83, 84, 85, 86, 87, 88, 89, 90, 9 92, 93, 94, 95, 96, 97, 98 or 99%. The concentration of sodium hydroxide in the molding fluid is about 3-10 wt%, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 wt%. . Further, the hydrophilic solvent used in the molding liquid may be methanol, ethanol, 201229339 water, acetone or a mixed solvent of at least two of them. By way of illustration and not limitation, in an optional embodiment, the concentration of acetic acid in the spinning solution is about 5 wt%, the oxime of chitosan is about 90%, and the concentration is about 5 wt%; Methanol and water in a volume ratio of about 1:1 are used, and the concentration of sodium argon is about 5 wt/hr. In a preferred embodiment, the resulting chitosan fibers can be rinsed with deionized water after step (3) to wash away residual base. After the appropriate chitosan fiber (fiber diameter of about 10-100 φ μηη) is obtained, it is immersed in an acid ethanol solution to acidify the surface of the chitosan fiber. In the examples of the present invention, an organic acid or a mineral acid may be used to prepare an ethanol solution of the above acid. Examples of organic acids include, but are not limited to, acetic acid, lactic acid, citric acid, succinic acid, malic acid, maleic acid, and acrylic acid; and examples of inorganic acids include, but are not limited to, hydrochloric acid and sulfuric acid. In the above acid ethanol solution, the weight percentage of the acid is about 3 to 60 wt%; preferably about 5 to 20 wt% ', such as about 3, 4, 5, 6, 7, 8, 9, φ 10, 1 Bu 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 4, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5, 52, 53, 54, 55, 56, 57, 58, 59, or 60 wt% . In general, the time of soaking depends on the type of acid used, the concentration, and the degree of acidification. However, the degree of acidification of the material is not easily quantified; therefore, in the present invention, the acidification of the material is illustrated by the process conditions and the effects exhibited by the product. By way of illustration and not limitation, when chitosan is immersed in acetic acid at a concentration of about 3-60 201229339 wt% acetic acid, a hemostatic material with desirable anti-solubility and coagulation efficacy can be obtained in about 1 to 18 minutes. The analysis of solubility and coagulation efficiency is detailed later. Specifically, the above soaking time may be about 1 〇, 15, 2 〇, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 1 〇 〇, 1〇5, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 minutes. In contrast, if other conventional process conditions are the same, if the conventional chitosan powder is soaked in the acidification solution, the solubility and coagulation properties of the material cannot be effectively improved. After the soaking is completed, the chitosan fibers can be taken out and dried. For example, chitosan fibers can be placed in an oven at about 40-7 Torr. (drying at a temperature of:: thereafter) pulverizing the acidified chitosan fiber to obtain a fibrous powder having a length of less than about 1 mm of chitosan. Generally, if the length of the fibrous powder is over When used on a wound, the patient may feel uncomfortable and may get stuck on the wound. Therefore, in the embodiment of the present invention, the length of the fibrous powder is limited to 1 mm or less. During the test, it was found that when the length of the fibrous powder is too small, the solubility resistance and coagulation efficiency of the fibrous powder may be greatly impaired; therefore, in an alternative embodiment of the present invention, the length of the fibrous powder is limited to Preferably, the length of the fibrous powder is from about 100 to about 500 μm. The powdering step can be carried out by any suitable means. For example, the chitosan fiber can be placed at a high speed. In the homogenizer, the powder of the fiber 201229339 is obtained by stirring, mixing, dispersing and shearing of a high-speed homogenizer. Test knot = according to the above steps A fibrous powder of chitosan which is not significantly cytotoxic can be obtained; in other words, the material can be directly applied to the animal as a hemostatic material, and (4) a rational step is required. According to a specific embodiment of the present invention, the above method is utilized. It is possible to obtain a micron-sized fiber=blood material which mainly comprises a diced powder of a partially acidified chitosan (four) whose fiber straight is about ι 〇 哗 哗 and has a length of less than about 1 Å. The test results show that The micron-sized fibrous hemostatic material is suitable for the treatment of f-type scars; it has a better anti-dissolving sex for wounds with a large amount of bleeding. Specifically, 'in the case of hemostasis, when such micron-sized fibrous hemostatic material is in After contact with blood, the fibrous hemostasis material of the cloth part will produce a blocky shape of various molecular centimeters in the blood. However, the (4) fibrous hemostatic material still retains the original fiber wheel structure, because = a fiber-condensation Block structure. ... Take this fiber-clot structure is roughly a kind of network structure, by linking the original fibrous materials together. Hard plastic Π 'Test results prove that absorbed The blood will be encapsulated in the gel, and it is not easy to be repeated. 2 The gel that is produced after the hemostatic material meets the blood: The transmission fiber-clot structure has better resistance to dissolution. 11 Under the car, Zhou The clot structure is less likely to be dissolved (or dissipated) by the blood, so that the stagnation time of the hemostatic material of the invention in the scar can be improved, thereby enhancing the ashing effect. After the hemostatic material contacts 201229339, it will form a network-like fiber-clot structure. When used, the fibrous hemostatic material can be used without any additional supporting substrate. A bleeding condition that is effective to stop the wound.
為了驗證此處所提出的微米級纖維狀止血材料的抗溶 解特性與凝血效能’根據本發明上述實施例中提出的方法 來製備不同的纖維狀止血材料’並分析其抗溶解特性與凝 血效能。下表一摘要整理了各實驗例中所用的部分製程條 件，以及各止血材料的抗溶解性與凝血效能（ 2 間與吸液倍率）。 時In order to verify the anti-dissolution property and coagulation efficiency of the micron-sized fibrous hemostatic material proposed herein, different fibrous hemostatic materials were prepared according to the method proposed in the above examples of the present invention and their anti-dissolution characteristics and clotting efficacy were analyzed. Table 1 below summarizes some of the process conditions used in each of the experimental examples, as well as the anti-solubility and coagulation efficacy of each hemostatic material (2 and aspiration rates). Time
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實驗例3B >1800 65 686 對照例3 613 > 1800 65 實驗例4A 20 1508 31 698 實驗例4B 1478 54 674 對照例4 377 875 34 實驗例5A 30 1203 43 683 實驗例5B 1243 49 657 對照例5 153 295 0 實驗例6A 40 1131 42 673 實驗例6B 1123 45 631 對照例6 113 78 0 實驗例7A 50 1085 43 660 實驗例7B 1051 46 614 對照例7 86 63 0 實驗例8A 60 1059 40 638 實驗例8B 1003 44 582 對照例8 57 62 0 實驗例9A 3 > 1800 66 757 實驗例9B > 1800 106 755 對照組9 > 1800 > 1800 118 201229339 CELOX™ 未浸泡 124 65 0 以實驗例1A為例，其製備方式係將直徑約10-20 μιη 的幾丁聚醣纖維浸泡於約5 wt%的醋酸之乙醇溶液中，浸 泡時間約30分鐘。之後，過濾出幾丁聚醣纖維並將其烘 乾，而後利用高速均質機（型號：RT02 ;凌廣工業股份有 限公司），將幾丁聚醣纖維粉碎，即可得到實驗例1的微米 • 級纖維狀止血材料，其長度約10-100 /xm之微米級纖維狀 止血材料，且其外觀如第1圖之掃描式電子顯微（SEM) 照片所示。表一所示的實驗例2A至8A之製備方式與實驗 例1大致相同，僅改變了醋酸之乙醇溶液的濃度。此外， 實驗例1B至8B係將幾丁聚醣纖維浸泡於不同濃度的琥珀 酸之乙醇溶液中。 在對照例1至8中，則將市售的幾丁聚醣粉末（世展 科技股份有限公司）分別浸泡於不同濃度的琥珀酸之乙醇 φ 溶液中3〇分鐘。 實驗例9A與9B分別將幾丁聚醣纖維浸泡於3 wt%的 醋酸之乙醇溶液與琥珀酸之乙醇溶液中，浸泡時間長達5 小時。對照例9則是將上述市售幾丁聚醣粉末浸泡於3 wt% 的琥珀酸之乙醇溶液中，浸泡時間也是5小時。 另外，還將市售CELOX™止血粉（開封後未經額外酸 化處理，然包裝顯示，此材料在製備時已浸泡過琥珀酸溶 液）進行了相同的測試’以作為參考。上述CELOX止jk 粉外觀為不規則的片狀顆粒，其尺寸在約100-900 /mi之 12 201229339 間。 、在取仔各，實驗例、對照例的止血材料之後，分別利 用以下方法來#估各材料的抗溶解性與止血效能。 由於▲液中含有約5G%以上的水分，因此，在進行抗 溶解性测試時，將約1 g止血材料與約35 mL的生理食鹽 水放置於樣品瓶中’接著以震ϋ震盪樣本瓶並開始計 時。當肉眼無法觀察到樣品瓶中有止血材料之明顯沉澱物 時，此-時間即為溶解該止血材料所需的時間。— • 第與2Β圖分別為實驗例2Α之止血材料與 CELOX™止血粉在試驗完成時的外觀。由第2Α圖可以發 現，在震盪了約30分鐘之後，樣本瓶底部仍有相當大的沉 澱物，這個沉澱物就是此處所述的纖維·凝塊結構。據估 計，在實驗例2中，有約42%的止血材料溶解形成了水凝 膠，而其餘的部分則仍是以纖維狀材料的形式存在於纖維_ 凝塊結構中。在第2Β圖中，在經過約124秒之後，樣本瓶 底部就幾乎沒有沉澱物的存在，此時認定CELOX™止血粉 _ 已完全溶解而形成凝膠並懸浮於生理食鹽水中。 單就材料的抗溶解性來看，溶解時間越長的材料，其 抗溶解性也越佳。未經酸化處理的幾丁聚醣材料（如，空 白實驗例或空白對照例）在試驗條件下幾乎不會溶解；亦 即，材料經過長時間震盪後，仍呈現原本的纖維狀或粉末 狀外觀。然而，此種未經酸化之幾丁聚醣材料的凝血性能 很不理想（詳如後述）。 由表—所示的結果可以發現，當酸化溶液中酸的濃度 約為3 Wt%時，本發明實驗例材料的溶解時間即大於3〇分 13 201229339 鐘（請見實驗例ΙΑ、IB、9A與9B)。此外，隨著酸化溶 液中酸的濃度逐漸提高，其溶解時間會略微下降。然而， 即使當酸化溶液中酸的濃度高達約6〇%的時候，本發明實 驗例材料的溶解時間也仍然高於16分鐘（請見實驗例8A、 8B)。相較之下，市售幾丁聚醣粉末在相同的試驗條件下， 只有在酸化溶液巾酸喊度低於5 wt%時，才能展現出抗 溶解的性質（請見對照例卜2、9);當酸化溶液中酸的濃 度高於10 wt%時，其溶解時間只剩下約1〇分鐘（請見對 照例3) ’且當進一步提升酸化溶液中酸的濃度時，其抗溶 解性會大幅降低（請見對照例4·8，材料的溶解時間從6 分鐘左右減少到不到1分鐘）。 另1，由表一與第2圖所示的結果可以看出，市售的 CELOX止血粉的抗溶解性不夠理想，在試驗條件下，約2 分鐘就會已實質上溶解而形成凝膠。 在凝血效能的部分，使用李氏-白氏測定法 (Lee-White’sMethod)來測定樣本的止血時間（血液凝固 時間）；並採用ELISA檢測法來測試止血材料是否能夠固 定血液。 根據李氏-白氏測定法，將約0.5 g的止血材料和約3 5 ml的血液（含有抗凝血劑—肝抗凝血素）加入樣本瓶中， 而後鎖上瓶蓋並放置於震盪器中上下震盪，觀察瓶内血液 是否流動，停止流動的時間，即為該樣本材料的止血時間。 由表一所示的結果可以發現到，當經丁聚醣 酸化處理時，其凝血效能很不理想，在試驗條件下\要花 超過30分鐘才能止血（請見空白實驗例、空白對照例 201229339 ΐ發明實餘例1人至9人之微米級纖維狀止血材 為約^3!秒了時間、約19一65秒，較佳的止血時間 維狀止血材料在•條^==Β= 條件:測狀止血财㈣處理與測試 發現血時間可以 能粉演了 _的角色；亦即，U =抗溶解性與凝血效 以縮短材料的止血時間，=的濃度增加，雖然可 性。然而，一個理相 /時降低了材料的抗溶解 解性與較短止血時^種特質，應該要同時兼具較佳抗溶 -:===聚醣粉末浸泡在“。 3〇分鐘時間由 其溶解時間卻大幅降低到約57 將二$而， 用於止血的場合，在有效止血之前右：=材料實際運 先溶解殆盡，而難以發揮實際的I血=材料可能就已經 而本發明所提出的製備方法，觝 的濃度和這兩種特性之間的衝突。解决了认化溶液中酸 論::::兼:"的條件 血效能的土血㈣。 “ _兼顧抗溶解性與凝 然而’發明人採用此處提出的幾丁聚釀纖維並搭配適 15 201229339 當的酸化溶液濃度，卻無法預期地得到兼顧抗溶解特性與 凝血效能的纖維狀止血材料，此種材料能夠在短時間之内 凝結血液同時可以在傷口處留滯較長時間，因而也適用於 大量出血的傷口。 在吸液量武驗方面，將乾重約1 g的止血材料放置於 樣品瓶中並加入約100mL生理食鹽水，而放置於震盪器上 震盪約5分鐘，以使止血材料有充分時間吸收水分。而後 利用孔徑5 /xm的濾紙來滞留粒徑大於5从爪的成分，並量 •秤其重量，即為樣本的吸液重。而後再根據以下公式計算 出每一止血材料的吸液倍率： 吸液倍率（°/〇)=(吸液重/乾重）χ100〇/〇。 由表一可以看出，CEL0X™止血粉幾乎沒有吸液能 力。相較之下’此處提出之微米級纖維狀止血材料具有非 常理想的吸液能力，在試驗條件下，能夠吸收本身重量4 倍以上的液體。此種理想的吸液能力，也使得此處提出的 微米級纖維狀止血材料適用於處理大量出血的傷口。 # 此外，為了進一步確認此處提出之微米級纖維狀止血 材料在與血液接觸後’所形成的纖維_凝塊結構是否能夠有 效地將血液固定於其中而不會被析出，亦針對實驗例2A 的止血材料與市售CELOX™止血粉進行了 ELISA檢測。 ELISA檢測的步驟如下。首先，將約〇 5 g的止血材料 與約3.5 mL的全血接觸並靜置約9〇秒，以利止血材料吸 收血液並發揮凝血效果。其後，在樣本中加入約2〇⑺丨的 生理食鹽水，再分別以震盪器震盪約〇 (不震盪）、丨、2、 3、5、1〇分鐘後測量生理食鹽水對波長54〇 nm之光線（參 201229339 考波長650 nm)的吸光值（opticai density，O.D·)，藉此判 斷受測物的凝血效能。一般來說，凝血效能越佳的材料， 血液較不容易被釋放到生理食鹽水中，那麼樣本對於波長 540 nm之光線的吸收度也會較低，表二呈現了相對於僅含 3.5 mL全血（將其〇.D.值設定為1)各止血材料之〇 d值。 表二 震盪時間 (分鐘） 0 1 2 3 5 10 實驗例2A 0.002 0.003 0.003 0.004 0.004 0.005 CELOX™ 1 1 1 1 1 1 全血 1 1 1 1 1 1 由表2可以看出，在震盪了 1〇分鐘後，實驗例2A之 樣本中的O.D_值約為0.005，亦即，約有99.5%以上的血液 被封包於凝膠中而未滲出。相較之下，CELOXTM止血粉在 # 與也液接觸90秒後，即便在未經震盪的情形下，都沒有辦 法有效地固定血液。由此可知，本發明實施例提出的微米 級纖維狀止企材料的血液固定效能優於市售CELOXtm止 血粉。 在不將本發明限定於特定理論的前提下，發明人認 為，根據本發明實施例的止血材料吸血後所產生的纖維·凝 塊結構’有助於將血液固定於其中而不致析出。 由於此處提出的微米級纖維狀止血材料主要的用途是 17 201229339 止血，因此所得到的材料不能具有顯著的細胞毒性，否則 在使用時反而會使得傷口處的細胞死亡。因此，根據ISO 10993-5標準，來分析實驗例2A之微米級纖維狀止血材料 和纖維母細胞（L-929)共培養24小時候的細胞存活率。 試驗結果顯示，對於實驗例2A之微米級纖維狀止血材料， 材料下方的細胞存活率約85-90% ;而在材料邊界（0線） 下方的細胞存活率高於99%。由此可知，根據本發明實施 例之方法所製得的微米級纖維狀止血材料對於纖維母細胞 不具有顯著的細胞毒性，因而此一材料適合作為動物體的 止血材料。 根據本發明的原理與精神，可選用各種既有的幾丁聚 醣纖維，搭配此處提出之適當的酸化溶液濃度，而後將經 酸化的幾丁聚醣纖維粉末化；即可得到兼具理想抗溶解性 與凝血效能之幾丁聚糖的纖維狀粉末。 值得注意的是，在上述方法中，雖然採用的手段並不 繁複，但發明人進行的一系列研究顯示，藉由採用適當的 酸化溶液濃度並搭配此處提出的幾丁聚醣纖維，能夠無法 預期地得到兼顧抗溶解特性與凝血效能的纖維狀止血材 料，此種材料能夠在短時間之内凝結血液同時可以在傷口 處留滯較長時間，因而也適用於大量出血的傷口。 雖然上文實施方式中揭露了本發明的具體實施例，然 其並非用以限定本發明，本發明所屬技術領域中具有通常 知識者，在不悖離本發明之原理與精神的情形下，當可對 其進行各種更動與修飾，因此本發明之保護範圍當以附隨 申請專利範圍所界定者為準。 18 201229339 【圖式簡單說明】 為讓本發明的上述與其他目的、特徵、優點與實施例 能更明顯易懂，所附圖式之說明如下： 第1圖是根據本發明一實施例之微米級纖維狀止血材 料的SEM照片； 第2A與2B圖分別為對本發明實驗例與對照例進行抗 溶解性測試的外觀照片。 【主要元件符號說明】 無Experimental Example 3B > 1800 65 686 Comparative Example 3 613 > 1800 65 Experimental Example 4A 20 1508 31 698 Experimental Example 4B 1478 54 674 Comparative Example 4 377 875 34 Experimental Example 5A 30 1203 43 683 Experimental Example 5B 1243 49 657 Comparative Example 5 153 295 0 Experimental Example 6A 40 1131 42 673 Experimental Example 6B 1123 45 631 Comparative Example 6 113 78 0 Experimental Example 7A 50 1085 43 660 Experimental Example 7B 1051 46 614 Comparative Example 7 86 63 0 Experimental Example 8A 60 1059 40 638 Experiment Example 8B 1003 44 582 Comparative Example 8 57 62 0 Experimental Example 9A 3 > 1800 66 757 Experimental Example 9B > 1800 106 755 Control Group 9 > 1800 > 1800 118 201229339 CELOXTM Unsoaked 124 65 0 Experimental Example 1A For example, it is prepared by soaking chitosan fibers having a diameter of about 10-20 μηη in a solution of about 5 wt% of acetic acid in ethanol for about 30 minutes. After that, the chitosan fiber is filtered and dried, and then the chitosan fiber is pulverized by a high-speed homogenizer (Model: RT02; Lingguang Industrial Co., Ltd.) to obtain the micrometer of Experimental Example 1. A fibrous hemostatic material having a micron-sized fibrous hemostatic material having a length of about 10-100 / xm and having an appearance as shown in the scanning electron microscopy (SEM) photograph of Fig. 1. The experimental examples 2A to 8A shown in Table 1 were prepared in the same manner as in Experimental Example 1, except that the concentration of the acetic acid solution in ethanol was changed. Further, Experimental Examples 1B to 8B were immersed in chitosan fibers in different concentrations of succinic acid in ethanol. In Comparative Examples 1 to 8, commercially available chitosan powder (Shizhan Technology Co., Ltd.) was separately immersed in different concentrations of succinic acid in ethanol φ solution for 3 minutes. In Experimental Examples 9A and 9B, chitosan fibers were respectively immersed in a 3 wt% ethanol solution of acetic acid and a solution of succinic acid in ethanol for a period of 5 hours. In Comparative Example 9, the above commercially available chitosan powder was immersed in a 3 wt% ethanol solution of succinic acid, and the soaking time was also 5 hours. In addition, commercially available CELOXTM hemostatic powder (the same test was carried out without additional acidification after opening, but the package showed that the material had been soaked in the succinic acid solution at the time of preparation) was used as a reference. The above CELOX stop jk powder has irregular sheet-like particles and has a size of about 100-900 /mi 12 201229339. After taking the hemostatic materials of each of the experimental and control examples, the following methods were used to estimate the anti-solubility and hemostasis of each material. Since ▲ liquid contains about 5G% or more of water, when performing the anti-solubility test, put about 1 g of hemostatic material and about 35 mL of physiological saline in the sample vial, and then shake the sample bottle with shock And start timing. This time is the time required to dissolve the hemostatic material when it is not visible to the naked eye that there is a significant deposit of hemostatic material in the vial. — • The first and second figures are the appearance of the hemostatic material of Experimental Example 2 and CELOXTM hemostatic powder at the completion of the test. It can be seen from Figure 2 that after about 30 minutes of shaking, there is still a considerable deposit at the bottom of the vial, which is the fiber-clot structure described here. It is estimated that in Experimental Example 2, about 42% of the hemostatic material dissolved to form a hydrogel, and the remaining portion remained in the fiber-clot structure in the form of a fibrous material. In Figure 2, after about 124 seconds, there was almost no sediment at the bottom of the vial, at which point CELOXTM Hemostatic Powder _ was completely dissolved to form a gel and suspended in physiological saline. In terms of the solubility resistance of the material alone, the longer the dissolution time, the better the solubility resistance of the material. The un-acidified chitosan material (eg, blank or blank) will hardly dissolve under the test conditions; that is, the material will still exhibit the original fibrous or powdery appearance after prolonged shaking. . However, the coagulation performance of such an unacidified chitosan material is not ideal (see below for details). From the results shown in Table--, it can be found that when the acid concentration in the acidified solution is about 3 Wt%, the dissolution time of the experimental material of the present invention is more than 3〇13 13 201229339 (see Experimental Example IB, IB, 9A). With 9B). In addition, as the concentration of the acid in the acidified solution is gradually increased, the dissolution time is slightly lowered. However, even when the acid concentration in the acidifying solution was as high as about 6%, the dissolution time of the material of the present invention was still higher than 16 minutes (see Experimental Examples 8A, 8B). In contrast, commercially available chitosan powders exhibit anti-dissolution properties only under the same test conditions, when the acidification solution has a acidity of less than 5 wt% (see Comparative Examples 2, 9). When the acid concentration in the acidified solution is higher than 10 wt%, the dissolution time is only about 1 minute (see Comparative Example 3)' and when it further increases the acid concentration in the acidified solution, its solubility resistance It will be greatly reduced (see Comparative Example 4·8, the dissolution time of the material is reduced from about 6 minutes to less than 1 minute). On the other hand, as can be seen from the results shown in Tables 1 and 2, the commercially available CELOX hemostatic powder has an insufficient solubility resistance, and under the test conditions, it is substantially dissolved to form a gel in about 2 minutes. In the part of coagulation efficacy, the Lee-White's Method was used to determine the hemostasis time (blood coagulation time) of the sample; and an ELISA test was used to test whether the hemostatic material can fix the blood. According to the Lee-Bai's method, about 0.5 g of hemostatic material and about 35 ml of blood (containing anticoagulant - hepatic anticoagulant) are added to the sample bottle, and then the cap is locked and placed in a shock The upper and lower sides of the device oscillate and observe whether the blood in the bottle flows or not, and the time for stopping the flow is the hemostasis time of the sample material. From the results shown in Table 1, it can be found that when the chitosan is acidified, its coagulation efficiency is not ideal, and it takes more than 30 minutes to stop bleeding under the test conditions (please see blank experiment, blank control case 201229339) ΐInventive example 1 to 9 people of micron-sized fibrous hemostatic material is about ^3! Second time, about 19-65 seconds, better hemostasis time, continuous hemostatic material in • Article ^==Β= conditions : Measurement of blood stasis (4) Treatment and testing found that blood time can be played in the role of _; that is, U = anti-solubility and coagulation effect to shorten the hemostasis time of the material, the concentration of = increased, although feasible. However, A phase/time reduces the anti-dissolving properties of the material and the short-term hemostatic properties, and should have a better anti-solvent -:=== glycan powder soaked in ". 3 minutes to dissolve from it The time is greatly reduced to about 57 will be two dollars, and in the case of hemostasis, before the effective hemostasis right: = the material actually dissolved first, and it is difficult to play the actual I blood = material may have been proposed by the present invention Preparation method, concentration of bismuth and these two characteristics The conflict between the two sides of the acid solution in the identification solution::::::" the condition of blood (4). " _ take into account the resistance to solubility and condensation." The inventor used the chitin brewed here. The fiber is matched with the concentration of the acidizing solution of 201229339, but it is unpredictable to obtain a fibrous hemostatic material which has both anti-dissolution properties and coagulation efficiency. This material can condense blood in a short time and can stay longer in the wound. Time, therefore also suitable for a large number of bleeding wounds. In the examination of the amount of liquid absorption, put a hemostatic material with a dry weight of about 1 g in a sample bottle and add about 100 mL of physiological saline, and placed on the oscillator to oscillate about 5 Minutes, so that the hemostatic material has sufficient time to absorb the water. Then use the filter paper with a pore size of 5 / xm to retain the component with a particle size greater than 5 from the claw, and weigh the weight, which is the weight of the sample. Then according to the following The formula calculates the liquid absorption ratio of each hemostatic material: Suction ratio (°/〇) = (absorbent weight/dry weight) χ100〇/〇. As can be seen from Table 1, CEL0XTM hemostatic powder has almost no liquid absorption capacity. .phase The micron-sized fibrous hemostatic material proposed here has a very good liquid absorption capacity and can absorb more than 4 times its own weight under the test conditions. This ideal liquid absorption capacity also makes the proposed The micron-sized fibrous hemostatic material is suitable for treating a large number of bleeding wounds. # In addition, in order to further confirm whether the micro-fibrous fibrous hemostatic material proposed here is in contact with blood, the formed fiber-clot structure can effectively treat blood. It was fixed in it without being precipitated, and the ELISA test was also performed on the hemostatic material of Experimental Example 2A and the commercially available CELOXTM hemostatic powder. The procedure of the ELISA test is as follows. First, about 5 g of hemostatic material is about 3.5 mL. The whole blood is contacted and allowed to stand for about 9 seconds, so that the hemostatic material absorbs blood and exerts a blood coagulation effect. Thereafter, about 2 〇 (7) 丨 of physiological saline was added to the sample, and then the physiological saline was measured at a wavelength of 54 震 after shaking with an oscillator about 〇 (not oscillating), 丨, 2, 3, 5, and 1 minute. The absorbance (opticai density, OD·) of the light of nm (refer to the wavelength of 650 nm at 201229339) is used to judge the coagulation efficiency of the test object. In general, the better the coagulation efficiency of the material, the blood is less likely to be released into the physiological saline solution, then the sample will have a lower absorbance for light with a wavelength of 540 nm. Table 2 shows that compared with only 3.5 mL of whole blood. (Set its 〇.D. value to 1) 〇d value of each hemostatic material. Table 2 Oscillation time (minutes) 0 1 2 3 5 10 Experimental example 2A 0.002 0.003 0.003 0.004 0.004 0.005 CELOXTM 1 1 1 1 1 1 Whole blood 1 1 1 1 1 1 As can be seen from Table 2, it is turbulent 1〇 After a minute, the O.D. value in the sample of Experimental Example 2A was about 0.005, that is, about 99.5% or more of the blood was encapsulated in the gel without oozing out. In contrast, CELOXTM hemostatic powder did not effectively fix the blood even after 90 seconds of contact with the liquid. It can be seen that the micron-sized fibrous material of the present invention has better blood fixation efficiency than the commercially available CELOXtm hemostatic powder. Without limiting the invention to a particular theory, the inventors believe that the fiber & clot structure produced by the hemostatic material of the embodiment of the present invention, which is produced after blood-sucking, helps to immobilize blood therein without precipitation. Since the main use of the micron-sized fibrous hemostatic material proposed here is 17 201229339 to stop bleeding, the obtained material cannot have significant cytotoxicity, otherwise the cells at the wound will be killed when used. Therefore, the cell viability of the micron-sized fibrous hemostatic material of Experimental Example 2A and fibroblasts (L-929) co-cultured for 24 hours was analyzed according to the ISO 10993-5 standard. The test results showed that for the micron-sized fibrous hemostatic material of Experimental Example 2A, the cell viability under the material was about 85-90%; and the cell survival rate below the material boundary (0 line) was higher than 99%. From this, it is understood that the micron-sized fibrous hemostatic material obtained by the method of the embodiment of the present invention does not have significant cytotoxicity to fibroblasts, and thus this material is suitable as a hemostatic material for animals. According to the principle and spirit of the present invention, various existing chitosan fibers can be selected, combined with the appropriate acidification solution concentration proposed herein, and then the acidified chitosan fiber is powdered; A fibrous powder of chitosan resistant to solubility and coagulation. It is worth noting that, although the method used is not complicated, the inventors conducted a series of studies showing that by using the appropriate acidification solution concentration and matching the chitosan fiber proposed here, it is impossible to It is expected to obtain a fibrous hemostatic material which combines anti-dissolution properties with coagulation efficacy, and which can condense blood in a short period of time while remaining in the wound for a long time, and thus is also suitable for a large number of bleeding wounds. Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS [0009] The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. SEM photograph of the graded fibrous hemostatic material; Figs. 2A and 2B are photographs showing the appearance of the anti-solubility test of the experimental examples and the comparative examples of the present invention, respectively. [Main component symbol description] None
[S] 19[S] 19