CN107569718A - A kind of preparation method of 3D printing PLA and bacteria cellulose compound support frame material - Google Patents
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Abstract
一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，属于组织工程技术领域。所述方法具体步骤为：1、菌株发酵制备细菌纤维素，依次进行水洗和碱洗；2、在室温条件下，制备细菌纤维素悬浮液，然后减压抽滤得到细菌纤维素薄膜，冷冻干燥得到细菌纤维素三维多孔支架；3、进行3D打印，得到3D打印聚乳酸和细菌纤维素复合支架材料。本发明的优点是：本发明制备方法简单，操作周期短，并可通过控制3D打印过程中的工艺参数精确调控支架表层微孔尺寸和形状。制备的3D打印聚乳酸和细菌纤维素的复合支架材料既具有良好的机械性能，又具有较高孔隙率以及优异的生物相容性，还可以提高细胞在支架材料表面的黏附能力，因此可用于组织工程领域。The invention discloses a method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material, which belongs to the technical field of tissue engineering. The specific steps of the method are as follows: 1. Bacterial cellulose is prepared by bacterial strain fermentation, followed by water washing and alkali washing; 2. Under room temperature, the bacterial cellulose suspension is prepared, and then the bacterial cellulose film is obtained by vacuum filtration, and freeze-dried Obtain a bacterial cellulose three-dimensional porous scaffold; 3. Perform 3D printing to obtain a 3D printed polylactic acid and bacterial cellulose composite scaffold material. The advantages of the present invention are: the preparation method of the present invention is simple, the operation period is short, and the size and shape of micropores on the surface of the support can be precisely regulated by controlling the process parameters in the 3D printing process. The prepared 3D printed polylactic acid and bacterial cellulose composite scaffold material not only has good mechanical properties, but also has high porosity and excellent biocompatibility, and can also improve the adhesion ability of cells on the surface of the scaffold material, so it can be used for field of tissue engineering.
Description
技术领域technical field
本发明属于组织工程技术领域，具体涉及一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法。The invention belongs to the technical field of tissue engineering, in particular to a method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material.
背景技术Background technique
组织工程，也称为“再生医学工程”，是一种将应用工程学、生物学和材料学相结合的原理和技术。通常实现的方式为，在体外构建与细胞外基质类似的三维多孔仿生结构，模拟细胞的生长环境，为细胞的附着和黏附提供适宜的环境，同时，相互贯穿的三维多孔结构能够为细胞的生长和增殖提供养分，并为代谢产物以及气体交换提供良好的通道和空间。Tissue engineering, also known as "regenerative medicine engineering", is a principle and technology that combines applied engineering, biology and materials science. The usual way to achieve this is to construct a three-dimensional porous bionic structure similar to the extracellular matrix in vitro, simulate the growth environment of cells, and provide a suitable environment for cell attachment and adhesion. Provide nutrients and proliferation, and provide good channels and spaces for metabolites and gas exchange.
理想的组织工程材料需要具有合适的形状和体积以满足实际的应用需求，同时还应具有较高的孔隙率、优异的生物相容性为细胞的生长提供良好的环境，同时，还应具备优异的机械性能、降解性以保证临床的应用。细菌纤维素（Bacterial Cellulose，BC）作为常用的医用高分子材料，具备以上的优异性能，是理想的组织工程材料之一。An ideal tissue engineering material needs to have a suitable shape and volume to meet the actual application requirements. It should also have high porosity and excellent biocompatibility to provide a good environment for cell growth. At the same time, it should also have excellent Excellent mechanical properties and degradability to ensure clinical application. Bacterial Cellulose (BC), as a commonly used medical polymer material, has the above excellent properties and is one of the ideal tissue engineering materials.
3D打印（3D Printing）技术是一种新型的快速成型技术，该技术是以计算机设计构建模型为基础，运用塑料或粉末状金属等可粘合材料，通过逐层打印的方式得到需求的复杂三维结构。它无需机械加工和模具，就能从计算机模型得到三维结构，从而大大缩短了产品的研制周期，生产率得到大幅提高的同时使得成本降低，因此在生物工程和医学领域具有很大的发展潜力。3D printing (3D Printing) technology is a new type of rapid prototyping technology, which is based on computer design and construction models, using adhesive materials such as plastics or powdered metals, and obtaining complex three-dimensional shapes required by layer-by-layer printing. structure. It can obtain a three-dimensional structure from a computer model without machining and molds, thereby greatly shortening the product development cycle, greatly improving productivity and reducing costs, so it has great development potential in the fields of bioengineering and medicine.
现在常用的支架材料的制备方法有：冷冻干燥法和溶液浇铸法。两种方法操作繁琐、生产周期长，支架材料的形状及厚度取决于模具，这使得其应用受到很大的限制。The commonly used preparation methods of scaffold materials include: freeze-drying method and solution casting method. The two methods are cumbersome to operate, have a long production cycle, and the shape and thickness of the scaffold material depend on the mold, which greatly limits their application.
发明内容Contents of the invention
本发明的目的是为了解决目前存在的操作繁琐、模具依赖性等问题，提供一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，所述方法利用3D打印技术，可制备一种多孔的组织工程复合支架材料。The purpose of the present invention is to provide a method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material in order to solve the existing problems of cumbersome operation and mold dependence. The method uses 3D printing technology to prepare a porous tissue engineering composite scaffolds.
为实现上述目的，本发明采取的技术方案如下：In order to achieve the above object, the technical scheme that the present invention takes is as follows:
一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，所述方法具体步骤如下：A preparation method for 3D printing polylactic acid and bacterial cellulose composite scaffold material, the specific steps of the method are as follows:
步骤一：选取菌株通过静态培养法发酵制备细菌纤维素，对制得的细菌纤维素依次进行水洗和碱洗；Step 1: Select bacterial strains to ferment and prepare bacterial cellulose by static culture method, and wash the bacterial cellulose with water and alkali in sequence;
步骤二：在室温条件下，利用步骤一得到的细菌纤维素制备细菌纤维素悬浮液，然后经过减压抽滤得到细菌纤维素薄膜，冷冻干燥得到细菌纤维素三维多孔支架；Step 2: At room temperature, use the bacterial cellulose obtained in Step 1 to prepare a bacterial cellulose suspension, then filter under reduced pressure to obtain a bacterial cellulose film, and freeze-dry to obtain a bacterial cellulose three-dimensional porous scaffold;
步骤三：对步骤二的细菌纤维素三维多孔支架进行3D打印，得到具有高孔隙率的3D打印聚乳酸和细菌纤维素复合支架材料。Step 3: 3D printing the bacterial cellulose three-dimensional porous scaffold in step 2 to obtain a 3D printed polylactic acid and bacterial cellulose composite scaffold material with high porosity.
本发明相对于现有技术的有益效果是：本发明制备的支架可作为组织工程支架，应用于组织修复和重建领域。本发明制备方法简单，操作周期短，并可通过控制3D打印过程中的工艺参数精确调控支架表层微孔尺寸和形状。制备的3D打印聚乳酸和细菌纤维素的复合支架材料既具有良好的机械性能，又具有较高孔隙率（60~95%）以及优异的生物相容性，还可以提高细胞在支架材料表面的黏附能力，因此可用于组织工程领域。Compared with the prior art, the beneficial effect of the present invention is that the scaffold prepared by the present invention can be used as a tissue engineering scaffold and applied in the field of tissue repair and reconstruction. The preparation method of the invention is simple, the operation cycle is short, and the size and shape of micropores on the surface of the support can be precisely regulated by controlling the process parameters in the 3D printing process. The prepared composite scaffold material of 3D printed polylactic acid and bacterial cellulose not only has good mechanical properties, but also has high porosity (60-95%) and excellent biocompatibility, and can also improve the stability of cells on the surface of the scaffold material. Adhesion ability, so it can be used in the field of tissue engineering.
具体实施方式detailed description
下面结合实施例对本发明的技术方案作进一步的说明，但并不局限于此，凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。The technical solution of the present invention will be further described below in conjunction with the examples, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered by the present invention within the scope of protection.
具体实施方式一：本实施方式记载的是一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，所述方法具体步骤如下：Specific embodiment one: what this embodiment records is a kind of preparation method of 3D printing polylactic acid and bacterial cellulose composite support material, and the specific steps of described method are as follows:
步骤一：选取菌株通过静态培养法发酵制备细菌纤维素，对制得的细菌纤维素依次进行水洗和碱洗；Step 1: Select bacterial strains to ferment and prepare bacterial cellulose by static culture method, and wash the bacterial cellulose with water and alkali in sequence;
步骤二：在室温条件下，利用步骤一得到的细菌纤维素制备细菌纤维素悬浮液，然后经过减压抽滤得到细菌纤维素薄膜，冷冻干燥得到细菌纤维素三维多孔支架；Step 2: At room temperature, use the bacterial cellulose obtained in Step 1 to prepare a bacterial cellulose suspension, then filter under reduced pressure to obtain a bacterial cellulose film, and freeze-dry to obtain a bacterial cellulose three-dimensional porous scaffold;
步骤三：对步骤二的细菌纤维素三维多孔支架进行3D打印，得到具有更高孔隙率的3D打印聚乳酸和细菌纤维素复合支架材料。Step 3: 3D printing the bacterial cellulose three-dimensional porous scaffold in step 2 to obtain a 3D printed polylactic acid and bacterial cellulose composite scaffold material with higher porosity.
具体实施方式二：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤一中，所述菌株为根瘤菌属（Rhizobium）、醋酸菌属（Acetobacter）、八叠球菌属（Sarcina）、土壤杆菌属（Agrobacterium）、固氮菌属（Azotobacter）、无色杆菌属（Achromobacter）或产碱菌属（Alcaligenes）中的一种或几种。Specific embodiment two: the preparation method of a 3D printing polylactic acid and bacterial cellulose composite scaffold material described in specific embodiment one, in step one, the strains are Rhizobium, Acetobacter , Sarcina, Agrobacterium, Azotobacter, Achromobacter or Alcaligenes.
具体实施方式三：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤一中，所述的水洗方法是用去离子水冲洗3~5次或用去离子水多次浸泡10~30 min；所述的碱洗方法是将细菌纤维素置于质量百分比为2~9 %的NaOH溶液中，在30~100 ℃温度下加热2~7 h。Specific embodiment three: the preparation method of a kind of 3D printing polylactic acid and bacterial cellulose composite support material described in specific embodiment one, in step one, the described water washing method is to rinse with deionized water 3 to 5 times or with Soak in deionized water for 10 to 30 minutes several times; the alkaline washing method is to place the bacterial cellulose in a NaOH solution with a mass percentage of 2 to 9%, and heat it at 30 to 100 °C for 2 to 7 hours.
具体实施方式四：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤二中，所述的细菌纤维素悬浮液的制备方法是，在室温条件下，先将纯化后的细菌纤维素剪成小块，置于搅拌机中，然后加入适量水，使得细菌纤维素的重量百分比为2.6%、0.66%或0.33%，搅拌10~30 min得到均匀的细菌纤维素悬浮液。Specific embodiment four: the preparation method of a kind of 3D printing polylactic acid and bacterial cellulose composite support material described in specific embodiment one, in step 2, the preparation method of described bacterial cellulose suspension is, at room temperature First, cut the purified bacterial cellulose into small pieces, place them in a mixer, and then add appropriate amount of water so that the weight percentage of bacterial cellulose is 2.6%, 0.66% or 0.33%, and stir for 10-30 minutes to obtain uniform bacterial cellulose Cellulose suspension.
具体实施方式五：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤二中，所述的减压抽滤时间为5~10 min，减压压力为-0.08~-0.1MPa。Embodiment 5: A method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material described in Embodiment 1. In step 2, the decompression suction filtration time is 5-10 min, and the decompression pressure is 5-10 min. -0.08~-0.1MPa.
具体实施方式六：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤二中，所述的冷冻干燥温度为-20℃~ -60℃，干燥时间为12~48 h。Embodiment 6: A method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material described in Embodiment 1. In step 2, the freeze-drying temperature is -20°C~-60°C, and the drying time is 12~48 hours.
具体实施方式七：具体实施方式一所述的一种3D打印聚乳酸和细菌纤维素复合支架材料的制备方法，步骤三中，所述的3D打印为：在计算机中建立所需多孔膜材料三维多孔支架的3D模型，然后将3D打印用聚乳酸粉料加入3D打印机料筒中，通过逐层打印的方式将聚乳酸打印在置于三维波动平台上的细菌纤维素膜材料上，得到3D打印聚乳酸和细菌纤维素的复合支架材料。Embodiment 7: A method for preparing a 3D printed polylactic acid and bacterial cellulose composite scaffold material described in Embodiment 1. In step 3, the 3D printing is: establish the required three-dimensional porous membrane material in the computer The 3D model of the porous scaffold, and then add the polylactic acid powder for 3D printing into the barrel of the 3D printer, and print the polylactic acid on the bacterial cellulose membrane material placed on the three-dimensional undulating platform by layer-by-layer printing to obtain a 3D printed poly Composite scaffold material of lactic acid and bacterial cellulose.
实施例1 ：Embodiment 1:
利用静态培养法，由根瘤菌属发酵培养得到细菌纤维素，用去离子水冲洗3~5次，除去粘附在细菌纤维素表面的培养基和杂质，之后浸泡于重量百分比为1%的NaOH水溶液中，在100℃温度下加热2 h，取出继续用去离子水反复冲洗至pH值为中性。Using the static culture method, bacterial cellulose is obtained from the fermentation culture of Rhizobium, rinsed with deionized water for 3 to 5 times to remove the medium and impurities adhering to the surface of bacterial cellulose, and then soaked in 1% NaOH by weight In the aqueous solution, heat at 100°C for 2 h, take it out and continue to rinse with deionized water repeatedly until the pH value is neutral.
将清洗过的细菌纤维素机械切割成小块形状，置于搅拌机中搅拌成细菌纤维素的均匀浆液。取10~30 g均匀浆液抽滤5~10 min得到细菌纤维素膜。在-20℃~ -60℃条件下，冷冻干燥后进行3D打印。The washed bacterial cellulose is mechanically cut into small pieces, placed in a mixer and stirred to form a uniform slurry of bacterial cellulose. Take 10-30 g of the homogeneous slurry and filter it with suction for 5-10 min to obtain the bacterial cellulose membrane. 3D printing after freeze-drying at -20°C~-60°C.
所述浆液浓度为细菌纤维素的重量百分比为2.6 %。The concentration of the slurry is 2.6% by weight of bacterial cellulose.
所述3D打印快速成型方法的具体操作步骤为：（1）建模：在计算机中建立所需多孔膜材料支架的3D模型，上述3D模型为规整的圆孔结构，圆孔的直径为1 mm，圆孔之间间隔0.5 mm，同时将模型分解为5层20 μm的薄片，最终聚乳酸厚度总计1 mm；（2）加料：将3D打印用聚乳酸粉料加入3D打印机料筒中；（3）打印：通过逐层打印的方式将聚乳酸打印在置于三维波动平台上的细菌纤维素膜材料上。The specific operation steps of the 3D printing rapid prototyping method are: (1) Modeling: establish a 3D model of the required porous membrane material support in the computer, the above 3D model is a regular circular hole structure, and the diameter of the circular hole is 1 mm , the distance between the round holes is 0.5 mm, and the model is decomposed into 5 layers of 20 μm sheets, and the final polylactic acid thickness is 1 mm in total; (2) Feeding: Add polylactic acid powder for 3D printing into the 3D printer barrel; (3 ) printing: Polylactic acid is printed on the bacterial cellulose membrane material placed on the three-dimensional undulating platform by layer-by-layer printing.
实施例2：Example 2:
利用静态培养法，由木醋杆菌属发酵培养得到细菌纤维素，用去离子水冲洗3~5次，除去粘附在细菌纤维素表面的培养基和杂质，之后浸泡于重量百分比为3%的NaOH水溶液中，在80 ℃温度下加热3h，取出继续用去离子水反复冲洗至pH值为中性。Using the static culture method, bacterial cellulose is obtained from the fermentation of Acetobacter xylinum, rinsed with deionized water for 3 to 5 times to remove the culture medium and impurities adhering to the surface of the bacterial cellulose, and then soaked in 3% by weight In NaOH aqueous solution, heat at 80°C for 3h, take it out and rinse repeatedly with deionized water until the pH value is neutral.
将清洗过的细菌纤维素机械切割成小块形状置于搅拌机中，并加入适量去离子水，搅拌成细菌纤维素的均匀浆液。取10~30 g均匀浆液抽滤5~10 min得到细菌纤维素膜。在-20℃~ -60℃条件下，冷冻干燥后进行3D打印。Mechanically cut the washed bacterial cellulose into small pieces and place it in a blender, add an appropriate amount of deionized water, and stir to form a uniform slurry of bacterial cellulose. Take 10-30 g of the homogeneous slurry and filter it with suction for 5-10 min to obtain the bacterial cellulose membrane. 3D printing is performed after freeze-drying at -20°C~-60°C.
所述浆液细菌纤维素的重量百分比为0.66 %。The percentage by weight of the bacterial cellulose in the slurry is 0.66%.
所述3D打印快速成型方法的具体操作步骤为：（1）建模：在计算机中建立所需多孔膜材料支架的3D模型，上述3D模型为规整的圆孔结构，圆孔的直径为0.5mm，圆孔之间间隔0.5 mm，同时将模型分解为5层20 μm的薄片，最终聚乳酸厚度总计100 μm；（2）加料：将3D打印用聚乳酸粉料加入3D打印机料筒中；（3）打印：通过逐层打印的方式将聚乳酸打印在置于三维波动平台上的细菌纤维素膜材料上。The specific operation steps of the 3D printing rapid prototyping method are: (1) Modeling: establish a 3D model of the required porous membrane material support in the computer, the above 3D model is a regular circular hole structure, and the diameter of the circular hole is 0.5mm , the distance between the round holes is 0.5 mm, and the model is decomposed into 5 layers of 20 μm sheets, and the final polylactic acid thickness is 100 μm in total; (2) Feeding: Add polylactic acid powder for 3D printing into the 3D printer barrel; (3 ) printing: Polylactic acid is printed on the bacterial cellulose membrane material placed on the three-dimensional undulating platform by layer-by-layer printing.
实施例3：Example 3:
利用静态培养法，由八叠球菌属发酵培养得到细菌纤维素，用去离子水冲洗3~5次，除去粘附在细菌纤维素表面的培养基和杂质，之后浸泡于重量百分比为5%的NaOH水溶液中，在60 ℃温度下加热4 h，取出后继续用去离子水反复冲洗至pH值为中性。Bacterial cellulose was obtained from the fermentation culture of Sarcina by static culture method, rinsed with deionized water for 3 to 5 times to remove the culture medium and impurities adhering to the surface of bacterial cellulose, and then soaked in 5% by weight In NaOH aqueous solution, heated at 60 °C for 4 h, and then rinsed repeatedly with deionized water until the pH value was neutral.
将清洗过的细菌纤维素机械切割成小块形状置于搅拌机中，并加入适量去离子水，搅拌成细菌纤维素的均匀浆液。取10~30 g均匀浆液抽滤5~10 min得到细菌纤维素膜。在-20℃~ -60℃条件下，冷冻干燥后进行3D打印。Mechanically cut the washed bacterial cellulose into small pieces and place it in a blender, add an appropriate amount of deionized water, and stir to form a uniform slurry of bacterial cellulose. Take 10-30 g of the homogeneous slurry and filter it with suction for 5-10 min to obtain the bacterial cellulose membrane. 3D printing is performed after freeze-drying at -20°C~-60°C.
所述浆液细菌纤维素的重量百分比为0.33 %。The percentage by weight of the bacterial cellulose in the slurry is 0.33%.
所述3D打印快速成型方法的具体操作步骤为：（1）建模：在计算机中建立所需多孔膜材料支架的3D模型，上述3D模型为规整的圆孔结构，圆孔的直径为0.8mm，圆孔之间间隔0.8 mm，同时将模型分解为5层20 μm的薄片，最终聚乳酸厚度总计1 mm；（2）加料：将3D打印用聚乳酸粉料加入3D打印机料筒中；（3）打印：通过逐层打印的方式将聚乳酸打印在置于三维波动平台上的细菌纤维素膜材料上。The specific operation steps of the 3D printing rapid prototyping method are: (1) Modeling: establish a 3D model of the required porous membrane material support in the computer, the above 3D model is a regular circular hole structure, and the diameter of the circular hole is 0.8mm , the distance between the round holes is 0.8 mm, and the model is decomposed into 5 layers of 20 μm sheets, and the final polylactic acid thickness is 1 mm in total; (2) Feeding: Add polylactic acid powder for 3D printing into the 3D printer barrel; (3 ) printing: Polylactic acid is printed on the bacterial cellulose membrane material placed on the three-dimensional undulating platform by layer-by-layer printing.
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