CN103147229A

Movatterモバイル変換

Info

Publication number: CN103147229A
Application number: CN2013100985206A
Authority: CN
Inventors: 莫秀梅; 吴晶磊
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2013-03-25
Filing date: 2013-03-25
Publication date: 2013-06-12
Anticipated expiration: 2033-03-25
Also published as: CN103147229B

Abstract

本发明涉及一种胶原蛋白/乳酸-己内酯共聚物复合纤维支架的制备方法，包括：（1）将胶原蛋白溶于六氟异丙醇，均匀搅拌，制得浓度为4%-10%克/毫升的胶原蛋白溶液；将乳酸-己内酯共聚物溶于六氟异丙醇，均匀搅拌，制得浓度为4-10%克/毫升的乳酸-己内酯共聚物溶液；（2）将胶原蛋白溶液和乳酸-己内酯共聚物溶液混合，得到总溶质占溶剂4vol%-10vol%的共混静电纺丝溶液；（3）将上述共混静电纺丝溶液进行静电纺丝，得到胶原蛋白/乳酸-己内酯共聚物复合纤维支架。本发明制备的支架的孔径大小随着纤维直径的增加而增大，同时具有纳米和微米纤维的结构特点，仿生细胞外基质，具有良好的应用前景。

The invention relates to a preparation method of a collagen/lactic acid-caprolactone copolymer composite fiber scaffold, comprising: (1) dissolving collagen in hexafluoroisopropanol and stirring evenly to obtain a concentration of 4%-10% Collagen solution of gram/ml; Lactic acid-caprolactone copolymer is dissolved in hexafluoroisopropanol, stirred evenly, and the obtained concentration is the lactic acid-caprolactone copolymer solution of 4-10% g/ml; (2 ) mixing the collagen solution and the lactic acid-caprolactone copolymer solution to obtain a blended electrospinning solution in which the total solute accounts for 4vol%-10vol% of the solvent; (3) electrospinning the above blended electrospinning solution, A collagen/lactic acid-caprolactone copolymer composite fiber scaffold is obtained. The pore diameter of the scaffold prepared by the invention increases with the increase of the fiber diameter, has the structural characteristics of nanometer and micrometer fibers at the same time, biomimetic extracellular matrix, and has good application prospects.

Description

Translated fromChinese

胶原蛋白/乳酸-己内酯共聚物复合纤维支架的制备方法Preparation method of collagen/lactic acid-caprolactone copolymer composite fiber scaffold

技术领域technical field

本发明属于复合纤维支架的制备领域，特别涉及一种胶原蛋白/乳酸-己内酯共聚物复合纤维支架的制备方法。The invention belongs to the field of preparation of composite fiber scaffolds, in particular to a preparation method of collagen/lactic acid-caprolactone copolymer composite fiber scaffolds.

背景技术Background technique

组织工程的核心在于构建由生物材料和细胞组成的三维空间复合体，即组织工程支架复合种子细胞。它的最大优点是可形成具有生命力的活体组织，仿生人体组织器官，对病损组织从形态、结构和功能上进行重建并达到永久性替代；用较少的组织细胞完成病损组织的修复和再生。在这一过程中，组织工程支架为细胞生长和迁移提供了适当的微环境，包括粘附位点和生长信号等。The core of tissue engineering is to construct a three-dimensional space complex composed of biomaterials and cells, that is, tissue engineering scaffold composite seed cells. Its biggest advantage is that it can form living tissues with vitality, bionic human tissues and organs, reconstruct the shape, structure and function of damaged tissues and achieve permanent replacement; use less tissue cells to complete the repair and repair of damaged tissues. regeneration. In this process, tissue engineering scaffolds provide an appropriate microenvironment for cell growth and migration, including adhesion sites and growth signals.

组织工程支架是决定组织修复成功与否的关键因素之一。理想的支架（Liu C,Xia Z,Czernuszka JT.Design and Development of Three-Dimensional Scaffolds for Tissue Engineering.Chemical Engineering Research and Design2007;85:1051-64）应该具备良好的生物相容性、可降解性、适当的力学性能和多孔结构等特点，即应该从组份和结构上仿生。静电纺技术为组织工程支架的制备提供了新的思路。Zhang等（Zhang KH,Wang HS,Huang C,Su Y,Mo XM,Ikada Y.Fabrication of silk fibroin blended P(LLA-CL)nanofibrous scaffolds for tissue engineering.Journal of Biomedical Materials Research Part A2010;93A:984-93）以六氟异丙醇为溶剂制备了丝素蛋白与乳酸-己内酯共聚物复合纳米纤维膜，并证明该纤维膜能够促进内皮细胞和成纤维细胞生长。这种通过层层叠加纳米纤维的方法得到的致密纤维膜，由于其孔径大小是纳米级的，不能完全满足细胞生长的要求，如细胞不能长入其内部。Tissue engineering scaffold is one of the key factors to determine the success of tissue repair. An ideal scaffold (Liu C, Xia Z, Czernuszka JT. Design and Development of Three-Dimensional Scaffolds for Tissue Engineering. Chemical Engineering Research and Design2007;85:1051-64) should have good biocompatibility, degradability, Appropriate mechanical properties and porous structure should be biomimetic from the composition and structure. Electrospinning technology provides a new idea for the preparation of tissue engineering scaffolds. Zhang et al (Zhang KH, Wang HS, Huang C, Su Y, Mo XM, Ikada Y. Fabrication of silk fibroin blended P(LLA-CL) nanofibrous scaffolds for tissue engineering. Journal of Biomedical Materials Research Part A2010;93A:984- 93) The composite nanofibrous membrane of silk fibroin and lactic acid-caprolactone copolymer was prepared by using hexafluoroisopropanol as solvent, and it was proved that the fibrous membrane can promote the growth of endothelial cells and fibroblasts. The dense fibrous membrane obtained by stacking nanofibers layer by layer cannot fully meet the requirements of cell growth because the pore size is nanoscale, such as cells cannot grow into its interior.

发明内容Contents of the invention

本发明所要解决的技术问题是提供一种胶原蛋白/乳酸-己内酯共聚物复合纤维支架的制备方法，该方法制备的支架的孔径大小随着纤维直径的增加而增大，这有利于营养物质在支架内部的传输和代谢废物的排出，也利于细胞长入支架内部，形成再生组织。The technical problem to be solved by the present invention is to provide a preparation method of collagen/lactic acid-caprolactone copolymer composite fiber scaffold, the pore size of the scaffold prepared by the method increases with the increase of fiber diameter, which is beneficial to nutrition The transmission of substances inside the scaffold and the discharge of metabolic waste are also conducive to the growth of cells into the inside of the scaffold to form regenerative tissues.

本发明的一种胶原蛋白/乳酸-己内酯共聚物复合纤维支架的制备方法，包括：A kind of preparation method of collagen/lactic acid-caprolactone copolymer composite fiber scaffold of the present invention comprises:

（1）将胶原蛋白溶于六氟异丙醇，均匀搅拌，制得浓度为4%-10%克/毫升的胶原蛋白溶液；将乳酸-己内酯共聚物溶于六氟异丙醇，均匀搅拌，制得浓度为4-10%克/毫升的乳酸-己内酯共聚物溶液；(1) Dissolve collagen in hexafluoroisopropanol and stir evenly to obtain a collagen solution with a concentration of 4%-10% g/ml; dissolve lactic acid-caprolactone copolymer in hexafluoroisopropanol, Stir evenly to obtain a lactic acid-caprolactone copolymer solution with a concentration of 4-10% g/ml;

（2）将胶原蛋白溶液和乳酸-己内酯共聚物溶液混合，得到总溶质占溶剂4vol%-10vol%的共混静电纺丝溶液；(2) Mix the collagen solution and the lactic acid-caprolactone copolymer solution to obtain a blended electrospinning solution in which the total solute accounts for 4vol%-10vol% of the solvent;

（3）将上述共混静电纺丝溶液进行静电纺丝，得到胶原蛋白/乳酸-己内酯共聚物复合纤维支架。(3) Electrospinning the above blended electrospinning solution to obtain a collagen/lactic acid-caprolactone copolymer composite fiber scaffold.

所述步骤（2）中的胶原蛋白占总溶质的10wt%-40wt%，乳酸-己内酯共聚物占总溶质的60wt%-90wt%。The collagen in the step (2) accounts for 10wt%-40wt% of the total solute, and the lactic acid-caprolactone copolymer accounts for 60wt%-90wt% of the total solute.

所述步骤（3）中的静电纺丝参数为：电压10-20千伏，注射速率0.5-1.5毫升/小时，接收距离5-25厘米，小孔直径5-10毫米，转轴转速30-100转/分钟，电场距离100～200毫米。The electrospinning parameters in the step (3) are: voltage 10-20 kV, injection rate 0.5-1.5 ml/hour, receiving distance 5-25 cm, hole diameter 5-10 mm, shaft speed 30-100 revolution/minute, the electric field distance is 100-200mm.

本发明以动态水溶液接收胶原蛋白和乳酸-己内酯共聚物的静电纺纳米纤维，并利用水中的漩涡将纳米纤维抱合成微米纤维束，再用转轴收集得到疏松的纤维膜支架。这种含胶原的由纳米纤维组成的微米纤维束从组份上更好的模拟肌腱细胞外基质中的纤维束，而且支架的大孔结构利于细胞生长、迁移和组织再生。The invention uses a dynamic aqueous solution to receive electrospun nanofibers of collagen and lactic acid-caprolactone copolymer, uses vortexes in water to coagulate the nanofibers into micron fiber bundles, and collects them with a rotating shaft to obtain a loose fibrous membrane support. The collagen-containing microfiber bundles composed of nanofibers better simulate the fiber bundles in the tendon extracellular matrix in composition, and the macroporous structure of the scaffold is conducive to cell growth, migration and tissue regeneration.

有益效果Beneficial effect

(1)本发明制备的支架的孔径大小随着纤维直径的增加而增大，这有利于营养物质在支架内部的传输和代谢废物的排出，也利于细胞长入支架内部，形成再生组织；(1) The pore size of the scaffold prepared by the present invention increases with the increase of the fiber diameter, which is conducive to the transmission of nutrients inside the scaffold and the discharge of metabolic waste, and is also conducive to the growth of cells into the interior of the scaffold to form regenerative tissues;

(2)本发明制备的支架是由微米纤维束构成的，而微米纤维束是通过漩涡将纳米纤维抱合而得到的，因而该支架同时具有纳米和微米纤维的结构特点，仿生细胞外基质；(2) The support prepared by the present invention is composed of micron fiber bundles, and the micron fiber bundles are obtained by entanglement of nanofibers by vortex, so the support has the structural characteristics of nanofibers and microfibers, bionic extracellular matrix;

(3)本发明制备的支架是由生物相容性良好的胶原蛋白和力学性能良好的乳酸-己内酯共聚物制备，通过改变二者的比例来调整支架的力学性能，以更好的满足实际应用的需求。(3) The stent prepared by the present invention is prepared from collagen with good biocompatibility and lactic acid-caprolactone copolymer with good mechanical properties, and the mechanical properties of the stent are adjusted by changing the ratio of the two to better meet practical application needs.

附图说明Description of drawings

图1为动态水溶液接收纤维的静电纺装置；Figure 1 is an electrospinning device for receiving fibers in a dynamic aqueous solution;

图2为胶原蛋白/乳酸-己内酯共聚物复合纤维支架的扫描电镜图片；Fig. 2 is the scanning electron microscope picture of collagen/lactic acid-caprolactone copolymer composite fiber support;

图3为胶原蛋白/乳酸-己内酯共聚物复合纤维支架照片；Fig. 3 is the photo of collagen/lactic acid-caprolactone copolymer composite fiber scaffold;

图4为苏木精-伊红染色法观察肌腱细胞在复合纤维支架上生长。Figure 4 shows the observation of tenocyte growth on the composite fiber scaffold by hematoxylin-eosin staining.

具体实施方式Detailed ways

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

实施例1Example 1

将0.8克胶原蛋白溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为8%（克/毫升）的胶原蛋白溶液；将0.8克聚乳酸-己内酯共聚物溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为8%（克/毫升）的乳酸-己内酯共聚物溶液；将两种溶液按体积比为10：90混合，得到胶原蛋白与乳酸-己内酯共聚物质量比为10：90的混合溶液；将混合溶液进行静电纺丝，纺丝条件：电压，15千伏；电场距离150毫米；注射速率，1毫升/小时；转轴转速，60转/分钟；小孔直径8毫米，得到复合纤维支架。支架中的纳米纤维平均直径为650纳米，微米纤维平均直径为24微米。Dissolve 0.8 g of collagen in 10 ml of hexafluoroisopropanol and stir until uniform to obtain a collagen solution with a concentration of 8% (g/ml); dissolve 0.8 g of polylactic acid-caprolactone copolymer in 10 ml In hexafluoroisopropanol, stir until uniform to obtain a lactic acid-caprolactone copolymer solution with a concentration of 8% (g/ml); mix the two solutions at a volume ratio of 10:90 to obtain collagen and lactic acid- A mixed solution with a caprolactone copolymer mass ratio of 10:90; the mixed solution was subjected to electrospinning, spinning conditions: voltage, 15 kV; electric field distance, 150 mm; injection rate, 1 ml/hour; shaft speed, 60 rotation/minute; the diameter of the small hole is 8 mm, and the composite fiber scaffold is obtained. The nanofibers in the scaffold have an average diameter of 650 nanometers, and the microfibers have an average diameter of 24 micrometers.

实施例2Example 2

将0.8克胶原蛋白溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为8%（克/毫升）的胶原蛋白溶液；将0.8克乳酸-己内酯共聚物溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为8%（克/毫升）的聚乳酸-聚己内酯溶液；将两种溶液按体积比为25：75混合，得到胶原蛋白与乳酸-己内酯共聚物质量比为25：75的混合溶液；将混合溶液进行静电纺丝，纺丝条件：电压，15千伏；电场距离150毫米；注射速率，1毫升/小时；转轴转速，60转/分钟；小孔直径8毫米，得到复合纤维支架。支架中的纳米纤维平均直径为542纳米，微米纤维平均直径为20微米。Dissolve 0.8 g of collagen in 10 ml of hexafluoroisopropanol and stir until uniform to obtain a collagen solution with a concentration of 8% (g/ml); dissolve 0.8 g of lactic acid-caprolactone copolymer in 10 ml of hexafluoroisopropanol In fluoroisopropanol, stir until uniform to obtain a polylactic acid-polycaprolactone solution with a concentration of 8% (g/ml); mix the two solutions at a volume ratio of 25:75 to obtain collagen and lactic acid-caprolactone A mixed solution with a lactone copolymer mass ratio of 25:75; electrospinning the mixed solution, spinning conditions: voltage, 15 kV; electric field distance, 150 mm; injection rate, 1 ml/hour; shaft speed, 60 rpm /min; the diameter of the small hole is 8 mm, and the composite fiber support is obtained. The nanofibers in the scaffold have an average diameter of 542 nanometers, and the microfibers have an average diameter of 20 micrometers.

实施例3Example 3

将0.8克胶原蛋白溶解于10毫升六氟异丙醇溶液中，搅拌至均匀，得到浓度为8%（克/毫升）的胶原蛋白溶液；将0.8克乳酸-己内酯共聚物溶解于10毫升六氟异丙醇溶液中，搅拌至均匀，得到浓度为8%（克/毫升）的乳酸-己内酯共聚物溶液；将两种溶液按体积比为40：60混合，得到胶原蛋白与聚乳酸-聚己内酯质量比为40：60的混合溶液；将混合溶液进行静电纺丝，纺丝条件：电压，15千伏；电场距离150毫米；注射速率，1毫升/小时；转轴转速，60转/分钟；小孔直径8毫米，得到复合纤维支架。支架中的纳米纤维平均直径为450纳米，微米纤维平均直径为15微米。Dissolve 0.8 g of collagen in 10 ml of hexafluoroisopropanol solution and stir until uniform to obtain a collagen solution with a concentration of 8% (g/ml); dissolve 0.8 g of lactic acid-caprolactone copolymer in 10 ml Hexafluoroisopropanol solution, stirred until uniform to obtain a lactic acid-caprolactone copolymer solution with a concentration of 8% (g/ml); mix the two solutions at a volume ratio of 40:60 to obtain collagen and poly A mixed solution with a mass ratio of lactic acid-polycaprolactone of 40:60; electrospinning the mixed solution, spinning conditions: voltage, 15 kV; electric field distance, 150 mm; injection rate, 1 ml/hour; shaft speed, 60 revolutions/minute; the diameter of the small hole is 8 mm, and the composite fiber scaffold is obtained. The nanofibers in the scaffold have an average diameter of 450 nanometers, and the microfibers have an average diameter of 15 micrometers.

实施例4Example 4

将0.6克胶原蛋白溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为6%（克/毫升）的胶原蛋白溶液；将0.6克乳酸-己内酯共聚物溶解于10毫升六氟异丙醇中，搅拌至均匀，得到浓度为6%（克/毫升）的乳酸-己内酯共聚物溶液；将两种溶液按体积比为25：75混合，得到胶原蛋白与聚乳酸-聚己内酯质量比为25：75的混合溶液；将混合溶液进行静电纺丝，纺丝条件：电压，15千伏；电场距离150毫米；注射速率，1毫升/小时；转轴转速，60转/分钟；小孔直径8毫米，得到复合纤维支架。支架中的纳米纤维平均直径为550纳米，微米纤维平均直径为18微米。Dissolve 0.6 g of collagen in 10 ml of hexafluoroisopropanol and stir until uniform to obtain a collagen solution with a concentration of 6% (g/ml); dissolve 0.6 g of lactic acid-caprolactone copolymer in 10 ml of hexafluoroisopropanol In fluoroisopropanol, stir until uniform to obtain a lactic acid-caprolactone copolymer solution with a concentration of 6% (g/ml); mix the two solutions at a volume ratio of 25:75 to obtain collagen and polylactic acid- A mixed solution with a polycaprolactone mass ratio of 25:75; the mixed solution was subjected to electrospinning, spinning conditions: voltage, 15 kV; electric field distance, 150 mm; injection rate, 1 ml/hour; shaft speed, 60 rpm /min; the diameter of the small hole is 8 mm, and the composite fiber support is obtained. The nanofibers in the scaffold have an average diameter of 550 nanometers, and the microfibers have an average diameter of 18 micrometers.

实施例5Example 5

将200微升肌腱细胞悬液（约10000个肌腱细胞）种植到直径为15毫米的圆形复合纤维支架上，经过7天的培养后，取出培养细胞的支架，并用4%多聚甲醛在4℃固定30分钟，经脱水、透明、浸蜡和包埋后，用切片机将样品切成10微米厚，贴在玻片上染色，于显微镜下观察并拍照，见图4。200 microliters of tenocyte suspension (approximately 10,000 tenocytes) was planted on a circular composite fiber scaffold with a diameter of 15 mm. After 7 days of culture, the scaffold for cultured cells was taken out and treated with 4% paraformaldehyde at 4 After fixing at ℃ for 30 minutes, after dehydration, transparency, wax dipping and embedding, the sample was cut into 10 micron thick with a microtome, attached to a glass slide for staining, observed under a microscope and photographed, see Figure 4.

Claims

1. the preparation method of collagen/lactic acid-caprolactone copolymer composite fibre support comprises:

(1) collagen is dissolved in hexafluoroisopropanol, uniform stirring makes the collagen solution that concentration is the 4%-10% grams per milliliter; Lactic acid-caprolactone copolymer is dissolved in hexafluoroisopropanol, and uniform stirring makes concentration and is the lactic acid of 4-10% grams per milliliter-caprolactone copolymer solution;

(2) collagen solution and lactic acid-caprolactone copolymer solution are mixed, obtain the blend electrostatic spinning solution that total soluble matters accounts for solvent 4vol%-10vol%;

(3) above-mentioned blend electrostatic spinning solution is carried out electrostatic spinning, obtain collagen/lactic acid-caprolactone copolymer composite fibre support.

2. the preparation method of a kind of collagen/lactic acid according to claim 1-caprolactone copolymer composite fibre support, it is characterized in that: the collagen in described step (2) accounts for the 10wt%-40wt% of total soluble matters, and lactic acid-caprolactone copolymer accounts for the 60wt%-90wt% of total soluble matters.

3. the preparation method of a kind of collagen/lactic acid according to claim 1-caprolactone copolymer composite fibre support, it is characterized in that: the electrostatic spinning parameter in described step (3) is: voltage 10-20 kilovolt, injection rate 0.5-1.5 ml/hour, receiving range 5-25 centimetre, hole diameter 5-10 millimeter, 30-100 rev/min of rotating shaft rotating speed, 100～200 millimeters of electric field distance.