CN116139340A - Bone repair material with double-factor gradient slow release and preparation method thereof - Google Patents

Abstract

The invention relates to a bone repair material with a double-factor gradient slow release and a preparation method thereof. The preparation method comprises the following steps: mixing collagen with acetic acid solution and stirring to obtain collagen solution; mixing and stirring the collagen solution and a calcium-containing compound, and then sequentially pre-freezing and freeze-drying to obtain a freeze-dried porous mixture; mixing the freeze-dried porous mixture with a cross-linking agent, cross-linking, sequentially cleaning, freeze-drying, and immersing in PBS (phosphate buffered saline) solution containing bone growth factor BMP-2 to obtain an inner layer bone repair material; mixing the light-cured material solution with endothelial growth factor VEGF to obtain an outer layer bone repair material mixed solution; finally, photo-curing is carried out. The obtained bone repair material has a double-layer structure, the outer layer can be rapidly vascularized in the early stage and establish blood supply connection with the organism so as to meet the transportation of oxygen, nutrition and metabolic products of cells which migrate into the inside of the bracket, and the inner layer can continuously stimulate osteogenesis in the later stage to finally form living autologous bone tissues.

Description

Translated fromChinese

一种双因子梯度缓释的骨修复材料及其制备方法A kind of two-factor gradient sustained-release bone repair material and its preparation method

技术领域technical field

本发明属于医用材料领域，具体涉及一种双因子梯度缓释的骨修复材料及其制备方法。The invention belongs to the field of medical materials, in particular to a two-factor gradient sustained-release bone repair material and a preparation method thereof.

背景技术Background technique

临床中引起骨缺损的主要原因有创伤、先天性疾病、感染和肿瘤的外科切除等。保守估计，全世界每年因骨科疾病导致消耗数千亿美元，我国每年也有约百万患者需要进行植骨手术。而目前修复骨缺损的主要方法有：自体骨移植、异体骨移植以及组织工程骨进行手术治疗。尽管自体骨移植有愈合快、无排斥反应等优点，存在的最大问题是来源有限，并且一般自体骨移植需要额外的手术来获得移植自体骨，增加了患者的痛苦、失血量、手术感染率以及额外的长期住院费用。同种异体骨移植来源和数量也受到限制，移植后机体对移植物存在排斥反应，血管系统构建困难，从而造成了功能恢复的困难，同时也存在晚期感染、移植骨愈合缓慢以及可能导致交叉感染从而引发艾滋病、海绵状脑病、肝炎等风险。临床上已经证明组织工程骨在原位成骨和异位成骨具有可行性。理想的组织工程骨支架材料应具有以下特性：(1)具有较高的孔隙率和高度连通的三维结构，以利于细胞的长入和细胞之间的物质交换，以及营养和代谢物的运输；(2)具有与细胞/组织的生长相适应的降解速率和良好的生物相容性；(3)表面具有良好的生物活性，有利于细胞的粘附、增殖和分化。The main causes of bone defects in clinical practice are trauma, congenital diseases, infection and surgical resection of tumors. It is conservatively estimated that hundreds of billions of dollars are spent every year due to orthopedic diseases all over the world, and about one million patients in my country need bone grafting surgery every year. At present, the main methods for repairing bone defects are: autologous bone transplantation, allogeneic bone transplantation and tissue engineering bone for surgical treatment. Although autologous bone transplantation has the advantages of fast healing and no rejection, the biggest problem is that the source is limited, and generally autologous bone transplantation requires additional operations to obtain transplanted autologous bone, which increases the patient's pain, blood loss, surgical infection rate and Additional long-term hospital costs. The source and quantity of allogeneic bone grafts are also limited. After transplantation, the body will reject the graft, and the construction of the vascular system is difficult, resulting in difficulties in functional recovery. At the same time, there are also late infection, slow healing of grafted bones, and possible cross-infection. Thereby causing AIDS, spongiform encephalopathy, hepatitis and other risks. It has been clinically proven that tissue engineered bone is feasible for in situ and ectopic osteogenesis. An ideal tissue engineering bone scaffold material should have the following characteristics: (1) It has a high porosity and a highly connected three-dimensional structure to facilitate the growth of cells and the exchange of substances between cells, as well as the transportation of nutrients and metabolites; (2) It has a degradation rate compatible with the growth of cells/tissues and good biocompatibility; (3) The surface has good biological activity, which is conducive to cell adhesion, proliferation and differentiation.

然而，构建组织工程骨需要面临的一个重要问题是如何在骨材料植入体内后迅速血管化并与机体建立血供连接，以满足对迁入支架内部的细胞氧气、营养和代谢产物运输。另外，如何在成骨中后期可以不断的刺激成骨，最终形成活的自体骨组织。However, an important problem that needs to be faced in the construction of tissue engineered bone is how to rapidly vascularize the bone material and establish a blood supply connection with the body after implantation in the body, so as to satisfy the transportation of oxygen, nutrients and metabolites of cells that move into the scaffold. In addition, how to continuously stimulate osteogenesis in the middle and late stages of osteogenesis, and finally form living autologous bone tissue.

故基于此，提出本发明技术方案。Therefore based on this, propose technical scheme of the present invention.

发明内容Contents of the invention

为了解决现有技术存在的问题，本发明提供了一种双因子梯度缓释的骨修复材料及其制备方法。In order to solve the problems in the prior art, the invention provides a two-factor gradient sustained-release bone repair material and a preparation method thereof.

本发明的方案是提供一种双因子梯度缓释的骨修复材料的制备方法，所述制备方法包括如下步骤：The solution of the present invention is to provide a preparation method of a two-factor gradient sustained-release bone repair material, the preparation method comprising the following steps:

(1)将胶原与醋酸溶液混合并进行搅拌，得到胶原溶液；(1) Collagen and acetic acid solution are mixed and stirred to obtain a collagen solution;

(2)将所述胶原溶液与含钙化合物混合并进行搅拌，然后依次预冻、冻干，得到冻干多孔混合物；(2) mixing and stirring the collagen solution and the calcium-containing compound, then pre-freezing and freeze-drying in sequence to obtain a freeze-dried porous mixture;

(3)将所述冻干多孔混合物与交联剂混合并进行交联，然后依次清洗、冻干，再浸入至含有骨生长因子BMP-2的PBS溶液中，得到内层骨修复材料；(3) mixing the freeze-dried porous mixture with a cross-linking agent and performing cross-linking, then sequentially washing, freeze-drying, and then immersing in a PBS solution containing bone growth factor BMP-2 to obtain an inner bone repair material;

(4)将光固化材料溶液与内皮生长因子VEGF混合，得到外层骨修复材料混合溶液；(4) mixing the light-curing material solution with endothelial growth factor VEGF to obtain a mixed solution of the outer bone repair material;

(5)将所述外层骨修复材料混合溶液在所述内层骨修复材料外层进行光固化，完成后即得所述双因子梯度缓释的骨修复材料。(5) Light-cure the mixed solution of the outer layer bone repair material on the outer layer of the inner layer bone repair material, and then obtain the bone repair material with gradient slow release of the two factors.

优选地，步骤(1)中，所述搅拌的温度为2～8℃，搅拌的时间为12～24h；所述胶原溶液中，胶原的浓度为0.5～5wt.％。Preferably, in step (1), the stirring temperature is 2-8° C., and the stirring time is 12-24 hours; the collagen concentration in the collagen solution is 0.5-5 wt.%.

优选地，步骤(2)中，所述含钙化合物为羟基磷灰石、磷酸三钙或硫酸钙中的一种，也可选择珊瑚。Preferably, in step (2), the calcium-containing compound is one of hydroxyapatite, tricalcium phosphate or calcium sulfate, and coral can also be selected.

优选地，步骤(2)中，所述预冻的温度为-80℃，预冻的时间为4～24h。Preferably, in step (2), the pre-freezing temperature is -80°C, and the pre-freezing time is 4-24 hours.

优选地，步骤(2)中，所述冻干的方式为：依次在-44～-46℃下处理4h，在-19～-21℃下处理48h，在-9～-11℃下处理16h，在-4～-6℃下处理6h，在4～6℃下处理4h，在24～26℃下处理5h。Preferably, in step (2), the freeze-drying method is as follows: sequentially treat at -44 to -46°C for 4 hours, at -19 to -21°C for 48 hours, and at -9 to -11°C for 16 hours , Treat at -4~-6°C for 6h, at 4~6°C for 4h, and at 24~26°C for 5h.

优选地，步骤(3)中，所述交联剂为戊二醛、1-乙基-3-(3-二甲基氨基丙基)碳二亚胺、N-羟基琥珀酰亚胺、京尼平或肉桂醛中的一种或多种的组合。Preferably, in step (3), the crosslinking agent is glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, Beijing A combination of one or more of nipin or cinnamaldehyde.

优选地，步骤(3)中，所述BMP-2的浓度为20～500μg/mL。Preferably, in step (3), the concentration of BMP-2 is 20-500 μg/mL.

优选地，步骤(4)中，所述外层骨修复材料混合溶液中VEGF的浓度为20～400μg/mL。Preferably, in step (4), the concentration of VEGF in the mixed solution of the outer bone repair material is 20-400 μg/mL.

优选地，步骤(5)中，所述光固化采用紫外灯照射30～60s。Preferably, in step (5), the photocuring is irradiated with ultraviolet light for 30-60s.

基于相同的技术构思，本发明的再一方案是提供一种由上述制备方法得到的双因子梯度缓释的骨修复材料，所述骨修复材料为圆柱状的双层结构。Based on the same technical idea, another solution of the present invention is to provide a two-factor gradient sustained-release bone repair material obtained by the above preparation method, and the bone repair material has a cylindrical double-layer structure.

本发明的有益效果为：The beneficial effects of the present invention are:

本发明所述的双因子梯度缓释的骨修复材料，于材料外层将心血管组织工程材料的活性修饰物质进行负载，在骨修复的前期，能达到快速释放的效果，使材料快速血管化并有利于氧气和营养物质的运输和传递，同时可显著促进细胞向大体积骨修复材料深部的迁移，加快骨修复。在材料的内层，将促骨再生的活性修饰物质进行负载，在骨修复的中后期，达到缓慢释放的效果，有助于不断地刺激成骨，达到天然骨取代植入物的目的。The two-factor gradient slow-release bone repair material of the present invention loads the active modification substance of cardiovascular tissue engineering material on the outer layer of the material, and can achieve the effect of rapid release in the early stage of bone repair, so that the material can be rapidly vascularized It is also beneficial to the transport and transmission of oxygen and nutrients, and can significantly promote the migration of cells to the deep part of the large-volume bone repair material and accelerate bone repair. In the inner layer of the material, the active modification substances that promote bone regeneration are loaded, and in the middle and late stages of bone repair, the effect of slow release is achieved, which helps to continuously stimulate osteogenesis and achieve the purpose of replacing implants with natural bone.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单的介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

图1是本发明所述双因子梯度缓释的骨修复材料的结构示意图。Fig. 1 is a schematic structural view of the bone repair material with two-factor gradient sustained release according to the present invention.

图2是试验例中，内层骨修复材料的SEM图。Fig. 2 is a SEM image of the inner layer bone repair material in the test example.

图3是试验例中，外层骨修复材料的SEM图。Fig. 3 is a SEM image of the outer bone repair material in the test example.

图4是试验例中，外层骨修复材料中VEGF的累计释放曲线图。Fig. 4 is a graph showing the cumulative release curve of VEGF in the outer bone repair material in the test example.

图5是试验例中，内层骨修复材料中BMP-2的累计释放曲线图。Fig. 5 is a graph showing the cumulative release curve of BMP-2 in the inner bone repair material in the test example.

图6是试验例中，细胞的增殖情况图。Fig. 6 is a graph showing the proliferation of cells in the test example.

图中附图说明：Description of the drawings in the figure:

1、外层骨修复材料；2、内层骨修复材料；3、VEGF内皮生长因子；4、BMP-2骨生长因子。1. Outer bone repair material; 2. Inner bone repair material; 3. VEGF endothelial growth factor; 4. BMP-2 bone growth factor.

具体实施方式Detailed ways

为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other implementations obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

实施例1Example 1

本实施例提供一种双因子梯度缓释的骨修复材料的制备方法，所述制备方法包括如下步骤：This embodiment provides a preparation method of a two-factor gradient sustained-release bone repair material, the preparation method comprising the following steps:

(1)在2℃低温反应釜中，将胶原溶解在醋酸溶液中，启动自动搅拌器，搅拌12小时，得到浓度为1％的胶原溶液。(1) Dissolve collagen in acetic acid solution in a low-temperature reaction kettle at 2° C., start an automatic stirrer, and stir for 12 hours to obtain a collagen solution with a concentration of 1%.

(2)称取胶原溶液重量2％、直径为20～50nm的羟基磷灰石粉末，并加入低温反应釜中，再启动搅拌机充分搅拌；然后将上述混合液体倒入带夹层的不锈钢容器中，采用自动灌装机，将上述混合液分装入事先准备好的硅胶模具中，在-80℃的温度下进行预冻，预冻时间为4小时。(2) Take 2% by weight of the collagen solution and hydroxyapatite powder with a diameter of 20-50nm, add it into the low-temperature reaction kettle, start the mixer and stir thoroughly; then pour the above-mentioned mixed liquid into a stainless steel container with an interlayer, Using an automatic filling machine, the above-mentioned mixed solution is divided into pre-prepared silica gel molds, and pre-freezing is carried out at a temperature of -80° C., and the pre-freezing time is 4 hours.

(3)将预冻得到的材料放入冻干机进行冻干，具体为：依次在-44℃下处理4h，在-19℃下处理48h，在-9℃下处理16h，在-4℃下处理6h，在4℃下处理4h，在24℃下处理5h，得到多孔混合物。(3) Put the pre-frozen material into a lyophilizer for lyophilization, specifically: treat at -44°C for 4h, at -19°C for 48h, at -9°C for 16h, and at -4°C Treated at low temperature for 6h, at 4°C for 4h, and at 24°C for 5h to obtain a porous mixture.

(4)将所述多孔混合物浸入浓度为2wt.％的戊二醛水溶液中交联12小时，完成后用纯化水清洗72小时，期间每1小时换水一次，然后重复步骤(3)中的冻干步骤，再将其浸入含有BMP-2骨生长因子4的20μg/mL的PBS溶液中24小时，得到内层骨修复材料2。(4) immerse the porous mixture in a 2wt.% glutaraldehyde aqueous solution for cross-linking for 12 hours, after completion, wash with purified water for 72 hours, during which the water is changed once every 1 hour, and then repeat the steps in step (3) The step of lyophilization, and then immersing it in a 20 μg/mL PBS solution containing BMP-2bone growth factor 4 for 24 hours to obtain the inner layerbone repair material 2.

(5)将所述内层骨修复材料放入模具正中间加以固定、备用。(5) Put the inner bone repair material into the middle of the mold to fix it and set it aside.

(6)配制浓度为2wt.％的甲基丙烯酸酰化明胶溶液，再加入VEGF内皮生长因子3，得到VEGF含量为20μg/mL的外层骨修复材料1混合溶液。(6) Prepare a methacrylic acylated gelatin solution with a concentration of 2 wt.%, and then add VEGFendothelial growth factor 3 to obtain a mixed solution of outer layerbone repair material 1 with a VEGF content of 20 μg/mL.

(7)将所述外层骨修复材料1混合溶液倒入步骤(5)所述模具中，其在内层骨修复材料2周围充满，并快速用紫外灯照射30s，将其固化成型，即得到所述双因子梯度缓释的骨修复材料。(7) Pour the mixed solution of the outer layerbone repair material 1 into the mold described in step (5), which is filled around the inner layerbone repair material 2, and quickly irradiate it with an ultraviolet lamp for 30 seconds to cure it into shape, namely The bone repair material with gradient sustained release of the two factors is obtained.

实施例2Example 2

本实施例提供一种双因子梯度缓释的骨修复材料的制备方法，所述制备方法包括如下步骤：This embodiment provides a preparation method of a two-factor gradient sustained-release bone repair material, the preparation method comprising the following steps:

(1)在8℃低温反应釜中，将丝素蛋白溶解在醋酸溶液中，启动自动搅拌器，搅拌24小时，得到浓度为2.5％的丝素蛋白溶液。(1) Dissolve silk fibroin in acetic acid solution in a low-temperature reaction kettle at 8° C., start an automatic stirrer, and stir for 24 hours to obtain a silk fibroin solution with a concentration of 2.5%.

(2)称取丝素蛋白溶液重量2.5％的磷酸三钙粉末，并加入低温反应釜中，再启动搅拌机充分搅拌；然后将上述混合液体倒入带夹层的不锈钢容器中，采用自动灌装机，将上述混合液分装入事先准备好的硅胶模具中，在-80℃的温度下进行预冻，预冻时间为12小时。(2) Weigh tricalcium phosphate powder with 2.5% weight of silk fibroin solution, add it to the low-temperature reaction kettle, start the mixer and stir thoroughly; then pour the mixed liquid into a stainless steel container with an interlayer, and use an automatic filling machine , put the mixture above into pre-prepared silica gel molds, and pre-freeze at -80°C for 12 hours.

(3)将预冻得到的材料放入冻干机进行冻干，具体为：依次在-46℃下处理4h，在-21℃下处理48h，在-11℃下处理16h，在-6℃下处理6h，在6℃下处理4h，在26℃下处理5h，得到多孔混合物。(3) Put the pre-frozen material into a lyophilizer for lyophilization, specifically: treat at -46°C for 4h, at -21°C for 48h, at -11°C for 16h, and at -6°C Treated at low temperature for 6h, at 6°C for 4h, and at 26°C for 5h to obtain a porous mixture.

(4)将所述多孔混合物浸入浓度为1wt.％的N-羟基琥珀酰亚胺水溶液中交联24小时，完成后用纯化水清洗48小时，期间每2小时换水一次，然后重复步骤(3)中的冻干步骤，再将其浸入含有BMP-2骨生长因子4的200μg/mL的PBS溶液中24小时，得到内层骨修复材料2。(4) immerse the porous mixture into a 1wt.% aqueous solution of N-hydroxysuccinimide to cross-link for 24 hours, after completion, wash with purified water for 48 hours, during which the water is changed once every 2 hours, and then repeat the steps ( In the freeze-drying step in 3), it was immersed in a 200 μg/mL PBS solution containing BMP-2bone growth factor 4 for 24 hours to obtain the inner layerbone repair material 2.

(5)将所述内层骨修复材料放入模具正中间加以固定、备用。(5) Put the inner bone repair material into the middle of the mold to fix it and set it aside.

(6)配制浓度为5wt.％的甲基丙烯酸酯化透明质酸溶液，再加入VEGF内皮生长因子3，得到VEGF含量为200μg/mL的外层骨修复材料1混合溶液。(6) Prepare a methacrylated hyaluronic acid solution with a concentration of 5 wt.%, and then add VEGFendothelial growth factor 3 to obtain a mixed solution of outer layerbone repair material 1 with a VEGF content of 200 μg/mL.

(7)将所述外层骨修复材料1混合溶液倒入步骤(5)所述模具中，其在内层骨修复材料2周围充满，并快速用紫外灯照射60s，将其固化成型，即得到所述双因子梯度缓释的骨修复材料。(7) Pour the mixed solution of the outer layerbone repair material 1 into the mold described in step (5), which is filled around the inner layerbone repair material 2, and quickly irradiate it with an ultraviolet lamp for 60 seconds to cure it into shape, namely The bone repair material with gradient sustained release of the two factors is obtained.

实施例3Example 3

本实施例提供一种双因子梯度缓释的骨修复材料的制备方法，所述制备方法包括如下步骤：This embodiment provides a preparation method of a two-factor gradient sustained-release bone repair material, the preparation method comprising the following steps:

(1)在5℃低温反应釜中，将壳聚糖溶解在醋酸溶液中，启动自动搅拌器，搅拌24小时，得到浓度为5％的壳聚糖溶液。(1) Dissolve chitosan in an acetic acid solution in a low-temperature reaction kettle at 5° C., start an automatic stirrer, and stir for 24 hours to obtain a chitosan solution with a concentration of 5%.

(2)称取壳聚糖溶液重量2％的硫酸钙粉末，并加入低温反应釜中，再启动搅拌机充分搅拌；然后将上述混合液体倒入带夹层的不锈钢容器中，采用自动灌装机，将上述混合液分装入事先准备好的硅胶模具中，在-80℃的温度下进行预冻，预冻时间为24小时。(2) take by weighing the calcium sulfate powder of 2% of chitosan solution weight, and add in the low-temperature reactor, then start the stirrer and fully stir; Then above-mentioned mixed liquid is poured in the stainless steel container with interlayer, adopts automatic filling machine, The above mixed solution was divided into pre-prepared silica gel molds and pre-frozen at a temperature of -80°C for 24 hours.

(3)将预冻得到的材料放入冻干机进行冻干，具体为：依次在-45℃下处理4h，在-20℃下处理48h，在-10℃下处理16h，在-5℃下处理6h，在5℃下处理4h，在25℃下处理5h，得到多孔混合物。(3) Put the pre-frozen material into a lyophilizer for lyophilization, specifically: treat at -45°C for 4h, at -20°C for 48h, at -10°C for 16h, and at -5°C Under the treatment of 6h, at 5°C for 4h, at 25°C for 5h, a porous mixture was obtained.

(4)将所述多孔混合物浸入浓度为0.5wt.％的京尼平水溶液中交联8小时，完成后用纯化水清洗48小时，期间每2小时换水一次，然后重复步骤(3)中的冻干步骤，再将其浸入含有BMP-2骨生长因子4的500μg/mL的PBS溶液中24小时，得到内层骨修复材料2。(4) immerse the porous mixture in a genipin aqueous solution with a concentration of 0.5wt.% for cross-linking for 8 hours, wash with purified water for 48 hours after completion, change the water once every 2 hours during this period, then repeat step (3) The freeze-drying step was followed by immersing it in a 500 μg/mL PBS solution containing BMP-2bone growth factor 4 for 24 hours to obtain the inner layerbone repair material 2.

(5)将所述内层骨修复材料放入模具正中间加以固定、备用。(5) Put the inner bone repair material into the middle of the mold to fix it and set it aside.

(6)配制浓度为2wt.％的甲基丙烯酸酯化丝素蛋白溶液，再加入VEGF内皮生长因子3，得到VEGF含量为400μg/mL的外层骨修复材料1混合溶液。(6) Prepare a methacrylated silk fibroin solution with a concentration of 2 wt.%, and then add VEGFendothelial growth factor 3 to obtain a mixed solution of outer layerbone repair material 1 with a VEGF content of 400 μg/mL.

(7)将所述外层骨修复材料1混合溶液倒入步骤(5)所述模具中，其在内层骨修复材料2周围充满，并快速用紫外灯照射45s，将其固化成型，即得到所述双因子梯度缓释的骨修复材料。(7) Pour the mixed solution of the outer layerbone repair material 1 into the mold described in step (5), which is filled around the inner layerbone repair material 2, and quickly irradiate it with an ultraviolet lamp for 45 seconds to solidify and form it, namely The bone repair material with gradient sustained release of the two factors is obtained.

试验例Test case

试验例选取的实验对象为实施例1得到的双因子梯度缓释的骨修复材料。The test object selected in the test example is the bone repair material obtained in Example 1 with gradient slow release of two factors.

(一)骨修复材料的孔径(1) Pore diameter of bone repair material

采用扫描电子显微镜(SEM)对骨修复材料的内层和外层分别进行观测，内层材料的电镜图如图2所示，外层材料的电镜图如图3所示，由图可看出，所述骨修复材料的孔径尺寸约为50～300μm。The inner layer and the outer layer of the bone repair material were observed respectively by scanning electron microscopy (SEM). The electron micrograph of the inner layer material is shown in Figure 2, and the electron micrograph of the outer layer material is shown in Figure 3. It can be seen from the figure , the pore size of the bone repair material is about 50-300 μm.

(二)骨修复材料的孔隙率(2) Porosity of bone repair materials

孔隙率的数据如表1所示。The porosity data are shown in Table 1.

表1孔隙率数据Table 1 Porosity data

组别groupM₁(g)M₁ (g)M₂(g)M₂ (g)V(cm³)V(cm³ )孔隙率(％)Porosity(%)110.04990.04990.2210.2210.250.2586.7486.74220.04920.04920.23350.23350.260.2693.4393.43330.05240.05240.23510.23510.270.2792.6292.62

孔隙率的计算方法：取三组平行的骨修复材料试样(形状为圆柱体)，其直径尺寸约为10mm，厚度约为3mm(具体尺寸用游标卡尺进行测量)，根据圆柱形体积计算出三组试样的原始体积V，且干态称重记作M₁，然后将其浸泡在无水乙醇中24小时。完成后取出，并将骨材料的表面多余的酒精用滤纸轻轻擦掉，然后称重并记作M₂。则孔隙率的计算公式为＝(M₂-M₁)÷0.789÷V×100％，即为孔隙率。其中，0.789为无水乙醇的密度(cm³/mL)。Calculation method of porosity: take three groups of parallel bone repair material samples (in the shape of a cylinder), the diameter of which is about 10mm, and the thickness is about 3mm (the specific size is measured with a vernier caliper), and three groups are calculated according to the volume of the cylinder. The original volume V of the sample group is weighed in dry state and recorded as M₁ , and then soaked in absolute ethanol for 24 hours. After completion, take it out, and gently wipe off excess alcohol on the surface of the bone material with filter paper, then weigh it and record it as M₂ . The formula for calculating the porosity is =(M₂ -M₁ )÷0.789÷V×100%, which is the porosity. Wherein, 0.789 is the density of absolute ethanol (cm³ /mL).

由电镜图片和孔隙率数据可知，骨修复材料的孔径尺寸约为50～300μm，孔隙率大于85％，这有利于氧气和营养物质的运输和传递，同时可显著促进细胞向大体积骨修复材料深部的迁移，加快骨修复。According to the electron microscope pictures and porosity data, the pore size of the bone repair material is about 50-300 μm, and the porosity is greater than 85%, which is conducive to the transportation and transmission of oxygen and nutrients, and can significantly promote the transfer of cells to the large-volume bone repair material. Deep migration, accelerated bone repair.

(三)双因子的释放时效(3) The release time of the two factors

外层骨材料中VEGF的累计释放曲线如图4所示，结果显示在24小时内释放速度比较快，后续缓慢释放，一直持续时间超过了10天，为前期的血管化形成起到积极的作用。The cumulative release curve of VEGF in the outer bone material is shown in Figure 4. The results show that the release rate is relatively fast within 24 hours, and the subsequent slow release lasts for more than 10 days, which plays a positive role in the formation of early vascularization .

内层骨材料中BMP-2的累计释放曲线如图5所示，结果显示在整个过程中，BMP-2能长期持续缓慢释放超过28天，为成骨的形成起到重要的作用。The cumulative release curve of BMP-2 in the inner bone material is shown in Figure 5. The results show that during the whole process, BMP-2 can be released slowly and continuously for more than 28 days, which plays an important role in the formation of osteogenesis.

(四)细胞增殖情况(4) Cell proliferation

细胞增殖情况如图6所示，由细胞在第1、第3、第5天的增殖情况可知，说明具有良好的生物相容性，以及满足细胞毒性0～2级。The cell proliferation is shown in Figure 6, and it can be known from the cell proliferation on the 1st, 3rd, and 5th day that it has good biocompatibility and meets the cytotoxicity level 0-2.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (10)

1. The preparation method of the bone repair material with the double-factor gradient slow release is characterized by comprising the following steps of:

(1) Mixing collagen with acetic acid solution and stirring to obtain collagen solution;

(2) Mixing the collagen solution with a calcium-containing compound, stirring, and then sequentially pre-freezing and freeze-drying to obtain a freeze-dried porous mixture;

(3) Mixing the freeze-dried porous mixture with a cross-linking agent, performing cross-linking, sequentially cleaning, freeze-drying, and immersing in a PBS (phosphate buffered saline) solution containing bone growth factor BMP-2 to obtain an inner layer bone repair material;

(4) Mixing the light-cured material solution with endothelial growth factor VEGF to obtain an outer layer bone repair material mixed solution;

(5) And (3) carrying out photo-curing on the outer layer of the outer layer bone repair material by the outer layer bone repair material mixed solution, and obtaining the double-factor gradient slow-release bone repair material after the photo-curing is completed.

2. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (1), the stirring temperature is 2-8 ℃, and the stirring time is 12-24 hours; the concentration of collagen in the collagen solution is 0.5 to 5wt.%.

3. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (2), the calcium-containing compound is one of hydroxyapatite, tricalcium phosphate or calcium sulfate.

4. The method for preparing a bone repair material with a dual-factor gradient slow release according to claim 1, wherein in the step (2), the pre-freezing temperature is-80 ℃ and the pre-freezing time is 4-24 hours.

5. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (2), the freeze-drying method is as follows: sequentially treating for 4 hours at-44 to-46 ℃, 48 hours at-19 to-21 ℃, 16 hours at-9 to-11 ℃, 6 hours at-4 to-6 ℃, 4 hours at 4 to 6 ℃ and 5 hours at 24 to 26 ℃.

6. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (3), the cross-linking agent is one or more of glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, genipin or cinnamaldehyde.

7. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (3), the concentration of BMP-2 is 20-500 μg/mL.

8. The method for preparing a dual-factor gradient slow-release bone repair material according to claim 1, wherein in the step (4), the concentration of VEGF in the outer layer bone repair material mixed solution is 20-400 μg/mL.

9. The method for preparing a bone repair material with a dual-factor gradient slow release according to claim 1, wherein in the step (5), the light curing is performed by irradiating with an ultraviolet lamp for 30-60 s.

10. The bone repair material of the dual-factor gradient slow release obtained by the preparation method of any one of claims 1 to 9.

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