本发明涉及一种纳米相钙磷盐/胶原/高分子骨复合多孔材料的制备方法，属生物工程技术领域。The invention relates to a preparation method of a nano-phase calcium phosphate salt/collagen/polymer bone composite porous material, which belongs to the technical field of bioengineering.
骨修复材料的研制开发一直是生物医学工程领域大批科学工作者努力的方向。现在手术中实际使用的骨修复材料效果最好的是自体骨和异体骨。但是自体骨数量有限，而异体骨又有免疫排斥和感染疾病的危险。人们已经开发了由金属、陶瓷、高分子以及其复合材料制成的多种骨修复材料。但是这些材料各有不足，但是这些材料各有不足，没有那一种能在临床中与自体骨或异体骨比美。考虑到自体骨和异体骨的生物学优势主要在于它们的成分和结构与天然骨完全相同，骨组织工程的原理认为一个具有和天然骨相似的组成和微观结构的框架材料将会具有优异的生物学性能。The research and development of bone repair materials has always been the direction of a large number of scientists in the field of biomedical engineering. The best bone repair materials actually used in surgery are autologous bone and allogeneic bone. However, the amount of autologous bone is limited, while allogeneic bone has the risk of immune rejection and infection. People have developed a variety of bone repair materials made of metals, ceramics, polymers and their composite materials. However, these materials have their own shortcomings, none of which can be compared with autologous bone or allogeneic bone in clinical practice. Considering that the biological advantages of autologous bone and allogeneic bone lie in that their composition and structure are exactly the same as those of natural bone, the principle of bone tissue engineering holds that a framework material with similar composition and microstructure to natural bone will have excellent biological properties. academic performance.
天然骨是具有精密分级结构的胶原和钙磷盐的复合物。骨中的钙磷盐主要是形状不规则的片状纳米晶，这些晶体的c轴大致平行。晶体宽度大约30-45nm，厚度4-6nm。临近的片晶构成钙磷盐晶体层，在空间上与胶原层交替排列，片层间距在纳米数量级，周期为10nm左右。排列高度有序的胶原作为一种细胞外基质对细胞活动也有重要影响。除了成分上的特点以外，天然骨还具有很高的孔隙率，孔洞之间是开放互联的。这对于营养物质的输运，骨细胞的迁移繁殖，旧骨的吸收和新骨的生成都至关重要。Natural bone is a complex of collagen and calcium phosphate with a finely graded structure. Calcium-phosphorus salts in bone are mainly irregularly shaped sheet-like nanocrystals with roughly parallel c-axes. The crystal width is about 30-45nm and the thickness is 4-6nm. Adjacent lamellar crystals constitute calcium-phosphorus salt crystal layers, which are alternately arranged with collagen layers in space, and the lamellar spacing is on the order of nanometers, and the period is about 10nm. Collagen, which is highly ordered, acts as an extracellular matrix and also has an important effect on cell activity. In addition to the characteristics of composition, natural bone also has a high porosity, and the pores are open and interconnected. It is crucial for the transportation of nutrients, the migration and reproduction of bone cells, the resorption of old bone and the formation of new bone.
poly(lactide-co-glycolide)(以下简称PLGA)是poly(lactic acid)(以下简称PLA)与poly(glycolic acid)(以下简称PGA)的共聚物，长期的医疗实践和实验室的研究都表明这种材料的生物相容性非常优异，而且由于PLA和PGA降解行为的不同，它们的共聚物PLGA的降解速度可以通过调整共聚物中两者的比例来控制。Poly(lactide-co-glycolide) (hereinafter referred to as PLGA) is a copolymer of poly(lactic acid) (hereinafter referred to as PLA) and poly(glycolic acid) (hereinafter referred to as PGA). Long-term medical practice and laboratory research have shown that The biocompatibility of this material is excellent, and due to the different degradation behaviors of PLA and PGA, the degradation rate of their copolymer PLGA can be controlled by adjusting the ratio of the two in the copolymer.
本发明的目的是提出一种纳米相钙磷盐/胶原/高分子骨复合多孔材料的制备方法，通过把生物相容性好、降解速度可控、易于成型的高分子材料与纳米晶磷酸钙胶原基复合材料整合，成为具有高度孔隙率、合适孔径尺寸的框架材料，得到具有优异生物相容性和生物活性的可降解骨替代材料，作为可降解骨替代材料在医疗方面得到广泛应用。还将框架与BMP等生长因子结合，最大限度的提高材料的生物活性。The purpose of the present invention is to propose a preparation method of nano-phase calcium phosphate salt/collagen/polymer bone composite porous material, by combining a polymer material with good biocompatibility, controllable degradation speed and easy molding with nanocrystalline calcium phosphate The integration of collagen-based composite materials becomes a framework material with high porosity and suitable pore size, and a degradable bone substitute material with excellent biocompatibility and bioactivity is obtained. As a degradable bone substitute material, it has been widely used in medical treatment. The framework is also combined with growth factors such as BMP to maximize the biological activity of the material.
本发明提出的用于骨修复的纳米相钙磷盐/胶原/高分子骨复合多孔材料的制备方法，包括下列各步骤：The preparation method of the nanophase calcium phosphate salt/collagen/polymer bone composite porous material for bone repair proposed by the present invention comprises the following steps:
(1)在酸溶的胶原溶液中缓慢滴加含有钙离子的溶液，滴加量为每克胶原滴加钙离子0.01～0.16mol，滴加的同时搅拌，其中的酸为盐酸、硝酸或乙酸中的任何一种，胶原溶液的浓度为5.0×10-5～5.0×10-3g/ml；(1) Slowly add a solution containing calcium ions to the acid-soluble collagen solution, the amount of which is 0.01 to 0.16 mol of calcium ions per gram of collagen, and stir while adding. The acid is hydrochloric acid, nitric acid or acetic acid Any one of them, the concentration of the collagen solution is 5.0×10-5 ~5.0×10-3 g/ml;
(2)在上述第一步的溶液中边搅拌边缓慢滴加含磷酸根离子的水溶液，加入的磷酸根离子的量与加入的钙离子的量的摩尔比为Ca∶P=1～2∶1；(2) Slowly add the aqueous solution containing phosphate ion while stirring in the solution of the above-mentioned first step, the mol ratio of the amount of phosphate ion added and the amount of calcium ion added is Ca: P=1～2: 1;
(3)在上述第一步的溶液中边搅拌边缓慢滴加NaOH溶液至pH值为6～8,PH值用试纸或pH计测定，在pH值为5～6时开始出现沉淀，pH值为7时出现白色悬浊液；(3) Slowly add NaOH solution dropwise to the solution in the first step above while stirring until the pH value is 6 to 8. The pH value is measured with test paper or a pH meter. When the pH value is 5 to 6, precipitation begins to appear. When it was 7, a white suspension appeared;
(4)将溶液静置1～5天，除去上清，离心分离出沉淀，用去离子水清洗后，放入冻干机内冰冻干燥，随后研磨制得干粉备用：(4) Let the solution stand for 1 to 5 days, remove the supernatant, centrifuge to separate the precipitate, wash it with deionized water, put it into a freeze dryer to freeze dry, and then grind it to obtain a dry powder for later use:
(5)将PLA∶PGA=50∶50～90∶10、分子量为50,000～150,000的PLGA置于烧瓶中，加入溶剂，在40～70℃的温度下配制质量体积浓度为0.02～0.15g/ml的溶液，其中的溶剂为1,4-二氧六环、氯仿或二甲基亚砜中的任何一种；(5) Put PLA:PGA=50:50～90:10 and PLGA with molecular weight of 50,000～150,000 in a flask, add solvent, and prepare a mass volume concentration of 0.02～0.15g/ml at a temperature of 40～70°C A solution in which the solvent is any one of 1,4-dioxane, chloroform or dimethyl sulfoxide;
(6)在第(5)步的溶液中加入第(4)步制得的干粉，并混合均匀，干粉与PLGA的质量比为1∶2～3∶2，制得PLGA/钙磷盐/胶原混合溶液；(6) Add the dry powder obtained in step (4) to the solution in step (5), and mix evenly. The mass ratio of dry powder to PLGA is 1: 2 to 3: 2 to obtain PLGA/calcium phosphorus salt/ Collagen mixed solution;
(7)采用溶盐法或热致分相并冻干溶剂的方法制备PLGA/钙磷盐/胶原复合多孔框架材料：(7) Preparation of PLGA/calcium phosphate salt/collagen composite porous framework material by the method of dissolving salt or heat-induced phase separation and freeze-drying solvent:
若使用溶盐法，首先将筛分过的100～400微米的NaCl晶体或蔗糖晶体加到PLGA/钙磷盐/胶原混合溶液中，加入的比例为NaCl/蔗糖∶PLGA/钙磷盐/胶原混合物=1～5∶1，在40～70℃下搅拌均匀，待溶剂快蒸干时将剩余物注模成型，随后用真空处理除去未挥发溶剂，用去离子水在25℃条件下浸泡48小时除去造孔剂，最后用真空除去水；If the salt-dissolving method is used, first add the sieved 100-400 micron NaCl crystals or sucrose crystals to the PLGA/calcium phosphate/collagen mixed solution, and the ratio of addition is NaCl/sucrose: PLGA/calcium phosphate/collagen Mixture = 1～5:1, stir evenly at 40～70°C, inject the residue when the solvent evaporates quickly, then use vacuum treatment to remove non-volatile solvent, soak in deionized water at 25°C for 48 hour to remove the pore forming agent, and finally remove the water by vacuum;
若采用热致分相并冻干溶剂的方法，则将上述第六步中配成的溶液倒入模具中冷冻，温度为-20℃～4℃，充分冷冻后，将模具转移到冰冻干燥机中进行冷冻干燥除去溶剂晶体，为了将彻底清除溶剂，冷冻干燥两天以后材料放入真空烘箱中三天；If the method of heat-induced phase separation and freeze-drying of the solvent is used, pour the solution prepared in the sixth step above into the mold and freeze at a temperature of -20°C to 4°C. After fully freezing, transfer the mold to a freeze dryer Freeze-drying was carried out to remove the solvent crystals. In order to completely remove the solvent, the material was placed in a vacuum oven for three days after two days of freeze-drying;
(8)将上述第七步的制品用环氧乙烷蒸气消毒2～4小时，或参照相关国标用Co60照射消毒后保存，即得骨修复的纳米相钙磷盐/胶原/高分子骨复合多孔材料。(8) Sterilize the product in the seventh step above with ethylene oxide steam for 2 to 4 hours, or store it after irradiating and sterilizing with Co60 according to the relevant national standard, and then get the nano-phase calcium phosphate salt/collagen/polymer bone composite for bone repair porous material.
本发明的方法中用生物自组装合成了纳米晶磷酸钙胶原基复合材料，该材料由纳米相的钙磷盐和胶原分子自组装而成。它在纳米尺度上具有重复片层结构，周期为10-15nm，由胶原层和钙磷盐层交替排列而成，由于其从成分和结构上仿天然骨，因此具有很好的生物相容性和很高的生物活性。In the method of the invention, the nanocrystalline calcium phosphate collagen-based composite material is synthesized by biological self-assembly, and the material is self-assembled by nano-phase calcium phosphate salt and collagen molecules. It has a repeating lamellar structure on the nanometer scale, with a period of 10-15nm, and is composed of collagen layers and calcium-phosphorus salt layers alternately. Because it imitates natural bone in terms of composition and structure, it has good biocompatibility and high biological activity.
本发明方法制备的用于骨修复的纳米相钙磷盐/胶原/PLGA骨复合多孔材料，具有优异的生物相容性，而且结构上也具有仿骨性。其钙磷盐晶体尺寸在纳米量级，与有机成分胶原的结合紧密，排列有一定规律。框架整体具有大量连通的60～400微米微孔，且孔隙率很高约为65％～90％，孔隙率与溶液浓度或筛分的造孔剂颗粒大小有关。此材料的强度和生物相容性也很好。很有希望作为骨材料得到应用。The nano-phase calcium phosphate salt/collagen/PLGA bone composite porous material for bone repair prepared by the method of the invention has excellent biocompatibility and bone-like property in structure. The crystal size of the calcium phosphate salt is at the nanometer level, closely combined with the organic component collagen, and arranged in a certain order. The overall framework has a large number of interconnected micropores of 60-400 microns, and the porosity is as high as 65%-90%. The porosity is related to the solution concentration or the particle size of the sieved pore-forming agent. The material is also very strong and biocompatible. It is very promising to be applied as a bone material.
下面介绍本发明的实施例：Introduce the embodiment of the present invention below:
本发明实施例中所用的胶原为益而康公司的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛皮胶原)，CELLON公司购买的液态Ⅰ型胶原(浓度：0.3％溶液PH值由HCl调整所含胶原为纯化的牛皮胶原)，天津第二附属医院的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛跟腱胶原)；所用的含PO43-的溶液为Na2HPO4,H3PO4,(NH4)2HPO4的水溶液；所用的含钙离子的溶液为CaCl26H2O,Ca(NO3)2,CaCl22H2O的水溶液。使用的PLGA为PLA∶PGA 50∶50 70∶30 85∶15几种不同比例的共聚物，分子量分别为80,000 100,000 150,000。实施例1：The collagen used in the examples of the present invention is the type I collagen gel (concentration: 1% solid content, the contained collagen is purified cowhide collagen) of Yierkang Company, and the liquid type I collagen (concentration: 0.3% The pH value of the solution was adjusted by HCl, and the collagen contained was purified bovine skin collagen), type I collagen gel from the Second Affiliated Hospital of Tianjin (concentration: solid content 1%, contained collagen was purified bovine Achilles tendon collagen); The solution of PO43- is the aqueous solution of Na2 HPO4 , H3 PO4 , (NH4 )2 HPO4 ; the solution containing calcium ions used is CaCl2 6H2 O, Ca(NO3 )2 , CaCl2 2H2 O in water. The PLGA used is PLA:PGA 50:50, 70:30, 85:15 copolymers with several different ratios, and the molecular weights are 80,000, 100,000, 150,000, respectively. Example 1:
所用材料为从益而康公司购买的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛皮胶原)、分析纯CaCl26H2O、分析纯Na2HPO4、分析纯1,4-二氧六环、PLGA(PLA∶PGA 70∶30)分子量∶100,000。The materials used are type I collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure CaCl2 6H2 O, analytically pure Na2 HPO4 , analytically pure 1,4-dioxane, PLGA (PLA:PGA 70:30) molecular weight: 100,000.
(1)将20g胶原凝胶溶于300ml 0.5M乙酸溶液中，缓慢滴加18.3ml 1mol/l CaCl2和11ml 1mol/l Na2HPO4，滴加的同时用磁力搅拌器搅拌；(1) Dissolve 20g collagen gel in 300ml 0.5M acetic acid solution, slowly add 18.3ml 1mol/l CaCl2 and 11ml 1mol/l Na2 HPO4 dropwise, and stir with a magnetic stirrer while adding dropwise;
(2)继续搅拌，同时缓慢滴加0.5mol/l的NaOH溶液至PH值为7；(2) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.5mol/l to pH value 7;
(3)静置溶液1天，除去上清，离心分离出沉淀用去离子水反复清洗三次后放入冻干机内进行冰冻干燥，研磨后制得干粉备用；(3) Let the solution stand for 1 day, remove the supernatant, centrifuge and separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder for subsequent use;
(4)60℃取1克PLGA溶于10ml 1,4-二氧六环中，初步溶解后加入第(3)步制备的干粉1克。(4) Dissolve 1 gram of PLGA in 10ml of 1,4-dioxane at 60°C, and add 1 gram of dry powder prepared in step (3) after initial dissolution.
(5)溶液在60℃下搅拌10小时后补足挥发的溶剂量，继续搅拌10分钟。(5) After the solution was stirred at 60°C for 10 hours, the amount of volatilized solvent was replenished, and the stirring was continued for 10 minutes.
(6)将(4)中的溶液倒入聚四氟乙烯模具中，于0℃冷冻2小时。(6) Pour the solution in (4) into a polytetrafluoroethylene mold and freeze at 0° C. for 2 hours.
(7)将冷冻成型的材料转移到冻干机中冷冻干燥48小时除去1,4-二氧六环。(7) Transfer the freeze-formed material to a freeze dryer for freeze-drying for 48 hours to remove 1,4-dioxane.
(8)将材料放入真空烘箱中三天，温度为37℃，真空度为-0.1MPa。(8) Put the material into a vacuum oven for three days, the temperature is 37° C., and the vacuum degree is -0.1 MPa.
(9)使用环氧乙烷蒸气消毒3小时后收存。(9) Sterilize with ethylene oxide steam for 3 hours and then store.
实施例2：Example 2:
所用材料为天津第二附属医院的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛跟腱胶原)、分析纯CaCl22H2O、分析纯(NH4)2HPO4、分析纯1,4-二氧六环、PLGA(PLA∶PGA 50∶50)分子量：80,000。The materials used are type Ⅰ collagen gel from Tianjin Second Affiliated Hospital (concentration: solid content 1%, the collagen contained is purified bovine Achilles tendon collagen), analytically pure CaCl2 2H2 O, analytically pure (NH4 )2 HPO4. Molecular weight of analytically pure 1,4-dioxane, PLGA (PLA:PGA 50:50): 80,000.
(1)20g胶原凝胶溶于300ml 0.1M HNO3溶液中，缓慢滴加10ml 1mol/l CaCl2和10ml 1mol/l(NH4)2HPO4，滴加的同时用磁力搅拌器搅拌；(1) Dissolve 20g collagen gel in 300ml 0.1M HNO3 solution, slowly add 10ml 1mol/l CaCl2 and 10ml 1mol/l(NH4 )2 HPO4 dropwise, and stir with a magnetic stirrer while adding dropwise;
(2)继续搅拌，同时缓慢滴加0.5mol/l的NaOH溶液至PH值为7；(2) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.5mol/l to pH value 7;
(3)静置溶液4天，除去上清，离心分离出沉淀用去离子水反复清洗三次后放入冻干机内进行冰冻干燥，研磨后制得干粉备用；(3) Let the solution stand for 4 days, remove the supernatant, centrifuge and separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder for subsequent use;
(4)50℃下取0.6克PLGA溶于10ml 1,4-二氧六环中，初步溶解后加入第(3)步制备的干粉0.3克。(4) Dissolve 0.6 g of PLGA in 10 ml of 1,4-dioxane at 50°C, and add 0.3 g of the dry powder prepared in step (3) after initial dissolution.
(5)溶液在50℃下搅拌10小时后补足挥发的溶剂量，继续搅拌10分钟。(5) After the solution was stirred at 50°C for 10 hours, the amount of volatilized solvent was replenished, and the stirring was continued for 10 minutes.
(6)将(4)中的溶液倒入聚四氟乙烯模具中，于-10℃冷冻2小时。(6) Pour the solution in (4) into a polytetrafluoroethylene mold and freeze at -10°C for 2 hours.
(7)将冷冻成型的材料转移到冻干机中冷冻干燥48小时除去1,4-二氧六环。(7) Transfer the freeze-formed material to a freeze dryer for freeze-drying for 48 hours to remove 1,4-dioxane.
(8)将材料放入真空烘箱中三天，温度为37℃，真空度为-0.1MPa。(8) Put the material into a vacuum oven for three days, the temperature is 37° C., and the vacuum degree is -0.1 MPa.
(9)使用环氧乙烷蒸气消毒2小时后收存。(9) Sterilize with ethylene oxide steam for 2 hours and then store.
实施例3：Example 3:
所用材料为CELLON公司购买的液态Ⅰ型胶原(浓度：0.3％溶液PH值由HCl调整所含胶原为纯化的牛皮胶原)、分析纯二甲基亚砜、分析纯的CaCl2·6H2O、分析纯H3PO4(含量＞=85％密度1.689g/ml)。PLGA(PLA∶PGA70∶30)分子量：100,000The materials used are liquid type Ⅰ collagen purchased by Cellon Company (concentration: the pH value of the 0.3% solution is adjusted by HCl and the contained collagen is purified cowhide collagen), analytically pure dimethyl sulfoxide, analytically pure CaCl2 ·6H2 O, Analytical pure H3 PO4 (content>=85% density 1.689 g/ml). PLGA (PLA:PGA70:30) molecular weight: 100,000
(1)100ml胶原溶液中滴加溶于10ml去离子水中的1.125ml H3PO4，滴加的同时用磁力搅拌器搅拌；(1) Add dropwise 1.125ml H3 PO4 dissolved in 10ml deionized water to 100ml collagen solution, and stir with a magnetic stirrer while adding dropwise;
(2)称取结晶良好的CaCl26H2O 6.01g溶于20ml去离子水使其完全溶解将其滴加入上一步制得的溶液中后，继续搅拌1小时；(2) Weigh 6.01 g of well-crystallized CaCl2 6H2 O and dissolve it in 20 ml of deionized water to dissolve it completely, add it dropwise to the solution prepared in the previous step, and continue stirring for 1 hour;
(3)继续搅拌，同时缓慢滴加0.75mol/l的NaOH溶液至PH值为7；(3) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.75mol/l to pH value 7;
(4)静置溶液1天，除去上清，离心分离出沉淀用去离子水反复清洗三次后放入冻干机内进行冰冻干燥，研磨后制得干粉备用；(4) Let the solution stand for 1 day, remove the supernatant, centrifuge and separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder for subsequent use;
(5)取0.6克PLGA溶于12ml二甲基亚砜中，初步溶解后加入第(3)步制备的干粉0.4克。(5) Dissolve 0.6 g of PLGA in 12 ml of dimethyl sulfoxide, and add 0.4 g of dry powder prepared in step (3) after preliminary dissolution.
(6)溶液在40℃下搅拌10小时后补足挥发的溶剂量，继续搅拌10分钟。(6) After the solution was stirred at 40°C for 10 hours, the amount of volatilized solvent was replenished, and the stirring was continued for 10 minutes.
(7)将(4)中的溶液倒入聚四氟乙烯模具中，于0℃冷冻2小时。(7) Pour the solution in (4) into a polytetrafluoroethylene mold and freeze at 0° C. for 2 hours.
(8)将冷冻成型的材料转移到冻干机中冷冻干燥48小时除去二甲基亚砜。(8) Transfer the freeze-formed material to a freeze dryer for freeze-drying for 48 hours to remove dimethyl sulfoxide.
(9)将材料放入真空烘箱中三天，温度为37℃，真空度为-0.1MPa。(9) Put the material into a vacuum oven for three days, the temperature is 37° C., and the vacuum degree is -0.1 MPa.
(10)使用环氧乙烷蒸气消毒4小时后收存。(10) Sterilize with ethylene oxide steam for 4 hours and then store.
实施例4：Example 4:
所用材料为益而康公司购买的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛皮胶原)、分析纯氯仿、分析纯Ca(NO3)2、分析纯Na2HPO4、PLGA(PLA∶PGA 85∶15)分子量：150,000。The materials used are type Ⅰ collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure chloroform, analytically pure Ca(NO3 )2 , analytically pure Na2 HPO purchased from Yierkang Company4. PLGA (PLA:PGA 85:15) molecular weight: 150,000.
(1)20g胶原凝胶溶于300ml 0.5M乙酸溶液中，缓慢滴加16.5ml 1mol/lCa(NO3)2和11ml 1mol/l H3PO4，滴加的同时用磁力搅拌器搅拌；(1) Dissolve 20g collagen gel in 300ml 0.5M acetic acid solution, slowly add 16.5ml 1mol/l Ca(NO3 )2 and 11ml 1mol/l H3 PO4 dropwise, and stir with a magnetic stirrer while adding dropwise;
(2)继续搅拌，同时缓慢滴加0.5mol/l的NaOH溶液至PH值为7；(2) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.5mol/l to pH value 7;
(3)静置溶液1天，除去上清，离心分离出沉淀用去离子水反复清洗三次后放入冻干机内进行冰冻干燥，研磨后制得干粉备用；(3) Let the solution stand for 1 day, remove the supernatant, centrifuge and separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for lyophilization, and grind it to obtain a dry powder for subsequent use;
(4)40℃下取0.6克PLGA溶于15ml氯仿中，充分溶解后加入第(3)步制备的干粉0.4克以及3g尺寸在100～300微米之间的NaCl晶体。(4) Dissolve 0.6 g of PLGA in 15 ml of chloroform at 40° C., and add 0.4 g of the dry powder prepared in step (3) and 3 g of NaCl crystals with a size between 100 and 300 microns after fully dissolving.
(5)溶液在此温度下搅拌至溶剂快挥发完毕时将剩余物注模成型，随后用真空烘箱在40℃烘烤1天，除去未挥发溶剂。(5) The solution was stirred at this temperature until the solvent evaporated quickly, and the residue was injection molded, and then baked in a vacuum oven at 40° C. for 1 day to remove the non-volatile solvent.
(6)烘烤后用去离子水在25℃条件下浸泡此材料48小时除去造孔剂，随后再用真空烘箱在37℃，真空度-0.1MPa,烘干3天除去水分。(6) After baking, soak the material with deionized water at 25°C for 48 hours to remove the pore-forming agent, and then dry it in a vacuum oven at 37°C with a vacuum of -0.1MPa for 3 days to remove moisture.
(7)使用环氧乙烷蒸气消毒4小时。(7) Sterilize with ethylene oxide steam for 4 hours.
实施例5：Example 5:
所用材料为益而康公司购买的Ⅰ型胶原凝胶(浓度：固含量1％，所含胶原为纯化的牛皮胶原)、分析纯CaCl26H2O、分析纯Na2HPO4、分析纯二甲基亚砜，PLGA(PLA∶PGA 85∶15)分子量：150,000。The materials used are type Ⅰ collagen gel (concentration: 1% solid content, the collagen contained is purified cowhide collagen), analytically pure CaCl2 6H2 O, analytically pure Na2 HPO4 , and analytically pure diuretic gel purchased by Yierkang Company. Methylsulfoxide, PLGA (PLA:PGA 85:15) molecular weight: 150,000.
(1)30g胶原凝胶溶于300ml 0.5M乙酸溶液中，缓慢滴加18.3ml 1mol/l CaCl2和11ml 1mol/l Na2HPO4，滴加的同时用磁力搅拌器搅拌；(1) Dissolve 30g collagen gel in 300ml 0.5M acetic acid solution, slowly add 18.3ml 1mol/l CaCl2 and 11ml 1mol/l Na2 HPO4 dropwise, and stir with a magnetic stirrer while adding dropwise;
(2)继续搅拌，同时缓慢滴加0.5mol/l的NaOH溶液至PH值为7；(2) continue to stir, slowly dropwise simultaneously the NaOH solution of 0.5mol/l to pH value 7;
(3)静置溶液5天，除去上清，离心分离出沉淀用去离子水反复清洗三次后放入冻干机内进行冰冻干燥，研磨后制得干粉备用；(3) Let the solution stand for 5 days, remove the supernatant, centrifuge and separate the precipitate, wash it repeatedly with deionized water three times, put it into a lyophilizer for freeze-drying, and grind it to obtain a dry powder for subsequent use;
(4)60℃下取0.6克PLGA溶于15ml二甲基亚砜中，初步溶解后加入第(3)步制备的干粉0.4克以及0.6g尺寸在100～300微米之间的蔗糖晶体。(4) Dissolve 0.6 g of PLGA in 15 ml of dimethyl sulfoxide at 60°C, and add 0.4 g of the dry powder prepared in step (3) and 0.6 g of sucrose crystals with a size between 100 and 300 microns after preliminary dissolution.
(5)溶液在60℃下搅拌10小时后补足挥发的溶剂量，继续搅拌10分钟。(5) After the solution was stirred at 60°C for 10 hours, the amount of volatilized solvent was replenished, and the stirring was continued for 10 minutes.
(6)将(4)中的溶液倒入聚四氟乙烯模具中，于4℃冷冻2小时。(6) Pour the solution in (4) into a polytetrafluoroethylene mold and freeze at 4°C for 2 hours.
(7)将冷冻成型的材料转移到冻干机中冷冻干燥48小时除去二甲基亚砜。(7) Transfer the freeze-formed material to a freeze dryer for freeze-drying for 48 hours to remove dimethyl sulfoxide.
(8)随后用去离子水在25℃条件下浸泡此材料48小时除去蔗糖，随后再用真空烘箱在37℃，真空度-0.1MPa，烘烤3天除去水分。(8) Soak the material with deionized water at 25°C for 48 hours to remove sucrose, and then bake it in a vacuum oven at 37°C with a vacuum of -0.1MPa for 3 days to remove moisture.
(9)使用环氧乙烷蒸气消毒2小时后收存。(9) Sterilize with ethylene oxide steam for 2 hours and then store.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011296992ACN1164338C (en) | 2001-06-29 | 2001-06-29 | Preparation method of nanophase calcium phosphate salt/collagen/polymer bone composite porous material |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011296992ACN1164338C (en) | 2001-06-29 | 2001-06-29 | Preparation method of nanophase calcium phosphate salt/collagen/polymer bone composite porous material |
| Publication Number | Publication Date |
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| CN1325734Atrue CN1325734A (en) | 2001-12-12 |
| CN1164338C CN1164338C (en) | 2004-09-01 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011296992AExpired - LifetimeCN1164338C (en) | 2001-06-29 | 2001-06-29 | Preparation method of nanophase calcium phosphate salt/collagen/polymer bone composite porous material |
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| CN100338118C (en)* | 2005-12-16 | 2007-09-19 | 西南交通大学 | Preparation method of biodegradable calcium phosphorus inorganic biological particle/polymer composite material |
| CN100348479C (en)* | 2005-11-07 | 2007-11-14 | 浙江大学 | Method for preparing prefab of porous carbon nanometer tube |
| CN101954122A (en)* | 2010-09-13 | 2011-01-26 | 陕西瑞盛生物科技有限公司 | Preparation method of natural bone repairing material with pre-plasticity |
| CN102154786A (en)* | 2011-04-25 | 2011-08-17 | 东南大学 | Mineralized corn protein fibrous membrane and preparation method thereof |
| CN102205150A (en)* | 2011-05-19 | 2011-10-05 | 清华大学 | Preparation method for anti-infectious nano collagen/ calcium phosphate bone repair material |
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| CN103142337A (en)* | 2002-06-03 | 2013-06-12 | 韦克福里斯特大学健康科学院 | Directed tissue growth employing reduced pressure |
| CN106421883A (en)* | 2016-11-10 | 2017-02-22 | 武汉医佳宝生物材料有限公司 | Preparation method of medical collagen sponge |
| CN110292661A (en)* | 2016-10-31 | 2019-10-01 | 北京奥精医药科技有限公司 | Compound porous bone material of mineralized collagen/macromolecule and preparation method thereof |
| CN112652734A (en)* | 2019-10-11 | 2021-04-13 | 广州汽车集团股份有限公司 | Thick negative pole piece and preparation method thereof, lithium ion battery cell, lithium ion battery pack and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103142337A (en)* | 2002-06-03 | 2013-06-12 | 韦克福里斯特大学健康科学院 | Directed tissue growth employing reduced pressure |
| CN100348479C (en)* | 2005-11-07 | 2007-11-14 | 浙江大学 | Method for preparing prefab of porous carbon nanometer tube |
| CN100338118C (en)* | 2005-12-16 | 2007-09-19 | 西南交通大学 | Preparation method of biodegradable calcium phosphorus inorganic biological particle/polymer composite material |
| CN101954122A (en)* | 2010-09-13 | 2011-01-26 | 陕西瑞盛生物科技有限公司 | Preparation method of natural bone repairing material with pre-plasticity |
| CN102154786A (en)* | 2011-04-25 | 2011-08-17 | 东南大学 | Mineralized corn protein fibrous membrane and preparation method thereof |
| CN102205150A (en)* | 2011-05-19 | 2011-10-05 | 清华大学 | Preparation method for anti-infectious nano collagen/ calcium phosphate bone repair material |
| CN102764455A (en)* | 2012-07-20 | 2012-11-07 | 清华大学 | Anti-infection mineralized collagen and calcium sulfate bone repair material and preparation method thereof |
| CN102764455B (en)* | 2012-07-20 | 2014-11-26 | 清华大学 | Anti-infection mineralized collagen and calcium sulfate bone repair material and preparation method thereof |
| CN110292661A (en)* | 2016-10-31 | 2019-10-01 | 北京奥精医药科技有限公司 | Compound porous bone material of mineralized collagen/macromolecule and preparation method thereof |
| CN106421883A (en)* | 2016-11-10 | 2017-02-22 | 武汉医佳宝生物材料有限公司 | Preparation method of medical collagen sponge |
| CN112652734A (en)* | 2019-10-11 | 2021-04-13 | 广州汽车集团股份有限公司 | Thick negative pole piece and preparation method thereof, lithium ion battery cell, lithium ion battery pack and application thereof |
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| EE01 | Entry into force of recordation of patent licensing contract | Assignee:Aojing Medicine, Sci. & Tech. Co., Ltd., Beijing Assignor:Tsinghua University Contract record no.:2010110000205 Denomination of invention:Process for preparing nm-phase calcium-phosphorus salt/collagen/high-molecular bone compounded porous material Granted publication date:20040901 License type:Exclusive License Open date:20011212 Record date:20101119 | |
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