CN102935246B - Three-dimensional cell culture scaffold, its preparation method and application - Google Patents

Three-dimensional cell culture scaffold, its preparation method and application Download PDF

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CN102935246B
CN102935246B CN 201110233215 CN201110233215A CN102935246B CN 102935246 B CN102935246 B CN 102935246B CN 201110233215 CN201110233215 CN 201110233215 CN 201110233215 A CN201110233215 A CN 201110233215A CN 102935246 B CN102935246 B CN 102935246B
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CN102935246A (en )
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宫建茹
崔金磊
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国家纳米科学中心
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Abstract

本发明提供一种用于细胞培养的三维培养支架,所述三维培养支架包含多孔磷酸钙支架和包被于所述多孔磷酸钙支架上的海藻酸钠。 The present invention provides a cell culture three-dimensional culture scaffold, the scaffold comprises a porous three-dimensional culture scaffold and calcium alginate coated in porous calcium phosphate on the scaffold. 该支架具有制备简单,便于操作和运输等优点。 The holder has a simple preparation, ease of operation and transportation advantages. 本发明还提供该三维培养支架的制备方法和应用。 The present invention also provides the preparation method and application of three-dimensional culture scaffold.

Description

一种三维细胞培养支架及其制备方法和应用 A three-dimensional cell culture scaffold and preparation method and application

技术领域 FIELD

[0001] 本发明属于生物组织工程领域,特别涉及一种应用于三维细胞培养支架的制备方法和应用。 [0001] The present invention belongs to the field of biological tissue engineering, applied particularly to a three-dimensional cell culture preparation method and application of the stent. 本发明以纳米磷酸三钙为初始原料,通过聚甲基丙烯酸甲酯模板合成多孔磷酸钙支架。 In the present invention, nano-tricalcium phosphate as starting material, synthetic porous calcium phosphate scaffold template by polymethyl methacrylate. 将磷酸钙支架浸入酸性海藻酸钠溶液中形成磷酸三钙/海藻酸钠薄膜的复合物,最终制备了三维细胞培养支架。 The calcium alginate stent is immersed in an acidic solution to form tricalcium phosphate / sodium alginate film composite, the final three-dimensional cell culture scaffolds.

背景技术 Background technique

[0002] 二维(2D)细胞培养是一种制备、观察细胞、并于体外研究细胞与药物、生物因子和生物材料相互作用的技术。 [0002] a two-dimensional (2D) preparing a cell culture, cells were observed and cells in vitro studies with the drug, biological factors and the interaction of biological materials in the art. 但是,它与细胞在体内的生长方式并不相仿。 However, it is not similar to the cells in vivo growth pattern. 在动物体内,细胞是三维(3D)生长并构建形成活组织或器官。 In animals, the cell is a three-dimensional (3D) form a construct were grown and living tissues or organs. 因此,细胞三维培养与二维培养相比,更加相似于动物体内的细胞微环境。 Thus, three-dimensional cell culture as compared with two-dimensional culture is more similar to animal cells microenvironment. 有报道指出,通过体外的三维细胞培养,我们可以了解组织在正常和病理的组织条件下的结构-功能。 It reported that, by the three-dimensional cell culture in vitro, we can understand the organization under normal and pathological conditions of the tissue structure - function. 因此,三维细胞培养支架具有很高的应用价值。 Thus, three-dimensional cell culture scaffold has a high application value.

[0003] 传统的三维细胞培养支架制备中采用的是有机聚合支架,如明胶-戊二醛,但其受制备工艺限制往往采用特定有机成分,成形机器复杂或强度低,不便作为产品。 [0003] conventional three-dimensional cell culture scaffold employed in the preparation of organic polymeric scaffold, such as gelatin - glutaraldehyde, but its preparation process often used by limiting a specific organic component, forming a complex machine or a low intensity, the inconvenience as a product.

发明内容 SUMMARY

[0004] 因此,本发明的目的在于克服上述制备工艺的不足,提供一种新型的三维细胞培养支架。 [0004] Accordingly, an object of the present invention to overcome the above-described manufacturing process is insufficient to provide a novel 3D cell culture scaffold.

[0005] 本发明的另一个目的是提供所述三维细胞培养支架的制备方法,该制备过程简便易行。 [0005] Another object of the present invention to provide a method of preparing the three-dimensional cell culture scaffold, the manufacturing process is simple. 并提供上述三维细胞培养支架在制备细胞培养装置中的应用。 And providing application of the three-dimensional cell scaffold in a cell culture preparation in the culture device.

[0006] 用于实现上述目的的技术方案如下: [0006] for achieving the above object, the technical solution as follows:

[0007] —方面,本发明提供一种用于细胞培养的三维细胞培养支架,其包含多孔磷酸钙支架和包被于多孔磷酸钙支架上的海藻酸钠层,所述多孔磷酸钙支架设有均匀分布的微孔。 [0007] - aspect, the present invention provides a cell culture scaffold for culturing three-dimensional cell, comprising a porous scaffold and calcium alginate layer coated on a porous calcium phosphate scaffold, the scaffold has a porous calcium phosphate uniform pore distribution.

[0008] 另一方面,本发明提供上述三维细胞培养支架的制备方法,该制备方法包括以下步骤: [0008] In another aspect, the present invention provides a method of preparing the three-dimensional cell culture scaffold, the preparation process comprises the steps of:

[0009] 步骤1:固相磷酸钙含量为50〜55% (重量体积比)的料浆的配制; [0009] Step 1: Solid calcium content of 50~55% (weight by volume) slurry formulation;

[0010] 步骤2:聚甲基丙烯酸甲酯微球的制备; [0010] Step 2: polymethylmethacrylate microspheres prepared;

[0011] 步骤3:多孔磷酸钙支架的制备; 3 [0011] Step: Preparation of porous calcium phosphate scaffold;

[0012] 步骤4:将步骤3制备的多孔磷酸钙支架放入质量百分含量为4〜7%的海藻酸钠盐酸溶液中,真空浸泡l_2h,用超纯水反复洗涤三次,即得。 [0012] Step 4: The porous calcium phosphate scaffold prepared in step 3 into the mass percentage of sodium alginate 4~7% hydrochloric acid solution, soaked in vacuo l_2h, repeatedly washed with deionized water three times, that is, too.

[0013] 优选地,在步骤I中,所述磷酸钙为纳米磷酸钙。 [0013] Preferably, in step I, the nano-calcium phosphate.

[0014] 优选地,在步骤I中,所述料浆的配制包括以下步骤: [0014] Preferably, in step I, the slurry formulation comprising the steps of:

[0015] 步骤1.1:纳米磷酸三钙粉末的配制; [0015] Step 1.1: preparing nano-tricalcium phosphate powder;

[0016] 步骤1.2:将纳米磷酸三钙粉末、分散剂及超纯水球磨0.5_2h,即得。 [0016] 1.2 Step: tricalcium phosphate nano powder, dispersant and milling ultrapure 0.5_2h, ie.

[0017] 优选地,所述纳米磷酸三钙粉末的配制包括以下步骤: [0017] Preferably, the nano-tricalcium phosphate powder formulation comprising the steps of:

[0018] 步骤1.1.1:将硝酸钙、磷酸氢二胺和尿素溶于超纯水中,优选地,所述超纯水溶液中各原料的浓度为:硝酸钙0.5-0.6摩尔/L、磷酸氢二胺0.4-0.5摩尔/L和尿素0.56-0.6 摩尔/L ; [0018] Step 1.1.1: calcium nitrate, diammonium hydrogen phosphate and urea were dissolved in ultrapure water, preferably, the concentration of the ultra-pure aqueous solution of each material: calcium nitrate 0.5-0.6 mol / L, phosphoric acid hydrogen diamine 0.4-0.5 mol / L urea and 0.56-0.6 mol / L;

[0019] 步骤1.1.2:将步骤1.1.1制得的溶液放置于烘箱中,于70_80°C干燥3_7h ; [0019] Step 1.1.2: 1.1.1 Step prepared solution was placed in an oven, dried at 70_80 ° C 3_7h;

[0020] 步骤1.1.3:将干燥后的物质放于马弗炉中,于700-800°C温度,煅烧l_2h,得到白色的纳米磷酸三钙粉末。 [0020] Step 1.1.3: The dried material was placed in a muffle furnace at a temperature of 700-800 ° C, calcined l_2h, to give a white powder of nano-tricalcium phosphate.

[0021] 优选地,在步骤2中,所述聚甲基丙烯酸甲酯微球的制备包括以下步骤: [0021] Preferably, in step 2, preparing the polymethylmethacrylate microspheres comprising the steps of:

[0022] 步骤2.1:用质量分数为5-6%氢氧化钠溶液缓慢清洗甲基丙烯酸甲酯两次,再用无水氯化钙干燥洗涤后的甲基丙烯酸甲酯12-24h,优选地,用25mL质量分数为5_6%氢氧化钠溶液缓慢清洗甲基丙烯酸甲酯两次; [0022] Step 2.1: mass fraction of 5-6% sodium hydroxide solution was slowly washed twice with methyl methacrylate, methyl methacrylate, dried over anhydrous calcium chloride drying after washing 12-24h, preferably with 5_6 25mL mass fraction% sodium hydroxide solution was slowly washed twice with methyl methacrylate;

[0023] 步骤2.2:将碱式碳酸镁溶于质量百分含量为0.3%的聚乙烯醇溶液中,再加入甲基丙烯酸甲酯溶液、引发剂、二甲基丙烯酸乙二醇酯,在N2保护下,将反应体系至于66-70°C下搅拌5-6h,搅拌速度为240-440r/min,优选地,所述碱式碳酸镁溶液的浓度为0.0142-0.015摩尔/L ;优选地,所述甲基丙烯酸甲酯溶液与聚乙烯醇溶液的体积比为I: 12.5-1: 13,更为优选地,所述甲基丙烯酸甲酯溶液的密度为0.9410g/mL;优选地,所述引发剂为过氧化苯甲酰,所述过氧化苯甲酰的质量浓度为0.3% ;优选地,所述二甲基丙烯酸乙二醇酯与聚乙烯醇溶液的体积比为1: 625-1: 667,更为优选地,所述二甲基丙烯酸乙二醇酯的密度为1.0151g/mL。 [0023] Step 2.2: basic magnesium carbonate is dissolved in the mass percentage of 0.3% polyvinyl alcohol solution was added a solution of methyl methacrylate, an initiator, ethylene glycol dimethacrylate, in N2 under protection, as for the reaction system was stirred for 5-6h at 66-70 ° C, stirring speed 240-440r / min, preferably, the concentration of the basic magnesium carbonate solution is 0.0142-0.015 mol / L; preferably, the solution was mixed with methyl methacrylate polyvinyl alcohol solution volume ratio of I: 12.5-1: 13, more preferably, the density of the solution of methyl methacrylate was 0.9410g / mL; preferably, the said initiator is benzoyl peroxide, said benzoyl peroxide concentration of 0.3%; volume preferably, the ethylene glycol dimethacrylate and polyvinyl alcohol solution as 1: 625- 1: 667, more preferably, the density of the ethylene glycol dimethacrylate was 1.0151g / mL.

[0024] 步骤2.3:反应结束后,用滤纸过滤粗产物除去残留溶液,并用超纯超纯水洗涤5次; [0024] Step 2.3: After completion of the reaction, the crude product was filtered through filter paper to remove residual solution, and washed 5 times with ultrapure water, ultrapure;

[0025] 步骤2.4:在60_65°C烘箱中,真空干燥得到直径为200〜300 μ m的聚甲基丙烯酸甲酯微球。 [0025] Step 2.4: 60_65 ° C in an oven, and dried in vacuo to give a diameter of 200~300 μ m polymethyl methacrylate microspheres.

[0026] 优选地,在步骤3中,所述多孔磷酸钙支架的制备包括以下步骤: [0026] Preferably, in step 3, the prepared porous calcium phosphate scaffold comprising the steps of:

[0027] 步骤3.1:将亚硫酸铝、尿素、聚乙烯醇吡咯烷酮溶液超纯水中,配制得到纳米氧化铝悬浊液,优选地,所述各原料溶于超纯水中的浓度分别为:亚硫酸铝0.05-0.06摩尔/L、尿素0.56-0.6摩尔/L、聚乙烯醇吡咯烷酮6.5xl0_6摩尔/L ; [0027] Step 3.1: The aluminum sulfite, urea, polyvinyl pyrrolidone solution in ultrapure water to prepare a suspension of nano-alumina, preferably, the respective raw materials was dissolved in the ultrapure water concentrations were: aluminum sulfite 0.05-0.06 mol / L, urea 0.56-0.6 mol / L, polyvinyl pyrrolidone 6.5xl0_6 mol / L;

[0028] 步骤3.2:将聚甲基星稀酸甲酯微球放置于烧杯中,于干燥箱中,于180-200°C下,干燥l-2h ; [0028] Step 3.2: The star dilute methyl polymethacrylate microspheres placed in a beaker, in the oven at 180-200 ° C, dried l-2h;

[0029] 步骤3.3:室温下,将纳米氧化铝悬浊液缓慢铺满聚甲基丙烯酸甲酯微球,于真空干燥箱中干燥,得到聚甲基丙烯酸甲酯微球支架。 [0029] Step 3.3: A solution of nano-alumina suspension slowly confluence polymethyl methacrylate microspheres, and dried in a vacuum oven to give polymethylmethacrylate microspheres stent.

[0030] 步骤3.4:将步骤3.3制得的聚甲基丙烯酸甲酯/支架进行真空处理,注入步骤I中所制备的磷酸三钙料浆,混合均匀得到料浆/聚甲基丙烯酸甲酯/支架复合物。 [0030] Step 3.4: Poly (methyl methacrylate) prepared in step 3.3 / stent vacuum treatment, injection phosphate prepared in step I tricalcium slurry uniformly mixed to obtain a slurry / PMMA / scaffold complex.

[0031] 步骤3.5:将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中烧结形成多孔磷酸钙支架。 [0031] Step 3.5: The slurry / PMMA / scaffold was placed in a muffle furnace to form a porous sintered calcium phosphate scaffold.

[0032] 优选地,所述烧结步骤为:依次置于室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟。 [0032] Preferably, the sintering steps of: sequentially kept at room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400-1300 ° C uniformly heated and sintered 400 minutes Finally, sintering 1250 ° C for 120 minutes.

[0033] 又一方面,本发明提供了上述三维细胞培养支架在制备细胞培养装置中的应用。 [0033] In yet another aspect, the present invention provides the above use three-dimensional cell scaffold in a cell culture preparation in the culture device.

[0034] 本发明提供了一种新型的细胞三维培养支架,其包含多孔磷酸钙支架和包被于所述多孔磷酸钙支架上的海藻酸钠,与现有技术相比,本发明的三维培养支架强度高,便于操作和运输;并且制备工艺简单,成本低,无需特殊设备。 [0034] The present invention provides a novel three-dimensional cell culture scaffold, which scaffold comprises a porous calcium phosphate and sodium alginate coated on said porous calcium phosphate scaffold, as compared with the prior art, three-dimensional culture of the invention high strength bracket, easy to operate and transport; and the preparation process is simple, low cost, without special equipment. 本发明制备的海藻酸钠支架是一种用于细胞培养的生长模版,它能够引导组织的再生,同时控制组织的形状。 Alginate scaffolds of the present invention is a template for cell culture growth, it can be guided tissue regeneration, while controlling the shape of the tissue. 本发明的支架是由聚甲基丙烯酸甲酯作为主体的,具有制作简单、抗压能力强的优点;并且聚甲基丙烯酸甲酯具有无毒,无免疫原性的优点,在血液储存等生物医药领域有广泛的应用。 Stent of the present invention is composed of polymethyl methacrylate as a main component, having a simple production, the advantages of compression capability; and polymethyl methacrylate having a non-toxic, non-immunogenic advantages of immunization, blood storage in biological the field of medicine have a wide range of applications. 具有合适通道尺寸的三维细胞培养架更有利于血管和组织的形成。 Suitable three-dimensional cell culture carrier having channel dimensions and is more conducive to the formation of vascular tissue.

附图说明 BRIEF DESCRIPTION

[0035] 以下,结合附图来详细说明本发明的实施方案,其中: [0035] Hereinafter, detailed description in conjunction with the accompanying drawings embodiments of the present invention, wherein:

[0036] 图1为向聚甲基丙烯酸甲酯微球模板中注入料浆烧结前的扫描电镜照片; [0036] Figure 1 is the polymethylmethacrylate microspheres injected into the template scanning electron micrograph of the slurry prior to sintering;

[0037] 图2为本发明的用于细胞培养的三维细胞培养支架的显微镜照片。 [0037] FIG 2 is a micrograph of a 3D cell culture cell culture scaffold of the invention.

具体实施方式 detailed description

[0038] 下面结合具体的实施例,并参照数据进一步详细描述本发明。 [0038] Next, with reference to specific embodiments, the reference data and the present invention is described in further detail. 应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制发明的范围。 It should be understood that these examples are only to illustrate the invention, not to limit its scope in any way.

[0039] 在以下的实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。 [0039] In the following examples, the various processes and methods not described in detail are conventional methods well known in the art.

[0040] 实施例1 [0040] Example 1

[0041] I)固相磷酸三钙为50%的料浆配制步骤如下: [0041] I) a solid phase tricalcium phosphate of 50% slurry formulation step as follows:

[0042] (a)固相磷酸三钙粉末制备:将硝酸钙9.8412g、磷酸氢二胺5.2836g、尿素3.3665g溶于10mL超纯水中。 [0042] (a) Preparation of solid tricalcium phosphate powder: calcium nitrate 9.8412g, diammonium hydrogen phosphate 5.2836g, 3.3665g of urea dissolved in 10mL of ultrapure water.

[0043] (b)将上述水溶液放置于70°C烘箱中干燥3h。 [0043] (b) the aqueous solution was placed in a 70 ° C oven for 3h.

[0044] (c)干燥后,将固体放于马弗炉中。 [0044] (c) After drying, the solid was placed in a muffle furnace. 在700°C温度下,煅烧lh,得到白色的纳米磷酸二隹丐粉末。 At a temperature of 700 ° C, calcined LH, to give a white powder of nano-phosphodiester short-tailed hack.

[0045] (d)将纳米磷酸三钙粉末3.0106g和分散剂聚丙烯酸铵1.2mL溶于3mL超纯水中,用行星式球磨机球磨2h,制备得到固相三钙的料浆。 [0045] (d) the nano-tricalcium phosphate powder and a dispersant 3.0106g ammonium polyacrylate were dissolved in 3mL 1.2mL ultrapure water, using a planetary ball milling 2h, tricalcium prepared solid phase slurry.

[0046] 2)聚甲基丙烯酸甲酯微球制备步骤如下: [0046] 2) PMMA microspheres prepared the following steps:

[0047] (a)配制质量分数为5%的氢氧化钠水溶液。 [0047] (a) formulated mass fraction of 5% aqueous sodium hydroxide.

[0048] (b)首先,取上述氢氧化钠溶液25mL缓慢清洗甲基丙烯酸甲酯两次。 [0048] (b) First, take the above sodium hydroxide solution was slowly 25mL washed twice with methyl methacrylate. 然后,用上述氢氧化钠溶液25mL清洗甲基丙烯酸甲酯两次。 Then, with the above sodium hydroxide solution was washed twice with 25 mL of methyl methacrylate. 最后,用无水氯化钙干燥洗涤后的甲基丙烯酸甲酯12h。 Finally, 12h methyl methacrylate after washing and drying over anhydrous calcium chloride.

[0049] (c)配制质量分数为0.3%的聚乙烯醇水溶液。 [0049] (c) formulating a mass fraction of 0.3% aqueous polyvinyl alcohol solution.

[0050] (d)在直三角烧瓶中,将碱式碳酸镁0.5182g溶解于10mL质量分数为0.3%的聚乙烯醇溶液。 [0050] (d) a straight Erlenmeyer flask, the basic magnesium carbonate was dissolved in 10mL 0.5182g mass fraction of 0.3% polyvinyl alcohol solution. 然后,加入8mL甲基丙烯酸甲酯溶液(密度P =0.9410g/mL)、引发剂过氧化苯甲酰0.0282g、0.16mL 二甲基丙烯酸乙二醇酯(密度P = 1.051g/mL)。 Then, a solution of methyl methacrylate was added 8mL (density P = 0.9410g / mL), benzoyl peroxide initiator 0.0282g, 0.16mL ethylene glycol dimethacrylate (density P = 1.051g / mL). 在N2保护下,将反应体系至于66°C下搅拌5h,搅拌速度为240r/min。 Under N2 protection, As for the reaction was stirred at 66 ° C 5h, stirring speed of 240r / min.

[0051] (d)反应结束后,用滤纸过滤粗产物除去残留溶液,并用超纯水洗涤5次。 After [0051] (d) the reaction, the crude product was filtered with filter paper to remove residual solution, and washed 5 times with ultrapure water.

[0052] (e)在60°C烘箱中,真空干燥得到直径为200〜300 μ m的聚甲基丙烯酸甲酯微球。 [0052] (e) at 60 ° C in an oven, and dried in vacuo to give a diameter of 200~300 μ m polymethyl methacrylate microspheres.

[0053] 3)聚甲基丙烯酸甲酯/支架的制备步骤如下: [0053] 3) the step of preparing a polymethyl methacrylate / stent following:

[0054] (a)将亚硫酸铝3.6752g、尿素8.4084g、聚乙烯醇吡咯烷酮0.0462g溶解于250mL超纯水中,配制得到纳米氧化铝悬浊液。 [0054] (a) The aluminum sulfite 3.6752g, urea 8.4084g, 0.0462g polyvinyl pyrrolidone was dissolved in 250mL ultrapure water to prepare a suspension of nano-alumina.

[0055] (b)将聚甲基星稀酸甲酯微球放置于烧杯中,于干燥箱中。 [0055] (b) The methyl polymethacrylate star dilute microspheres placed in a beaker, in the drybox. 在180°C下,干燥lh。 At 180 ° C, and dried lh.

[0056] (c)室温下,将纳米氧化铝悬浊液慢慢的铺满聚甲基丙烯酸甲酯微球,于真空干燥箱中干燥,得到聚甲基丙烯酸甲酯微球支架。 [0056] (c) A room temperature, the nano-alumina suspension slowly covered polymethyl methacrylate microspheres, and dried in a vacuum oven to give polymethylmethacrylate microspheres stent.

[0057] 4)将3)步骤中制得的聚甲基丙烯酸甲酯/支架进行真空处理,注入第I)步中所制备的磷酸三钙料浆,混合均匀得到料浆/聚甲基丙烯酸甲酯/支架复合物。 [0057] 4) 3) prepared in step a polymethyl methacrylate / stent vacuum treatment, the first injection I) prepared in Step phosphate tribasic calcium slurry uniformly mixed to obtain a slurry / polymethacrylate methacrylate / scaffold complex.

[0058] 5)将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中,室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟,得到孔磷酸钙支架。 [0058] 5) The slurry / PMMA / scaffold was placed in a muffle furnace from room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400-1300 ° C and warmed uniformly sintered 400 minutes, and finally sintering 1250 ° C for 120 minutes to give a calcium phosphate scaffold aperture.

[0059] 6)用于细胞培养的三维培养支架即海藻酸钠支架的制备步骤如下: [0059] 6) a step of preparing alginate stent i.e., three-dimensional culture scaffold for cell culture is as follows:

[0060] (a)配制质量浓度为4%的海藻酸钠凝胶; [0060] (a) formulated in a concentration of 4 mass% sodium alginate gels;

[0061] (b)将第5)步中制备的多孔磷酸钙支架置于4%海藻酸钠盐酸溶液中真空浸泡lh,用超纯水冲洗3次,制备得到海藻酸钠支架。 [0061] (b) a porous calcium phosphate scaffolds step 5) was placed in a hydrochloric acid solution 4% sodium alginate LH soaked in vacuo, washed three times with ultrapure water, prepared alginate stent.

[0062] 实施例2 [0062] Example 2

[0063] I)固相磷酸三钙为55%的料浆配制步骤如下: [0063] I) a solid phase of 55% tricalcium phosphate slurry formulation steps:

[0064] (a)固相磷酸三钙粉末制备:将硝酸钙12.0222g、磷酸氢二胺6.8940g、尿素3.7730g溶于10mL超纯水中。 [0064] (a) Preparation of solid tricalcium phosphate powder: calcium nitrate 12.0222g, diammonium hydrogen phosphate 6.8940g, 3.7730g of urea dissolved in 10mL of ultrapure water.

[0065] (b)将上述水溶液放置于80°C烘箱中干燥7h。 [0065] (b) the aqueous solution was placed in a 80 ° C oven for 7h.

[0066] (c)干燥后,将固体放于马弗炉中。 [0066] (c) After drying, the solid was placed in a muffle furnace. 在800°C温度下,煅烧2h,得到白色的纳米磷酸二隹丐粉末。 At a temperature of 800 ° C, calcined 2h, to give a white powder of nano-phosphodiester short-tailed hack.

[0067] (d)将纳米磷酸三钙粉末3.0259g和分散剂聚丙烯酸铵1.2mL溶于3mL超纯水中,用行星式球磨机球磨0.5h,制备得到固相三•丐的料楽:。 [0067] (d) the nano-tricalcium phosphate powder and a dispersant 3.0259g ammonium polyacrylate were dissolved in 3mL 1.2mL ultrapure water, using a planetary ball milling 0.5h, three prepared solid phase material yue • hack:

[0068] 2)聚甲基丙烯酸甲酯微球制备步骤如下: [0068] 2) PMMA microspheres prepared the following steps:

[0069] (a)配制质量分数为6%的氢氧化钠水溶液。 [0069] (a) formulated mass fraction of 6% aqueous sodium hydroxide.

[0070] (b)首先,取上述氢氧化钠溶液40mL缓慢清洗甲基丙烯酸甲酯两次。 [0070] (b) First, take the above sodium hydroxide solution was slowly 40mL washed twice with methyl methacrylate. 最后,用无水氯化钙干燥洗涤后的甲基丙烯酸甲酯24h。 Finally, anhydrous calcium chloride with methyl methacrylate after washing and drying 24h.

[0071] (c)配制质量分数为0.4%的聚乙烯醇水溶液。 [0071] (c) formulated with 0.4% by mass aqueous polyvinyl alcohol solution.

[0072] (d)在直三角烧瓶中,将碱式碳酸镁0.5482g溶解于10mL质量分数为0.4%的聚乙烯醇溶液。 [0072] (d) a straight Erlenmeyer flask, the basic magnesium carbonate was dissolved in 10mL 0.5482g mass fraction of 0.4% polyvinyl alcohol solution. 然后,加入7.7mL甲基丙烯酸甲酯溶液(密度P =0.9410g/mL)、引发剂过氧化苯甲酰0.0282g、0.15mL 二甲基丙烯酸乙二醇酯(密度P = 1.051g/mL)。 Then, a solution of methyl methacrylate was added 7.7mL (density P = 0.9410g / mL), benzoyl peroxide initiator 0.0282g, 0.15mL ethylene glycol dimethacrylate (density P = 1.051g / mL) . 在N2保护下,将反应体系至于70°C下搅拌6h,搅拌速度为440r/min。 Under N2 protection, As for the reaction was stirred 70 ° C for 6h, stirring speed of 440r / min.

[0073] (d)反应结束后,用滤纸过滤粗产物除去残留溶液,并用超纯水洗涤5次。 After [0073] (d) the reaction, the crude product was filtered with filter paper to remove residual solution, and washed 5 times with ultrapure water.

[0074] (e)在65°C烘箱中,真空干燥得到直径为200〜300 μ m的聚甲基丙烯酸甲酯微球。 [0074] (e) at 65 ° C in an oven, and dried in vacuo to give a diameter of 200~300 μ m polymethyl methacrylate microspheres.

[0075] 3)聚甲基丙烯酸甲酯/支架的制备步骤如下: [0075] 3) the step of preparing a polymethyl methacrylate / stent following:

[0076] (a)将亚硫酸铝4.4109g、尿素9.0OlOg、聚乙烯醇吡咯烷酮0.0475g溶解于250mL超纯水中,配制得到纳米氧化铝悬浊液。 [0076] (a) The aluminum sulfite 4.4109g, urea 9.0OlOg, 0.0475g polyvinyl pyrrolidone was dissolved in 250mL ultrapure water to prepare a suspension of nano-alumina.

[0077] (b)将聚甲基星稀酸甲酯微球放置于烧杯中,于干燥箱中。 [0077] (b) The methyl polymethacrylate star dilute microspheres placed in a beaker, in the drybox. 在200°C下,干燥2h。 At 200 ° C, drying 2h.

[0078] (c)室温下,将纳米氧化铝悬浊液慢慢的铺满聚甲基丙烯酸甲酯微球,于真空干燥箱中干燥,得到聚甲基丙烯酸甲酯微球支架。 [0078] (c) A room temperature, the nano-alumina suspension slowly covered polymethyl methacrylate microspheres, and dried in a vacuum oven to give polymethylmethacrylate microspheres stent.

[0079] 4)将第3)步制得的聚甲基丙烯酸甲酯/支架进行真空处理,注入第I)步中所制备的磷酸三钙料浆,混合均匀得到料浆/聚甲基丙烯酸甲酯/支架复合物。 [0079] 4) The step 3) made of a polymethyl methacrylate / stent vacuum treatment, the first injection I) prepared in Step phosphate tribasic calcium slurry uniformly mixed to obtain a slurry / polymethacrylate methacrylate / scaffold complex.

[0080] 5)将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中,室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟,得到孔磷酸钙支架。 [0080] 5) The slurry / PMMA / scaffold was placed in a muffle furnace from room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400-1300 ° C and warmed uniformly sintered 400 minutes, and finally sintering 1250 ° C for 120 minutes to give a calcium phosphate scaffold aperture.

[0081] 6)用于细胞培养的三维培养支架即海藻酸钠支架的制备步骤如下: [0081] 6) a step of preparing alginate stent i.e., three-dimensional culture scaffold for cell culture is as follows:

[0082] (a)配制质量浓度为7 %的海藻酸钠凝胶; [0082] (a) formulated in a concentration of 7 mass% sodium alginate gels;

[0083] (b)将第5)步中制备的多孔磷酸钙支架置于7%海藻酸钠盐酸溶液中浸泡lh,用超纯水冲洗3次,制备得到海藻酸钠支架。 [0083] (b) a porous calcium phosphate scaffolds step 5) was placed in a hydrochloric acid solution of 7% sodium alginate LH immersed, rinsed with ultrapure water three times, prepared alginate stent.

[0084] 实施例3 [0084] Example 3

[0085] I)固相磷酸三钙为52%的料浆配制步骤如下: [0085] I) a solid phase tricalcium phosphate of 52% slurry formulation step as follows:

[0086] (a)固相磷酸三钙粉末制备:将硝酸钙10.6191g、磷酸氢二胺6.0986g、尿素3.6099g溶于10mL超纯水中。 [0086] (a) Preparation of solid tricalcium phosphate powder: calcium nitrate 10.6191g, diammonium hydrogen phosphate 6.0986g, 3.6099g of urea dissolved in 10mL of ultrapure water.

[0087] (b)将上述水溶液放置于75°C烘箱中干燥5h。 [0087] (b) the aqueous solution was placed in a 75 ° C oven for 5h.

[0088] (c)干燥后,将固体放于马弗炉中。 [0088] (c) After drying, the solid was placed in a muffle furnace. 在780°C温度下,煅烧1.5h,得到白色的纳米磷酸三钙粉末。 At a temperature of 780 ° C, calcined 1.5h, to give a white powder of nano-tricalcium phosphate.

[0089] (d)将纳米磷酸三钙粉末3.0119g和分散剂聚丙烯酸铵1.2mL溶于3mL超纯水中,用行星式球磨机球磨0.5h,制备得到固相三•丐的料楽:。 [0089] (d) the nano-tricalcium phosphate powder and a dispersant 3.0119g ammonium polyacrylate were dissolved in 3mL 1.2mL ultrapure water, using a planetary ball milling 0.5h, three prepared solid phase material yue • hack:

[0090] 2)聚甲基丙烯酸甲酯微球制备步骤如下: [0090] 2) PMMA microspheres prepared the following steps:

[0091] (a)配制质量分数为5.5%的氢氧化钠水溶液。 [0091] (a) formulating a mass fraction of 5.5% aqueous sodium hydroxide.

[0092] (b)首先,取上述氢氧化钠溶液40mL缓慢清洗甲基丙烯酸甲酯两次。 [0092] (b) First, take the above sodium hydroxide solution was slowly 40mL washed twice with methyl methacrylate. 最后,用无水氯化钙干燥洗涤后的甲基丙烯酸甲酯20h。 Finally, anhydrous calcium chloride with methyl methacrylate after washing and drying 20h.

[0093] (c)配制质量分数为0.36%的聚乙烯醇水溶液。 [0093] (c) formulating the mass fraction of 0.36% polyvinyl alcohol aqueous solution.

[0094] (d)在直三角烧瓶中,将碱式碳酸镁0.5295g溶解于10mL质量分数为0.3%的聚乙烯醇溶液。 [0094] (d) a straight Erlenmeyer flask, the basic magnesium carbonate was dissolved in 10mL 0.5295g mass fraction of 0.3% polyvinyl alcohol solution. 然后,加入8mL甲基丙烯酸甲酯溶液(密度P =0.9410g/mL)、引发剂过氧化苯甲酰0.0282g、0.16mL 二甲基丙烯酸乙二醇酯(密度P = 1.051g/mL)。 Then, a solution of methyl methacrylate was added 8mL (density P = 0.9410g / mL), benzoyl peroxide initiator 0.0282g, 0.16mL ethylene glycol dimethacrylate (density P = 1.051g / mL). 在N2保护下,将反应体系至于68°C下搅拌5.5h,搅拌速度为300r/min。 Under N2 protection, As for the reaction system was stirred for 5.5 h at 68 ° C, stirring speed of 300r / min.

[0095] (d)反应结束后,用滤纸过滤粗产物除去残留溶液,并用超纯水洗涤5次。 After [0095] (d) the reaction, the crude product was filtered with filter paper to remove residual solution, and washed 5 times with ultrapure water.

[0096] (e)在65°C烘箱中,真空干燥得到直径为200〜300 μ m的聚甲基丙烯酸甲酯微球。 [0096] (e) at 65 ° C in an oven, and dried in vacuo to give a diameter of 200~300 μ m polymethyl methacrylate microspheres.

[0097] 3)聚甲基丙烯酸甲酯/支架的制备步骤如下: [0097] 3) the step of preparing a polymethyl methacrylate / stent following:

[0098] (a)将亚硫酸铝4.1452g、尿素8.8762g、聚乙烯醇吡咯烷酮0.0456g溶解于250mL超纯水中,配制得到纳米氧化铝悬浊液。 [0098] (a) The aluminum sulfite 4.1452g, urea 8.8762g, 0.0456g polyvinyl pyrrolidone was dissolved in 250mL ultrapure water to prepare a suspension of nano-alumina.

[0099] (b)将聚甲基星稀酸甲酯微球放置于烧杯中,于干燥箱中。 [0099] (b) The methyl polymethacrylate star dilute microspheres placed in a beaker, in the drybox. 在190°C下,干燥1.5h。 At 190 ° C, and dried 1.5h.

[0100] (c)室温下,将纳米氧化铝悬浊液慢慢的铺满聚甲基丙烯酸甲酯微球,于真空干燥箱中干燥,得到聚甲基丙烯酸甲酯微球支架。 [0100] (c) A room temperature, the nano-alumina suspension slowly covered polymethyl methacrylate microspheres, and dried in a vacuum oven to give polymethylmethacrylate microspheres stent.

[0101] 4)将第3)步制得的聚甲基丙烯酸甲酯/支架进行真空处理,注入第I)步中所制备的磷酸三钙料浆,混合均匀得到料浆/聚甲基丙烯酸甲酯/支架复合物。 [0101] 4) The step 3) made of a polymethyl methacrylate / stent vacuum treatment, the first injection I) prepared in Step phosphate tribasic calcium slurry uniformly mixed to obtain a slurry / polymethacrylate methacrylate / scaffold complex.

[0102] 5)将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中,室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟,得到孔磷酸钙支架。 [0102] 5) The slurry / PMMA / scaffold was placed in a muffle furnace from room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400-1300 ° C and warmed uniformly sintered 400 minutes, and finally sintering 1250 ° C for 120 minutes to give a calcium phosphate scaffold aperture.

[0103] 6)用于细胞培养的三维培养支架即海藻酸钠支架的制备步骤如下: [0103] 6) a step of preparing alginate stent i.e., three-dimensional culture scaffold for cell culture is as follows:

[0104] (a)配制质量浓度为5%的海藻酸钠凝胶; [0104] (a) formulated in a concentration of 5 mass% sodium alginate gels;

[0105] (b)将第5)步中制备的多孔磷酸钙支架置于5%海藻酸钠盐酸溶液中浸泡lh,用超纯水冲洗3次,制备得到海藻酸钠支架。 [0105] (b) a porous calcium phosphate scaffolds step 5) was placed in a hydrochloric acid solution of 5% sodium alginate LH immersed, rinsed with ultrapure water three times, prepared alginate stent.

[0106] 实施例4 [0106] Example 4

[0107] I)固相磷酸三钙为54%的料浆配制步骤同实施例1 [0107] I) a solid phase of 54% tricalcium phosphate slurry preparation step in Example 1

[0108] 2)聚甲基丙烯酸甲酯微球制备步骤同实施例1: [0108] 2) PMMA microspheres The same procedure as in Example 1:

[0109] 3)聚甲基丙烯酸甲酯/支架的制备步骤同实施例1: [0109] 3) polymethyl methacrylate / scaffold preparation step in Example 1:

[0110] 4)料浆/聚甲基丙烯酸甲酯/支架复合物制备步骤同实施例1。 [0110] 4) the slurry / PMMA / scaffold was prepared in the same procedure as in Example 1.

[0111] 5)将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中,室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟,得到孔磷酸钙支架。 [0111] 5) The slurry / PMMA / scaffold was placed in a muffle furnace from room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400-1300 ° C and warmed uniformly sintered 400 minutes, and finally sintering 1250 ° C for 120 minutes to give a calcium phosphate scaffold aperture.

[0112] 6)用于细胞培养的三维培养支架即海藻酸钠支架的制备步骤同实施例1。 [0112] 6) for cell culture i.e. three-dimensional culture scaffold bracket alginate preparation step in Example 1.

[0113] 实施例5 [0113] Example 5

[0114] I)固相磷酸三钙为55%的料浆配制步骤同实施例1 [0114] I) a solid phase of 55% tricalcium phosphate slurry preparation step in Example 1

[0115] 2)聚甲基丙烯酸甲酯微球制备步骤同实施例1: [0115] 2) PMMA microspheres The same procedure as in Example 1:

[0116] 3)聚甲基丙烯酸甲酯/支架的制备步骤同实施例1: [0116] 3) polymethyl methacrylate / scaffold preparation step in Example 1:

[0117] 4)料浆/聚甲基丙烯酸甲酯/支架复合物制备步骤同实施例1。 [0117] 4) the slurry / PMMA / scaffold was prepared in the same procedure as in Example 1.

[0118] 5)将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中,在1250°C下烧结120分钟,得到多孔磷酸钙支架。 [0118] 5) The slurry / PMMA / scaffold was placed in a muffle furnace and sintered at 1250 ° C 120 minutes to obtain a porous calcium phosphate scaffold.

[0119] 6)用于细胞培养的三维培养支架即海藻酸钠支架的制备步骤同实施例1。 [0119] 6) for cell culture i.e. three-dimensional culture scaffold bracket alginate preparation step in Example 1.

[0120] 用于细胞培养三维细胞培养支架的应用:本发明制备的用于细胞培养的三维培养即海藻酸钠支架是一种用于细胞培养的生长模版,它能够引导组织的再生,同时控制组织的形状。 [0120] Applications for the three-dimensional culture scaffold Cell culture: preparation of the present invention for cell culture i.e., three-dimensional culture scaffold is a growth Alginate stencil for cell culture, it is capable of directing tissue regeneration, while the control the shape of the organization. 本发明的支架是由聚甲基丙烯酸甲酯作为主体的,具有制作简单、抗压能力强的优点;并且聚甲基丙烯酸甲酯具有无毒,无免疫原性的优点,在血液储存等生物医药领域有广泛的应用。 Stent of the present invention is composed of polymethyl methacrylate as a main component, having a simple production, the advantages of compression capability; and polymethyl methacrylate having a non-toxic, non-immunogenic advantages of immunization, blood storage in biological the field of medicine have a wide range of applications. 具有合适通道尺寸的三维细胞培养架更有利于血管和组织的形成。 Suitable three-dimensional cell culture carrier having channel dimensions and is more conducive to the formation of vascular tissue.

[0121] 实验表明:本发明自制的聚甲基丙烯酸甲酯具有很好的三维取向。 [0121] Experimental results show that: the present invention is made of polymethyl methacrylate having a good three-dimensional orientation. 该聚合物在烧结前的结构通过扫描电子显微镜(附图1)表征,该三维支架的结构通过显微镜(附图2)观察。 The polymer structure before sintering by a scanning electron microscope (FIG. 1) to characterize the structure of the three-dimensional scaffold is observed by a microscope (Fig. 2). 从图中可以看出,三维细胞支架显示出多孔性和三维取向。 As can be seen from the figure, three-dimensional cell scaffold exhibits porosity and three-dimensional orientation.

Claims (15)

  1. 1.一种三维培养支架的制备方法,其特征在于,所述制备方法包括: 步骤1:固相磷酸I丐含量为50〜55% (重量体积比)的料楽:的配制; 步骤2:聚甲基丙烯酸甲酯微球的制备; 步骤3:多孔磷酸钙支架的制备; 步骤4:将步骤3制备的多孔磷酸钙支架放入质量百分含量为4〜7%的海藻酸钠盐酸溶液中,真空浸泡l_2h,用超纯水反复洗涤三次,即得, 其中,在步骤3中,所述多孔磷酸钙支架的制备包括以下步骤: 步骤3.1:将亚硫酸铝、尿素、聚乙烯醇吡咯烷酮超纯水中,配制得到纳米氧化铝悬浊液; 步骤3.2:将聚甲基丙稀酸甲酯微球放置于烧杯中,于干燥箱中,于180-200°C下,干燥l-2h ; 步骤3.3:室温下,将纳米氧化铝悬浊液缓慢铺满聚甲基丙烯酸甲酯微球,于真空干燥箱中干燥,得到聚甲基丙烯酸甲酯微球支架; 步骤3.4:将步骤3.3制得的聚甲基丙烯酸甲酯微球支架进行真 1. A process for producing three-dimensional culture scaffold, wherein the manufacturing method comprises: Step 1: Solid Phase I Hack phosphate content 50~55% (weight by volume) of material yue: formulation; Step 2: preparation of poly (methyl methacrylate) microspheres; step 3: preparation of porous calcium phosphate scaffold; step 4: the step of preparing porous calcium phosphate scaffold placed in the mass percentage of 3% sodium alginate 4~7 hydrochloric acid solution in vacuo soaking l_2h, repeatedly washed with deionized water three times, to obtain which, in step 3, the prepared porous calcium phosphate scaffold comprising the following steps: step 3.1: the aluminum sulfite, urea, polyvinyl pyrrolidone ultrapure water to prepare a suspension of nano-alumina; step 3.2: the polymethylmethacrylate microspheres placed in a beaker, in the oven at 180-200 ° C, dried l-2h ; step 3.3: a solution of nano-alumina suspension slowly confluence polymethyl methacrylate microspheres, and dried in a vacuum oven to give a polymethyl methacrylate microsphere holder; step 3.4: step 3.3 the resulting polymethyl methacrylate microspheres stent true 处理,注入步骤I中所制备的磷酸三钙料浆,混合均匀得到料浆/聚甲基丙烯酸甲酯/支架复合物; 步骤3.5:将料浆/聚甲基丙烯酸甲酯/支架复合物放于马弗炉中烧结形成多孔磷酸钙支架。 Process, the implantation step I is prepared tricalcium phosphate slurry uniformly mixed to obtain a slurry / PMMA / stent complex; Step 3.5: The slurry / PMMA / discharge composite stent in a muffle furnace to form a porous sintered calcium phosphate scaffold.
  2. 2.根据权利要求1所述的制备方法,其特征在于,在步骤I中,所述磷酸钙为纳米磷酸钙。 2. The production method according to claim 1, wherein, in step I, the nano-calcium phosphate.
  3. 3.根据权利要求2所述的制备方法,其特征在于,在步骤I中,所述料浆的配制包括以下步骤: 步骤1.1:纳米磷酸三钙粉末的配制; 步骤1.2:将纳米磷酸三钙粉末、分散剂及超纯水球磨0.5-2h,即得。 3. The production method according to claim 2, characterized in that, in step I, the slurry formulation comprising the following steps: Step 1.1: tricalcium phosphate nano powder formulation; Step 1.2: tricalcium phosphate nano powder, dispersant and milling ultrapure 0.5-2h, i.e., too.
  4. 4.根据权利要求3所述的制备方法,其特征在于,所述纳米磷酸三钙粉末的配制包括以下步骤: 步骤1.1.1:将硝酸钙、磷酸氢二胺和尿素溶于超纯水中; 步骤1.1.2:将步骤1.1.1制得的溶液放置于烘箱中,于70-80°C干燥3_7h ; 步骤1.1.3:将干燥后的物质放于马弗炉中,于700-800°C温度,煅烧l_2h,得到白色的纳米磷酸三钙粉末。 4. The method of preparation according to claim 3, wherein the nano-formulation tricalcium phosphate powder comprises the following steps: Step 1.1.1: calcium nitrate, diammonium hydrogen phosphate and urea were dissolved in ultrapure water ; step 1.1.2: 1.1.1 step prepared solution was placed in an oven at 70-80 ° C drying 3_7h; step 1.1.3: the dried material was placed in a muffle furnace at 700-800 ° C temperature, calcination l_2h, to give a white powder of nano-tricalcium phosphate.
  5. 5.根据权利要求4所述的制备方法,其特征在于,在步骤1.1.1中,所述超纯水溶液中各原料的浓度为:硝酸钙0.5-0.6摩尔/L、磷酸氢二胺0.4-0.5摩尔/L和尿素0.56-0.6摩尔/L。 The production method as claimed in claim 4, wherein, in step 1.1.1, the concentration of the ultra-pure aqueous solution of each material: calcium nitrate 0.5-0.6 mol / L, hydrogen phosphate diamine 0.4 0.5 mol / L urea and 0.56-0.6 mol / L.
  6. 6.根据权利要求1至5任一项所述的制备方法,其特征在于,在步骤2中,所述聚甲基丙烯酸甲酯微球的制备包括以下步骤: 步骤2.1:用质量分数为5-6%氢氧化钠溶液缓慢清洗甲基丙烯酸甲酯两次,再用无水氯化钙干燥洗涤后的甲基丙烯酸甲酯12_24h, 步骤2.2:将碱式碳酸镁溶于质量百分含量为0.3%的聚乙烯醇溶液中,再加入甲基丙烯酸甲酯溶液、引发剂、二甲基丙烯酸乙二醇酯,在N2保护下,将反应体系置于66-70°C下搅拌6h,搅拌速度为240-440r/min,步骤2.3:反应结束后,用滤纸过滤粗产物除去残留溶液,并用超纯水洗涤5次; 步骤2.4:在60-65°C烘箱中,真空干燥得到直径为200〜300 μ m的聚甲基丙烯酸甲酯微球。 1 to 6. The production method according to any one of claim 5, wherein, in Step 2, the preparation of polymethylmethacrylate microspheres comprising the following steps: Step 2.1: 5 with a mass fraction of -6% sodium hydroxide solution was slowly washed twice with methyl methacrylate, methyl methacrylate, dried over anhydrous 12_24h calcium chloride drying after washing, step 2.2: basic magnesium carbonate is dissolved in the mass percentage of 0.3% polyvinyl alcohol solution, and then added a solution of methyl methacrylate, an initiator, ethylene glycol dimethacrylate, under N2 protection, was placed the reaction was stirred at 66-70 ° C 6h, stirring speed 240-440r / min, step 2.3: after completion of the reaction, the crude product was filtered with filter paper to remove residual solution, and washed 5 times with ultrapure water; step 2.4: 60-65 ° C in an oven, and dried in vacuo to give a diameter of 200 ~300 μ m of polymethylmethacrylate microspheres.
  7. 7.根据权利要求6所述的制备方法,其特征在于,在步骤2.1中,用25mL质量分数为5-6%氢氧化钠溶液缓慢清洗甲基丙烯酸甲酯两次。 7. The method of preparation according to claim 6, wherein, in step 2.1, the mass fraction of 25mL with 5-6% sodium hydroxide solution was slowly washed twice with methyl methacrylate.
  8. 8.根据权利要求6所述的制备方法,其特征在于,在步骤2.2中,所述碱式碳酸镁溶液的浓度为0.0142-0.015摩尔/L。 8. The method of preparation according to claim 6, wherein, in step 2.2, the concentration of basic magnesium carbonate solution is 0.0142-0.015 mol / L.
  9. 9.根据权利要求6所述的制备方法,其特征在于,在步骤2.2中,所述甲基丙烯酸甲酯溶液与聚乙烯醇溶液的体积比为1:12.5-1:13。 9. A method of preparation according to claim 6, wherein, in step 2.2, the methyl methacrylate solution of polyvinyl alcohol solution with a volume ratio of 1: 12.5-1: 13.
  10. 10.根据权利要求6所述的制备方法,其特征在于,在步骤2.2中,所述甲基丙烯酸甲酯溶液的密度为0.9410g/mL。 10. The production method according to claim 6, wherein, in step 2.2, the density of the solution of methyl methacrylate was 0.9410g / mL.
  11. 11.根据权利要求6所述的制备方法,其特征在于,在步骤2.2中,所述引发剂为过氧化苯甲酰,所述过氧化苯甲酰的质量浓度为0.3%。 The production method according to claim 6, wherein, in step 2.2, the initiator is benzoyl peroxide, said benzoyl peroxide concentration of 0.3 mass%.
  12. 12.根据权利要6所述的制备方法,其特征在于,在步骤2.2中,所述二甲基丙烯酸乙二醇酯与聚乙烯醇溶液的体积比为1:625-1:667。 12. The production method according to claim 6, wherein, in step 2.2, the ethylene glycol dimethacrylate and polyvinyl alcohol solution in a volume ratio of 1: 625-1: 667.
  13. 13.根据权利要求6所述的制备方法,其特征在于,在步骤2.2中,所述二甲基丙烯酸乙二醇酯的密度为1.0151g/mL。 13. A method of preparation according to claim 6, wherein, in step 2.2, the density of the ethylene glycol dimethacrylate ester of 1.0151g / mL.
  14. 14.根据权利要求1所述的制备方法,其特征在于,在步骤3.1中,所述各原料溶于超纯水中的浓度分别为:亚硫酸铝0.05-0.06摩尔/L、尿素0.56-0.6摩尔/L、聚乙烯醇吡咯烷丽6.5x10 6 摩尔/L0 14. The production method according to claim 1, wherein, in step 3.1, the concentration of each material was dissolved in ultrapure water are: aluminum sulfite 0.05-0.06 mol / L, urea 0.56-0.6 mol / L, polyvinyl pyrrolidine Korea 6.5x10 6 mol / L0
  15. 15.根据权利要求1所述的制备方法,其特征在于,所述烧结步骤为:依次置于室温到200°C均匀升温并烧结50分钟,200-400°C均匀升温并烧结600分钟,400-1300°C均匀升温并烧结400分钟,最后1250°C烧结120分钟。 15. A method of preparation according to claim 1, wherein said sintering step is: turn left at room temperature to 200 ° C uniformly heated and sintered 50 minutes, 200-400 ° C uniformly heated and sintered 600 minutes, 400 -1300 ° C and sintered uniformly heated 400 minutes, and finally sintering 1250 ° C for 120 minutes.
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