CN102397586A - Preparation method of bone repair hydrogel and fiber pipe composite material loading growth factors - Google Patents

Preparation method of bone repair hydrogel and fiber pipe composite material loading growth factors Download PDF

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CN102397586A
CN102397586A CN 201110344893 CN201110344893A CN102397586A CN 102397586 A CN102397586 A CN 102397586A CN 201110344893 CN201110344893 CN 201110344893 CN 201110344893 A CN201110344893 A CN 201110344893A CN 102397586 A CN102397586 A CN 102397586A
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alginate
hydrogel
sodium alginate
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许杉杉
韩志超
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无锡中科光远生物材料有限公司
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Abstract

The invention discloses a preparation method of a bone repair hydrogel and fiber pipe composite material loading growth factors. Firstly, a spinning solution is prepared, the spinning solution is arranged in a syringe, a nanometer fiber reticular layer is prepared by high-voltage electrospinning, sodium alginate modified by RGD (Arg-Gly-Asp) is irradiated with gamma rays, and after irradiation, the RGD-sodium alginate is filtered to be degermed and is stored at the room temperature of -20 DEG C so as to prepare hydrogel. According to the method disclosed by the invention, the sodium alginate degraded by gamma ray irradiation can be combined with adhesive peptide in covalence to promote cell attachment; a nanometer fiber pipe is prepared through electrostatic spinning, the fiber has a diameter being the same as that of an extracellular matrix (ECM) and a large specific surface area, can improve cell attachment and migration, and is suitable for osteoblast differentiation and stem cell in vitro regeneration; and the composite material consisting of the bone repair hydrogel and the fiber pipe can guide bone regeneration by hydrogel injected with the sodium alginate and the protein electrostatic spinning nanometer fiber reticular film, promotes the regeneration and repair functions of vessels, can be used as a bone repair transmission system, has a simple production process and is easy to operate.

Description

负载生长因子的骨修复水凝胶和纤维管复合材料制备方法 Hydrogel and method for preparing the composite fiber tube supported bone growth factors to repair

技术领域: FIELD:

[0001] 本发明涉及一种生物复合材料,尤其涉及一种负载生长因子的骨修复水凝胶和纤维管复合材料的制备方法。 [0001] The present invention relates to a biocomposite material, in particular a hydrogel and method for preparing fiber composite material relates to a tube loading of bone growth factors to repair.

背景技术: Background technique:

[0002] 自体和异体骨移植是治疗骨折和大范围骨缺陷的最常规方式,然而,这一技术有许多弊端,其中包括可用的骨移植材料有限、移植故障率高等等。 [0002] Autologous and allogeneic bone graft in a conventional manner is the most widespread treatment of fractures and bone defects, however, this technique has many drawbacks, including the limited availability of bone graft materials, transplant failure rate and the like. 通过组织工程/再生医学(TE/RM),研发了一些有潜力的骨移植替代品,结合生物生化和物理棚架结构,使用三维支架以支持体内装载,然而,这些棚架结构难以设计和制造高孔隙率产品,他们通常无法为受损细胞提供一个最佳的环境,从而妨碍了受损组织功能的恢复。 By tissue engineering / regenerative medicine (TE / RM), has developed a number of potential bone graft substitute, biochemical and biological binding physical scaffolding structure, a three-dimensional scaffold to support in vivo loading, however, these structures difficult to design and manufacture scaffolding high porosity products, they often can not provide an optimal environment for the damaged cells, thus preventing restoration of damaged tissue function.

[0003] 静电纺丝纳米纤维膜,具有多项功能适合组织再生,他们的纤维直径与细胞外基质(ECM)的大小相同,规模成分(纤维直径从纳米到亚微米)和大表面积,可提高细胞附着,迁移和功能化,纳米纤维网格,已被证明可以支持成骨细胞分化和干细胞在体外再生, 并已在体内颅骨缺损模型中进行测试。 [0003] electrospun nanofibrous membrane, having a suitable number of functional tissue regeneration, their fiber diameter of the same size with the extracellular matrix (ECM), the scale component (fiber diameter ranging from nano to submicron) and a large surface area, can improve the cell attachment, migration and functionalized nano fiber grid, has been shown to support osteogenic cell differentiation and regeneration of stem cells in vitro, and has been tested in vivo cranial defect model.

[0004] 虽然棚架提供了一个引导骨再生的模板,为了促进骨骼快速愈合,通常还需提供生长因子,像rhBMP-2已经被临床证明可以成功诱导成骨,促进骨愈合的,但需要大剂量, 所以需要改进配送系统,提供缓释,减低给药剂量,从而最大程度地减少并发症,降低治疗成本;海藻酸钠水凝胶,褐藻多糖衍生物,是一种被广泛用作棚架材料和运载工具的蛋白质,但是哺乳动物细胞缺乏海藻酸钠聚合物的受体,所以我们急需制备一种适合的TE/RM 促进细胞和组织再生的物质,从而开发和生产负载生长因子的用于骨修复的传递系统。 [0004] While the scaffolding provides a template to guide bone regeneration, in order to promote rapid bone healing, growth factors generally need to provide, as rhBMP-2 has been clinically proven to successfully induce bone and promote bone healing, but requires a large dose, it is necessary to improve the delivery system to provide sustained release, reducing the dosage, and thus minimize complications, reducing the cost of treatment; alginate hydrogels, alginate polysaccharide derivative is a widely used as scaffolds protein materials and means of delivery, but mammalian cells lack receptors alginate polymer is an urgent need to prepare a suitable TE / RM cell and tissue regeneration-promoting substances, so that the development and production of growth factors for the load delivery system bone repair.

发明内容: SUMMARY:

[0005] 本发明针对现有技术的不足,提供了一种负载生长因子的骨修复水凝胶和纤维管复合材料的制备方法。 [0005] The present invention addresses deficiencies of the prior art by providing a method for producing a bone growth factor and load repair hydrogel composite fiber tube.

[0006] 负载生长因子的骨修复水凝胶和纤维管复合材料制备方法,其步骤如下: [0006] Load a bone growth factor and a method for preparing hydrogel composite fiber tube repair, the following steps:

[0007] (1)配制纺丝溶液;将高分子材料溶于水或有机溶剂,配制成质量百分浓度为10-30%的高分子溶液。 [0007] (1) Preparation of spinning solution; the polymer material dissolved in water or an organic solvent to prepare a mass percent concentration of 10 to 30% polymer solution.

[0008] (2)制备纳米纤维网状管;纺丝溶液被装在l_5mL注射器中,并在尾端连接不锈钢针头,注射器与注射泵相连,其流速为0. 5-1毫升/小时,得到的纤维用一个距离针尖20-23cm的平面铜箔板进行收集,用13-20kv的高压来电纺3_6h,以获得的较厚的纳米纤维网状层,得到的纤维网状层在干燥箱中蒸发过夜以去除残留溶剂,用上面制得的纳米纤维网制造可植入的管状材料,从网上裁剪出一个13-19mm的矩形样品,在一些样品中,使用直径为0. 5-lmm的针来打一些间距为1. 5-2. 5mm的孔,矩形网格样品用钢芯轴缠为一个直径5-15毫米和长度10-20毫米的管,重叠的部分用UV胶固定在一起,纳米纤维网管用50-80%的酒精冲洗两次,然后侵泡在乙醇中,带溶剂蒸发后可保证无菌,保存直到进行植入。 [0008] (2) Preparation of a nanofiber web tube; l_5mL spinning solution is contained in a syringe, and is connected at the end of a stainless steel needle, connected to a syringe and a syringe pump, a flow rate of 0. 5-1 ml / hr, to give fibers for a distance of 20-23cm tip collection plane copper plate, high pressure 13-20kv 3_6h call spinning, the thicker layer to obtain a nanofiber web, the fibrous web layer was evaporated in a drying cabinet overnight to remove residual solvent using the above prepared nanofiber web manufacturing implantable tubular material, a rectangular sample cut from a web of 13-19mm, in some samples, with a diameter of 0. 5-lmm needle play some holes pitch 1. 5-2. 5mm, the rectangular grid of sample steel mandrel wound length of 5-15 mm and a diameter of 10-20 mm tube, the fixing portion overlapping with UV glue, nano NM fiber rinsed with 50-80% alcohol twice, and then soaked in ethanol, the solvent was evaporated with a guaranteed sterile storage until implantation. [0009] (3)含生长因子的海藻酸钠水凝胶的制备;用Y射线照射R6D修饰的海藻酸钠,使得聚合物分子量降低以适合用于进行体内材料,照射后的海藻酸钠使用标准的碳二亚胺化学共价连接上了G4RGDASSP肽序列,其密度为每条聚合物链有1_3个序列,得到的RGD-海藻酸钠过滤除菌,冻干并在室温为_20°C储存,将RGD-藻酸盐溶解于MEM得到1_3% W/V的溶液,以制备水凝胶,冻干的rhBMP-2溶解在含0. 1 %大鼠血清白蛋白的4mM盐酸中, 其终浓度为100-400mg/mL,海藻酸钠溶液与rhBMP-2溶液以5 : 1-8 : 1的比例混合,含rhBMP-2的海藻酸钠溶液与0.2-0. 4%的硫酸钙浆15 : 1-25 : 1的比例进行交联,海藻酸钠溶液在注射器中室温保持30分钟以变为凝胶,然后转移到4°C保存。 [0009] (3) Preparation of alginate hydrogels containing growth factors; R6D modified by Y-ray irradiation sodium alginate, such that the molecular weight of the polymer is lowered to materials suitable for use in vivo, the use of sodium alginate after irradiation standard carbodiimide chemistry on covalently linked G4RGDASSP peptide sequence, a density of each polymer chain has 1_3 sequences, resulting RGD- alginate filter sterilized and lyophilized at room temperature _20 ° C stored in the RGD- alginate was dissolved in MEM to give a solution 1_3% W / V to prepare hydrogels, lyophilized rhBMP-2 was dissolved in 4mM HCl containing 0.1% rat serum albumin, which final concentration of 100-400mg / mL, and sodium alginate solution to a solution of rhBMP-2 5: 1-8: 1 ratio, rhBMP-2 solution containing sodium alginate and 0.2-0 4% calcium sulfate slurry. 15: 1-25: 1 ratio of crosslinked sodium alginate solution for 30 minutes at room temperature to become a gel in the syringe, and then transferred to a at 4 ° C.

[0010] 所述高分子材料所对应的溶剂分别为:聚己内酯(PCL)溶于丙酮或六氟异丙醇(HFP)和二甲基甲酰胺(DMF)的混合液;聚醋酸乙烯酯(PVAc)溶于二甲基甲酰胺(DMF); PEO溶于水;PVA溶于水或丙酮或水和丙酮的混合液;聚乳酸(PLA)溶于二甲基甲酰胺(DMF)或二甲基甲酰胺(DMF)和丙酮的混合液;聚氨酯(PU)溶于的氯仿和乙醇的混合液。 [0010] The polymeric material corresponding solvents are: polycaprolactone (PCL) or hexafluoroisopropanol was dissolved in acetone (HFP) and dimethylformamide (DMF) mixed solution; polyvinyl acetate ester (of PVAc) was dissolved in dimethylformamide (DMF); the PEO was dissolved in water; the PVA soluble in water or acetone or a mixture of water and acetone; polylactic acid (PLA) was dissolved in dimethylformamide (DMF) or dimethylformamide (DMF) and acetone mixture; polyurethane (PU) dissolved in a mixture of chloroform and ethanol.

[0011] 本发明采用Y射线照射降解的海藻酸钠,与粘附肽共价结合可以促进细胞附着; 通过静电纺丝制备纳米纤维管,纤维直径与细胞外基质(ECM)的大小相同,比表面积大,可提高细胞附着和迁移,适合成骨细胞分化和干细胞在体外再生;此两者组成复合材料,用注射海藻酸钠水凝胶和蛋白质静电纺丝纳米纤维网状膜可引导骨再生,促进血管的再生和修复功能,可以应做骨修复的传递系统,生产工艺简单、易于操作,可实现大规模产业化。 [0011] The present invention employs Y-ray irradiation degradation of alginate, combined with adhesion promoting peptides can be covalently attached to the cell; through the fiber tube, the same preparation of nano-sized electrostatic spinning fiber diameter of extracellular matrix (ECM), the ratio of large surface area, can enhance cell adhesion and migration, for osteoblast differentiation and regeneration of stem cells in vitro; both, composition of the composite material, injection of protein and alginate hydrogels electrospun nanofiber web can be guided bone regeneration membrane promoting vascular regeneration and repair function, bone repair should be done transfer system, the production process is simple, easy to operate, can achieve large-scale industrialization.

具体实施方式: detailed description:

[0012] 为了加深对本发明的理解,下面结合实施例对本发明作进一步详述。 [0012] For better understanding of the present invention, the following in connection with embodiments of the present invention will be described in further detail.

[0013] 负载生长因子的骨修复水凝胶和纤维管复合材料制备方法,其步骤如下: [0013] Load a bone growth factor and a method for preparing hydrogel composite fiber tube repair, the following steps:

[0014] (1)配制纺丝溶液;将聚己内酯(PCL)溶于六氟异丙醇(HFP)和二甲基甲酰胺(DMF)的混合液(体积比90 : 10),配制成质量百分浓度为12%的高分子溶液。 [0014] (1) Preparation of spinning solution; and polycaprolactone (PCL) was dissolved in hexafluoroisopropanol (HFP) and dimethylformamide (DMF) mixed solution (volume ratio 90: 10), formulated to 12% by weight percent of the polymer solution.

[0015] (2)制备纳米纤维网状管;纺丝溶液被装在3mL注射器中,并在尾端连接不锈钢针头,注射器与注射泵相连,其流速为0. 7毫升/小时。 [0015] (2) Preparation of a nanofiber web tube; 3mL spinning solution in the syringe is mounted and connected at the end of a stainless steel needle, connected to a syringe and a syringe pump, a flow rate of 0.7 ml / hr. 得到的纤维用一个距离针尖22cm的平面铜箔板进行收集,用15kv的高压来电纺5h,以获得的较厚的纳米纤维网状层,得到的纤维网状层在干燥箱中蒸发过夜以去除残留溶剂,用上面制得的纳米纤维网制造可植入的管状材料,从网上裁剪出一个15mm的矩形样品,在一些样品中,使用直径为Imm的针来打一些间距为2. 5mm的孔,矩形网格样品用钢芯轴缠为一个直径10毫米和长度15毫米的管,重叠的部分用UV胶固定在一起,纳米纤维网管用70%的酒精冲洗两次,然后侵泡在乙醇中, 带溶剂蒸发后可保证无菌,保存直到进行植入。 The resulting fibers for a distance of 22cm needle collection plane copper plate, a thicker layer of high pressure nanofiber web spun 5h 15kv call to obtain the fibrous web layer was evaporated overnight in an oven to remove residual solvent using the above prepared nanofiber web manufacturing implantable tubular material, a rectangular sample cut from a web of 15mm, in some samples, using a needle having a diameter of Imm to play some holes 2. 5mm pitch , a rectangular grid of samples of steel wrapped around a mandrel diameter of 10 mm and a length of 15 mm pipe, overlapping portions secured together with UV glue nanofiber network rinsed twice with 70% ethanol and then soaked in ethanol after evaporation of the solvent can be guaranteed with a sterile storage until implantation.

[0016] (3)含生长因子的海藻酸钠水凝胶的制备;用Y射线照射RGD修饰的海藻酸钠, 使得聚合物分子量降低以适合用于进行体内材料,照射后的海藻酸钠使用标准的碳二亚胺化学共价连接上了G4RGDASSP肽序列,其密度为每条聚合物链有2个序列,得到的RGD-海藻酸钠过滤除菌,冻干并在室温为-20°C储存,将RGD-藻酸盐溶解于MEM得到2% W/V的溶液,以制备水凝胶。 [0016] (3) Preparation of alginate hydrogels containing growth factors; Y-ray irradiation with the RGD-modified alginate, so that the decrease in polymer molecular weight materials suitable for use in vivo, the use of sodium alginate after irradiation standard carbodiimide chemistry on covalently linked G4RGDASSP peptide sequence, a density of 2 per polymer chain with a sequence resulting RGD- alginate filter sterilized, and lyophilized at -20 ° C up rt storing the RGD- alginate was dissolved in a solution of MEM 2% W / V obtained to prepare a hydrogel. 冻干的rhBMP-2溶解在含0. 大鼠血清白蛋白的4mM盐酸中,其终浓度为200mg/mL,海藻酸钠溶液与rhBMP-2溶液以5 : 1的比例混合,含rhBMP-2的海藻酸钠溶液与0.3%的硫酸钙浆25 : 1的比例进行交联,海藻酸钠溶液在注射器中室温保持30 分钟以变为凝胶,然后转移到4°C保存。 lyophilized rhBMP-2 was dissolved in hydrochloric acid containing 0. 4mM rat serum albumin at a final concentration of 200mg / mL, sodium alginate solution with a solution of rhBMP-2 to 5: 1 mixing ratio, containing rhBMP-2 0.3% sodium alginate solution and calcium sulphate pulp 25: 1 ratio of crosslinked sodium alginate solution in a syringe at room temperature for 30 minutes to become a gel, and then transferred to a at 4 ° C.

Claims (2)

  1. 1.负载生长因子的骨修复水凝胶和纤维管复合材料制备方法,其特征在于:其步骤如下:(1)配制纺丝溶液;将高分子材料溶于水或有机溶剂,配制成质量百分浓度为10-30% 的高分子溶液。 1. Load bone growth factors hydrogel and method for preparing fiber composites repair tube, characterized in that: the following steps: (1) Preparation of spinning solution; the polymer material dissolved in water or an organic solvent to prepare a mass of one hundred content concentration of 10 to 30% polymer solution. (2)制备纳米纤维网状管;纺丝溶液被装在l_5mL注射器中,并在尾端连接不锈钢针头,注射器与注射泵相连,其流速为0. 5-1毫升/小时,得到的纤维用一个距离针尖20-23cm 的平面铜箔板进行收集,用13_20kv的高压来电纺3-6h,以获得的较厚的纳米纤维网状层,得到的纤维网状层在干燥箱中蒸发过夜以去除残留溶剂,用上面制得的纳米纤维网制造可植入的管状材料,从网上裁剪出一个13-19mm的矩形样品,在一些样品中,使用直径为0. 5-lmm的针来打一些间距为1. 5-2. 5mm的孔,矩形网格样品用钢芯轴缠为一个直径5_15 毫米和长度10-20毫米的管,重叠的部分用UV胶固定在一起,纳米纤维网管用50-80%的酒精冲洗两次,然后侵泡在乙醇中,带溶剂蒸发后可保证无菌,保存直到进行植入。 (2) Preparation of a nanofiber web tube; l_5mL spinning solution is contained in a syringe, and is connected at the end of a stainless steel needle, connected to a syringe and a syringe pump, a flow rate of 0. 5-1 ml / hr, and the resulting fibers collected from a flat tip 20-23cm copper plate, the high pressure calls spun 13_20kv 3-6h, thicker layer to obtain a nanofiber web, the fiber network layer was evaporated overnight in an oven to remove residual solvent using the above prepared nanofiber web manufacturing implantable tubular material, a rectangular sample cut from a web of 13-19mm, in some samples, a diameter of 0. 5-lmm needle to play some spacing 5-2 to 1. 5mm aperture., a rectangular grid of samples wound steel mandrel having a diameter 10-20 mm 5_15 mm and a length of the tube, partially overlapping with UV glue together with the nanofiber network 50 rinsed twice with 80% alcohol, and then soaked in ethanol, the solvent was evaporated with a guaranteed sterile storage until implantation. (3)含生长因子的海藻酸钠水凝胶的制备;用Y射线照射RGD修饰的海藻酸钠,使得聚合物分子量降低以适合用于进行体内材料,照射后的海藻酸钠使用标准的碳二亚胺化学共价连接上了G4RGDASSP肽序列,其密度为每条聚合物链有1_3个序列,得到的RGD-海藻酸钠过滤除菌,冻干并在室温为-20°C储存,将RGD-藻酸盐溶解于MEM得到1_3% W/V的溶液,以制备水凝胶,冻干的rhBMP-2溶解在含0. 大鼠血清白蛋白的4mM盐酸中,其终浓度为100-400mg/mL,海藻酸钠溶液与rhBMP-2溶液以5 : 1-8 : 1的比例混合,含rhBMP-2 的海藻酸钠溶液与0.2-0. 4%的硫酸钙浆15 : 1-25 : 1的比例进行交联,海藻酸钠溶液在注射器中室温保持30分钟以变为凝胶,然后转移到4°C保存。 (3) Preparation of alginate hydrogels containing growth factors; Y-ray irradiation with the RGD-modified alginate, so that the decrease in polymer molecular weight material suitable for use in vivo, alginate carbon after irradiation using the standard carbodiimide chemistry on covalently linked G4RGDASSP peptide sequence, a density of each polymer chain has 1_3 sequences, resulting RGD- alginate filter sterilized and lyophilized at -20 ° C up storage at room temperature, the in RGD- alginate was dissolved in MEM to give a solution 1_3% W / V to prepare hydrogels, lyophilized rhBMP-2 was dissolved in hydrochloric acid containing 0. 4mM rat serum albumin, a final concentration of 100 400mg / mL, and sodium alginate solution to a solution of rhBMP-2 5: 1-8: 1 ratio, rhBMP-2 solution containing sodium alginate and 0.2-0 4% calcium sulfate slurry 15: 1-25 : 1 ratio crosslinked alginate solution was kept at room temperature for 30 minutes to the syringe to a gel, and then transferred to a at 4 ° C.
  2. 2.根据权利要求1所述的负载生长因子的骨修复水凝胶和纤维管复合材料制备方法, 其特征在于:所述高分子材料所对应的溶剂分别为:聚己内酯溶于丙酮或六氟异丙醇和二甲基甲酰胺的混合液;聚醋酸乙烯酯溶于二甲基甲酰胺;PEO溶于水;PVA溶于水或丙酮或水和丙酮的混合液;聚乳酸溶于二甲基甲酰胺或二甲基甲酰胺和丙酮的混合液;聚氨酯溶于的氯仿和乙醇的混合液。 2. The bone growth factor of the load according to claim 1 and fibers produced hydrogel composite pipe repair method, wherein: said polymer material corresponding solvents are: polycaprolactone dissolved in acetone or hexafluoroisopropanol, and a mixture of dimethylformamide; polyvinyl acetate was dissolved in dimethyl formamide; the PEO was dissolved in water; the PVA soluble in water or acetone or a mixture of water and acetone; polylactic acid dissolved in diethyl dimethylformamide or dimethyl formamide and acetone mixture; the mixture was dissolved in chloroform and the polyurethane ethanol.
CN 201110344893 2011-11-04 2011-11-04 Preparation method of bone repair hydrogel and fiber pipe composite material loading growth factors CN102397586A (en)

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