CN1799647A - Nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明公开一种纳米羟基磷灰石/壳聚糖/明胶复合多孔骨组织工程支架材料及其制备方法。该材料包括质量比为3∶7－7∶3壳聚糖和明胶以及在二者形成的多孔支架材料上原位沉积的40－80nm的纳米羟基磷灰石组成。运用相分离技术制备成孔径为100－300μm，孔隙率大于90％的多孔支架材料，然后将其浸入Ca(NO₃) ₂的Tris缓冲溶液中浸泡10－12小时，用去离子水冲洗3次后在将其浸入的Na₃PO₄的Tris缓冲溶液中浸泡10－12小时，并用NaOH溶液调整pH值在11－13之间，再用去离子水冲洗3次后冷冻干燥。如此循环多次次制得纳米羟基磷灰石/壳聚糖/明胶复合多孔材料，其羟基磷灰石的粒径在50nm左右，本发明制备的材料具有良好的力学性能和生物相容性。

The invention discloses a nano-hydroxyapatite/chitosan/gelatin composite porous bone tissue engineering scaffold material and a preparation method thereof. The material comprises chitosan and gelatin with a mass ratio of 3:7-7:3 and nano-hydroxyapatite of 40-80nm deposited in situ on the porous support material formed by the two. Use phase separation technology to prepare a porous scaffold material with a pore size of 100-300 μm and a porosity greater than 90%, then immerse it in a Ca(NO₃ )₂ Tris buffer solution for 10-12 hours, and rinse it with deionized water for 3 times Then soak it in the Tris buffer solution of Na₃ PO₄ for 10-12 hours, adjust the pH value between 11-13 with NaOH solution, rinse with deionized water three times, and freeze-dry. The nano-hydroxyapatite/chitosan/gelatin composite porous material is obtained through repeated cycles in this way, and the particle size of the hydroxyapatite is about 50nm. The material prepared by the invention has good mechanical properties and biocompatibility.

Description

Nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof

Technical field

The present invention relates to a kind of nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof, belong to the bone reparing biological material technology.

Background technology

The bone reparation is a general and complicated clinical problem with regeneration in the plastic surgery operation, and the whole world has the millions of people to suffer from orthopaedics class disease owing to wound every year, and many people death owing to lack ideal substitution material.Present bone-grafting material mainly contains from body bone, homogeneous allogenic bone, through the xenogenesis bone of special handling and synthetic bone material etc.But limited and need carry out second operation from body bone source, post-transplantation complication can reach 8%; Homogeneous allogenic bone also exists immunogenicity and shortcoming such as pathogenic, exists during heteroplastic transplantation and infects and immunoreation, and may cause other health problems.Seeking ideal osseous tissue substitution material is the key that solves orthopaedics class disease, therefore makes up the focus that has become people's concern with the synthetic material of nature bone similar.

Nano level hydroxyapatite crystal and collagen stroma closely combine, and produce a high complexity and orderly composite construction, and this composite construction has constituted the primary structure framework of nature bone.Natural hydroxyapatite exists with the form of nanocrystal in the organism, is the acicular crystals of 65-80nm.The biological nature of hydroxyapatite is closely related with granular size, and it is active that nano-grade hydroxy apatite has unique biological.According to " nano effect " theory, the surface area of the nanometer particle of unit mass makes the atom number that is in particle surface obviously increase obviously greater than micro-size particles, has improved the activity of particle, very the combination that helps organizing

In order to prepare comparatively ideal bone tissue engineer substitution material, recently, research worker has been developed many and the nanometer hydroxyapatite composite nature bone similar.People (biomaterials such as Kikuchi M, 22 (2003): 1705-1711), the hydroxyapatite/collagen composite that has prepared the class bone under bionical condition by self assembly mechanism has bone conduction performance and a whole biological property preferably though his mechanical property and biodegradability are relatively poor; In order to improve its mechanical strength, people such as Cui Fuzhai have added alginate (Journal of biomedicalmaterials research.Part B in collagen/nanometer hydroxyapatite, Applied biomaterials, 69 (2004): 159-165), people such as H.W.Kim are that host material has made gelatin/nanometer hydroxyapatite composite that biocompatibility is better, be suitable for cell adhesion and growth by original position is synthetic with phase detachment technique with the gelatin; In addition, people such as Li Yubao has prepared chitosan/nanometer hydroxyapatite composite; Except the complex of these natural high polymers and nanometer hydroxyapatite, the composite of synthetic high polymer material and nanometer hydroxyapatite is also developed, and polylactic acid/nano hydroxy-apatite porous support materials is adopted by most scholar that (Acta Biomaterialia 1 (2005): 653-662; Biomaterials, 25 (2004): 4749-4757; ), the mechanical property of polyamide/nano hydroxyapatite composite material is better than polylactic acid/hydroxy apatite material (the materials in medicine 14 (2003): 655-660) that equally also receives publicity.

But these composites all also have its weak point, the bone tissue engineering stent material of preparation and nature bone structural similarity, improve its mechanical property and biology performance, improve and dispersion situation and the size research emphasis that be bone tissue engineer of control hydroxyapatite in support.

Summary of the invention

The object of the present invention is to provide a kind of nanometer hydroxyapatite/chitosan/gelatin porous scaffold material and preparation method thereof, the nanometer hydroxyapatite/chitosan/gelatin porous material that makes with this method has the structure similar to nature bone and excellent mechanical property and bio-compatible performance.

The present invention is realized by following technical proposals, a kind of nanometer hydroxyapatite/chitosan/gelatin porous scaffold material, it is characterized in that this material comprises that mass ratio is 3: 7-7: 3 chitosans and gelatin and on the porous support materials of the two formation the nanometer hydroxyapatite of the 40-80nm of in-situ deposition form.

The preparation method of above-mentioned nanometer hydroxyapatite/chitosan/gelatin porous scaffold material is characterized in that comprising following process:

(1) chitosan is dissolved in the acetic acid solution of 1%--3%, being prepared into mass concentration is the acetic acid solution solution of 2%--4% chitosan.

(2) with gelatin at 50 ℃--with in its dissolved ions water, being mixed with mass concentration is the 2%--4% aqueous gelatin solution under 60 ℃ the temperature.

(3) with (1) and (2) preparation solution with chitosan bright with the glue mass ratio be 3: 7-7: 3 mixed, stirred 10-12 hour.

(4) glutaraldehyde solution with (3) prepared mixed solution and 0.25% is 3 by volume also: 1-0.5: 1 mixed stirs and forms gel, and it is added in mould, puts into-20 ℃--and pre-freeze is more than 8 hours under 60 ℃ the low temperature.Gel lyophilization in vacuum freeze drier that pre-freeze is good.

(5) be to soak 3-8 hour in the sodium hydroxide solution of 1%--5% the support after the lyophilizing in mass concentration, remove residual acetic acid.Be placed in the sodium borohydride solution that mass concentration is 2%--5% again and soaked 3-8 hour, remove free glutaraldehyde, wash, be dipped to neutrality repeatedly with deionized water, the secondary lyophilizing obtains chitosan/gelatin porous scaffold material.

(6) preparation Tris (Tris) biological buffer, and take by weighing Ca (NO respectively₃)₂6H₂O and Na₃PO₄12H₂O dissolves in the Tris biological buffer respectively, and being mixed with amount concentration respectively is the Ca (NO of 0.06-0.2mol/L₃)₂And Na₃PO₄The Tris solution for standby.

(7) chitosan that (5) are prepared/gelatin timbering material is at Ca (NO₃)₂Tris solution in soaked 10--12 hour, use deionized water rinsing 2-3 time then; Again at Na₃PO₄Tris solution in soaked 10--12 hour, and adjust pH value between 11-13 with NaOH, it is same with deionized water rinsing 2-3 time to take out the back, carries out lyophilizing once more.

(8) the deposition process cyclic deposition that repeats (7) reaches requirement until the content of hydroxyapatite for several times, can obtain the bone repair porous material of bionic nano hydroxyapatite/chitosan/gelatin of the present invention.

The composite porosity of the simple and preparation of this procedure is generally greater than 90%, aperture with a large amount of connections is the micropore of being fit to of 100--300 μ m and cell growth, porosity is by the solution concentration decision of chitosan and gelatin, the size in aperture is determined by the pre-freeze temperature, sedimentary nano-grade hydroxy apatite is to not influence of pore size, and the hydroxyapatite crystal grain diameter that obtains of deposition is the synthos structural similarity in 50nm left and right sides (see figure 1) and the nature bone.This material has excellent biological compatibility and mechanical property, is expected as osseous tissue renovating material.

Description of drawings

Fig. 1 is the transmission electron microscope photo with the nanometer hydroxyapatite/chitosan/gelatin composite material behind this method embodiment one prepared cyclic deposition eight times.

The specific embodiment

Implementing used primary raw material is chitosan (deacetylation 95% molecular weight 200,000; Qingdao Hai Hui biological product company limited), gelatin (Sigma company; the U.S.), the acetic acid (CH of Tris (tris, Gibico company, the U.S.) and analytical pure level₃COOH), lime nitrate (Ca (NO₃)₂), tertiary sodium phosphate (Na₃PO₃), glutaraldehyde (OHC (CH₂)₃CHO), sodium hydroxide (NaOH), sodium borohydride (NaBH) or the like.

Embodiment one:

(1) the preparation mass percent concentration is 1% acetic acid solution 50ml, takes by weighing the 1g chitosan and dissolves in wherein that magnetic agitation 20min fully dissolves it, and the mass concentration of preparation is 2% chitosan solution.

(2) take by weighing the 1g gelatin in 50 ℃ of deionized waters that are dissolved in 25ml, be mixed with 4% gelatin solution.

(3) (1) (2) two solution are mixed, the room temperature lower magnetic force stirred 10 hours.

(4) the glutaraldehyde solution 100ml of preparation 0.25% gets (3) preparation mixed liquor 7ml mixed solution and adds the 3ml glutaraldehyde solution, and the back splashes into them in 24 well culture plates with glue head dropper rapidly about magnetic agitation 50s.4 ℃ leave standstill 12h, remove the bubble in the solution.Then culture plate was put into cryogenic refrigerator (60 ℃) pre-freeze 12 hours.

(5) pre-freeze is good gel placed the freezer dryer lyophilization 48 hours, formed porous support materials.

(6) be to soak 4h in 2% the sodium hydroxide solution with the mass concentration that is placed on after the lyophilizing, remove residual acetic acid.Be placed on mass concentration behind the deionized water rinsing again and be 2% sodium borohydride solution and soak 3h, wash, be dipped to neutrality repeatedly with deionized water, the secondary lyophilizing obtains chitosan/gelatin porous scaffold material.

(7) the Tris aqueous slkali with 100ml0.1mol/L mixes with the 0.1mol/LHCL of 86.8ml, adds water volume is transferred to 200ml, obtains pH and be 7.3 Tris biological buffer.

(8) take by weighing the Ca (NO of 2.36g respectively₃)₂6H₂The Na of O crystal and 3.80g₃PO₄12H₂O is dissolved in respectively in the 100mlTris biological buffer, and obtaining amount concentration is 0.1mol/L Ca (NO₃)₂And Na₃PO₄Solution; With chitosan/gelatin porous scaffold material at Ca (NO₃)₂Tris biological buffer solution in soak after 10 hours with deionized water rinsing 2-3 time; To be soaked into Na at it again₃PO₄The Tris biological buffer in and with NaOH the solution pH value is adjusted between the 11-13, take out after 10 hours and with deionized water rinsing 2-3 time; Lyophilization once more.

(9) the deposition process cyclic deposition of repetition (2) can obtain the bone repair porous material of bionic nano hydroxyapatite/chitosan/gelatin of the present invention for 8 times.

Embodiment two:

Raw materials used and embodiment are together.

Embodiment one (8) is adjusted into takes by weighing 2.36gCa (NO₃)₂6H₂O and 2.28gNa₃PO₄12H₂O is dissolved in respectively that (making amount concentration is the Ca (NO of 0.1mol/L in the 100mlTris biological buffer₃)₂The Na of solution and 0.06mol/L₃PO₄Solution); With chitosan/gelatin porous scaffold material at Ca (NO₃)₂Tris biological buffer solution in soak after 10 hours with deionized water rinsing 2-3 time; To be soaked into Na at it again₃PO₄The Tris biological buffer in and with NaOH the solution pH value is adjusted between the 11-13, take out after 10 hours and with deionized water rinsing 2-3 time; Lyophilization once more.

All the other steps are identical with embodiment one.

Embodiment three:

Raw materials used and embodiment are together

To implement the chitosan/gelatin mixed solution in one (4) and the volume ratio of glutaraldehyde solution and be adjusted into 1: 1, and promptly get the 5ml mixed liquor and mix, with glue head dropper it be splashed in 24 orifice plates rapidly about magnetic agitation 40s with the 5ml glutaraldehyde solution.

All the other steps are identical with embodiment one.

Embodiment four:

Raw materials used and embodiment are together

(1) the preparation mass percent concentration is 1% acetic acid solution 50ml, takes by weighing the 0.5g chitosan and dissolves in wherein that magnetic agitation 20min fully dissolves it.The mass concentration of preparation is 1% chitosan solution

(2) take by weighing the 1g gelatin in 50 ℃ of deionized waters that are dissolved in 25ml, be mixed with 4% gelatin solution.

(3) (1) (2) two solution are mixed, the room temperature lower magnetic force stirred 10 hours.

All the other steps are identical with embodiment one.

Embodiment five:

(1) the preparation mass percent concentration is 1% acetic acid solution 50ml, takes by weighing the 1g chitosan and dissolves in wherein that magnetic agitation 20min fully dissolves it.The mass concentration of preparation is 1% chitosan solution

(2) take by weighing the 0.5g gelatin in 50 ℃ of deionized waters that are dissolved in 25ml, be mixed with 2% gelatin solution.

(3) (1) (2) two solution are mixed, the room temperature lower magnetic force stirred 10 hours.

All the other steps are identical with embodiment one.

Claims (2)

Translated fromChinese

1.一种纳米羟基磷灰石/壳聚糖/明胶多孔支架材料，其特征在于，该材料包括质量比为3∶7-7∶3壳聚糖和明胶以及在二者形成的多孔支架材料上原位沉积的40-80nm的纳米羟基磷灰石组成。1. a nanometer hydroxyapatite/chitosan/gelatin porous scaffold material, is characterized in that, this material comprises mass ratio and is 3: 7-7: 3 chitosan and gelatin and the porous scaffold material formed in both It is composed of 40-80nm nano-hydroxyapatite deposited in situ.2.一种制备权利要求1所述的纳米羟基磷灰石/壳聚糖/明胶多孔支架材料的方法，其特征在于包括以下过程：2. a method for preparing nano-hydroxyapatite/chitosan/gelatin porous support material according to claim 1, is characterized in that comprising the following process:(1)将壳聚糖溶解于1％--3％的乙酸溶液中，制备成质量浓度为2％--4％壳聚糖的乙酸溶液溶液；(1) chitosan is dissolved in 1%--3% acetic acid solution, and it is prepared into an acetic acid solution solution whose mass concentration is 2%--4% chitosan;(2)将明胶在50℃--60℃的温度下将其溶解离子水中，配制成质量浓度为2％--4％明胶水溶液；(2) dissolving gelatin in deionized water at a temperature of 50°C--60°C to prepare an aqueous gelatin solution with a mass concentration of 2%--4%;(3)将(1)和(2)配制的溶液以壳聚糖明与胶质量比为3∶7-7∶3的比例混合，搅拌10-12小时；(3) The solution prepared by (1) and (2) is mixed in the ratio of chitosan gelatin to gum mass ratio of 3:7-7:3, and stirred for 10-12 hours;(4)将(3)所制得的混合溶液和0.25％的戊二醛溶液并按体积比为3∶1-0.5∶1比例混合搅拌形成凝胶，将其加入模具中，放入-20℃--60℃的低温下预冻8小时以上，将预冻好的凝胶在真空冷冻干燥机中冷冻干燥；(4) The mixed solution prepared in (3) and 0.25% glutaraldehyde solution are mixed and stirred at a volume ratio of 3:1-0.5:1 to form a gel, which is added to the mold and placed in -20 Prefreeze at a low temperature of ℃--60℃ for more than 8 hours, and freeze-dry the pre-frozen gel in a vacuum freeze dryer;(5)将冻干后的支架在质量浓度为1％--5％的氢氧化钠溶液中浸泡3-8小时，除去残留的乙酸。再将其置于质量浓度为2％--5％的硼氢化钠溶液中浸泡3-8小时，除去游离的戊二醛，用去离子水反复冲洗、浸泡至中性，二次冻干获得壳聚糖/明胶多孔支架材料；(5) Soak the lyophilized stent in a sodium hydroxide solution with a mass concentration of 1%-5% for 3-8 hours to remove residual acetic acid. It is then placed in a sodium borohydride solution with a mass concentration of 2%--5% and soaked for 3-8 hours to remove free glutaraldehyde, repeatedly rinsed with deionized water, soaked to neutrality, and freeze-dried twice to obtain Chitosan/gelatin porous scaffold material;(6)配制三羟甲基氨基甲烷(Tris)生物缓冲液，并分别称取Ca(NO₃)₂6H₂O和Na₃PO₄12H₂O分别溶入Tris生物缓冲液，分别配制成物质量浓度为0.06-0.2mol/L的Ca(NO₃)₂和Na₃PO₄的Tris溶液备用；(6) Prepare tris (Tris) biological buffer, and weigh Ca(NO₃ )₂ 6H₂ O and Na₃ PO₄ 12H₂ O respectively and dissolve them into Tris biological buffer to prepare the product The Tris solution of Ca(NO₃ )₂ and Na₃ PO₄ with a mass concentration of 0.06-0.2mol/L is ready for use;(7)将(5)制备好的壳聚糖/明胶支架材料在Ca(NO₃)₂的Tris溶液中浸泡10--12小时，然后用去离子水冲洗2-3次；再在Na₃PO₄的Tris溶液中浸泡10--12小时，并用NaOH调整pH值在11-13之间，取出后同样用去离子水冲洗2-3次，再次进行冻干；(7) Soak the chitosan/gelatin scaffold material prepared in (5) in the Tris solution of Ca(NO₃ )₂ for 10--12 hours, then rinse_2-3 times with deionized water; Soak in the Tris solution of PO₄ for 10--12 hours, and adjust the pH value between 11-13 with NaOH, after taking it out, rinse it with deionized water for 2-3 times, and freeze-dry it again;(8)重复(7)的沉积过程循环沉积数次，直至羟基磷灰石的含量达到要求，即可获得本发明的仿生纳米羟基磷灰石/壳聚糖/明胶骨修复多孔材料。(8) Repeat the deposition process of (7) for several times until the content of hydroxyapatite reaches the requirement, and then the biomimetic nano-hydroxyapatite/chitosan/gelatin bone repair porous material of the present invention can be obtained.