CN100441755C - Preparation method of gelatin/chitosan blend for biomimetic extracellular matrix fibrous scaffold - Google Patents

Abstract

The invention relates to a gelatin-chitosan complex manufacture method for fiber scaffold of bionic extra-cellular matrix, comprising (1) chitosan solution with 4-10% (g/ml) concentration is obtained after it is dissolved into the mixed solvent of HFIP and TFA; (2) gelatin solution with 4-10% concentration is obtained after it is dissolved into HFIP; (3) gelatin-chitosan complex solution with 4-10% concentration is obtained by mixing the chitosan and gelatin solution in the same cubage and gelatin-chitosan nano composite fiber coat is manufactured by electrostatic spinning.

Description

Preparation method of gelatin/chitosan blend for biomimetic extracellular matrix fibrous scaffold

technical field

The invention belongs to the field of electrospinning, and in particular relates to a preparation method for gelatin/chitosan blending used for bionic extracellular matrix fiber supports.

Background technique

The failure, defect, or dysfunction of tissues and organs is one of the main hazards facing human health. The traditional treatment model "repairing wounds with trauma" has defects such as insufficient donor sources. The proposal of tissue engineering provides a technology for tissue regeneration means to promote the development of regenerative medicine. There have been many reports on chitosan-gelatin composites used as extracellular scaffold materials. Mao J S et al[1] prepared chitosan-gelatin porous composite scaffolds by freeze-drying pore-forming method. Fibroblast L929 cells enter the normal cell cycle, promote cell proliferation, and reduce cell apoptosis. Xia Wanyao et al[2] planted pig ear chondrocytes with a chitosan-gelatin composite scaffold prepared by freeze-drying method, and transplanted them into the abdomen subcutaneously of pigs after in vitro expansion. After 16 weeks, the chondrogenic scaffolds were completely degraded without inflammatory reaction. It shows that the porous chitosan-gelatin composite scaffold is a suitable scaffold for tissue engineered cartilage, and the chitosan-gelatin composite material constructed with positively charged chitosan and amphoteric gelatin has become a promising material. The size range of collagen fibers in natural extracellular matrix is 50-500nm[3]. Electrospinning is one of the techniques for processing nanomaterials. The fiber diameter ranges from a few nanometers to a few microns[4,5], which can mimic natural cells to the greatest extent. Collagen structures of the exogenous matrix, chitosan [6, 7, 8] and gelatin [9, 10, 11] have been successfully electrospun into nanofibers. But so far, the preparation of the chitosan-gelatin blend material by electrospinning has not been reported. The present invention is to prepare the biomimetic extracellular matrix fiber scaffold by electrospinning the gelatin-chitosan blend system. The references are as follows:

[11]Mao J S, Cui Y L, Wang X H, et al.A preliminary study on chitosanand gelation polyelectrolyte complex cytocompatibility by cell cycle andapoptosis analysis.Biomaterials.2004;25:3973-3981

[2] Xia Wanyao, Liu Wei, Cui Lei, etc. Experimental study on the construction of autologous tissue-engineered cartilage tissue by chitosan-gelatin porous composite scaffold. Chinese Journal of Medicine. 2003; 83(7): 577-579

[3]Min B M, You Y, Kim J M, et al.Formation of nanostructuredpoly(lactic-co-glycolic acid)/chitin matrix and its cellular response tonormal human keratinocytes and fibroblasts.Carbohydrate Polymers2004;57:285-292

[4] Huang Z M, Zhang Y Z, kotakic M, et al. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Composites Science and Technology 2003; 63: 2223-2253

[5] Subbiah T, Bhat G S, Tock R W, et al. Electrospinning of Nanofibers. Journal of Applied Polymer Science. 2005; 96: 557-569

[6] Mina B M, Leeb S W, Limb J N, et al. Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers. Polymer2004; 45: 7137-7142

[7] K Ohkawa, D Cha, H Kim, et al. Electrospinning of Chitosan. Macromol. Rapid Commun. 2004; 25: 1600-1605

[8] Xinying Geng, Oh-Hyeong Kwon, Jinho Jang. Electrospinning of chitosandissolved in concentrated acetic acid solution. Biomaterials 2005; 26:5427-5432

[9] Z M. Huang, Y Z. Zhang, S Ramakrishna, et al. Electrospinning and mechanical characterization of gelatin nanofibers. Polymer. 2004; 45: 5361-5368

[10] C S. Ki, D H. Baek, K D. Gang, et al. Characterization of gelatin nanofiber prepared from gelatin-formic acid solution. Polymer. 2005; 46: 5094-5102

[11] M Y. Li, J Mondrinos, M R, Gandhi, et al. Electrospun protein fibersas matrices for tissue engineering. Biomaterials. 2005; 26: 5999-6008

Contents of the invention

The preparation method of the present invention comprises the steps:

(1) Prepare chitosan solution: dissolve chitosan in a mixed solvent of hexafluoroisopropanol (HFIP) and trifluoroacetic acid (TFA), the volume ratio of HFIP/TFA is 8/2, stir until transparent with slight heat, Obtaining concentration is the chitosan solution of 4-10% (gram/milliliter);

(2) Prepare the gelatin solution: dissolve the gelatin in hexafluoroisopropanol, heat and stir until completely dissolved, and obtain a gelatin solution with a concentration of 4-10% (g/ml);

(3) Prepare gelatin and chitosan mixed solution: chitosan solution and gelatin solution are mixed with equal volumes to obtain a total concentration of 4%-10% (g/ml) gelatin and chitosan blended solution;

(4) The gelatin/chitosan composite nanofiber membrane can be prepared by electrospinning.

The above method adopts respectively disposing chitosan and gelatin solution, after being completely dissolved, take equal volume solutions to mix, stir evenly and let stand for ten minutes, then carry out spinning, the electrostatic spinning process of chitosan solution and gelatin mixed solution Parameters: voltage: 12-40 kV; electric field distance: 60-200 mm; spinning rate, 0.1-1 ml/hour.

The present invention prepares gelatin/chitosan blended nanofiber nonwoven material by electrospinning, and the chitosan-gelatin composite material constructed with positively charged chitosan and amphoteric gelatin becomes a promising material, which can be used as Biomimetic extracellular matrix materials.

Description of drawings

Figure 1 is the scanning electron micrograph of gelatin/chitosan blend electrospinning

Figure 2 is the film formation photo of glue/chitosan blend electrospinning

Detailed ways

Example one

Take by weighing 0.12 gram of chitosan (deacetylation 85%) with electronic analytical balance and be dissolved in 2 milliliters of hexafluoroisopropanol and trifluoroacetic acid (volume ratio is 8/2) mixed solvent, obtain concentration and be 6% (gram/ml) ) chitosan solution; 0.12 gram of gelatin was dissolved in 2 milliliters of hexafluoroisopropanol to obtain a gelatin solution with a concentration of 6% (gram/ml); after magnetic stirring at normal temperature was completely dissolved, equal volumes were mixed and Stir to obtain a total concentration of 6% chitosan/gelatin blended solution, statically spin the blended solution after ten minutes, the voltage is 24kv, the syringe pump advance speed is 0.6ml/h, the receiving distance is 13cm, select 7 No. needle, received by aluminum foil or coarse cotton base cloth, to obtain a non-woven matrix with an average diameter of 101 nm of electrospun gelatin/chitosan blended nanofibers.

Example two

Take by weighing 0.16 gram of chitosan (deacetylation 85%) with electronic analytical balance and be dissolved in 2 milliliters of hexafluoroisopropanol and trifluoroacetic acid (volume ratio is 8/2) mixed solvent, obtain concentration and be 8% (gram/ml) ) chitosan solution; 0.16 gram of gelatin was dissolved in 2 milliliters of hexafluoroisopropanol to obtain a gelatin solution with a concentration of 8% (gram/ml); after magnetic stirring at normal temperature was completely dissolved, equal volumes were mixed and Stir to obtain the chitosan/gelatin blend solution that total concentration is 8%, leave standstill for ten minutes and then electrospin the blend solution, the voltage is 24kv, the injection pump advance speed is 0.6ml/h, the receiving distance is 13cm, select 7 No. needles, received by aluminum foil or coarse cotton base cloth, to obtain a non-woven fabric matrix with an average diameter of 245 nm of electrospun gelatin/chitosan blended nanofibers.

Example three

Take by weighing 0.20 gram of chitosan (deacetylation 85%) with electronic analytical balance and be dissolved in 2 milliliters of hexafluoroisopropanol and trifluoroacetic acid (volume ratio is 8/2) mixed solvent, obtain concentration and be 10% (gram/ml) ) chitosan solution; 0.20 gram of gelatin was dissolved in 2 milliliters of hexafluoroisopropanol to obtain a gelatin solution with a concentration of 10% (gram/ml); after magnetic stirring at normal temperature was completely dissolved, equal volumes were mixed and Stir to obtain a total concentration of 10% chitosan/gelatin blended solution, statically spin the blended solution after ten minutes, the voltage is 24kv, the syringe pump advance speed is 0.6ml/h, the receiving distance is 13cm, select 7 No. needle, received by aluminum foil or coarse cotton base cloth, to obtain a non-woven fabric matrix with an average diameter of electrospun gelatin/chitosan blended nanofibers of 335 nm.

Example four

Take by weighing 0.08 gram of chitosan (deacetylation 85%) with electronic analytical balance and be dissolved in 2 milliliters of hexafluoroisopropanol and trifluoroacetic acid (volume ratio is 8/2) mixed solvent, obtain concentration and be 4% (gram/ml) ) chitosan solution; 0.24 gram of gelatin was dissolved in 2 milliliters of hexafluoroisopropanol to obtain a gelatin solution with a concentration of 12% (gram/ml); after magnetic stirring at normal temperature was completely dissolved, equal volumes were mixed and Stir to obtain a chitosan/collagen blended solution with a total concentration of 8%. After standing for ten minutes, the blended solution is electrospun. The voltage is 24kv, the advancing speed of the syringe pump is 0.6ml/h, and the receiving distance is 13cm. Needle No. 7, received by aluminum foil or coarse cotton base cloth, to obtain a non-woven matrix with an average diameter of electrospun gelatin/chitosan blended nanofibers of 112 nm.

Claims (2)

1. A preparation method for electrospinning of gelatin/chitosan blended nanofiber membranes for biomimetic extracellular matrix fiber scaffolds, comprising the steps of: (1) prepare chitosan solution: chitosan is dissolved in the mixed solvent of hexafluoroisopropanol HFIP and trifluoroacetic acid TFA, and its HFIP/TFA volume ratio is 8/2; Gentle heat is stirred to transparent, obtains concentration as 4-10% g/ml chitosan solution; (2) Prepare gelatin solution: dissolve gelatin in hexafluoroisopropanol, heat and stir until completely dissolved, and obtain a gelatin solution with a concentration of 4-10% g/ml; (3) Prepare gelatin and chitosan mixed solution: chitosan solution and gelatin solution are mixed with equal volumes to obtain a total concentration of 4%-10% g/ml gelatin and chitosan blended solution; (4) The gelatin/chitosan composite nanofiber membrane was prepared by electrospinning. 2. the electrospinning preparation method of the gelatin/chitosan blending nanofiber membrane that is used for biomimetic extracellular matrix fiber scaffold as claimed in claim 1, is characterized in that: the total concentration of gelatin and chitosan blending solution is 6% g/ml, 8% g/ml or 10% g/ml.

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