CN102949751A - Preparation method for tissue engineering collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning bracket - Google Patents
Preparation method for tissue engineering collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning bracket Download PDFInfo
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- CN102949751A CN102949751A CN2012104936986A CN201210493698A CN102949751A CN 102949751 A CN102949751 A CN 102949751A CN 2012104936986 A CN2012104936986 A CN 2012104936986A CN 201210493698 A CN201210493698 A CN 201210493698A CN 102949751 A CN102949751 A CN 102949751A
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Abstract
The invention discloses a preparation method for a tissue engineering collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning bracket, which is characterized by comprising the following steps: preparing a solvent, a glue solution, and the electrical spinning solution, and then performing electrostatic spinning. The bracket prepared by the method not only has a three-dimensional porous structure, but also has extracellular matrix highly bionic property with high tissue compatibility, controllable size and shape, and controllable porosity and diameter of spun nanofibers in a certain range; and the majority of extracellular matrix is a network comprising water, the collagen, polysaccharide (hyaluronic acid, chondrotin sulfate and the like), inorganic substance and the like, so that the collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning bracket has a wide application prospect in the field of the tissue engineering bracket, in particular the soft tissue engineering bracket.
Description
Technical field
The invention belongs to biomedical tissue engineering material field, is a kind of organizational project collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning support preparation method.
Background technology
In recent years, organizational project is reconstituted in the fields such as cartilage, bone, skin, blood vessel and develops and obtained significant achievement rapidly, also has Related product such as blood vessel, skin, bone to be applied to clinical.The timbering material of breeding carrier as the adhesion of tissue engineering seed cell is of a great variety, is hot topic and the emphasis of present technical field of biological material.Timbering material provides the place that obtains nutrition, gas exchange, drainage refuse, Growth and Differentiation propagation for cell, also be the material base that organizational project is rebuild histoorgan.Desirable tissue engineering bracket should possess following primary condition: 1, good biocompatibility and bio-mechanical performance; 2, suitable biological degradability; 3, good plasticity; 4, be easy to sterilization, sterilization.Tissue engineering bracket mainly is divided into natural biologic material and synthetic material at present, such as collagen, gelatin, chitosan, alginate, bioceramic, hydroxyapatite, polylactic acid etc.Wherein collagen, gelatin, chitosan have good biocompatibility, degradability etc. and are favored by vast researcher, especially in organizational project and reconstruction field.The common method of preparation collagen class support is freeze-drying, be about to the mixed solution frozen drying of collagen or collagen and other biological material, prepare the three-dimensional porous rack of given shape, such as Chinese patent CN1394655 through freezing-lyophilizing, further prepare collagen/chitosan porous rack by vacuum dry heating method, aldehyde compound or Carbodiimides compound crosslink again, be mainly used in the tissue engineering support; This kind support has the general characteristic of collagen scaffold, but its porosity and homogeneity are relatively not good enough.Electrostatic spinning technique is a revolutionary technology of rising at Material Field in recent years, has particularly obtained broad research aspect tissue engineering material, also is a focus of research.Electrostatic spinning is by the mode spinning of high-pressure electrostatic, prepare porosity high, aperture and fibre diameter be controlled three-dimensional porous rack within the specific limits, at present relevant with collagen electrostatic spinning patent is considerably less, prepare the biomembrane of a kind of low-molecular-weight collagen peptide and Chitosan Oligosacchaides such as Chinese patent CN102477590A through electrostatic spinning technique, be mainly used in pharmaceuticals industry; And for example Chinese patent CN1944724 has prepared a kind of collagen protein and chitosan composite nano fibre through electrostatic spinning technique, is mainly used in bionical histiocyte epimatrix and medicament slow release.Above-mentioned patent technique is complicated, and controllability is relatively poor, needs more catalyst.
Summary of the invention
The object of the present invention is to provide a kind of preparation technology simply controlled, reaction condition is gentle, need not the novel collagen-hyaluronic acid of the used in tissue engineering of catalyst-chondroitin sulfate electrostatic spinning support preparation method.
The preparation method of collagen-hyaluronic acid of the present invention-chondroitin sulfate support follows these steps to carry out:
(1) preparation solvent: prepare 2,2 by 1-10:1 volume ratio, 2-trifluoroethanol and distillation water as solvent;
(2) preparation electrospinning liquid: add collagen, hyaluronic acid and chondroitin sulfate, collagen in solvent: hyaluronic acid: the weight ratio between the chondroitin sulfate is 4-16:1:0.5-3, and mixing speed 100-3000rpm stirs and obtains electrospinning liquid after 1-10 days;
(3) electrostatic spinning: electrospinning liquid carries out electrostatic spinning in the high-voltage electrostatic spinning device that is comprised of high voltage power supply, solution storage device, injection apparatus and receiving system, 10-40 ℃ of the temperature of electrostatic spinning, electrostatic spinning voltage 10-30kv, required syringe needle model is 5-No. 8 in the injection apparatus, the distance of syringe needle and receiving system is 5-15cm, injection apparatus liquor charging speed 5-25ml/h namely obtains collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning support.
Among the present invention, used solvent is that the volume ratio optimal proportion of trifluoroethanol and distilled water is 1:1.
Described collagen macromolecule collagen, molecular weight is between 1 ten thousand-30 ten thousand.
The best mixing time of electrospinning liquid is 3 days among the present invention, and best mixing speed is 500rpm.
30 ℃ of described electrostatic spinning optimum temperatures.
The best model of required syringe needle is No. 6 in the described injection apparatus.
The optimum distance of described syringe needle and receiving system is 10-12cm.
Described injection apparatus liquor charging optimum speed 9-11ml/h.
The optium concentration of described collagen-hyaluronic acid-chondroitin sulfate electrospinning liquid is 8% to 10%.
Optimal proportion between described collagen-hyaluronic acid-chondroitin sulfate is 6-8:0.5-1:0.5-2.
Key of the present invention is with 2,2, and 2-trifluoroethanol and distilled water mix by a certain percentage, and the hyaluronic acid, the chondroitin sulfate that make under normal atmosphere temperature neutral water-fast collagen and be insoluble to organic solvent are dissolved in this solvent altogether, and can carry out smoothly electrostatic spinning.This is that other organic solvents can't be finished such as organic solvents such as 6-fluorine isopropyl alcohols at present.
The support of the inventive method preparation, not only have three-dimensional porous structure, also have simultaneously the bionical character of extracellular matrix height, histocompatibility is good, size shape is controlled, and the diameter of brace aperture rate and spun nanofiber also can be controlled within the specific limits.And tissue most cells epimatrix is the network that is made of water, collagen protein, polysaccharide (hyaluronic acid and chondroitin sulfate etc.) and inanimate matter etc., so collagen-hyaluronic acid-chondroitin sulfate support particularly is with a wide range of applications at soft tissue engineering scaffold in the tissue engineering bracket field.
The present invention has following characteristics and advantage:
(1) preparation technology is simply controlled, and reaction condition is gentle, and equipment is simple;
(2) the brace aperture rate is higher, even pore distribution, and can regulate as required;
(3) this support be can be used as various kinds of cell and cultivate adhesion propagation carrier, conserve space, the cell proliferation increment can reach the several more than 10 times of general carrier on the support, can be used for the amplification of cell;
(4) this support composition has the bio-imitability of height near the main solid phase composition of cartilage cell epimatrix, and bio-compatible is good, and no cytotoxicity is a kind of good tissue engineering bracket material.
The specific embodiment
Below by embodiment the present invention is further described.
Embodiment 1:
Prepare 2 by the 1:1 volume ratio in advance, 2,2-trifluoroethanol and distillation water as solvent, after getting collagen 80mg solubilizer 1ml soaked overnight+hyaluronic acid 10mg+ chondroitin sulfate 10mg is made into the mixed liquor of total concentration 10%, 100 rpm, stir and be electrospinning liquid in 10 days, electrospinning liquid is by high voltage power supply, the solution storage device, carry out electrostatic spinning, electrostatic spinning voltage 20kv in the high-voltage electrostatic spinning device that injection apparatus and receiving system form, the present embodiment 5ml glass syringe, No. 6 syringe needles, syringe needle apart from dash receiver apart from 10cm, syringe pump liquor charging speed 10ml/h, 30 ℃ of ambient temperatures.Carry out obtaining collagen-hyaluronic acid-chondroitin sulfate three-dimensional electrostatic spinning ternary nano biological support behind the electrostatic spinning.
Embodiment 2:
Prepare 2 by the 2:1 volume ratio in advance, 2,2-trifluoroethanol and distillation water as solvent, after the collagen 80mg solubilizer 1ml soaked overnight+hyaluronic acid 20mg+ chondroitin sulfate 10mg is made into the mixed liquor of total concentration 11%, 500rpm, stir and be electrospinning liquid in 3 days, electrospinning liquid carries out electrostatic spinning in the high-voltage electrostatic spinning device, electrostatic spinning voltage 30kv, the present embodiment with 5ml glass syringe, No. 8 syringe needles, syringe needle apart from dash receiver apart from 10 ℃ of 15cm, syringe pump liquor charging speed 15ml/h, ambient temperatures.Carry out obtaining collagen-hyaluronic acid-chondroitin sulfate three-dimensional electrostatic spinning ternary nano biological support behind the electrostatic spinning.
Embodiment 3:
Prepare 2 by the 10:1 volume ratio in advance, 2,2-trifluoroethanol and distillation water as solvent, after the collagen 80mg solubilizer 1ml soaked overnight+hyaluronic acid 5mg+ chondroitin sulfate 15mg is made into the mixed liquor of total concentration 10%, 3000rpm, stir and be electrospinning liquid in 1 day, electrospinning liquid carries out electrostatic spinning in the high-voltage electrostatic spinning device, electrostatic spinning voltage 30kv, the present embodiment with 5ml glass syringe, No. 5 syringe needles, syringe needle apart from dash receiver apart from 20 ℃ of 5cm, syringe pump liquor charging speed 5ml/h, ambient temperatures.Carry out obtaining collagen-hyaluronic acid-chondroitin sulfate three-dimensional electrostatic spinning ternary nano biological support behind the electrostatic spinning.
Embodiment 4:
Prepare 2 by the 5:1 volume ratio in advance, 2,2-trifluoroethanol and distillation water as solvent, after the collagen 87.3mg solubilizer 1ml soaked overnight+hyaluronic acid 10.9mg+ chondroitin sulfate 21.8mg is made into the mixed liquor of total concentration 12%, 1000rpm, stir and be electrospinning liquid in 2 days, electrospinning liquid carries out electrostatic spinning in the high-voltage electrostatic spinning device, electrostatic spinning voltage 10kv, the present embodiment with 5ml glass syringe, No. 8 syringe needles, syringe needle apart from dash receiver apart from 40 ℃ of 5cm, syringe pump liquor charging speed 25ml/h, ambient temperatures.Carry out obtaining collagen-hyaluronic acid-chondroitin sulfate three-dimensional electrostatic spinning ternary nano biological support behind the electrostatic spinning.
Claims (10)
1. organizational project collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning support preparation method is characterized in that: follow these steps to carry out
(1) preparation solvent: prepare 2,2 by 1-10:1 volume ratio, 2-trifluoroethanol and distillation water as solvent;
(2) preparation electrospinning liquid: add collagen, hyaluronic acid and chondroitin sulfate, collagen in solvent: hyaluronic acid: the weight ratio between the chondroitin sulfate is 4-16:1:0.5-3, and mixing speed 100-3000rpm stirs and obtains electrospinning liquid after 1-10 days;
(3) electrostatic spinning: electrospinning liquid carries out electrostatic spinning in the high-voltage electrostatic spinning device that is comprised of high voltage power supply, solution storage device, injection apparatus and receiving system, 10-40 ℃ of the temperature of electrostatic spinning, electrostatic spinning voltage 1kv-30kv, required syringe needle model is 5-No. 8 in the injection apparatus, the distance of syringe needle and receiving system is 5-15cm, injection apparatus liquor charging speed 5-25ml/h namely obtains collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning support.
2. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: used solvent trifluoroethanol and the volume ratio ratio of distilled water are 1:1.
3. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: described collagen is macromolecule collagen, and molecular weight is between 1 ten thousand-30 ten thousand.
4. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: electrospinning liquid mixing time is 3 days, mixing speed is 500rpm.
5. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: described electrostatic spinning temperature is 30 ℃.
6. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: required syringe needle model is No. 6 in the described injection apparatus.
7. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: the distance of described syringe needle and receiving system is 10cm-12cm.
8. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: described injection apparatus liquor charging speed is 9-11ml/h.
9. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: the concentration of described collagen-hyaluronic acid-chondroitin sulfate electrospinning liquid is 8% to 10%.
10. a kind of organizational project collagen-hyaluronic acid according to claim 1-chondroitin sulfate electrostatic spinning support preparation method, it is characterized in that: the ratio between described collagen-hyaluronic acid-chondroitin sulfate is 6-8:0.5-1:0.5-2.
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| CN103603087A (en) * | 2013-11-15 | 2014-02-26 | 无锡中科光远生物材料有限公司 | Preparation method of nanofiber for soft tissue regeneration |
| CN105525441A (en) * | 2016-01-28 | 2016-04-27 | 华南理工大学 | Foldable multilayer cross-linked composite fiber net film as well as preparation method and application thereof |
| CN111441103A (en) * | 2020-05-27 | 2020-07-24 | 南京彼洲生物科技有限公司 | Medical nano-fiber composite material and preparation method and application thereof |
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| CN111441103A (en) * | 2020-05-27 | 2020-07-24 | 南京彼洲生物科技有限公司 | Medical nano-fiber composite material and preparation method and application thereof |
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Application publication date: 20130306 |