CN100536933C - Grafted protein hydrogel biological material and its preparing process - Google Patents
Grafted protein hydrogel biological material and its preparing process Download PDFInfo
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- CN100536933C CN100536933C CNB2006100766497A CN200610076649A CN100536933C CN 100536933 C CN100536933 C CN 100536933C CN B2006100766497 A CNB2006100766497 A CN B2006100766497A CN 200610076649 A CN200610076649 A CN 200610076649A CN 100536933 C CN100536933 C CN 100536933C
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Abstract
The invention relates to an aquagel material used to recover hurt nerve center. Wherein, it uses nerve center cross method to graft the layer adhesive protein (Laninin) with nerve grow activity to the molecule skeleton of transparent acid aquagel, to rpare the aquagel frame, which can accelerate the regeneration of nerve axon.
Description
Technical field
The present invention relates to biomaterial of a kind of central nervous system injury reparation usefulness and preparation method thereof.
Background technology
The damage of contemporary medical science application organizes engineering method repair tissue is exactly at damage or defect filled biomass material, and the three-dimensional rack of autogenous cell to damage location migration growth is provided, and reaches the purpose of reconstruction of damage location form and functional rehabilitation.
For the biomaterial that is applied to the central nervous system, except that should possessing general performances such as biocompatibility, machinability and sterilizability, also should have following performance characteristics: (1) is degradation in vivo and absorbed by body in time, and promptly the degradation speed of material and metabolism infiltration rate should be complementary with the speed of neuranagenesis reparation; (2) should have macroporous structure,, blood capillary and fibrous tissue can be grown into to guarantee the neural required three dimensions of repairing; (3) should guarantee the neural required nutrition supply of repairing, the required adjusting played nerve growth, differentiation and promotion nerve are repaired and the nerve growth factor of tissue regeneration promptly to provide injured nerve to regenerate.So far, the disclosed both at home and abroad tissue engineering material that is used for neural reparation has collagen stroma, acrylonitrile and vinyl chloride copolymer, Merlon, nitrocellulose, poly-alpha hydroxy acid fruit acid or silicones etc.These materials can promote the neurocyte axonal regeneration in varying degrees, but can't be satisfactory at aspects such as biocompatibility, physicochemical property, degradation rate and slow-releasing.
On the tissue engineering development history, (Hyaluronic acid HA) is a kind of biomaterial that early is employed to hyaluronic acid.Extraction separation is to hyaluronic acid in bovine vitreous body for Meyer in 1934 and Palmer, and the 1970's took the lead in being used for ophthalmology as the viscoelasticity protective agent, were used as the material for the treatment of osteoarthritis afterwards again.But because pure hyaluronic acid derivatives contains a large amount of polyanions, be not suitable for cell adhesion and as the substrate of nerve growth, cause hyaluronic acid as tissue renovation material not in central nervous system's extensive use.
Summary of the invention
The object of the present invention is to provide a kind of implantation brain injury position, be suitable for the migration growth of neurocyte, can provide nutrition, promote the biomaterial of nervous tissue's Regeneration and Repair for neurocyte.
The present invention is used for the biomaterial that central nervous system injury is repaired, and is the method that adopts covalent cross-linking, with hyaluronic acid and laminin (laminin) made loose porous hydrogel crosslinked together.It is to carry out crosslinked method with adipic dihydrazide (ADH) with hyaluronic acid (HA) under the mediation of carbodiimide hydrochloride (EDC) to prepare hyaluronic acid derivatives; Adopt the method for covalent cross-linking, hyaluronic acid and laminin is crosslinked together, adopt Freeze Drying Technique, preparation has active hydrogel, as the frame material of cerebral tissue engineering.Its concrete preparation method is:
1) with deionized water preparation mass percent be 0.8～1.2% hyaluronic acid solution, transferring pH value of solution with hydrochloric acid solution is acidity, and optimum pH value is 4.75; Successively add adipic dihydrazide and carbodiimide hydrochloride, stir.Be 6～8 with the sodium hydroxide solution adjust pH afterwards, get hyaluronic acid gel.Hydrogel is cleaned in ultrasound wave 2～3 times with deionized water, put into the freeze dryer lyophilization after freezing.
2) with the acetone gradient dehydration with variable concentrations of the hydrogel of swelling, form the desiccant gel polymer, add 1,1 '-carbonyl dimidazoles (CDI) solution to be to activate the oh group in the hydrogel, make hydrogel can with the combining of laminin; Wash to remove unconjugated carbonyl dimidazoles, with the acetone in the sodium carbonate buffer solution washing replacing water gel of the activatory gel usefulness of carbonyl dimidazoles 100mM with dry acetone; Laminin solution is at room temperature mixed stirring 24～48 hours with activatory hydrogel; The product that obtains was cleaned in phosphate buffer 4 hours fully; The sodium bicarbonate buffer solution sealing of the 100mM of pH=7～9 2 hours; Carry out lyophilization, make the activated hydrogel of getting everything ready, as the frame material of cerebral tissue engineering.
Wherein, the adding adipic dihydrazide is many more in preparation hyaluronic acid derivatives process, and the degree of cross linking is high more, and the hardness of gel is high more, and degradation time in vivo is long more.Therefore, can regulate final products degradation time in vivo by the consumption that changes adipic acid dihydrazide.Suitable adipic dihydrazide quality proportioning is adipic dihydrazide: hyaluronic acid=1: 4～1: 10; Best proportioning is adipic dihydrazide: hyaluronic acid=1:6.Used carbodiimide hydrochloride quality proportioning is carbodiimide hydrochloride: hyaluronic acid=1: 2.5～1: 5.0.
Can also in the hyaluronan molecule chain, introduce sulfonic acid group to prolong the hydrogel degradation in vivo time.
The present invention adopts 1, and 1 '-carbonyl dimidazoles with the activation of the hydroxyl in the gelatin polymer, and holds and the activated positions combination N of polypeptide as the polypeptide cross-linking agent.
The present invention adopts hyaluronic acid as frame material its unique advantage to be arranged; Therefore hyaluronic repetition disaccharide unit all is consistent in all species and tissue, does not cause the immunologic rejection of self; Hyaluronic acid solution has the viscoelasticity of height, is similar to vivo biological tissue; Hyaluronic acid has the chemical composition of regulating extracellular fluid, lubricated and functions such as promotion wound healing, the formation of inhibition scar tissue; The effect of regulating inflammation, promoting angiogenesis is arranged.It is a kind of possess hydrophilic property and water-swollen polymer, along with the generation of its loose structure, has had support and inductive potential to the cambium oriented growth.
Pure hyaluronic acid derivatives contains a large amount of polyanions, is not suitable for cell adhesion and as the substrate of nerve growth.And laminin is a kind of high molecular weight cell epimatrix glycoprotein, can promote adhesion, growth, migration, differentiation and the axon regeneration of cell.The present invention adopts the method for chemical graft, has just overcome the pure hyaluronic acid hydrogel and is unfavorable for the adherent drawback of neuron hyaluronic acid and laminin are crosslinked.The inventor has detected the neurocyte compatibility of this biomaterial by the method for neuronal cell cultures, and to the biocompatibility of this material in rat cerebral tissue with promote the ability of tissue repair to detect, confirm that this biomaterial can significantly promote the attaching and the neural axon regeneration of neurocyte, promote revascularization simultaneously in addition, suppress the effect that glial scar forms.
The specific embodiment
The invention will be further described by the following examples.
Embodiment 1 preparation hyaluronic acid gel
With deionized water preparation mass percent is 1% hyaluronic acid solution, and the 1M hydrochloric acid solution is dropwise added hyaluronic acid solution, stirs simultaneously, makes the two fully mixed, reaches 4.75 until its pH value; Take by weighing adipic dihydrazide by 10%～25% of used hyaluronic acid amount and add in the hyaluronic acid solution, fully stir; Take by weighing carbodiimide hydrochloride by 20%～40% of used hyaluronic acid amount again and add in the hyaluronic acid solution, stirred 10～20 minutes; Be 6～8 with the sodium hydroxide solution adjust pH again, get hyaluronic acid gel; Hydrogel is cleaned in ultrasound wave 2～3 times with deionized water, and putting into freeze dryer after freezing carries out lyophilization.
Embodiment 2 hydrogels and laminin covalent cross-linking
(1) get the aquogel polymer 3～4g of swelling, use acetone/DW solution of 30/70,60/40,80/20 respectively to wash once successively, pure acetone washes twice, and dry acetone is given a baby a bath on the third day after its birth time, progressively with the aquogel polymer dehydration, is kept in the dry acetone at last;
(2) with 5ml 1,1 '-carbonyl dimidazoles (CDI) solution joins in the desiccant gel polymer of step 1 preparation, and priming reaction is 15 minutes under gentle agitation.CDI is made into 150mg/mL with anhydrous propanone;
(3) wash more than 5 times with dry acetone, remove unconjugated CDI, the sodium carbonate buffer solution (PH=8.5) of the activatory gel of CDI with 100mM is washed 5 times, replace whole acetone solvents;
(4) the activatory hydrogel of CDI is immersed in the 100mM sodium bicarbonate buffer solution (0.6mg/ml) of pH=8.5;
(5) laminin solution is at room temperature mixed stirring 24～48 hours with activatory hydrogel;
(6) hydrogel was cleaned 4 hours in phosphate buffer (pH=7.4) fully;
(7) the further sodium bicarbonate buffer solution of the 100mM of pH=8.5 sealing at room temperature 2 hours;
(8) lyophilizing on freezer dryer.
The phenetic analysis of embodiment 3 hydrogels
The gel of getting after the lyophilization is observed under scanning electron microscope, and as seen it is a loose porous framework structure.Its hole is covered with whole gel, and hole wall is very thin, and direct or indirect connection is arranged between each hole.The aperture in each hole is about 100～200 μ m.Adopt mercury injection apparatus that the porosity and the pore-size distribution of the gel under the dry state are detected, measure liquid mercury is invaded under the dry state volume and pressure in hole, the result is aperture uneven distribution between 0～1000 μ m of this biomaterial, most hole concentrates between 20～200 μ m, wherein the Kongzui of aperture about 80 μ m is many, and mean porosities is 90%.
Above-mentioned testing result shows, can hold a large amount of cell migration by the hydrogel of the inventive method preparation and enter, and is beneficial to new vessels especially and grows into.
Embodiment 4 water absorption rates are measured
Take by weighing the dried hydrogel solid of constant weight, 4 ℃ of balances blotted with the water of filter paper with hydrogel surface after 2 hours in the phosphate buffer solution of pH=7.2, weighed, and calculated water absorption rate according to following formula.
H=(W
Aq-W
Dry)/W
AqH: water absorption rate, W
Aq: suction back weight, W
Dry: weight when dry
The water absorption rate of biomaterial of the present invention is 75-95%.This is the index that the reflection hydrogel comprises outlet capacity, also is the important indicator of the porosity of reflection gel.This illustrates that also the gel by the inventive method preparation has very high porosity.
Transplantation experiments detects in embodiment 5 cell culture and the body
The present invention confirms that by cell culture the hydrogel of grafting laminin can significantly promote the attaching of neurocyte, and can promote the regeneration of neural axon after hyaluronic acid gel is passed through grafting laminin modification.With grafting the hydrogel of laminin implant in the adult rat cerebral cortex of damage, find that gel can promote the inside of the migration gel of the regeneration of blood vessel and glial cell, and can suppress the formation of glial scar.In grafting the hydrogel of laminin can significantly promote the inside of the gel of growing into of aixs cylinder, and do not have not find in the hydrogel of grafting laminin that aixs cylinder grows into, just found fiber at the interface at gel and cerebral tissue.
Experiment in vitro confirms that cultivating through 5 days at the neurocyte of control material (pure hyaluronic acid) does not have adherent and sign axon growth, and in grafting the neurocyte cultivated on the gel of laminin attach fine and grow aixs cylinder.
Claims (4)
1. one kind is used for the biomaterial that central nervous system injury is repaired, it is characterized in that, it is under the mediation of carbodiimide hydrochloride hyaluronic acid and adipic dihydrazide to be carried out crosslinked preparation hyaluronic acid gel, reuse 1,1 '-carbonyl dimidazoles is crosslinked as polypeptide cross-linking agent and laminin (laminin), by cryodesiccated method, water is prepared into as pore creating material has active loose structure hydrogel; Wherein,
The hyaluronic quality proportioning of used carbodiimide hydrochloride is 1: 2.5～1: 5.0;
Used adipic dihydrazide and hyaluronic quality proportioning are 1: 4～1: 10.
2. biomaterial according to claim 1 is characterized in that, the aperture of this biomaterial that exists with the porous aquagel form is 20～200 μ m.
3. the preparation method of claim 1 or 2 described biomaterials is characterized in that, it comprises following processing step:
1) with deionized water preparation 0.8～1.2% hyaluronic acid solution, transferring the solution pH value with hydrochloric acid solution is acidity, successively add adipic dihydrazide and carbodiimide hydrochloride, with the sodium hydroxide solution adjust pH is 6～8, hyaluronic acid gel, in ultrasound wave with lyophilization after the washed with de-ionized water;
2) hydrogel with swelling carries out the dehydration of gradient acetone, adds 1,1 '-carbonyl dimidazoles solution, and with the dry acetone washing, the washing of reuse sodium carbonate buffer solution; Laminin solution at room temperature mixed with activatory hydrogel stirred 24～48 hours, in phosphate buffer, cleaned fully 4 hours, in the sodium bicarbonate buffer solution sealing of the 100mM of pH=7～9 2 hours, lyophilization.
4. according to the preparation method of the described biomaterial of claim 3, it is characterized in that transferring the hyaluronic acid solution pH value with hydrochloric acid solution is 4.75.
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| CNB2006100766497A CN100536933C (en) | 2006-04-28 | 2006-04-28 | Grafted protein hydrogel biological material and its preparing process |
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| CN101965357B (en) * | 2007-08-15 | 2014-11-05 | 特拉维夫大学拉莫特有限公司 | Polypeptides, matrices, hydrogels and methods of using same for tissue regeneration and repair |
| CN104491925B (en) * | 2014-12-17 | 2016-04-06 | 浙江大学 | A kind of gel stent implant system of combined with mesenchymal stem cells and application thereof |
| EP3165233B1 (en) * | 2015-08-28 | 2021-08-18 | Latvijas Universitate | Biomaterial for treatment of acute and chronic skin wounds |
| CN114904062B (en) * | 2022-04-29 | 2023-04-28 | 中国科学院金属研究所 | Vascular stent with selective biological functionalization and preparation method thereof |
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Non-Patent Citations (2)
| Title |
|---|
| 应用细胞外基质修复周围神经缺损的研究进展. 梁安霖，蒋电明.中国临床康复，第9卷第13期. 2005 |
| 应用细胞外基质修复周围神经缺损的研究进展. 梁安霖，蒋电明.中国临床康复，第9卷第13期. 2005 * |
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