CN102532566B - Preparation method of interpenetrating network composite hydrogel - Google Patents
Preparation method of interpenetrating network composite hydrogel Download PDFInfo
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- CN102532566B CN102532566B CN 201110433125 CN201110433125A CN102532566B CN 102532566 B CN102532566 B CN 102532566B CN 201110433125 CN201110433125 CN 201110433125 CN 201110433125 A CN201110433125 A CN 201110433125A CN 102532566 B CN102532566 B CN 102532566B
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Abstract
The invention relates to a preparation method of composite hydrogel with an interpenetrating network structure. The preparation method comprises the following process steps of: (1) preparing a collagen solution with acetic acid, and preparing methacrylated chondroitin sulfate solution and methacrylated hyaluronic acid solution with deionized water, distilled water or phosphate buffer solution; (2) adjusting the pH value of the collagen solution prepared in the step (1) to 7.0-7.4 with sodium hydroxide solution under stirring at normal pressure and 0-4 DEG C, then adding the methacrylated chondroitin sulfate solution and methacrylated hyaluronic acid solution prepared in the step (1) to the collagen solution under stirring, uniformly mixing three solutions, then adding phosphate buffer solution for diluting until the collagen concentration in the mixed solution is 5-9mg/mL, then adding an initiator and a catalyst, and uniformly mixing to form a precursor solution; and (3) injecting theprecursor solution prepared in the step (2) to a mold, and placing the mold holding the precursor solution in an environment of normal pressure and 37 DEG C for 30-60minutes.
Description
Technical field
The invention belongs to the tissue engineering material field, particularly the preparation method of a kind of collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel.
Background technology
Articular cartilage damage is one of clinical common difficult and complicated cases.Joint cartilage is limit by himself physiological structure, and damage back Chang Buneng repairs voluntarily.Though at present clinically to the certain short term efficacy of the method for articular cartilage damage treatment+have, but all be based on the fibrous cartilage reparation, the mechanical property and the wearing quality that lack normal hyaline, thereby repairing effect is relatively poor usually, is difficult to obtain satisfied clinical effectiveness at a specified future date.
Cartilage tissue engineered treatment to cartilage injury provides a kind of novel method.Cartilage tissue engineered be external with the seed cell embedding or the plantation in three-dimensional rack, approach such as the adhesion by cell, propagation, differentiation, construct structure and physiological property and cartilaginous tissue and similarly organize, be transplanted to the cartilage defect position again and carry out repairing and treating.Desirable tissue engineering bracket should imitate composition and the structure of extracellular matrix as much as possible.
Collagen, chondroitin sulfate, hyaluronic acid all are the important component of cartilage cell epimatrix.In cartilaginous tissue, extracellular matrix has constituted a complicated network structure.Wherein, proteoglycan is the covalent conjunct agent of core protein and glycosaminoglycan (as chondroitin sulfate, keratan sulfate etc.), and it is combined with hyaluronic acid by connection albumen and forms the proteoglycan polymer.Proteoglycan by glycosaminoglycan on its side chain with a large amount of negative charges and collegen filament twine mutually, and with a large amount of moisture cohesions wherein, constitute the stable netted gelling system of wetting ability, thereby give cartilaginous tissue good biomechanical property, realize stable, the motor function flexibly in joint.
Yet, in all sorts of ways at present not exist with natural cartilage in collagen/chondroitin sulfate/hyaluronic acid of obtaining (being called for short CCH) composite aquogel and organize similar complex network structures, the cross-linked network that only exists collegen filament network or CS and HA to form by linking agent and collagen, although thereby the CCH composite aquogel mechanical property and the degradation property that obtain make moderate progress, but still remain further to be improved.Therefore, need other method of research to obtain and the similar CCH-IPN composite aquogel of natural cartilage weave construction.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of novel composite aquogel to obtain collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, improves mechanical property and the degradation property of collagen/chondroitin sulfate/hyaluronic acid composite aquogel.
Technical scheme of the present invention: by hyaluronic acid (HA) and chondroitin sulfate (CS) are carried out modification, in its molecule, introduce the functional group that is easy to react, and the self-crosslinking reaction of modification CS and modified HA can be carried out under the condition of collagen fibroblast, thereby under the condition of gentleness, obtain collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks (CCH-IPN) composite aquogel by the self-assembly polymerization of collagen and the self-crosslinking polymerization of modification CS and modified HA.
The preparation method of interpenetrating(polymer)networks composite aquogel of the present invention, processing step is as follows:
(1) preparation of material solution
Material solution is the chondroitin sulfate cellulose solution of collagen solution, methacrylic acidifying and the hyaluronic acid solution of methacrylic acidifying;
Acetic acid with volumetric concentration 0.5%～2% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 9mg/mL～14mg/mL with the concentration of collagen in collagen solution and is limited;
Chondroitin sulfate with the methacrylic acidifying under normal pressure, room temperature is mixed with solution with solvent, and the consumption of solvent can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited;
Hyaluronic acid with the methacrylic acidifying under normal pressure, room temperature is mixed with solution with solvent, and the consumption of solvent can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
The solvent of the hyaluronic acid solution of the chondroitin sulfate cellulose solution of preparation methacrylic acidifying and the acidifying of preparation methacrylic is a kind of in deionized water, distilled water, the phosphate buffer soln;
The chondroitin sulfate of methacrylic acidifying is obtained by chondroitin sulfate (CS) modification, the methacrylic acidifying modification of chondroitin sulfate (CS), see document: Wang LF, Shen SS, Lu SC.Synthesis and characterization of chondroitinsulfate-methacrylate hydrogels.Carbohydr Polym.2003; 52:389-396;
The hyaluronic acid of methacrylic acidifying is obtained by hyaluronic acid (HA) modification, the methacrylic acidifying modification of hyaluronic acid (HA), see document: Smeds KA, Grinstaff MW.Photocrosslinkable polysaccharides in situhydrogel formation.J Biomed Mater Res.2001; 54:115-121.
(2) preparation of preceding solution
Under agitation in normal pressure, 0 ℃～4 ℃ are adjusted to 7.0～7.4 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.0～7.4 the collagen solution that 0 ℃～4 ℃ hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, adding phosphate buffer soln dilutes, the concentration that makes collagen in the mixed solution is 5mg/mL～9mg/mL, add initiator and catalyzer again and mix and namely form preceding solution, the add-on of initiator reaches 4mmol/L with initiator concentration in the solution before described and is limited, and the add-on of catalyzer reaches 4mmol/L with catalyzer concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.0～7.4 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=2: 1～1: 2, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=7: 1～1: 7;
(3) become glue
The preceding solution of step (2) preparation is injected mould, and the mould of solution was placed 30 minutes～60 minutes in normal pressure, 37 ℃ environment before will containing then, namely formed collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel.
In order to be easy to mix, in the preparation steps of preceding solution, preferably initiator and catalyzer are mixed with solution with deionized water or distilled water or phosphate buffer soln and add in the described mixed solution.
In the aforesaid method, the daltonian chondroitin sulfate in preferred molecular weight 1～50,000 carries out modification, and preferred molecular weight is that 150～2,000,000 daltonian hyaluronic acids carry out modification.
In the aforesaid method, described initiator is ammonium persulphate or Potassium Persulphate, and described catalyzer is Tetramethyl Ethylene Diamine.
In the aforesaid method, the preferred 0.01M of described concentration of phosphate buffer.
Collagen/the chondroitin sulfate with inierpeneirating network structure/hyaluronic acid (CCH-IPN) composite aquogel that makes with above-mentioned preparation method can be used for joint cartilage reparation field.
Compared with prior art, the present invention has following beneficial effect:
1, the method for the invention can obtain collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, provides a kind of more preferably material for cartilage tissue engineered.
2, compare with existing collagen/chondroitin sulfate/hyaluronic acid composite aquogel, the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel of the method for the invention preparation, its mechanical property obviously improves, and degradation property obviously improves.
3, experiment shows, the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel of the method for the invention preparation has good cell consistency (seeing accompanying drawing 2).
4, the method for the invention is owing to select for use composition in the natural cartilage extracellular matrix (ECM) as raw material, therefore prepared collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel has bionical function, the microenvironment of similar ECM can be provided for cell, be conducive to the sprawling of cell, propagation and matrix secretion.
Description of drawings
Fig. 1 is the stereoscan photograph of composite aquogel, and wherein, A figure is the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel of the method for the invention preparation; B figure be the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel among the A figure in collagenase, degrade 50% the back remaining hydrogel; C figure is the composite aquogel of Comparative Examples preparation.
Behind the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel parcel chondrocyte of Fig. 2 for the method for the invention preparation, at the Laser Scanning Confocal Microscope photo of vitro culture after 7 days and 14 days, wherein, A figure is at 7 days Laser Scanning Confocal Microscope photo of vitro culture, and B figure is at 14 days Laser Scanning Confocal Microscope photo of vitro culture.
Embodiment
Further specified below by the method for the invention of embodiment.Among the following embodiment, collagen, chondroitin sulfate and hyaluronic acid all can be bought by market, and all the other raw materials are conventional chemical reagent.
Embodiment 1
Present embodiment carries out modification to chondroitin sulfate (CS) and hyaluronic acid (HA), the chondroitin sulfate of preparation methacrylic acidifying and the hyaluronic acid of methacrylic acidifying.In the present embodiment, the molecular weight of chondroitin sulfate is 1～50,000 dalton, and hyaluronic molecular weight is 150～2,000,000 dalton.
Chondroitin sulfate is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 4% with deionized water, mol ratio according to the hydroxyl of contained chondroitin sulfate in the chondroitin sulfate cellulose solution and methacrylic anhydride (MA) is 1: 10 metering methacrylic anhydride (MA), and with in the described chondroitin sulfate cellulose solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the chondroitin sulfate of methacrylic acidifying.
Hyaluronic acid is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 0.5% with deionized water, mol ratio according to contained hyaluronic hydroxyl in the hyaluronic acid solution and methacrylic anhydride (MA) is 1: 10 metering methacrylic anhydride (MA), and with in the described hyaluronic acid solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the hyaluronic acid of methacrylic acidifying.
Embodiment 2
Present embodiment carries out modification to chondroitin sulfate (CS) and hyaluronic acid (HA), the chondroitin sulfate of preparation methacrylic acidifying and the hyaluronic acid of methacrylic acidifying.In the present embodiment, the molecular weight of chondroitin sulfate is 1～50,000 dalton, and hyaluronic molecular weight is 150～2,000,000 dalton.
Chondroitin sulfate is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 4% with deionized water, mol ratio according to the hydroxyl of contained chondroitin sulfate in the chondroitin sulfate cellulose solution and methacrylic anhydride (MA) is 1: 5 metering methacrylic anhydride (MA), and with in the described chondroitin sulfate cellulose solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the chondroitin sulfate of methacrylic acidifying.
Hyaluronic acid is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 0.5% with deionized water, mol ratio according to contained hyaluronic hydroxyl in the hyaluronic acid solution and methacrylic anhydride (MA) is 1: 5 metering methacrylic anhydride (MA), and with in the described hyaluronic acid solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the hyaluronic acid of methacrylic acidifying.
Embodiment 3
Present embodiment carries out modification to chondroitin sulfate (CS) and hyaluronic acid (HA), the chondroitin sulfate of preparation methacrylic acidifying and the hyaluronic acid of methacrylic acidifying.In the present embodiment, the molecular weight of chondroitin sulfate is 1～50,000 dalton, and hyaluronic molecular weight is 150～2,000,000 dalton.
Chondroitin sulfate is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 4% with deionized water, mol ratio according to the hydroxyl of contained chondroitin sulfate in the chondroitin sulfate cellulose solution and methacrylic anhydride (MA) is 1: 1 metering methacrylic anhydride (MA), and with in the described chondroitin sulfate cellulose solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the chondroitin sulfate of methacrylic acidifying.
Hyaluronic acid is mixed with the chondroitin sulfate cellulose solution of mass/volume concentration 0.5% with deionized water, mol ratio according to contained hyaluronic hydroxyl in the hyaluronic acid solution and methacrylic anhydride (MA) is 1: 1 metering methacrylic anhydride (MA), and with in the described hyaluronic acid solution of methacrylic anhydride (MA) adding, at normal pressure, 4 ℃ of stirring reaction 24h, dehydrated alcohol precipitates then, and with absolute ethanol washing 5～6 times, continue after reaction product after will washing be dissolved in deionized water, dialysed 4 days, and the reaction product lyophilize after the dialysis was namely obtained the hyaluronic acid of methacrylic acidifying.
Embodiment 4
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 0.5% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 9mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying of embodiment 1 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 4 ℃ are adjusted to 7.0 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.0 the collagen solution that 4 ℃ of hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 5mg/mL, add the ammonium persulfate solution that concentration is 1mol/L (preparing with deionized water) and concentration again and be the Tetramethyl Ethylene Diamine solution (preparing with deionized water) of 1mol/L and mix namely form before solution, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with ammonium persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.0 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=2: 1, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=7: 1;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 30 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 27.7 ± 1.8kPa, degraded about 50% after 60 hours in collagenase.The composite aquogel of present embodiment preparation, microscopic appearance after its freeze-drying is seen the A figure among Fig. 1, microscopic appearance after it was degraded in collagenase 60 hours is seen the B figure among Fig. 1, has proved the existence of inierpeneirating network structure in the collagen/chondroitin sulfate/hyaluronic acid composite aquogel of present embodiment preparation.
Comparative Examples
This Comparative Examples is the Comparative Examples of embodiment 2.
(1) preparation of material solution
Acetic acid with volumetric concentration 0.5% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 9mg/mL with the concentration of collagen in collagen solution and is limited,
Under normal pressure, room temperature chondroitin sulfate is mixed with solution with deionized water, the consumption of deionized water can dissolve fully with chondroitin sulfate and be limited,
Under normal pressure, room temperature hyaluronic acid is mixed with solution with deionized water, the consumption of deionized water can dissolve fully with hyaluronic acid and be limited;
(2) preparation of preceding solution
Under agitation with sodium hydroxide solution the pH value of the collagen solution of step (1) preparation is adjusted to 7.0 in normal pressure, 4 ℃, under agitation the chondroitin sulfate cellulose solution of step (1) preparation and hyaluronic acid solution being added the pH value in normal pressure, 4 ℃ then is in 7.0 the collagen solution, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, and the concentration that makes collagen in the mixed solution is that 5mg/mL namely forms preceding solution
Described pH value is that 7.0 collagen solution, chondroitin sulfate cellulose solution and the hyaluronic acid solution of step (1) preparation measure by following proportioning:
In the mixed solution, the quality of collagen: the quality of chondroitin sulfate+hyaluronic quality=2: 1, chondroitin sulfate quality: hyaluronic quality=7: 1;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 30 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid composite aquogel, its modulus of compression is 8.7 ± 0.7kPa, degraded fully after 24 hours in collagenase.Microscopic appearance after described collagen/chondroitin sulfate/hyaluronic acid composite aquogel freeze-drying is seen the C figure among Fig. 1, A figure among C among Fig. 1 figure and Fig. 1 is compared, and in conjunction with the degraded state, proved that the prepared collagen/chondroitin sulfate of Comparative Examples/hyaluronic acid composite aquogel does not have inierpeneirating network structure.
Embodiment 5
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 1% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 12mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with distilled water, and the consumption of distilled water can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with distilled water, and the consumption of distilled water can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 2 ℃ are adjusted to 7.4 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.4 the collagen solution that 2 ℃ of hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 5mg/mL, add the ammonium persulfate solution that concentration is 1mol/L (preparing with distilled water) and concentration again and be the Tetramethyl Ethylene Diamine solution (preparing with distilled water) of 1mol/L and mix namely form before solution, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with ammonium persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.4 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=2: 1, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=7: 1;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 40 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 39.9 ± 2.0kPa, degraded about 50% after 60 hours in collagenase.The composite aquogel of present embodiment preparation, microscopic appearance after its freeze-drying is similar to the A figure among Fig. 1, microscopic appearance after it was degraded in collagenase 60 hours is similar to the B figure among Fig. 1, with behind the composite aquogel parcel chondrocyte of present embodiment preparation vitro culture 7 days and 14 days, its Laser Scanning Confocal Microscope photo is seen Fig. 2, A figure from Fig. 2 and B figure are as can be seen, surpass 95% cell well-grown, proved that the collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel of the method for the invention preparation has the good cell consistency.
Embodiment 6
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 2% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 14mg/mL with the concentration of collagen in collagen solution and is limited,
The phosphate buffer soln that with the chondroitin sulfate concentration of the methacrylic acidifying of embodiment 2 preparation is 0.01M under normal pressure, room temperature is mixed with solution, and the consumption of described phosphate buffer soln can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
The phosphate buffer soln that with the hyaluronic acid concentration of the methacrylic acidifying of embodiment 3 preparation is 0.01M under normal pressure, room temperature is mixed with solution, and the consumption of described phosphate buffer soln can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 3 ℃ are adjusted to 7.4 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.4 the collagen solution that 3 ℃ of hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 7mg/mL, add the Tetramethyl Ethylene Diamine solution that the ammonium persulfate solution that concentration is 1mol/L (be the phosphate buffer soln preparation of 0.01M with concentration) and concentration are 1mol/L (be the phosphate buffer soln preparation of 0.01M with concentration) again and mix the preceding solution of i.e. formation, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with ammonium persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.4 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=1: 1, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=1: 7;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 60 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 24.9 ± 3.1kPa, and the microscopic appearance after its freeze-drying is similar to the A figure among Fig. 1.
Embodiment 7
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 0.5% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 9mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying of embodiment 3 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying of embodiment 3 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 0 ℃ is adjusted to 7.4 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.4 the collagen solution that 0 ℃ of hyaluronic acid solution with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying adds the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 5mg/mL, add the ammonium persulfate solution that concentration is 1mol/L (preparing with deionized water) and concentration again and be the Tetramethyl Ethylene Diamine solution (preparing with deionized water) of 1mol/L and mix namely form before solution, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with ammonium persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.4 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=1: 2, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=7: 1;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 30 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 36.8 ± 3.8kPa, and the microscopic appearance after its freeze-drying is similar to the A figure among Fig. 1.
Embodiment 8
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 2% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 14mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 2 ℃ are adjusted to 7.2 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.2 the collagen solution that 2 ℃ of hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 9mg/mL, add the ammonium persulfate solution that concentration is 1mol/L (preparing with deionized water) and concentration again and be the Tetramethyl Ethylene Diamine solution (preparing with deionized water) of 1mol/L and mix namely form before solution, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with Potassium Persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.2 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=1: 1, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=1: 1;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 50 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 28.7 ± 1.27kPa, and the microscopic appearance after its freeze-drying is similar to the A figure among Fig. 1.
Embodiment 9
Present embodiment prepares collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, and processing step is as follows:
(1) preparation of material solution
Acetic acid with volumetric concentration 1% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 12mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying of embodiment 2 preparation under normal pressure, room temperature is mixed with solution with deionized water, and the consumption of deionized water can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited;
(2) preparation of preceding solution
Under agitation in normal pressure, 4 ℃ are adjusted to 7.2 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.2 the collagen solution that 4 ℃ of hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, the phosphate buffer soln that adds concentration and be 0.01M dilutes, the concentration that makes collagen in the mixed solution is 7mg/mL, add the ammonium persulfate solution that concentration is 1mol/L (preparing with deionized water) and concentration again and be the Tetramethyl Ethylene Diamine solution (preparing with deionized water) of 1mol/L and mix namely form before solution, concentration is that the add-on of the ammonium persulfate solution of 1mol/L reaches 4mmol/L with Potassium Persulphate concentration in the solution before described and is limited, and the add-on of Tetramethyl Ethylene Diamine solution reaches 4mmol/L with Tetramethyl Ethylene Diamine concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.2 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying=2: 1, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality of methacrylic acidifying=2: 5;
(3) become glue
The preceding solution of step (2) preparation is added in the mould, the mould of solution was placed 30 minutes in normal pressure, 37 ℃ environment before will containing then, namely form collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel, its modulus of compression is 30.2 ± 2.26kPa, and the microscopic appearance after its freeze-drying is similar to the A figure among Fig. 1.
Claims (5)
1. the preparation method of an interpenetrating(polymer)networks composite aquogel is characterized in that processing step is as follows:
(1) preparation of material solution
Material solution is the chondroitin sulfate cellulose solution of collagen solution, methacrylic acidifying and the hyaluronic acid solution of methacrylic acidifying,
Acetic acid with volumetric concentration 0.5%～2% under normal pressure, room temperature is solvent preparation collagen solution, and the consumption of acetic acid reaches 9mg/mL～14mg/mL with the concentration of collagen in collagen solution and is limited,
Chondroitin sulfate with the methacrylic acidifying under normal pressure, room temperature is mixed with solution with solvent, and the consumption of solvent can dissolve fully with the chondroitin sulfate of methacrylic acidifying and be limited,
Hyaluronic acid with the methacrylic acidifying under normal pressure, room temperature is mixed with solution with solvent, and the consumption of solvent can dissolve fully with the hyaluronic acid of methacrylic acidifying and be limited,
The solvent of the hyaluronic acid solution of the chondroitin sulfate cellulose solution of preparation methacrylic acidifying and the acidifying of preparation methacrylic is a kind of in deionized water, distilled water, the phosphate buffer soln;
(2) preparation of preceding solution
Under agitation in normal pressure, 0 ℃～4 ℃ are adjusted to 7.0～7.4 with sodium hydroxide solution with the pH value of the collagen solution of step (1) preparation, then under agitation in normal pressure, it is in 7.0～7.4 the collagen solution that 0 ℃～4 ℃ hyaluronic acid solutions with the chondroitin sulfate cellulose solution of the methacrylic acidifying of step (1) preparation and methacrylic acidifying add the pH value, after three kinds of solution mix, adding phosphate buffer soln dilutes, the concentration that makes collagen in the mixed solution is 5mg/mL～9mg/mL, add initiator and catalyzer again and mix and namely form preceding solution, the add-on of initiator reaches 4mmol/L with initiator concentration in the solution before described and is limited, and the add-on of catalyzer reaches 4mmol/L with catalyzer concentration in the solution before described and is limited;
Described pH value be the hyaluronic acid solution of the chondroitin sulfate cellulose solution of methacrylic acidifying of 7.0～7.4 collagen solution, step (1) preparation and methacrylic acidifying by following proportioning metering:
In the mixed solution, the quality of collagen: the hyaluronic quality=2:1～1:2 of the quality of the chondroitin sulfate of methacrylic acidifying+methacrylic acidifying, the chondroitin sulfate quality of methacrylic acidifying: the hyaluronic quality=7:1～1:7 of methacrylic acidifying;
Described initiator is ammonium persulphate or Potassium Persulphate, and described catalyzer is Tetramethyl Ethylene Diamine;
(3) become glue
The preceding solution of step (2) preparation is injected mould, and the mould of solution was placed 30 minutes～60 minutes in normal pressure, 37 ℃ environment before will containing then, namely formed collagen/chondroitin sulfate/hyaluronic acid interpenetrating(polymer)networks composite aquogel.
2. according to the preparation method of the described collagen/chondroitin sulfate of claim 1/hyaluronic acid interpenetrating(polymer)networks composite aquogel, before it is characterized in that in the preparation steps of solution, initiator and catalyzer are mixed with solution with deionized water or distilled water or phosphate buffer soln add in the described mixed solution.
3. according to the preparation method of claim 1 or 2 described collagen/chondroitin sulfates/hyaluronic acid interpenetrating(polymer)networks composite aquogel, the molecular weight that it is characterized in that described chondroitin sulfate is 1～50,000 dalton, and hyaluronic molecular weight is 150～2,000,000 dalton.
4. according to the preparation method of claim 1 or 2 described interpenetrating(polymer)networks composite aquogels, it is characterized in that described concentration of phosphate buffer is 0.01M.
5. according to the preparation method of the described interpenetrating(polymer)networks composite aquogel of claim 3, it is characterized in that described concentration of phosphate buffer is 0.01M.
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| CN106620876B (en) * | 2017-01-12 | 2019-10-25 | 广东泰宝医疗器械技术研究院有限公司 | A kind of repair of cartilage hydrogel and preparation method thereof |
| CN108850764B (en) * | 2018-07-17 | 2021-07-20 | 南京林业大学 | Preparation method of hydrogel for improving stability of blueberry anthocyanin |
| US20220218868A1 (en) * | 2019-05-07 | 2022-07-14 | Roquette Freres | Novel polysaccharide-based hydrogel scaffolds for wound care |
| CN111214702A (en) * | 2020-03-04 | 2020-06-02 | 上海交通大学医学院附属第九人民医院 | Injectable bionic repair material for defects of temporomandibular joint disc and preparation method and application thereof |
| WO2022028565A1 (en) * | 2020-08-06 | 2022-02-10 | 华夏司印(上海)生物技术有限公司 | Bionic tissue stent, preparation method therefor and application thereof |
| CN112472368B (en) * | 2020-12-22 | 2023-04-21 | 广东广纳安疗科技有限公司 | Joint implant with cartilage tissue formation promoting functional coating and preparation method thereof |
| CN112999408B (en) * | 2021-03-02 | 2022-05-10 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of cryogel scaffold |
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