CN115770323A - Recombinant collagen gel dressing and preparation method and application thereof - Google Patents
Recombinant collagen gel dressing and preparation method and application thereof Download PDFInfo
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- CN115770323A CN115770323A CN202211656646.6A CN202211656646A CN115770323A CN 115770323 A CN115770323 A CN 115770323A CN 202211656646 A CN202211656646 A CN 202211656646A CN 115770323 A CN115770323 A CN 115770323A
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- recombinant collagen
- collagen
- recombinant
- phosphate
- solution
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 102000008186 Collagen Human genes 0.000 claims abstract description 116
- 108010035532 Collagen Proteins 0.000 claims abstract description 116
- 229920001436 collagen Polymers 0.000 claims abstract description 115
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 66
- 206010052428 Wound Diseases 0.000 claims abstract description 65
- 239000002245 particle Substances 0.000 claims abstract description 48
- 239000007864 aqueous solution Substances 0.000 claims abstract description 45
- 239000002562 thickening agent Substances 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- 238000004132 cross linking Methods 0.000 claims abstract description 25
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 21
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 21
- 239000010452 phosphate Substances 0.000 claims abstract description 21
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 20
- 238000001035 drying Methods 0.000 claims abstract description 18
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 51
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- 238000000034 method Methods 0.000 claims description 12
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 8
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- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 claims description 6
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- 208000008960 Diabetic foot Diseases 0.000 claims description 4
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 4
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
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- 150000003839 salts Chemical class 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000005057 Hexamethylene diisocyanate Substances 0.000 claims description 2
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- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 claims description 2
- 229940062672 calcium dihydrogen phosphate Drugs 0.000 claims description 2
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- 238000000855 fermentation Methods 0.000 claims description 2
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- 238000010353 genetic engineering Methods 0.000 claims description 2
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 claims description 2
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- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 2
- 235000019691 monocalcium phosphate Nutrition 0.000 claims description 2
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims description 2
- 235000019830 sodium polyphosphate Nutrition 0.000 claims description 2
- 229940048086 sodium pyrophosphate Drugs 0.000 claims description 2
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims description 2
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
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- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 2
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- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Materials For Medical Uses (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a recombinant collagen gel dressing and a preparation method and application thereof, belonging to the technical field of collagen gel dressings. The recombinant collagen gel dressing is prepared by adopting the recombinant collagen crosslinked particles obtained by crosslinking and crushing through the crosslinking agent as a matrix raw material, mixing the matrix raw material with an aqueous solution of a thickening agent, adding phosphate as a stabilizing agent, and drying without drying. The dressing can be directly applied to the wound surface, is convenient to use and is not limited by the position and the shape of the wound surface; the degradation resistance is good, and the repair requirement of the wound surface needing to be recovered for a longer time can be met; moderate humidity and good absorption performance, can quickly absorb tissue fluid exuded from wounds and promote the healing of the wounds; no subcutaneous irritation, no potential cytotoxicity and no sensitization reaction.
Description
Technical Field
The invention belongs to the technical field of collagen gel dressings, and particularly relates to a recombinant collagen gel dressing as well as a preparation method and application thereof.
Background
Skin tissue is often damaged by mechanical, physical, chemical and biological factors, and the repair process of the body is called tissue repair. According to the healing cycle of the wound surface, the wound surface can be divided into an acute wound surface and a chronic wound surface, wherein the acute wound surface generally refers to abrasion, knife wound, gun wound, operation incision, burn, chemical injury and the like, and the chronic wound surface refers to diabetic foot ulcer, leg ulcer, pressure sore and the like. For common wounds, a normal body can repair the wound by itself, but for some wounds with overlarge damaged area or chronic wounds, the body can not be completely repaired by itself, and medical treatment intervention is needed for auxiliary treatment. The tooth extraction wound is a wound left after tooth extraction, once the tooth extraction wound is not well healed, postoperative bleeding, pain, swelling, infection and other complications can be caused, the healing time of bone tissues is prolonged, and great influence is caused on the chewing function and daily life of a patient.
Various medical dressings are commonly used as skin repair materials, the traditional dressings are mostly solid inert dressings, comprise absorbent cotton, plaster, gauze and bandages, are used for covering wounds and preventing wound infection, have no promotion effect on wound healing, only have physical protection effect, and have the advantages of low price and simple manufacture; has the disadvantages of easy wound adhesion, secondary wound caused by dressing change, and prolonged wound healing time. With the development of science and technology, the horn of the head is exposed in interactive wound repair dressings which mainly comprise foam dressings, hydrogel dressings, hydrocolloid dressings and alginate dressings, and although the dressings have the characteristics of air permeability, bacteria isolation, high water content or high absorptivity and the like, the foam dressings and the hydrocolloid dressings need to be frequently changed, and foam fragments are easy to remain on the wound to cause more scars; the hydrogel dressing is easy to generate bacterial breeding and is not suitable for infected and severe drainage wounds; the alginate dressing can dehydrate the wound and delay healing, and is not suitable for dry wounds.
The wet wound healing theory is proposed by Winter in the early 60 s of the 20 th century, the development and application of skin wound healing materials are revolutionarily changed, and various novel wound repair materials are developed successively. The bioactive wound dressing is mostly made of natural materials or artificial synthetic materials, has natural bioactive components, can maintain the local moist environment of the wound, has the characteristics of good biocompatibility, biodegradability, extracellular matrix macromolecule similarity and low cytotoxicity, and can promote the healing of the wound and reduce scars.
The patent (CN 109985271A) discloses a composite collagen dressing for repairing a wound surface which is difficult to heal, I-type collagen and III-type collagen are dispersed in a solvent in proportion, and are homogenized and then are prepared into the composite collagen dressing by composite molding, so that the characteristics of all collagen components can be fully exerted, the repair of the wound is accelerated, and the wound healing quality is improved.
Patent (CN 113975448A) discloses a preparation method of collagen composite dressing, which comprises the following steps: dissolving chitosan in a solvent to prepare a chitosan solution; preparing a chitosan membrane from the chitosan solution through electrostatic spinning, and removing a solvent in the chitosan membrane after shaping to obtain a chitosan matrix material; adding collagen into the traditional Chinese medicine extracting solution, and completely swelling to prepare a collagen mixed solution; covering the surface of the chitosan matrix with the collagen mixed solution, drying, and repeating the operation for 3-10 times to obtain the collagen composite dressing. The composite dressing can effectively solve the problems of poor air permeability, high humidity of wound parts and poor wound healing effect of the existing dressing.
The patent (CN 108273122A) discloses a recombinant collagen hydrogel wound dressing, which is prepared by compounding recombinant collagen, sodium chloride, a thickening agent and deionized water and sterilizing, belongs to an external gel dressing, has good moisturizing, hemostasis and adhesion properties, can promote wound healing and protect wound regeneration tissues, reduces scar formation, is suitable for treatment of wounds such as ulcer, burn, postoperative wound and abrasion, and is simple to prepare, low in production cost and suitable for large-scale production.
Patent (CN 114470314A) discloses a recombinant humanized collagen gel dressing comprising: 0.001% -0.05% of recombinant humanized collagen; 0.005% -0.02% of fibronectin; 0.5 to 2 percent of sodium hyaluronate; heparan sulfate 0.1% -0.8%; 1% -10% of sugar alcohol compounds; 0.5 to 3 percent of 1, 2-pentanediol; polydatin 3% -8%; the balance being water. The collagen gel dressing is formed by selecting fibronectin, sodium hyaluronate, heparan sulfate, sugar alcohol compounds and polydatin which have good biocompatibility with recombinant humanized collagen, adding 1, 2-pentanediol and water (purified water), has no stimulation and no toxicity, can promote the slow release of materials such as the recombinant humanized collagen, the fibronectin, the polydatin and the like, promotes the cell growth at the wound, and has very mild effect on wound repair.
However, the collagen gel dressings disclosed in the above prior art also have some problems, such as poor mechanical strength, fragility, and inconvenience in use; the degradation resistance is poor, and the material is easy to degrade and erode under the condition of enzyme or aqueous solution, so that the retention time of the material in the body is not enough to support the recovery of the wound of a patient; poor absorption performance, unfavorable wound recovery, etc. Therefore, there is a need to provide a recombinant collagen gel dressing with good degradability, absorption and biocompatibility, and is convenient to use.
Disclosure of Invention
The invention provides a recombinant collagen gel dressing and a preparation method and application thereof, aiming at the problems in the prior art, the recombinant collagen gel dressing is prepared by adopting cross-linked particles obtained by cross-linking and crushing a cross-linking agent as a matrix raw material, mixing the matrix raw material with an aqueous solution of a thickening agent, adding phosphate as a stabilizing agent, obtaining the recombinant collagen gel dressing without drying, and adjusting the form and the performance of the dressing by adjusting the dosage of the cross-linked particles of the recombinant collagen and the thickening agent. The dressing can be directly coated on the wound surface, is convenient to use and is not limited by the position and the shape of the wound surface; the degradation resistance is good, and the repair requirement of the wound surface needing to be recovered for a longer time can be met; moderate humidity and good absorption performance, can quickly absorb tissue fluid exuded from the wound and promote the wound healing; no subcutaneous irritation, no potential cytotoxicity and no sensitization reaction.
In order to achieve the above objects, in a first aspect, the present application provides a recombinant collagen gel dressing comprising the following raw materials: the collagen crosslinking particles are recombined, the thickening agent aqueous solution and phosphate;
the recombinant collagen crosslinking particles are obtained by crosslinking and crushing recombinant collagen and a crosslinking agent, wherein the mass ratio of the recombinant collagen to the crosslinking agent is (4-20): 1, the mass concentration of the thickening agent aqueous solution is 0.5-5.0%, and the mass ratio of the recombinant collagen crosslinked particles to the thickening agent aqueous solution is 1 (2-15). After pulverization, the D90 of the recombinant collagen crosslinked particles is preferably 50-800 μm.
The cross-linked network is formed after the cross-linking agent cross-links the recombinant collagen, which can endow the recombinant collagen with certain mechanical strength and better degradation resistance, but the dosage of the cross-linking agent is moderate. If the dosage of the cross-linking agent is too small, the cross-linking effect is limited, and the mechanical strength and the degradation resistance of the dressing cannot be well improved. If the cross-linking agent is used in a large amount, on one hand, the chemical cross-linking agent has certain irritation and toxicity, and the use of the chemical cross-linking agent in a large amount can cause intradermal irritation, potential cytotoxicity and sensitization reaction, and on the other hand, after the recombinant collagen is excessively cross-linked, the absorption performance is limited, and the recombinant collagen cannot be well dispersed in a thickening agent aqueous solution, so that the dressing which is stable in performance and meets the expectation can be prepared.
Preferably, the recombinant collagen is collagen produced by fermentation by using a genetic engineering method, and comprises recombinant human collagen, recombinant human-like collagen and recombinant collagen-like protein, and more preferably type III humanized recombinant collagen.
Preferably, the crosslinking agent is at least one of carbodiimide, propylene oxide, diphenyl phosphate, hexamethylene diisocyanate, genipin, formaldehyde, and glutaraldehyde. More preferably at least one of genipin, formaldehyde and glutaraldehyde. Most preferably glutaraldehyde.
Preferably, the thickener is a cellulosic thickener. Further preferably, the cellulose-based thickener is at least one of hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose or a soluble salt of the above cellulose. Most preferred are the soluble salts of carboxymethyl cellulose. The addition of the thickening agent can enable the recombinant collagen crosslinked particles to swell in the thickening agent aqueous solution, and the dressing with required viscosity is prepared, so that the dressing is convenient to use.
Preferably, the phosphate is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the amount of the phosphate is 0.2-1% of the total amount of the recombinant collagen crosslinked particles, the thickener aqueous solution and the phosphate. The addition of phosphate can improve the stability of the recombinant collagen gel dressing.
Preferably, the water meets pharmaceutical or medicinal standards, such as water for injection.
In a second aspect, the present application provides a method for preparing the recombinant collagen gel dressing, comprising the following steps:
(1) Preparation of recombinant collagen crosslinked particles:
1a, dissolving recombinant collagen in water, stirring and foaming to obtain a collagen solution;
1b, adding a cross-linking agent into the collagen solution obtained in the step 1a for cross-linking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles;
(2) Preparation of aqueous thickener solution: dissolving the thickener with the formula amount in water to prepare a thickener aqueous solution;
(3) And (3) adding the recombinant collagen crosslinked particles and the phosphate obtained in the step (1) into the thickener aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Preferably, the stirring condition in step 1a is 500-5000r/min, the stirring speed is too low, the recombinant collagen is dissolved slowly, and the stirring speed is too high, which can cause the degradation of the recombinant collagen.
Preferably, the mass concentration of the collagen solution in the step 1a is 0.1-5%.
Preferably, step 1d is heat drying or freeze drying; the heating and drying temperature is preferably 50 to 90 ℃.
In a third aspect, the application provides an application of the recombinant collagen gel dressing in wound repair. The recombinant collagen gel dressing has wide applicability, and can meet the use requirements of various wound surfaces, such as tooth extraction wounds, oral ulcers, bruises, cutting wounds, scalds, burns, electric burns, diabetic feet, venous ulcers and the like.
The invention has the following beneficial effects:
1. the recombinant collagen gel dressing is prepared by adopting the recombinant collagen crosslinked particles obtained by crosslinking and crushing through the crosslinking agent as a matrix raw material, mixing the matrix raw material with an aqueous solution of a thickening agent, adding phosphate as a stabilizing agent, and drying without drying. The dressing can be directly applied to the wound surface, is convenient to use and is not limited by the position and the shape of the wound surface; the degradation resistance is good, and the repair requirement of the wound surface needing to be recovered for a longer time can be met; moderate humidity and good absorption performance, can quickly absorb tissue fluid exuded from the wound and promote the wound healing; no subcutaneous irritation, no potential cytotoxicity and no sensitization reaction.
2. The preparation process of the recombinant collagen gel dressing is simple and is suitable for mass production.
3. The recombinant collagen gel dressing has wide applicability, and can meet the use requirements of various wound surfaces, such as tooth extraction wounds, oral ulcers, bruises, cutting wounds, scalds, burns, electric burns, diabetic feet, venous ulcers and the like.
The specific implementation mode is as follows:
it should be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited. The technical solution of the present invention is illustrated by specific examples below.
The following sources of raw materials are exemplary:
recombinant collagen protein: the III type recombinant humanized collagen has the weight-average molecular weight of 10 ten thousand daltons and is sold in the market;
thickening agent: sodium carboxymethylcellulose, having a weight average molecular weight of 15 ten thousand daltons, commercially available;
a crosslinking agent: glutaraldehyde, genipin, commercially available;
phosphate salt: potassium dihydrogen phosphate, disodium hydrogen phosphate, commercially available;
water: water for injection, commercially available.
Example 1
The recombinant collagen gel dressing is prepared from 10g of recombinant collagen cross-linked particles, 100g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 3% and 0.3g of monopotassium phosphate, and the specific process comprises the following steps:
(1) Preparation of recombinant collagen crosslinked particles:
dissolving 20g of recombinant collagen in 380g of injection water, stirring and foaming to obtain a collagen solution with the mass concentration of 5%;
1b, adding 2g of glutaraldehyde into the collagen solution obtained in the step 1a for a crosslinking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles; wherein, the D90 of the recombinant collagen crosslinked particles is 251 μm.
(2) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 3g of sodium carboxymethylcellulose in 97g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%;
(3) And (3) adding 10g of the recombinant collagen crosslinked particles obtained in the step (1) and 0.3g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Example 2
The recombinant collagen gel dressing is prepared from 10g of recombinant collagen cross-linked particles, 60g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 2% and 0.2g of disodium hydrogen phosphate, and the specific process comprises the following steps:
(1) Preparation of recombinant collagen crosslinked particles:
dissolving 20g of recombinant collagen in 380g of injection water, stirring and foaming to obtain a collagen solution with the mass concentration of 5%;
1b, adding 1g of glutaraldehyde into the collagen solution obtained in the step 1a for crosslinking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles; wherein, the D90 of the recombinant collagen crosslinked particles is 276 mu m.
(2) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 1.2g of hydroxymethyl cellulose in 58.8g of water to prepare a sodium hydroxymethyl cellulose aqueous solution with the mass concentration of 2%;
(3) And (3) adding 10g of the recombinant collagen crosslinked particles obtained in the step (1) and 0.2g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Example 3
The recombinant collagen gel dressing is prepared from 10g of recombinant collagen cross-linked particles, 120g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 5% and 0.4g of monopotassium phosphate, and the specific process is as follows:
(1) Preparation of recombinant collagen crosslinked particles:
dissolving 20g of recombinant collagen in 380g of injection water, stirring and foaming to obtain a collagen solution with the mass concentration of 5%;
1b, adding 4g of genipin into the collagen solution obtained in the step 1a to perform a crosslinking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles; wherein, the D90 of the recombinant collagen crosslinked particles is 298 μm.
(2) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 6g of sodium carboxymethylcellulose in 114g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 5%;
(3) And (3) adding 10g of the recombinant collagen crosslinked particles obtained in the step (1) and 0.4g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Comparative example 1
The recombinant collagen gel dressing is prepared from 10g of recombinant collagen cross-linked particles, 100g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 3% and 0.3g of monopotassium phosphate, and the specific process comprises the following steps:
(1) Preparation of recombinant collagen crosslinked particles:
dissolving 20g of recombinant collagen in 380g of injection water, stirring and foaming to obtain a collagen solution with the mass concentration of 5%;
1b, adding 0.4g of glutaraldehyde into the collagen solution obtained in the step 1a for a crosslinking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles; wherein, the D90 of the recombinant collagen crosslinked particles is 246 μm.
(2) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 3g of sodium carboxymethylcellulose in 97g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%;
(3) And (3) adding 10g of the recombinant collagen crosslinked particles obtained in the step (1) and 0.3g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Comparative example 2
The recombinant collagen gel dressing is prepared from 10g of recombinant collagen cross-linked particles, 100g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 3% and 0.3g of monopotassium phosphate, and the specific process comprises the following steps:
(1) Preparation of recombinant collagen crosslinked particles:
dissolving 20g of recombinant collagen in 380g of injection water, stirring and foaming to obtain a collagen solution with the mass concentration of 5%;
1b, adding 8g of glutaraldehyde into the collagen solution obtained in the step 1a to perform a crosslinking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles; wherein, the D90 of the recombinant collagen crosslinked particles is 247 mu m.
(2) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 3g of sodium carboxymethylcellulose in 97g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%;
(3) And (3) adding 10g of the recombinant collagen crosslinked particles obtained in the step (1) and 0.3g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
Comparative example 3
The recombinant collagen gel dressing is prepared from 9.09g of recombinant collagen, 100g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 3% and 0.3g of monopotassium phosphate. The specific process comprises the following steps:
(1) Preparation of sodium carboxymethylcellulose aqueous solution: dissolving 3g of sodium carboxymethylcellulose in 97g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%;
(2) And (2) adding 9.09g of recombinant collagen and 0.3g of phosphate into the sodium carboxymethylcellulose aqueous solution prepared in the step (1), and uniformly stirring to obtain the recombinant collagen gel dressing.
Comparative example 4
The recombinant collagen gel dressing is prepared from 9.09g of recombinant collagen, 100g of sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%, 0.3g of monopotassium phosphate and 0.91g of glutaraldehyde. The specific process comprises the following steps:
(1) Preparation of aqueous thickener solution: dissolving 3g of sodium carboxymethylcellulose in 97g of water to prepare a sodium carboxymethylcellulose aqueous solution with the mass concentration of 3%;
(2) And (2) adding 9.09g of recombinant collagen and 0.91g of glutaraldehyde into the sodium carboxymethylcellulose aqueous solution prepared in the step (1), carrying out a crosslinking reaction, adding 0.3g of phosphate after the reaction is finished, and uniformly stirring to obtain the recombinant collagen gel dressing.
Test examples
The dressings of examples 1-3 and comparative examples 1-4 were subjected to performance tests, which included: the results of the absorption times, in vitro degradability, intradermal stimulation, cytotoxicity, delayed hypersensitivity are shown in Table 1.
1. And (3) measuring the water absorption times: 10g of dressing (m) 0 ) Soaking in 1000mL of normal saline for 24 hr, filtering to remove excessive normal saline, absorbing excessive water on the surface with filter paper, and weighing 1 ). Finally, the suction is carried outCalculating the water rate, measuring each sample for 3 times, and averaging, wherein the water absorption times = (m) 1 -m 0 )/m 0 X 100% is the saturated water absorption.
2. In vitro degradation performance: 10g of the dressing (mass m after freeze-drying) 0 ) The cells were immersed in 100mL of PBS (pH = 7.4) containing 12.5U/mL of collagenase, and digested at 37 ℃ for 7 days in an incubator. Filtering to remove the solution, washing with deionized water for 3 times, and freeze-drying to obtain the final product with mass (mt) and in vitro degradation rate: degradation rate = (m) 0 -m t )/m 0 ×100％。
3. Intradermal stimulation, delayed type hypersensitivity: reference standard GB/T16886.10-2017 part 10 of medical instrument biological evaluation: the test is carried out in the irritation and skin sensitization test, the sample extraction ratio is 0.1g/mL, two solvents of 0.9% sodium chloride solution (solvent) and sesame oil (non-polar) are used, before extraction, the sample is sterilized at 121 ℃ for 20min, and extracted for 72 +/-2 h in an incubator at 37 +/-1 ℃ for test.
4. Cytotoxicity: reference standard GB/T16886.5-2017 medical device biological evaluation part 5: in vitro cytotoxicity assay GB/T16886.12-2017 medical device biology evaluation part 12: sample preparation and reference material', the sample leaching ratio is 0.1g/mL, the sample is sterilized at 121 ℃ for 20min, DMEM medium containing 10% fetal calf serum is added according to the leaching ratio for leaching, the sample is leached at 37 ℃ for 24h in an incubator to prepare 100% leaching solution, then the 100% leaching solution is diluted to prepare 50% leaching solution, the cell survival rates of 100% leaching solution and 50% leaching solution are tested, the cell survival rate of 100% leaching solution is more than 70%, and the cell survival rate of 50% leaching solution is more than 100% leaching solution, and the sample represents no potential cytotoxicity.
TABLE 1
As can be seen from table 1, the recombinant collagen gel dressings prepared in examples 1 to 3 are extruded flowable viscous bodies, and can be conveniently applied to the wound surface; the wound dressing has good degradation resistance and good water absorption, can meet the requirement of longer service time, and absorbs tissue fluid exuded from the wound surface to promote the recovery of the wound surface; has no irritation and cytotoxicity, no sensitization, and can be directly used for wound surface.
It can be seen from comparison between example 1 and comparative example 1 that, when the amount of glutaraldehyde is too small, the crosslinking degree of the recombinant collagen crosslinked particles is limited, and the prepared recombinant collagen gel dressing has a certain viscosity, but is not viscous enough, so that the dressing can easily overflow and is not easy to shape after being directly coated on a wound surface, and the water absorption performance and the degradation performance are poor due to the limited crosslinking degree.
Comparing example 1 with comparative example 2, it can be seen that when the dosage of glutaraldehyde is too much, the recombinant collagen crosslinked particles are excessively crosslinked, on one hand, the prepared recombinant collagen gel dressing has certain sensitization and the cytotoxicity is also improved, on the other hand, because the recombinant collagen crosslinked particles are excessively crosslinked, the recombinant collagen gel dressing is difficult to swell in a sodium carboxymethylcellulose solution, the prepared dressing is a non-uniform fluid with visible particles with certain consistency, the viscosity is insufficient, the dressing cannot be directly used for a wound surface, and the water absorption performance is greatly reduced.
Comparing example 1 with comparative example 3, it can be seen that when glutaraldehyde is not used to crosslink recombinant collagen, the prepared product is dilute, cannot be directly applied to the wound surface without drying to remove water, and has poor water absorption and degradation properties.
Comparing example 1 with comparative example 4, it can be seen that when the recombinant collagen was directly dissolved in the sodium carboxymethylcellulose solution without crosslinking with glutaraldehyde, and then crosslinked, the prepared dressing was a lumpy aggregate, had no fluidity, could not be applied to the wound surface in an extruded form, and had poor water absorption and degradation resistance. On one hand, after the recombinant collagen is directly dissolved in the sodium carboxymethyl cellulose solution, the sodium carboxymethyl cellulose has fewer active groups capable of performing a crosslinking reaction with glutaraldehyde, and the crosslinking reaction of the recombinant collagen is interfered, so that the overall crosslinking degree of a reaction system is low; on the other hand, the recombinant collagen is directly dissolved in the sodium carboxymethylcellulose solution, and the concentration of the recombinant collagen in the prepared dispersion is higher, which is not beneficial to the crosslinking reaction, so that the finally obtained recombinant collagen gel dressing has low crosslinking degree, and poor water absorption performance and degradation resistance performance.
The technical scheme of the invention is not limited to the technical means disclosed by the technical means, and also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.
Claims (10)
1. The recombinant collagen gel dressing is characterized by comprising the following raw materials in parts by weight: the collagen crosslinking particles are recombined, the thickening agent aqueous solution and phosphate;
the recombinant collagen crosslinked particles are obtained by crosslinking and crushing recombinant collagen and a crosslinking agent, wherein the mass ratio of the recombinant collagen to the crosslinking agent is (4-20): 1, the mass concentration of the thickening agent aqueous solution is 0.5-5.0%, and the mass ratio of the recombinant collagen crosslinked particles to the thickening agent aqueous solution is 1 (2-15).
2. The recombinant collagen gel dressing of claim 1, wherein said recombinant collagen is collagen produced by fermentation using genetic engineering methods, including recombinant human collagen, recombinant human-like collagen, recombinant collagen-like.
3. The recombinant collagen gel dressing of claim 1, wherein said cross-linking agent is at least one of carbodiimide, propylene oxide, diphenyl phosphate, hexamethylene diisocyanate, genipin, formaldehyde and glutaraldehyde; the thickening agent is a cellulose thickening agent; the phosphate is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate, and the amount of the phosphate is 0.2-1% of the total amount of the recombinant collagen crosslinked particles, the thickener aqueous solution and the phosphate.
4. The recombinant collagen gel dressing of claim 3, wherein said cross-linking agent is at least one of genipin, formaldehyde and glutaraldehyde; the cellulose thickener is at least one of hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose or soluble salt of above cellulose.
5. The recombinant collagen gel dressing of claim 4, wherein said cross-linking agent is glutaraldehyde; the cellulose thickener is carboxymethyl cellulose soluble salt.
6. The method of preparing a recombinant collagen gel dressing according to any one of claims 1 to 5, comprising the steps of:
(1) Preparation of recombinant collagen crosslinked particles:
1a, dissolving recombinant collagen in water, stirring and foaming to obtain a collagen solution;
1b, adding a cross-linking agent into the collagen solution obtained in the step 1a for cross-linking reaction to obtain a reaction solution 1;
1c, drying and crushing the reaction solution 1 obtained in the step 1b to obtain recombinant collagen crosslinked particles;
(2) Preparation of aqueous thickener solution: dissolving the thickener with the formula amount in water to prepare a thickener aqueous solution;
(3) And (3) adding the recombinant collagen crosslinked particles and the phosphate obtained in the step (1) into the thickener aqueous solution prepared in the step (2), and uniformly stirring to obtain the recombinant collagen gel dressing.
7. The method of claim 6, wherein: in the step 1a, the mass concentration of the collagen solution is 0.1-5%, and the stirring condition is 500-5000r/min.
8. The method of claim 6, wherein: the step 1d adopts heating drying or freeze drying; the heating and drying temperature is 50-90 ℃.
9. Use of a recombinant collagen gel dressing according to any one of claims 1 to 5 or prepared according to the method of any one of claims 6 to 8 in wound repair.
10. The use of claim 9, wherein: the wound surface is dental extraction wound, oral ulcer, abrasion, cut wound, scald, burn, electric burn, diabetic foot, and venous ulcer.
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