A kind of method of the sodium alginate-modified acellular dermal matrix of epoxyTechnical field
The present invention relates to a kind of methods of the sodium alginate-modified acellular dermal matrix of epoxy, are applied to biomedical neckDomain.
Background technique
Acellular dermal matrix has the function of dermal template effect, tissue shielding action and guide tissue regeneration etc.,It is widely used in burn trauma reparation, scar treatment, ear-nose-throat department treatment, face-lifting, neurosurgical treatment etc..
In order to reduce the antigenicity of acellular dermal matrix, improve degradation resistance, improvement physical mechanical property, generally requireIt carries out cross-linking modified.Common glutaraldehyde cross-linking is also easy to produce that calcification phenomenon, carbodiimides cross-linked hydrophilic be poor, genipin cross-linkedColor is deeper.Common epoxide, such as glycol glycidyl ethers, butanediol glycidol ether have preferableizationLearn activity, the acellular dermal matrix increased hydrophilicity of crosslinking, the enhancing of anti-calcification capacity.But their poorly water-solubles, using notJust, and since they belong to exogenous anthropogenics, there are poor biocompatibility, containing by-product and after being crosslinkedThe disadvantages of reagent remains, influences its cross-linking properties.
Alginic acid is by ((1,4)-beta-D-mannuronic acid (M unit) and α-L- guluronic acid (G unit)) block copolymerizationIt forms.Alginic acid and its salt are cheap and easy to get, gel easy to form, hydrophilic moisture retention is high, biocompatibility is excellent, by widelyApplied to fields such as organizational project, artificial dressing, pharmaceutical carrier, control delivery systmes.It is big due to containing in alginic acid and its saltTherefore the hydroxyl and carboxyl of amount have good hydrophily and moisture retention, such as water absorption rate of pure alginate fiber is its ownThe water absorption rate of 2.2 times of quality, calcium alginate freeze-drying film may be up to 985%.Common alginate includes sodium alginate, seaweedSour calcium and alginic acid zinc etc..Alginate dressing has high-hygroscopicity, ease of removal, high oxygen permeability, gel blockage and excellentGood biological degradability and good biocompatibility are widely used in hemostasis, acute scald, bedsore, second-degree burn of all kinds of wounds etc.The surface of a wound sticks, it is possible to reduce the advantages that wound infection keeps the surface of a wound wet, promotes wound healing.
Be conducive to improve the hydrophilicity of acellular dermal matrix by introducing hydrophilic substance in acellular dermal matrix,Crosslinking is then conducive to improve its degradation resistance energy.By alginate and acellular dermal matrix it is compound be a kind of preferable selection.However, lacking stronger chemical interaction between common alginate and acellular dermal matrix, cross-linking reaction cannot be generated,Alginate after blending is easy to happen dissolution, migration, loss, cannot generate ideal moisturizing and cross-linking effect.In order to promoteThe two organically combines, and someone is aoxidized sodium alginate using sodium metaperiodate, to obtain the oxidized sodium alginate containing aldehyde radical.ByDivide neutron to contain the active aldehyde radical of chemistry in oxidized sodium alginate, therefore can occur with the amino isoreactivity group on collagen anti-It answers, forms Schiff, so that the two is generated stronger Covalent bonding together, to generate cross-linking effect.But the aldehyde radical of high-content is handed overAfter joining biomaterial, due to aldehyde radical residual etc., be easy to produce the big defect of calcification, cytotoxicity, especially when oxidizability is high,When reacting incomplete, these disadvantages are particularly evident.In addition, when forming oxidized sodium alginate, strand exists after sodium alginate oxidationIt is disconnected at o-dihydroxy, destroys the basic structure of sodium alginate sugar unit, thus the performances such as its hydrophily, gelation are generatedAdverse effect.
How alginate molecular structure can be introduced into acellular dermal matrix, increase acellular dermal matrixHydrophilic performance of keeping humidity, while good cross-linking effect can be generated again, and the drawbacks of avoid oxidized sodium alginate from being crosslinked, it is this hairIt is bright to solve the problems, such as.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of sides of the sodium alginate-modified acellular dermal matrix of epoxyMethod.
The present invention is achieved through the following technical solutions:
The method of the sodium alginate-modified acellular dermal matrix of epoxy includes following technique:
Acellular dermal matrix is placed in alkaline buffer solution by step (1), and epoxy alginate is then added and is handed overConnection, wherein the epoxy alginate, is dehydrated after forming epoxy group for the vicinal diamines structure on 2,3 in alginate moleculeObtained substance；The epoxy alginate is epoxy sodium alginate；
Step (2) is completed after being crosslinked, the liquid of falling dereaction, and is cleaned, then remove cleaning solution, and lactic acid pair is then addedRemaining epoxy group is hydrolyzed；
After the completion of step (3), hydrolysis, treatment fluid is removed, buffer solution is then added and is neutralized；
Treatment fluid is removed after step (4), processing, culture medium is added, utilizes the active material and remaining ring in culture mediumOxygroup is reacted；
Treatment fluid is removed after step (5), processing, water is added and is cleaned, is then dried, obtains epoxy alginateModified acellular dermal matrix.
Preferably, each specific technique of step is:
Step (1), the acellular dermal matrix for weighing 100 parts by weight, the alkaline buffer for being placed in 100~300 parts by weight are moltenIn liquid, the epoxy alginate of 1~20 parts by weight is added, is warming up to 25~35 DEG C, reacts 2~8 hours, then it is warming up to 35~42 DEG C are reacted 4~8 hours, then are warming up to 42~47 DEG C, are reacted 1~4 hour；The epoxy alginate is epoxy alginic acidSodium；Early period, acellular dermal matrix denaturation temperature was low, needs to carry out at a lower temperature, otherwise easily degrade；Later period withThe denaturation temperature of the progress of crosslinking, acellular dermal matrix increases, at this point, carrying out reaction at relatively high temperatures can be improved reactionRate.Therefore, it is crosslinked with using stage heating and is conducive to improve cross-linking efficiency.
The injection water of step (2), the liquid of falling dereaction, 100~200 parts by weight for being 25~35 DEG C with temperature cleans 20~30Minute；Cleaning solution is removed, the lactic acid solution that the mass fraction of 0.5~5 parts by weight is 10% is added, handles 30 at 20~25 DEG C~120 minutes；During epoxy alginate is crosslinked acellular dermal matrix, the characteristics of due to organic reaction, inevitably haveReact incomplete phenomenon, a part is has been grafted on acellular dermal matrix but the complete epoxy seaweed of epoxy group unreactedSour sodium, this, which will lead to, may remain a small amount of epoxy group on the acellular dermal matrix after crosslinking, influence its biocompatibility, and onePart is the epoxy alginate not reacted with acellular dermal matrix, is remained in reaction solution.Epoxy group is in acid conditionMeeting ring opening hydrolysis is therefore hydroxyl is handled using lactic acid, epoxy on the acellular dermal matrix after can both having eliminated crosslinkingBase improves biocompatibility, and can eliminate the epoxy group in the unreacted epoxy alginate in waste liquid, improves technique ringGuarantor property, and adjustable solution ph.Even if finally will form lactate just in case remaining a small amount of lactic acid, it is still with goodGood biocompatibility, and be a kind of preferable moisturizer.
Step (3) removes treatment fluid, the phosphate buffer of 150~200 parts by weight is added, at 20~25 DEG CProcessing 1~2 hour；The pH of acellular dermal matrix after being crosslinked can be further adjusted by phosphate buffer.
Step (4) removes treatment fluid, and the culture medium of 50~100 parts by weight is added, and it is small that 1~2 is handled at 10~25 DEG CWhen；The chemical active radical contained in culture medium, such as amino, can be linked on acellular dermal matrix epoxy seaThe epoxy group of alginates is reacted, while these active materials can be grafted on acellular dermal matrix, furtherWhile eliminating possible remaining a small amount of epoxy group on crosslinking acellular dermal matrix, the bioactive substance in culture medium is connectBranch further increases its biocompatibility into acellular dermal matrix.
Step (5) removes treatment fluid, and the injection water of 150~300 parts by weight is added, and 30~60 are handled at 20~25 DEG CMinute, 6~8 times repeatedly；Unreacted impurity is removed as far as possible, guarantees its biocompatibility.
Step (6): acellular dermal matrix, freeze-drying are taken out.Acellular dermal matrix is able to maintain after freeze-dryingThe microstructure of material is conducive to cell adherence, wound reparation, and the product after freeze-drying can store and transport at normal temperature, and extension is guaranteed the qualityPhase.
Preferably, the alkaline buffer solution includes NaOH-NaHCO3In buffer solution, borate buffer solutionIt is one or two kinds of.
Preferably, wherein the culture medium includes RPMI-1640 culture medium, DMEM culture medium and MCDB131 cultureOne of base is a variety of.
Preferably, the epoxy alginate can also be epoxy calcium alginate and epoxy in addition to epoxy sodium alginateOne of alginic acid zinc is a variety of.
The invention has the characteristics that:
(1) epoxy group in epoxy alginate molecule has stronger chemical activity due to the tension of three-membered ring,Cross-linking reaction can be generated with the amino etc. in acellular dermal matrix, so that the shrinkage temperature of acellular dermal matrix is improved,It is increased to 68~80 DEG C by 55~65 DEG C.Due to the formation of cross-linked structure, the steady of acellular dermal matrix is considerably increasedIt is qualitative, hence it is evident that improve the degradation resistance of acellular dermal matrix.
(2) different from oxidized sodium alginate crosslinking acellular matrix, the epoxy group of the epoxy alginate in the present invention isIt being dehydrated to be formed by the vicinal diamines structure on 2,3, after ring-opening reaction, the backbone structure of epoxy alginate is not turned off, thus,More remain the excellent performance of alginate.And oxidized sodium alginate is when forming dialdehyde base, especially when its oxidizabilityWhen higher, strand is oxidized fracture, and structural intergrity is destroyed, and affects its structure and performance.
(3) acellular dermal matrix being crosslinked through epoxy alginate increases due to introducing alginate structural unitHydroxyl and carboxyl make it have better hydrophily and water retention property, simultaneously because the gelation of alginate structural unitsCan, moisture can preferably " be lockked ", wound moist is kept.When being used as dressing or repair materials, by " wet healing is theoretical "It is found that this moisture retention is beneficial to accelerate the healing of wound.In addition, such " water lock " property, also has good resistance bacterium functionEnergy.
(4) property hydrolyzed in acid condition using epoxy group, is post-processed using lactic acid, by remaining epoxy groupIt is hydrolyzed to hydroxyl.Meanwhile also using the active material in culture medium, and remain epoxy reaction, to remove remaining ringOxygroup finally makes acellular dermal matrix after post treatment have excellent biological capacitive.The acellular dermal being crosslinked through itMatrix, cytotoxicity are lower than 1 grade.With previous oxidation alginate cross-linked phase ratio, it is issuable to avoid high aldehyde group contentCytotoxicity, thus there is better biocompatibility.
(5) acellular dermal matrix of oxidized sodium alginate crosslinking is in faint yellow, and the crosslinking of epoxy alginate is de- thinBorn of the same parents' dermal matrix is white.
The beneficial effects of the present invention are:
Epoxy alginate used in the present invention, 2,3 upper epoxy groups are formed after vicinal diamines are dehydrated, with oxidationSodium alginate is compared, during forming three-membered ring, 2,3 in strand and unbroken.Due to epoxy alginic acid salinityContain multiple epoxy groups in son, it is thus possible to react between the amino isoreactivity group of collagen, generate excellent crosslinkingEffect.Meanwhile after epoxy group is reacted with the amino etc. on acellular dermal matrix, by the alginate with excellent performance of keeping humidityIt is introduced into acellular dermal matrix, to assign its excellent hydrophilic performance of keeping humidity, it may be assumed that using epoxy alginate to de- thinBorn of the same parents' dermal matrix is crosslinked, and has obtained a kind of acellular dermal matrix material with excellent hydrophilic performance of keeping humidity, it is simultaneouslyExcellent degradation resistance energy is had both, low cytotoxicity is a kind of novel functional form acellular dermal matrix, social value and economyHuge value is worthy to be popularized.
Specific embodiment
The method of the sodium alginate-modified acellular dermal matrix of epoxy includes following technique: weighing the de- thin of 100 parts by weightBorn of the same parents' dermal matrix is placed in the alkaline buffer solution of 100~300 parts by weight, and the epoxy alginate of 1~20 parts by weight is added,25~35 DEG C are warming up to, is reacted 2~8 hours, 35~42 DEG C is then warming up to and reacts 4~8 hours, then be warming up to 42~47 DEG C,Reaction 1~4 hour；The injection water of the liquid of falling dereaction, 100~200 parts by weight for being 25~35 DEG C with temperature cleans 20~30 pointsClock；It removes cleaning solution, the lactic acid solution that the mass fraction of 0.5~5 parts by weight is 10% is added, handle 30 at 20~25 DEG C~120 minutes；Treatment fluid is removed, the phosphate buffer of 150~200 parts by weight is added, 1~2 is handled at 20~25 DEG CHour；Treatment fluid is removed, the culture medium of 50~100 parts by weight is added, is handled 1~2 hour at 10~25 DEG C；It goes to handleLiquid is added the injection water of 150~300 parts by weight, handles at 20~25 DEG C 30~60 minutes, repeatedly 6~8 times；It takes out de- thinBorn of the same parents' dermal matrix, freeze-drying, wherein the epoxy alginate, refers to the vicinal diamines in alginate molecule on 2,3Structure is dehydrated to form epoxy group after obtained substance.
The alkaline buffer solution includes NaOH-NaHCO3One of buffer solution, borate buffer solution or twoKind.
Wherein the culture medium includes one in 131 culture medium of RPMI-1640 culture medium, DMEM culture medium and MCDBKind is a variety of.
Preferably, the epoxy alginate can also be epoxy calcium alginate and epoxy in addition to epoxy sodium alginateOne of alginic acid zinc is a variety of.
Embodiment 1
1. weighing 100 grams of acellular allodermis matrix (butt).
2. weighing 8 grams of epoxy alginate.
3. 4.2 grams of sodium bicarbonates are dissolved in distilled water and are diluted to 1000ml (A liquid), 4.0 grams of sodium hydroxides are dissolvedIt in distilled water and is diluted to 1000ml (B liquid), 500ml A liquid and 107ml B liquid is uniformly mixed, the bicarbonate of pH10.0 is obtainedSodium-sodium hydroxide buffer solution, and weigh the buffer solution of 150 parts by weight.
4. acellular allodermis matrix is put into reactor, weighed buffer is added, epoxy alginate is added, adjustsTemperature is saved to 25 DEG C, is reacted 2 hours, 42 DEG C is then warming up to and reacts 8 hours, then be warming up to 47 DEG C, react 1 hour.
5. the injection water of the liquid of falling dereaction, 200 parts by weight for being 25 DEG C with temperature cleans 30 minutes.
6. removing cleaning solution, the lactic acid solution that the mass fraction of 1 parts by weight is 10% is added, is handled 2 hours at 25 DEG C.
7. removing treatment fluid, the phosphate buffer of the 0.02mol/L pH7.0 of 200 parts by weight is added, at 25 DEG CLower processing 2 hours.
8. removing treatment fluid, the RPMI-1640 culture medium of 100 parts by weight is added, is handled 2 hours at 10 DEG C.
9. removing treatment fluid, the injection water of 300 parts by weight is added, is handled at 20 DEG C 60 minutes, repeatedly 6 times.
10. taking out acellular dermal matrix, freeze-drying.
Embodiment 2
1. weighing 100 grams of acellular dermal matrix (butt).
2. weighing 5 grams of epoxy alginate.
3. 4.2 grams of sodium bicarbonates are dissolved in distilled water and are diluted to 1000ml (A liquid), 4.0 grams of sodium hydroxides are dissolvedIt in distilled water and is diluted to 1000ml (B liquid), 500ml A liquid and 165ml B liquid is uniformly mixed, the bicarbonate of pH10.4 is obtainedSodium-sodium hydroxide buffer solution, and weigh the buffer solution of 300 parts by weight.
4. acellular allodermis matrix is put into reactor, weighed buffer is added, epoxy alginate is added, adjustsReaction temperature is saved to 35 DEG C, is reacted 8 hours, 42 DEG C is then warming up to and reacts 4 hours, then be warming up to 47 DEG C, react 4 hours.
5. the injection water of the liquid of falling dereaction, 150 parts by weight for being 35 DEG C with temperature cleans 20 minutes.
6. removing cleaning solution, the lactic acid solution that the mass fraction of 0.5 parts by weight is 10% is added, it is small that 1 is handled at 20 DEG CWhen.
7. removing treatment fluid, the phosphate buffer of the 0.05mol/L pH7.1 of 150 parts by weight is added, at 20 DEG CLower processing 1 hour.
8. removing treatment fluid, the DMEM culture medium of 50 parts by weight is added, is handled 1 hour at 20 DEG C.
9. removing treatment fluid, the injection water of 150 parts by weight is added, is handled at 25 DEG C 30 minutes, repeatedly 7 times.
10. taking out acellular dermal matrix, freeze-drying.
Embodiment 3
1. weighing 100 grams of acellular dermal matrix (butt).
2. weighing 20 grams of epoxy alginate.
3. distilled water is dissolved in by H3BO312.4 grams and is diluted to 1000ml (A liquid), it is molten by Na2B4O7H2O19.1 gramsSolution is in distilled water and is diluted to 1000ml (B liquid), and 550ml A liquid and 450ml B liquid are uniformly mixed, the boric acid-of pH8.4 is obtainedBorax buffer solution, and weigh the buffer solution of 200 parts by weight.
4. acellular allodermis matrix is put into reactor, weighed buffer is added, epoxy alginate is added, adjustsReaction temperature is saved to 30 DEG C, is reacted 4 hours, 37 DEG C is then warming up to and reacts 1 hour, then be warming up to 47 DEG C, react 4 hours.
5. the injection water of the liquid of falling dereaction, 100 parts by weight for being 40 DEG C with temperature cleans 25 minutes.
6. removing cleaning solution, the lactic acid solution that the mass fraction of 2 parts by weight is 10% is added, 30 points are handled at 25 DEG CClock.
7. removing treatment fluid, the phosphate buffer of the 0.04mol/L pH7.2 of 180 parts by weight is added, at 20 DEG CLower processing 1 hour.
8. removing treatment fluid, 131 culture medium of MCDB of 80 parts by weight is added, is handled 90 minutes at 20 DEG C.
9. removing treatment fluid, the injection water of 200 parts by weight is added, is handled at 20 DEG C 40 minutes, repeatedly 8 times.
10. taking out acellular dermal matrix, freeze-drying.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, anyThe change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the inventionProtection scope should be determined by the scope of protection defined in the claims.