CN109847088A - Compound acellular dermal matrix biological dressing and preparation method thereof - Google Patents
Compound acellular dermal matrix biological dressing and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of compound acellular dermal matrix biological dressing and preparation method thereof, by using fatty alcohol polyoxyethylene ether, nonylphenol polyoxyethylene ether, dodecyl sodium sulfate mixes in proper proportions, after carrying out degreasing to skin, after carrying out de- cell processing to skin using Dispase and the Trypsin-EDTA complex enzyme of proper ratio, it is soaked in carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and keratan sulfate mixed solution simultaneously carry out cross-linking reaction with oxidized sodium alginate, the characteristics of based on degreasing rate height and maintaining the content of original skin collagen, and cooperate carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, the joint synergistic effect of calcium chloride and keratan sulfate, the compound acellular dermal matrix biological dressing being prepared, with good antibacterial , promoting healing the effect of, and the dressing can have both good mechanical performance simultaneously, and being easy to tile spreads on wound surface, not cracky.
Description
Technical field
The invention belongs to biomedical materials field, in particular to a kind of compound acellular dermal matrix biological dressing.
Background technique
Acellular dermal matrix dressing is exactly a kind of dermal substitute that can be used for full thickness dermal and for burning
The medical dressing of the skin injuries wound healings such as wound, it is that natural skin eliminates in epidermis and corium by a series of processing
Cell component, but dermal extracellular matrix ingredient and its three-D space structure are remained, such structure can be skin regeneration
There is provided " dermal template ", after inducing transplantation host cell contents such as fibroblast, endothelial cell break up in supporting structure,
Growth.But (ADM) dressing of acellular dermal matrix made from conventional method is there are poor permeability, anti-microbial property is low, bio-compatible
Property it is poor the disadvantages of, burning, diabetic foot ulcer, pressure sore, Venous Ulcers, more property operative incision, wound, chronic wound
In the treatment of all kinds of wounds such as mouth, skin donor site, the disadvantages of there are healing cycles long, easy scar hyperplasia, easy infection, wound is seriously affected
Face healing effect.
Based on this, it is necessary to providing one kind, can to take into account wound healing effect, good biocompatibility, irritation small,
And the compound acellular dermal matrix biological dressing of good mechanical property.
Summary of the invention
Based on this, the purpose of the present invention is to provide a kind of compound acellular dermal matrix biological dressings, can take into account
Wound healing effect, good biocompatibility, irritation are small, and the effect of good mechanical property.
To achieve the above object, the present invention provides the following technical scheme that
A kind of preparation method of compound acellular dermal matrix biological dressing, comprising the following steps:
(1) skin samples are soaked in degreasing agent and are ultrasonically treated degreasing, obtain degreased skin；It include rouge in the degreasing agent
Fat alcohol polyoxyethylene ether, nonylphenol polyoxyethylene ether, dodecyl sodium sulfate；
(2) it takes off cell: after degreased skin multigelation, being placed in composite enzyme solution and be incubated overnight, obtain de- cell skin；
(3) by the de- cell skin be impregnated in carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and
In the mixed solution of keratan sulfate；Be added dropwise oxidized sodium alginate solution be crosslinked to get.
In wherein some embodiments, the degreasing agent are as follows: 18wt%~22wt% fatty alcohol polyoxyethylene ether, 13wt%
The mixed liquor of~17wt% nonylphenol polyoxyethylene ether and 4wt%~6wt% dodecyl sodium sulfate.
In wherein some embodiments, in the degreasing agent: 18wt%~22wt% fatty alcohol polyoxyethylene ether, 13wt%
~17wt% nonylphenol polyoxyethylene ether and 4wt%~6wt% dodecyl sodium sulfate are 1:(1~5 with volume ratio): (1~3)
Mixing.
In wherein some embodiments, the time that skin samples are soaked in ultrasonic treatment degreasing in degreasing agent is 3
~5h.
In wherein some embodiments, mixed solution described in step (3) are as follows: 4wt%~6wt% carboxymethyl chitosan,
10wt%~20wt% hyaluronic acid, 5wt%~10wt% hydroxyethyl cellulose, 1wt%~5wt% calcium chloride, 5wt%~
7wt% keratan sulfate, and/or
Mixed solution 4wt%~6wt% carboxymethyl chitosan, 10wt%~20wt% hyaluronic acid described in step (3),
5wt%~10wt% hydroxyethyl cellulose, 1wt%~5wt% calcium chloride, 5wt%~7wt% keratan sulfate by volume 1:
(2~8): (2~5): (1~5): the mixing of (3~6).
In wherein some embodiments, dropwise addition oxidized sodium alginate solution described in step (3) is crosslinked are as follows: is added dropwise
10wt%~25wt% oxidized sodium alginate is crosslinked, and 20~60min is crosslinked；And/or
The oxidized sodium alginate solution of dropwise addition described in step (3) and the volume ratio of the mixed solution are (1~3): 50.
In wherein some embodiments, after the degreased skin multigelation, it is placed in composite enzyme solution and is incubated overnight are as follows:
From -80 DEG C to 37 after DEG C multigelation 2~4 times, it is placed in 4 DEG C and is incubated overnight in composite enzyme solution.
In wherein some embodiments, step (2) described composite enzyme solution are as follows: 2.5wt%~3.5wt%Dispase with
0.2wt%~0.3wt%trypsin-EDTA is with volume ratio 1:(2~5) it is mixed to get；And/or complex enzyme described in step (2)
It is further comprising the steps of after being incubated overnight in solution:
The DMEM culture medium containing serum is added and terminates digestion；
De- cell sample is placed in antibiotic solution to impregnate 4~6 minutes, PBS impregnates 8~12 minutes, repeats 3~5 times.
The present invention also provides a kind of compound acellular dermal matrix biological dressing, specific technical solution is as follows:
A kind of compound acellular dermal matrix biological dressing, is prepared by preparation method as described above.
The present invention also provides the application of compound acellular dermal matrix biological dressing, specific technical solution is as follows:
Above-mentioned compound acellular dermal matrix biological dressing is promoting the application in skin healing.
Based on the above-mentioned technical proposal, the invention has the following advantages:
The present invention is asked for be difficult to take into account between the wound healing effect of dressing, biocompatibility and mechanical performance
Topic, mixes in proper proportions by using fatty alcohol polyoxyethylene ether, nonylphenol polyoxyethylene ether, dodecyl sodium sulfate,
After carrying out degreasing to skin, skin is carried out at de- cell using Dispase and the Trypsin-EDTA complex enzyme of proper ratio
After reason, be soaked in carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and keratan sulfate mixed solution and with
The characteristics of oxidized sodium alginate carries out cross-linking reaction, based on degreasing rate height and maintains the content of original skin collagen, and cooperate
Carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and keratan sulfate joint synergistic effect, be prepared
Compound acellular dermal matrix biological dressing, have effects that good antibacterial, promoting healing, and the dressing can have both simultaneously
Good mechanical performance, being easy to tile spreads on wound surface, not cracky, good application effect.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to described herein
Embodiment.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.Embodiment
In used various common agents, be commercial product.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Used term is intended merely to describe specific reality in the description of the invention
Apply the purpose of example, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more relevant institutes
Any and all combinations of list of items.
The present invention provides a kind of preparation methods of compound acellular dermal matrix biological dressing, comprising the following steps:
(1) skin samples are soaked in degreasing agent and are ultrasonically treated degreasing, obtain degreased skin；It include fatty alcohol polyoxy in the degreasing agent
Vinethene, nonylphenol polyoxyethylene ether, dodecyl sodium sulfate；(2) it takes off cell: after degreased skin multigelation, being placed in multiple
It is incubated overnight in synthase solution, obtains de- cell skin；(3) the de- cell skin is impregnated in carboxymethyl chitosan, hyalomitome
Acid, hydroxyethyl cellulose, calcium chloride and keratan sulfate mixed solution in；Oxidized sodium alginate solution is added dropwise to be crosslinked,
To obtain the final product.
Preferably, the degreasing agent are as follows: 18wt%~22wt% fatty alcohol polyoxyethylene ether, 13wt%~17wt% nonyl
The mixed liquor of phenol polyethenoxy ether and 4wt%~6wt% dodecyl sodium sulfate；It is furthermore preferred that three with volume ratio be 1:
(1~5): (1~3) mixing.It is further preferred that using 20% fatty alcohol polyoxyethylene ether, 15% nonylphenol polyoxyethylene ether
It is mixed with 5% dodecyl sodium sulfate with the suitable ratio of 1:3:2, under its synergistic effect, is preparing acellular dermal base
During matter, good degreasing effect can be realized, and reduce the loss of skin collagen.
Preferably, the time for using above-mentioned degreasing agent to carry out ungrease treatment, for 3~5h, specifically, skin samples were impregnated
The time that degreasing is ultrasonically treated in degreasing agent is 3~5h.
Further, mixed solution described in step (3) are as follows: 4wt%~6wt% carboxymethyl chitosan, 10wt%~
20wt% hyaluronic acid, 5wt%~10wt% hydroxyethyl cellulose, 1wt%~5wt% calcium chloride, 5wt%~7wt% sulfuric acid
The mixed solution of keratan；Preferably, 4wt%~6wt% carboxymethyl chitosan, 10wt%~20wt% hyaluronic acid, 5wt%
~10wt% hydroxyethyl cellulose, 1wt%~5wt% calcium chloride, 5wt%~7wt% keratan sulfate mixed solution press body
Product is than 1:(2~8): (2~5): (1~5): the mixing of (3~6), preferably 1:5:3:3:4.
Preferably, dropwise addition oxidized sodium alginate solution described in step (3) is crosslinked are as follows: 10wt%~25wt% is added dropwise
Oxidized sodium alginate is crosslinked, and 20~60min is crosslinked.In some of embodiments, the oxidized sodium alginate solution of dropwise addition with
The volume ratio of the mixed solution is (1~3): 50.The characteristics of based on degreasing rate height and maintaining the content of original skin collagen,
And cooperate the joint synergistic effect of carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride, keratan sulfate, it prepares
Obtained compound acellular dermal matrix biological dressing, has effects that good antibacterial, promoting healing.
Optionally, further comprising the steps of after being incubated overnight in composite enzyme solution described in step (2): addition contains blood
Clear DMEM culture medium terminates digestion；De- cell sample is placed in antibiotic solution to impregnate 4~6 minutes, PBS impregnates 8~12 points
Clock repeats 3~5 times.
In wherein some embodiments, after the degreased skin multigelation, it is placed in composite enzyme solution and is incubated overnight are as follows:
From -80 DEG C to 37 after DEG C multigelation 2~4 times, it is placed in 4 DEG C and is incubated overnight in composite enzyme solution.Wherein, the complex enzyme is molten
Liquid are as follows: 2.5wt%~3.5wt%Dispase and 0.2wt%~0.3wt%trypsin-EDTA are with volume ratio 1:(2~5) it is mixed
Conjunction obtains, it is preferable that the composite enzyme solution are as follows: 3wt%Dispase and 0.25wt%trypsin-EDTA is with volume ratio 1:(2
~5) it is mixed to get, it is highly preferred that volume ratio is 1:3.
The present invention also provides compound acellular dermal matrix biological dressings as described above in promoting skin healing
Using.Preferably, which can be applied to burned skin.
The present invention is made a more thorough explanation combined with specific embodiments below:
Embodiment 1
A kind of preparation method of compound acellular dermal matrix biological dressing is present embodiments provided, specific steps are such as
Under:
(1) preparation of reagent needed for
Degreasing agent: 20wt% fatty alcohol polyoxyethylene ether, 15wt% nonylphenol polyoxyethylene ether, 5wt% dodecyl sulphur
Sour sodium configures the mixed solution of 50mL with volume ratio 1:3:2 mixing.
Complex enzyme: 3wt%Dispase and 0.25wt%trypsin-EDTA is with the mixed of volume ratio 1:3 mixed configuration 10mL
Close solution.
(2) cell technique is taken off
1) it samples: choosing the skin sample of ridge line two sides on pigskin, every piece is cut into 5cm × 5cm size, and hair processing is dry
Only.
2) degreasing: above-mentioned sample is soaked in 100mL degreasing agent and is ultrasonically treated 3-5h.
3) cell: the method combined using physics and chemically combined method, that is, multigelation and enzymatic reagent is taken off, specifically
Are as follows: the processed sample of step 2) is rinsed several times through PBS, DEG C multigelation 2~4 times from -80 DEG C to 37, this process is completed
Afterwards, sample is placed in 4 DEG C and is incubated overnight in complex enzyme.
4) termination digestion is carried out to it with the DMEM containing serum, is rinsed for several times with PBS later, it is spare.
5) it sterilizes: cell free sample being placed in antibiotic solution and is impregnated 5 minutes, PBS impregnates 10 minutes, this process repeats
3-5 times.
6) gnotobasis carries out closing packaging.
7) it irradiates, is stored at room temperature.
(3) preparation of complex biological dressing
1) by 5wt% carboxymethyl chitosan, 15wt% hyaluronic acid, 8wt% hydroxyethyl cellulose, 4wt% calcium chloride,
6wt% keratan sulfate, each solution are mixedly configured into the mixed solution of 50mL with volume ratio 1:5:3:3:4；Wherein, carboxymethyl shell
Glycan, which is dissolved in 1% acetic acid, is prepared carboxymethyl chitosan solution.
2) by the above-mentioned acellular matrix sample prepared, dipping and the 15wt% oxidation seaweed that 2mL in 1) solution, is added dropwise
Sour sodium is crosslinked, and 30min is crosslinked；
3) crosslinked sample is flushed three times with PBS, each 15min；
4) it is freeze-dried, irradiates, save.
Embodiment 2
The preparation method for present embodiments providing a kind of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than difference are as follows: (one) in the preparation of reagent needed for, degreasing agent are as follows: 20wt% fatty alcohol polyoxyethylene ether, 15wt% nonyl phenol are poly-
Ethylene oxide ether, 5wt% dodecyl sodium sulfate are with volume ratio1:1:1Mixing, configure the mixed solution of 50mL；Complex enzyme are as follows:
3wt%Dispase and 0.25%trypsin-EDTA are with volume ratio1:2The mixed solution of mixed configuration 10mL；(3) compound
In the preparation of biological dressing, 1) by 5wt% carboxymethyl chitosan, 10wt% hyaluronic acid, 5wt% hydroxyethyl cellulose, 1wt%
Calcium chloride, 6wt% keratan sulfate solution are with volume ratio1:2:2:5:3It is mixedly configured into the mixed solution of 50mL；It 2) will be above-mentioned
The acellular matrix sample prepared, dipping are added dropwise with 1) solution1mL15wt% oxidized sodium alginate be crosslinked, hand over
Connection20min。
Remaining step is same as Example 1.
Embodiment 3
The preparation method for present embodiments providing a kind of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than difference are as follows: (one) in the preparation of reagent needed for, degreasing agent are as follows: 20wt% fatty alcohol polyoxyethylene ether, 15wt% nonyl phenol are poly-
Ethylene oxide ether, 5wt% dodecyl sodium sulfate are with volume ratio1:5:3Mixing, configures the mixed solution of 50mL；Complex enzyme are as follows:
3wt%Dispase and 0.25wt%trypsin-EDTA are with volume ratio1:5The mixed solution of mixed configuration 10mL；(3) compound
In the preparation of type biological dressing, 1) by 5wt% carboxymethyl chitosan, 20wt% hyaluronic acid, 10wt% hydroxyethyl cellulose,
5wt% calcium chloride, 6wt% keratan sulfate solution are with volume ratio1:8:5:1:6It is mixedly configured into the mixed solution of 50mL；2) will
The above-mentioned acellular matrix sample prepared, dipping are added dropwise with 1) solution3mL15wt% oxidized sodium alginate handed over
Connection, crosslinking60min。
Remaining step is same as Example 1.
Embodiment 4
The preparation method for present embodiments providing a kind of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than difference are as follows: (one) in the preparation of reagent needed for, degreasing agent are as follows: 20wt% fatty alcohol polyoxyethylene ether, 15wt% nonyl phenol are poly-
Ethylene oxide ether, 5wt% dodecyl sodium sulfate are with volume ratio1:3:5Mixing, configures the mixed solution of 50mL；Complex enzyme are as follows:
3wt%Dispase and 0.25wt%trypsin-EDTA are with volume ratio1:5The mixed solution of mixed configuration 10mL.
Remaining step is same as Example 1.
Embodiment 5
The preparation method for present embodiments providing a kind of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than difference are as follows: (three) in the preparation of complex biological dressing, 1) by 5wt% carboxymethyl chitosan, 20wt% hyaluronic acid,
10wt% hydroxyethyl cellulose, 5wt% calcium chloride, 6wt% keratan sulfate solution are with volume ratio1:1:1:1:1It is mixedly configured into
The mixed solution of 50mL；2) by the above-mentioned acellular matrix sample prepared, dipping is added dropwise with 1) solution3mL15wt%
Oxidized sodium alginate is crosslinked, crosslinking60min。
Comparative example 1
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than this comparative example is without ungrease treatment.
Remaining step is same as Example 1.
Comparative example 2
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than this comparative example degreasing agent are as follows: 20wt% fatty alcohol polyoxyethylene ether, 5wt% dodecyl sodium sulfate are mixed with the volume ratio of 1:2
It closes, configures the mixed solution of 50mL.
Remaining step is same as Example 1.
Comparative example 3
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than this comparative example degreasing agent are as follows: the solution of the 15wt% nonylphenol polyoxyethylene ether of 50mL.
Remaining step is same as Example 1.
Comparative example 4
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than in the preparation of (three) complex biological dressing, 1) by 5wt% carboxymethyl chitosan, 15wt% hyaluronic acid, 3wt% chlorination
Calcium, each solution of 6wt% keratan sulfate are mixedly configured into the mixed solution of 50mL with the volume ratio of 1:5:3:4.
Remaining step is same as Example 1.
Comparative example 5
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than in the preparation of (three) complex biological dressing, 1) by 8wt% hydroxyethyl cellulose, 15wt% hyaluronic acid, 3wt% chlorination
Calcium, each solution of 6wt% keratan sulfate are mixedly configured into the mixed solution of 50mL with the volume ratio of 5:3:3:4.
Remaining step is same as Example 1.
Comparative example 6
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than in the preparation of (three) complex biological dressing, 1) by 15wt% hyaluronic acid, 3wt% calcium chloride, 6wt% keratan sulfate
Each solution is configured to the mixed solution of 50mL with the volume ratio of 5:3:4.
Remaining step is same as Example 1.
Comparative example 7
This comparative example provides a kind of preparation method of compound acellular dermal matrix biological dressing, with 1 phase of embodiment
Than, in the preparation of (three) complex biological dressing, without be soaked in 1) described in mixed solution in processing, directly be added dropwise
The 15wt% oxidized sodium alginate of 2mL is crosslinked, and 30min is crosslinked.
Remaining step is same as Example 1.
1 acellular dermal matrix degreasing rate of experimental example and collagen loss late
Referring to step in GB/T5009.6-2003 and ISO3496:1978 (E) detection Examples 1 to 3 and comparative example 1~3
(2) acellular dermal matrix degreasing rate and collagen loss late after taking off cell.The results are shown in Table 1.
1 degreasing rate of table and the detection of collagen loss late
As seen from the results in Table 1, degreasing rate is all larger than 90% in Examples 1 to 3, and degreasing effect is good, while having lower
Collagen loss late, collagen loss late is below 5%.It can be seen that poly- using 20% fatty alcohol polyoxyethylene ether, 15% nonyl phenol
Ethylene oxide ether and 5% sodium dodecyl sulfate solution are mixed with suitable volume ratio, under its synergistic effect, are preparing de- cell
During dermal matrix, good degreasing effect can be realized, and reduce the loss of skin collagen.Degreasing rate in embodiment 4
It is 89%, collagen loss late is 7% also a little higher than 5%, and effect is preferable on the whole, but opposite other embodiments are slightly worse, reason
Although being to use 20% fatty alcohol polyoxyethylene ether, 15% nonylphenol polyoxyethylene ether and 5% sodium dodecyl sulfate solution
Mixing, but the volume ratio of its mixing, not in optimum range, effect slightly declines.And degreasing agent is not used to handle in comparative example 1,
Degreasing rate is 0, and other processing are carried out under the protection of its each component in no degreasing agent, and collagen has to be lost to a certain degree,
But loss late is not high.And degreasing rate in comparative example 2 and comparative example 3 is relatively low, collagen loss late is very high, it is seen then that is not carried out three
When the coordinated of kind of substance, while having played surfactant certain degreasing effect, the loss late of collagen also pole
Height cannot achieve the effect for taking into account high degreasing rate and low collagen loss late not there are three types of the coordinated of substance.Therefore, it uses
20% fatty alcohol polyoxyethylene ether, 15% nonylphenol polyoxyethylene ether and 5% dodecyl sodium sulfate mix in proper proportions,
It plays good degreasing effect for skin on the one hand, on the other hand, under the cooperation of three, realizes mild degreasing,
Skin collagen is not easy to be lost with degreasing, to realize lower collagen loss late.In the embodiment of the present invention, in embodiment 1
Degreasing rate is 100%, and collagen loss late is 1, and effect is best, it is seen then that 20% fatty alcohol polyoxyethylene ether, 15% polyoxyethylene nonyl phenyl
Vinethene, 5% dodecyl sodium sulfate are mixed with the ratio of 1:3:2, have optimal synergy.
The compound acellular dermal matrix biological dressing of experimental example 2 promotes wound healing assay
This experiment detects influence of the compound acellular dermal matrix biological dressing for new zealand rabbit wound healing situation,
Specific step is as follows:
1) 30 healthy new zealand rabbits are chosen, 3% Nembutal sodium solution is slowly injected in auricular vein disinfection, completely to it
It is tested after anesthesia；
2) shaving disinfection is carried out with huckle of the Iodophor to new zealand rabbit, then uses scalpel as the wound of 1cm × 1cm
Face, the surface of a wound are deep to skin corium；
3) after bleeding, blood first is absorbed with common sterile gauze, it will be compound made from Examples 1 to 3 and comparative example 1~7
Type acellular dermal matrix biological dressing, is cut to 1cm × 1cm size, spreads on the surface of a wound, gauze wrapping；Every group each 3 new west
Blue rabbit, each new zealand rabbit single cage raising；
4) variation of observation wound daily, observes the healing state of each wound, and with the reaction of wound no inflammation, wound healing is complete
Whole is judgment criteria, counts the number of days of new zealand rabbit wound healing, the results are shown in Table 2.
The compound acellular dermal matrix biological dressing of table 2 promotes wound healing situation
As it can be seen that in Examples 1 to 3, using 20% fatty alcohol polyoxyethylene ether, 15% nonylphenol polyoxyethylene ether, 5%0
Dialkyl sulfonates mix in proper proportions, to skin carry out degreasing after, using proper ratio 3%Dispase with
After 0.25%Trypsin-EDTA complex enzyme carries out de- cell processing to skin, it is fine to be soaked in chitosan, hyaluronic acid, ethoxy
It ties up element, calcium chloride, keratan sulfate and cross-linking reaction is carried out with oxidized sodium alginate, based on degreasing rate height and maintain original skin
The characteristics of content of skin collagen, and cooperate the joint of chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride, keratan sulfate
Synergistic effect, the compound acellular dermal matrix biological dressing being prepared, has effects that good antibacterial, promoting healing.
Without ungrease treatment in comparative example 1, collagen loss late is relatively low, and healing effect connects with Examples 1 to 3
Closely, but it does not carry out ungrease treatment, when will lead to the compound acellular dermal matrix biological dressing being prepared for wound
With certain immunogenicity.
For comparative example 2~3 compared with Examples 1 to 3, the time needed for promoting wound healing is slightly long, is primarily due to compare
In example 2~3 when carrying out ungrease treatment to skin, skin degreasing rate is low and collagen loss late height promotes the effect of wound healing not have
There is Examples 1 to 3 good.
Carboxymethyl chitosan, the hyaluronic acid, hydroxyethyl cellulose, calcium chloride, sulphur being crosslinked in comparative example 4~6 with skin
Synergistic mating reaction between tamarind quality component is broken, compared with Examples 1 to 3 promote wound healing effect also compared with
Weak, wherein on the one hand carboxymethyl chitosan has antibacterial effect, on the other hand cooperates with hydroxyethyl cellulose, and chlorination is added
De- cell skin and oxygenation sodium alginate cross-linking are impregnated in calcium, hyaluronic acid and keratan sulfate mixing, are obviously improved corium base
The promoting healing of matter dressing acts on.
Comparative example 7 compared with Examples 1 to 3, directly by skin be added crosslinking agent oxidized sodium alginate without with other groups
Distribution is closed, and does not have antibacterial effect, and promote the effect of wound healing most weak.In the above description embodiment of the present invention 1~3
It realizes the effect quickly to heal for promoting skin wound, needs to carry out skin samples reasonable degreasing and take off cell processing, and match
It closes and synergistic, each component is mixed in proper proportions with chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride, keratan sulfate
Close fit, collective effect, are just achieved.
The compound acellular dermal matrix biological dressing performance test of experimental example 3
This experiment is to the disconnected of compound acellular dermal matrix biological dressing described in Examples 1 to 3 and comparative example 1~7
Resistance to spalling (GB/T 24218.3-2010), elongation at break (GB/T 24218.3-2010), vitro cytotoxicity (GB/T
16886.5-2003), delayed allergy (GB/T 16886.10-2005), intradermal reaction (GB/T 16886.10-2005)
It is detected.
The performance test results of biological support in 1 medical dressing of table
Seen from table 1, the testing result for the biological support that prepared by the embodiment of the present invention 1~5 shows that each group is pierced without skin
Swash property, sensitivity response and cytotoxicity, harmless, the mechanical performance of embodiment 1 is best.And in comparative example 1, because it is not adopted
Ungrease treatment is carried out with degreasing agent, therefore its degreasing rate is extremely low, there is immunogenicity, causes delayed allergy and intradermal anti-
It answers, shows as severe erythema.And in comparative example 2 and comparative example 3, the degreasing agent of use does not form rationally matching between three
Than degreasing rate is low and collagen loss late is very high, and mechanical performance is poor, and also can cause delayed super quick to a certain extent
Reaction and intradermal reaction, show as moderate erythema.And the mechanical performance decline of 4~6 dressing of comparative example, reason are to lack carboxymethyl
Chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride, the synergistic effect between keratan sulfate.Wherein, carboxymethyl chitosan
Sugar not only have fungistatic effect, but also on the one hand can with hydroxyethyl cellulose cooperate realize enhancing dressing mechanical performance to
Be conducive to that wound surface not cracky can be laid in when application, on the other hand can also play synergistic effect, improves promoting healing effect
Fruit.Comparative example 7 does not add chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride, keratan sulfate, and mechanical performance is most
Difference.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to the above reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of compound acellular dermal matrix biological dressing, which comprises the following steps:
(1) skin samples are soaked in degreasing agent and are ultrasonically treated degreasing, obtain degreased skin；It include fatty alcohol in the degreasing agent
Polyoxyethylene ether, nonylphenol polyoxyethylene ether, dodecyl sodium sulfate；
(2) it takes off cell: after degreased skin multigelation, being placed in composite enzyme solution and be incubated overnight, obtain de- cell skin；
(3) the de- cell skin is impregnated in carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and sulfuric acid
In the mixed solution of keratan；Be added dropwise oxidized sodium alginate solution be crosslinked to get.
2. preparation method according to claim 1, which is characterized in that the degreasing agent are as follows: 18wt%~22wt% fat
The mixing of alcohol polyoxyethylene ether, 13wt%~17wt% nonylphenol polyoxyethylene ether and 4wt%~6wt% dodecyl sodium sulfate
Liquid.
3. preparation method according to claim 2, which is characterized in that in the degreasing agent: 18wt%~22wt% fat
Alcohol polyoxyethylene ether, 13wt%~17wt% nonylphenol polyoxyethylene ether and 4wt%~6wt% dodecyl sodium sulfate are with volume
Than for 1:(1~5): (1~3) mixing.
4. preparation method according to claim 1, which is characterized in that described that skin samples are soaked in ultrasound in degreasing agent
The time for handling degreasing is 3~5h.
5. preparation method according to claim 1, which is characterized in that mixed solution described in step (3) are as follows: 4wt%~
6wt% carboxymethyl chitosan, 10wt%~20wt% hyaluronic acid, 5wt%~10wt% hydroxyethyl cellulose, 1wt%~
5wt% calcium chloride, 5wt%~7wt% keratan sulfate, and/or
Mixed solution 4wt%~6wt% carboxymethyl chitosan, 10wt%~20wt% hyaluronic acid described in step (3),
5wt%~10wt% hydroxyethyl cellulose, 1wt%~5wt% calcium chloride, 5wt%~7wt% keratan sulfate by volume 1:
(2~8): (2~5): (1~5): the mixing of (3~6).
6. described in any item preparation methods according to claim 1~5, which is characterized in that the oxidation of dropwise addition described in step (3) sea
Solution of sodium alginate is crosslinked are as follows: 10wt%~25wt% oxidized sodium alginate is added dropwise and is crosslinked, 20~60min is crosslinked；With/
Or
The oxidized sodium alginate solution of dropwise addition described in step (3) and the volume ratio of the mixed solution are (1~3): 50.
7. described in any item preparation methods according to claim 1~5, which is characterized in that after the degreased skin multigelation,
It is placed in composite enzyme solution and is incubated overnight are as follows: from -80 DEG C to 37 after DEG C multigelation 2~4 times, be placed in 4 DEG C in composite enzyme solution
Middle overnight incubation.
8. described in any item preparation methods according to claim 1~5, which is characterized in that step (2) described composite enzyme solution
Are as follows: 2.5wt%~3.5wt%Dispase is with 0.2wt%~0.3wt%trypsin-EDTA with volume ratio 1:(2~5) it mixes
It obtains；It is further comprising the steps of and/or after being incubated overnight in composite enzyme solution described in step (2):
The DMEM culture medium containing serum is added and terminates digestion；
De- cell sample is placed in antibiotic solution to impregnate 4~6 minutes, PBS impregnates 8~12 minutes, repeats 3~5 times.
9. a kind of compound acellular dermal matrix biological dressing, which is characterized in that as described in any one of claim 1~9
The preparation method of medical dressing be prepared.
10. compound acellular dermal matrix biological dressing as claimed in claim 9 is promoting the application in skin healing.
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