CN105288702B - A kind of acellular polysaccharide dermal matrix material and its preparation method and application - Google Patents
A kind of acellular polysaccharide dermal matrix material and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of acellular polysaccharide dermal matrix materials and its preparation method and application.The material includes cell-less corium ground substance material and polysaccharide, which is the composite solution of carboxymethyl chitosan, chondroitin sulfate, hyaluronic acid.The preparation method of acellular polysaccharide dermal matrix material is：The PADM after encapsulation disinfection is selected, by weighing, compound carboxymethyl chitosan, sulfate composite chondroitin, glutaraldehyde cross-linking, composite transparent matter acid, PBS buffer solution wash, encapsulate, sterilize.Addition functional form material can be used to prepare biological dressing, cograft holder, tissue engineering epidermis holder in this is acellular polysaccharide dermal matrix material.This is acellular, and polysaccharide dermal matrix material has good external degradation, high-tensile, high permeability steam, high porosity and excellent anti-microbial property.
Description
Technical field
The present invention relates to bioengineering field is related generally to, especially a kind of acellular polysaccharide dermal matrix material and its system
Standby and application.
Background technology
Acellular matrix (acellular dermal matrix, ADM) is to utilize the methods of physics, chemistry, biology by skin
Thoroughly removal only retains the extracellular matrix of the rack containing collagen in corium and obtains for epidermis in skin and the cell containing antigenic component
It arrives, has and resist originality, good biology performance, good mechanical property, be ideal biomaterial, xenogenesis (pig)
Cell-less corium ground substance has with respect to allosome cell-less corium ground substance to be derived from a wealth of sources, advantage of high quality and at a reasonable price, and is contained in collagen
There is very high tissue homology to human skin in amount, establishment structure and height is similar, it can be by modified and crosslinking
One step reduces its antigenicity, improves mechanical property, increases material antibiotic property, has huge application prospect.
Chitin is that a kind of distributed pole is wide, is easy to the high molecular polymer of extraction, is present in the first of the crustaceans such as shrimp, crab
In shell and the cell wall of bacterium, insect algae and higher plant, chitosan is production of the chitin deacetylation 60% or more
Object is the derivative that a kind of linear, semi-crystal shape polysaccharide carboxymethyl chitosan is chitosan, passes through and introduces the small changes such as carboxymethyl
Group is learned, makes it that there is good water solubility in neutral and alkaline environment, while not influencing the inherent characteristic of chitosan.Carboxylic first
Base enclosure glycan has better water-retaining property, biocompatibility, adhesiveness, antibiotic property, biological degradability and low compared with chitosan
Toxicity, while carboxymethyl chitosan can accelerate fibroblast proliferation, wound healing, these advantageous properties make carboxymethyl
Chitosan is more suitable for bio-medical wound repair material, is with a wide range of applications.
Chondroitin sulfate (CS) is to be covalently attached a kind of glycosaminoglycan that proteoglycans is formed on protein, it divides extensively
It is distributed in the extracellular matrix and cell surface of animal tissue, there is blood coagulation resisting function.Being compounded in stromal surface can increase
The biocompatibility of dressing is conducive to cell adherence and proliferation, accelerates wound healing.
Hyaluronic acid also known as sodium hyaluronate, HA, are a kind of acid mucopolysaccharides, belong to high molecular polymer, are by N- acetyl Portugal
What osamine and D-Glucose aldehydic acid were linked to be by glycosidic bond, this polysaccharide has high-moisture-retention, can play and keep cell moisture,
It protects cell not encroached on by pathogen, prevents from infecting, while the recovery of skin histology can be accelerated, improve Wound healing Regenerated energy
Power.The composite transparent matter acid in matrix, can be improved the wound repair ability of dressing, accelerate epithelialization, regulate and control collage synthesis, effectively
Cicatrization is reduced, makes the tissue after healing closer to normal skin.
Biomaterial has good biocompatibility relative to synthetic material, the advantages of being easy to cell attachment and grow, but its
Have the shortcomings that degradation rate is too fast, micro-structure is not easy to control, mechanical strength is insufficient simultaneously.As patent CN1387923A is disclosed
A kind of heterogeneous cell-free hypodermal framework and preparation method thereof, the carriage support trypsase and glutaraldehyde processing sucking pig skin and obtain,
This method treated dermis scaffold have tensile strength is high, good biocompatibility a little, but its poor permeability, the surface of a wound are easy
Hydrops, pneumatosis are generated, vascularization is slow.Patent CN1343523A discloses a kind of micropore allosome or xenogenesis can be used as wound repair
Acellular dermis substitute regularly stamps intensive penetrability using the method for machinery or laser on acellular dermis
Hole, although this can solve the disadvantage that the poor permeability of acellular dermis substitute, aperture is excessive or overstocked, can increase anti-
The exposure put in situ and antigen presenting cell etc. contact the chance of ADM, cause cell compatibility poor, and can break to a certain extent
The structure of bad collagen scaffold.Patent CN1775189A discloses a kind of acellular dermal matrix and preparation method thereof.The de- cell
Dermal matrix is by mammal skin successively with the product obtained after sodium hydroxide solution and detergent-treatment, at this method
Acellular dermal matrix after reason has the advantages that histocompatbility is good, degradability is good, but its tensile strength is to be improved, resists
Bacterium performance is insufficient.
Invention content
Present invention aims to overcome that above-mentioned the deficiencies in the prior art, provide a kind of with low antigenicity, excellent biology
The preparation method of the acellular polysaccharide dermal matrix material of compatibility, good mechanical property and excellent antibiotic property.
A kind of acellular polysaccharide dermal matrix material：The compound water-soluble polysaccharide on the acellular polysaccharide dermal matrix,
The water-soluble polysaccharide includes that carboxymethyl chitosan, chondroitin sulfate, three kinds of hyaluronic acid collectively constitute.
It is used for the first time by the compound method of three kinds of carboxymethyl chitosan, chondroitin sulfate, hyaluronic acid agents in combination,
A kind of preparation method of acellular polysaccharide dermal matrix material further includes specifically that steps are as follows：
(1) the pig acellular dermis matrix (PADM) Jing Guo packaged sterile is selected, and correct amount is as following each process
Meter material foundation；
(2) compound carboxymethyl chitosan：The PADM made is put into rotary drum, will with PADM quality of materials ratios be 1.5~
3.0：1,25~45 DEG C of temperature, pH value 5.5~6.0, the carboxymethyl chitosan solution that mass concentration is 0.50~1.20%, by several times
Rotary drum is added, reaction adjusts pH value to 4.5~5.5 after 30 minutes, then reacts 60~90 minutes, adjusting pH value to 6.5~7.0,
It puts net waste liquid after being disposed, then by treated material with its mass ratio is 4.0：1 distilled water washes 2 times, and every time 15
Minute, drain water after washing；
(3) glutaraldehyde cross-linking：Under conditions of 25 DEG C of temperature, pH value 3.0~5.0 to step (2) resulting materials be added with
PADM quality of materials ratios are 2.0：1 mass concentration is 0.1~0.20% glutaraldehyde solution, is added by several times, gradually adjusts pH value
It to 8.0, reacts 30~60 minutes, takes out material, it is 7.0~7.3 to be washed to pH value with distilled water；
(4) sulfate composite chondroitin：To be 1.5~3.0 with PADM quality of materials ratios:1,25~45 DEG C of temperature, quality are dense
Degree is 0.80~1.00% chondroitin sulfate solution, is added by several times in the rotary drum added with step (3) product, is reacted after sixty minutes,
PH value is adjusted to 6.0~7.0, then by treated material with its mass ratio is 2.0：1 distilled water washes 2 times, and every time 15
Minute, drain water after washing；
(5) composite transparent matter acid：1.00g hyaluronic acids are completely dissolved in acetum, 100ml is diluted to, are made
The solution of a concentration of 1.00% (w/v) will be compounded with the cell-less corium ground substance material after carboxymethyl chitosan, chondroitin sulfate
Centrifugal dehydration makes its volumetric(al) moisture content 70~90%, and hyaluronic acid solution is then sprayed to acellular dermis with small spray bottle
Host surface, quantity for spray are 0.5~1g/m2；
(6) PBS liquid cleans：With with above-mentioned resulting materials mass ratio it is 3.0 at 25 DEG C of temperature：1 PBS solution rinses 3
It is secondary, 30 minutes every time.
(7) it encapsulates：Material pack sealing；
(8) it sterilizes：Packaged material -60 100Gy dose irradiations of cobalt are sterilized, -4 DEG C of gnotobasis is put into and preserves.
The application of above-mentioned acellular polysaccharide dermal matrix material：Material addition functional material can be used to prepare biology
Dressing, cograft holder, tissue engineering epidermis holder, wherein the functional material is antiseptic, antifungal agent, wound are cured
Mixture, growth factor, skin basement membrane are formed in accelerating agent, glycerine.For example, within this material addition volume content be 1.0~
Antibacterial biological dressing is then made in 3.0% glycerine, and one face paste can then be played the protection surface of a wound in the surface of a wound, absorb body fluid
Medical purpose.For example, adding 3~6 μ g/m in acellular polysaccharide dermal matrix material2Growth factor, 8~12 μ g/m2Skin
Skin basilar memebrane formed accelerating agent, 1.0~3.0% glycerine be then formed with the labor statement cutaneous branch frame of antibacterial effect.
Cell-less corium ground substance material has stronger tensile strength, suitable growing space is provided for cell, favorably
Be grown on arrangement naturally in cell, carboxymethyl chitosan have good water-retaining property, biocompatibility, adhesiveness, antibiotic property,
Biological degradability and hypotoxicity, while carboxymethyl chitosan can accelerate fibroblast proliferation, wound healing.Sulfuric acid is soft
Ossein (CS) has blood coagulation resisting function, and the biocompatibility of material can be increased by being compounded in stromal surface, viscous conducive to cell
Attached proliferation accelerates wound healing.Hyaluronic acid has high-moisture-retention, can play and keep cell moisture, protect cell not by disease
The infringement of opportunistic pathogen prevents from infecting, while can accelerate the recovery of skin histology, improves Wound healing power of regeneration.It is multiple in matrix
Hyaluronic acid is closed, the wound repair ability of material can be improved, accelerates epithelialization, regulates and controls collage synthesis, effectively reduces cicatrization,
Make the tissue after healing closer to normal skin.
Compared with prior art, the present invention haing the following advantages：
1. acellular polysaccharide dermal matrix material has good degradability.Experiment shows carboxymethyl chitosan dosage to applying
Material degradation time in vitro has a certain impact, and with the increase of carboxymethyl chitosan dosage, the degradation time in vitro of dressing increases,
Material has degradable property.
2. acellular polysaccharide dermal matrix material has good water vapor permeability.Experiment shows to use with carboxymethyl chitosan
The vapor pervious rate of the increase of amount, dressing increases, and the use of carboxymethyl chitosan improves the water vapor permeability of material, statistics indicate that
For carboxymethyl chitosan dosage when between 0.00~1.50%, the vapor pervious rate of material is in 3400~3700g.m-2.24h-1It
Between, vapor pervious rate is significantly improved.
3. acellular polysaccharide dermal matrix material has the advantages that high porosity.With the increasing of carboxymethyl chitosan dosage
Add, the porosity of dressing increases, when carboxymethyl chitosan dosage is 0.00~1.50%, the porosity of the material 73~
Between 82%, the porosity of material is remarkably reinforced.
4. acellular polysaccharide dermal matrix material has excellent tensile strength.With the increasing of carboxymethyl chitosan dosage
Add, the tensile strength of dressing is remarkably reinforced.When carboxymethyl chitosan dosage is 0.00~1.50%, the tensile strength of the material
Between 9.40~10.80MPa.
5. acellular polysaccharide dermal matrix material has high anti-microbial property.Experiment shows not meet carboxymethyl chitosan
Material, antibiotic rate 3.10%, after 1.00% carboxymethyl chitosan of Material cladding, the antibiotic rate of material is more than 70%,
Illustrate that material has good anti-microbial property.
6. acellular polysaccharide dermal matrix material has wide purposes.We can will be acellular according to different requirements
Polysaccharide dermal matrix material preparation is at biological dressing, cograft holder, tissue engineering epidermis holder.
Description of the drawings
Fig. 1 is the production technological process of the present invention；
Fig. 2 is dressing grain figure SEM × 500；
Fig. 3 is dressing grain figure SEM × 1000.
Specific implementation mode
The present invention is specifically described below by embodiment.Embodiment 1：
The present embodiment provides a kind of methods preparing acellular polysaccharide dermal matrix dressing, are prepared using following steps：
(1) the pig acellular dermis matrix (PADM) Jing Guo packaged sterile is selected, and correct amount is as following each process
Meter material foundation；
(2) compound carboxymethyl chitosan：The cell-less corium ground substance made is put into rotary drum, in 40 DEG C of temperature, pH value
Under conditions of 6.0, it is 2.5 to be added by several times with PADM quality of materials ratios：1 mass concentration is that 1.00% carboxymethyl chitosan is molten
Liquid, reaction adjusts pH value to 5.0 after 30 minutes, then reacts after sixty minutes, adjusts pH value to 7.0.By treated, material is used again
Distilled water is washed 2 times, 15 minutes every time, is washed and is finished control solid carbon dioxide；
(3) glutaraldehyde cross-linking：It is added with PADM quality of materials under conditions of 25 DEG C of temperature, pH value 5.0 than being 2.0
0.20% glutaraldehyde, be added has in the rotary drum of step (2) product by several times, gradually adjusts pH value to 8.0, reacts 30 minutes, take out
Material, it is 7.0~7.3 to be washed to pH value with distilled water；
(4) sulfate composite chondroitin：In 36 DEG C of temperature, pH value is to be added with PADM quality of materials ratios under the conditions of 7.5 to be
2.5：1 1.00% chondroitin sulfate solution of mass concentration, be added has in the rotary drum of step (3) product by several times, reacts 60 minutes
Afterwards, pH value is adjusted to 7.0.Treated material washes 2 times with distilled water, 15 minutes every time, washes to finish and drains to not dripping；
(5) composite transparent matter acid：1.00g hyaluronic acids are completely dissolved in acetum, 100ml is diluted to, are made
The solution of a concentration of 1.00% (w/v).The centrifugation of the cell-less corium ground substance after carboxymethyl chitosan, chondroitin sulfate will be compounded with
Dehydration, makes its volumetric(al) moisture content 80% or so.Then hyaluronic acid solution is sprayed into cell-less corium ground substance with small spray bottle
Surface, quantity for spray are 1g/m2；
(6) PBS liquid cleans：With the distilled water flushing for being 3.0 with above-mentioned material mass ratio 3 times at 25 DEG C of temperature, every time
30 minutes.
(7) it encapsulates：Material pack sealing；
(8) it sterilizes：Packaged material -60 100Gy dose irradiations of cobalt are sterilized, -4 DEG C of gnotobasis is put into and preserves；
It is more than 70% by acellular polysaccharide dermal matrix material antibiotic rate prepared by the above method, tensile strength is more than
10MPa, vapor pervious rate are more than 3600g.m-2.24h-1, porosity is more than 80%.By above-mentioned statistics indicate that the material has
Good antibacterial effect, excellent mechanical property, high vapor pervious rate, high porosity, good biocompatibility.
Antibacterial biological dressing is then made in addition 2wt.% glycerine within this material, and one face paste can then be played in the surface of a wound
The medical purpose protected the surface of a wound, absorb body fluid.
SEM observations are carried out to dressing, as a result as shown in Figure 2 and Figure 3：
It will be seen that the collagenous fibres of this acellular polysaccharide dermal matrix antiseptic dressing are in reticular structure from figure
Arrangement, short texture have a large amount of hole to be distributed, and are conducive to steam volatilization, dressing is made to have good permeable steam permeability.And
Collagenous fibres thickness is uneven, and collagenous fibres interstitial is removed, and has larger hole between fiber, this makes this kind of dressing profit again
It is grown into and vascularization in histocyte, promotes wound healing.
Tensile strength test is carried out to dressing, with reference to GB/T1040.3-2006, tensile strength is on unit cross-sectional area
Tension stress, calculation formula：
TS=F/S
In formula：TS- tensile strength (MPa), the maximum tension (N) that whens F- sample fractures bears, cross when S- sample fractures
Sectional area (mm2).Sample is measured in the case that water content is identical, grip separation speed 100 ± 10mm of movement about per minute,
Sample is not subjected to displacement in drawing process.
As a result as follows：
The tensile strength of 1 acellular polysaccharide dermal matrix antiseptic dressing of table
The result shows that this acellular polysaccharide dermal matrix antiseptic dressing has good tensile strength, that is, have good
Mechanical property.
Vapor pervious rate test is carried out to dressing, is measured using the cup water law of similar Aiba methods, takes the examination of a diameter of 2cm
Pipe, fills physiological saline (0.9%), film is made to be contacted with liquid level, and centre does not interspace, and sealing is weighed along test tube side, by body
System is put into 36 ± 1 DEG C of drier, weighs after 24 hours, and steam transport is calculated by following formula：
Wr=(M0-M1)/S
Wr in formula：Vapor pervious rate (gm-2·24h-1)；M0：Original weight amount (g)；M1:Weight (g) after 24 hours；S：Test tube
Area (the m of mouth2)
As a result as follows：
The vapor pervious rate of 2 acellular polysaccharide dermal matrix antiseptic dressing of table
The experimental results showed that this acellular polysaccharide dermal matrix antiseptic dressing has excellent vapor pervious performance.
Porosity test is carried out to dressing, by -20 DEG C of freeze-dryings of sample warp, weighs 5-6g (W1), accurately to 0.001g,
It is placed in 50ml volumetric flasks, toluene is added in volumetric flask to graticule.Cycle is evacuated to sponge, and there is no bubble spillings, claim also
There is the volumetric flask (W of toluene and timbering material2), then timbering material of the inside containing toluene is taken out, by remaining toluene and appearance
Measuring bottle (W-3).Porosity (P) is calculated according to the following formula：
P=(W2-W3-W1)/(W2-W3) × 100%
As a result as follows：
The porosity of 3 acellular polysaccharide dermal matrix antiseptic dressing of table
The experimental results showed that this acellular polysaccharide dermal matrix antiseptic dressing has high porosity, it is logical to be conducive to steam
It crosses.
Anti-microbial property test is carried out to dressing, it is big that acellular polysaccharide dermal matrix antiseptic dressing is cut into 2.0cm × 2.0cm
It is small, it sets and is used as print group in sterile conical flasks, and the matrix dressing of non-polysaccharide print group as a contrast as stated above, simultaneously
Preparation is not added with print group.It is separately added into 70mL phosphate buffers (0.03molL by 3 groups-1) and 5mL staphylococcus aureuses
The bacterium solution of (or Candida albicans), in 25 DEG C, 200rmin-1Under the conditions of, shake 1h.Respectively take 100ul drops flat respectively at 0h, 1h
Plate, after liquid penetrates into culture medium, 37 DEG C are cultivated 12-24 hours, and bacterium colony counting is carried out.Bacteriostasis rate calculation formula：
X=(A-B)/A × 100%
X is bacteriostasis rate, and A is average colony number before test agent oscillation, and B is average colony number after test agent oscillation.
Criterion：Be not added with the front and back clump count difference of print group shaking will within 10%, test specimen bacteriostasis rate with
Difference >=26% of control sample bacteriostasis rate can determine that the product has antibacterial action.
As a result as follows：
The bacteriostasis rate of 4 acellular polysaccharide dermal matrix antiseptic dressing of table
The experimental results showed that this acellular polysaccharide dermal matrix antiseptic dressing has outstanding anti-microbial property.
Embodiment 2：
The present embodiment provides a kind of methods preparing acellular polysaccharide dermal matrix epidermis holder, take following steps：
(1) according to the acellular polysaccharide dermal matrix material of 1 operation preparation of example；
(2) in the compound 5 μ g/m of material surface2Growth factor, 10 μ g/m2Skin basement membrane formed accelerating agent, 2.0%
Glycerine.
Protection wound can be played by preparing epidermis holder by this method, and can play the work for promoting skin to increase
With raising Wound healing power of regeneration.
Claims (1)
1. a kind of preparation method of acellular polysaccharide dermal matrix, it is characterised in that：It is compound water-soluble on cell-less corium ground substance
Property polysaccharide, the water-soluble polysaccharide include that carboxymethyl chitosan, chondroitin sulfate, three kinds of hyaluronic acid collectively constitute；
Specifically preparation method step is：
(1) the pig acellular dermis matrix PADM Jing Guo packaged sterile, and meter material of the correct amount as following each process are selected
Foundation；
(2) compound carboxymethyl chitosan：The PADM made is put into rotary drum, will be 1.5~3.0 with PADM quality of materials ratios：
1,25~45 DEG C of temperature, the carboxymethyl chitosan solution of pH value 5.5~6.0, are added rotary drum by several times, and reaction adjusts pH after 30 minutes
It is worth 4.5~5.5, then reacts 60~90 minutes, adjust pH value to 6.5~7.0, net waste liquid is put after being disposed, then will processing
Material afterwards is 4.0 with its mass ratio：1 distilled water is washed 2 times, and 15 minutes every time, water is drained after washing；
(3) glutaraldehyde cross-linking：To the addition of step (2) resulting materials and PADM under conditions of 25 DEG C of temperature, pH value 3.0~5.0
Quality of materials ratio is 2.0：1 mass concentration is 0.1~0.20% glutaraldehyde solution, is added by several times, gradually adjusts pH value and arrives
8.0, it reacts 30~60 minutes, takes out material, it is 7.0~7.3 to be washed to pH value with distilled water；
(4) sulfate composite chondroitin：To be 1.5~3.0 with PADM quality of materials ratios:1, the chondroitin sulfate of 25~45 DEG C of temperature
Solution is added by several times in the rotary drum added with step (3) product, and reaction after sixty minutes, adjusts pH value to 6.0~7.0, then will processing
Material afterwards is 2.0 with its mass ratio：1 distilled water is washed 2 times, and 15 minutes every time, water is drained after washing；
(5) composite transparent matter acid：Hyaluronic acid is completely dissolved in acetum and is made into hyaluronic acid solution, carboxylic will be compounded with
Cell-less corium ground substance material centrifugal dehydration after methyl chitosan, chondroitin sulfate makes its volumetric(al) moisture content 70~90%,
Then hyaluronic acid solution is sprayed into cell-less corium ground substance material surface with small spray bottle；
(6) PBS liquid cleans：With with above-mentioned resulting materials mass ratio it is 3.0 at 25 DEG C of temperature：1 PBS solution flushing 3 times, often
Secondary 30 minutes；
(7) it encapsulates：Material pack sealing；
(8) it sterilizes：Packaged material -60 100Gy dose irradiations of cobalt are sterilized, -4 DEG C of gnotobasis is put into and preserves；
In step (2), the carboxymethyl chitosan Sugar concentration is between 0.50~1.20%；
In step (3), the glutaraldehyde mass concentration is between 0.1~0.2%；
In step (4), the chondroitin sulfate mass concentration is between 0.80~1.00%；
In step (5), the solution of a concentration of 1.00%w/v of hyaluronic acid solution, hyaluronic acid solution quantity for spray is 0.5~
1.0g/m2。
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| CN106390180A (en) * | 2016-09-19 | 2017-02-15 | 广东泰宝医疗科技股份有限公司 | Composite dressing having biological activity and preparation method thereof |
| CN106421868A (en) * | 2016-09-19 | 2017-02-22 | 广东泰宝医疗科技股份有限公司 | Chitosan quaternary ammonium salt porcine acellular dermal matrix dressing material and preparation method thereof |
| CN106310352A (en) * | 2016-11-01 | 2017-01-11 | 广东泰宝医疗科技股份有限公司 | Preparation method of antibacterial acellular dermal matrix dressing material |
| CN107029293B (en) * | 2017-03-03 | 2022-06-21 | 济南金泉生物科技有限公司 | Pericardium collagen membrane for guiding bone regeneration and preparation method and application thereof |
| CN107412867B (en) * | 2017-05-10 | 2020-09-01 | 广州润虹医药科技股份有限公司 | Preparation method of heterogenous acellular dermal matrix |
| CN107412868A (en) * | 2017-08-10 | 2017-12-01 | 广州润虹医药科技股份有限公司 | A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix |
| WO2019079672A1 (en) * | 2017-10-19 | 2019-04-25 | Lifecell Corporation | Flowable acellular tissue matrix products and methods of production |
| CN109847088B (en) * | 2019-01-18 | 2022-04-01 | 广州润虹医药科技股份有限公司 | Composite acellular dermal matrix biological dressing and preparation method thereof |
| CN109777765A (en) * | 2019-02-19 | 2019-05-21 | 武汉普诺赛生命科技有限公司 | Stem cell media and its application and stem cell culture method |
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