CN105031730A - Preparation method of artificial heterogeneous antibacterial skin - Google Patents

Preparation method of artificial heterogeneous antibacterial skin Download PDF

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CN105031730A
CN105031730A CN201510410473.3A CN201510410473A CN105031730A CN 105031730 A CN105031730 A CN 105031730A CN 201510410473 A CN201510410473 A CN 201510410473A CN 105031730 A CN105031730 A CN 105031730A
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skin
pig
thoracic aorta
solution
artificial
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CN105031730B (en
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邓超
付海田
王秀秀
陈敬华
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Shanghai Zechong Biotechnology Co ltd
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Jiangnan University
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Abstract

The invention discloses a preparation method of artificial heterogeneous antibacterial skin. The preparation method includes: taking an endangium after a pig thoracic aorta is treated; rinsing the endangium clean after being treated by 1M of sodium chloride solution, 10mg/mL of lauryl sodium sulfate solution and 2.5mg/mL of pancreatin solution; soaking the endangium in a nano silver solution, and then cutting the endangium into proper size according to clinical needs to obtain the artificial heterogeneous antibacterial skin. The artificial skin is rich in raw material source, short in production period, remarkable in antibacterial activity, long in preservation time and low in cost and has huge potential in clinical use.

Description

The preparation method of the antibacterial skin of a kind of artificial xenogenesis
Technical field
The present invention relates to tissue engineering technical field of biological materials, especially relate to a kind of with pig thoracic aorta for the method for the antibacterial skin of artificial xenogenesis prepared by raw material.
Background technology
Skin covers the organ that human body surface has high-level organization structure; there is protection human body, prevent antibacterial from corroding and infect; the effects such as regulate body temperature and excretion waste liquid; when skin be subject to wound, burn or other infringement time; the loss organizing body fluid can be caused; thus cause human body water, electrolyte and acid base imbalance, affect human normal and absorb and metabolic balance.In addition, be exposed to the wound very easily bacterial infection of air, shock or septicemia time serious, can be caused.Therefore the reparation of skin must solve.
Desirable artificial skin should possess following characteristics: (1) can cover and close skin wound, protects from infection; (2) there is pliability and certain mechanical strength, transplant after can adhere to well with wound surface, prevent moisture and body fluid from wound surface drain evaporation; (3) promote veins beneath the skin growth, form Autologous epidermis layer and skin corium structure gradually; (4) transplanting survival rate high, forever to be accepted by receptor and to survive, farthest close to the physiological healing quality of normal skin; (5) economy is easy to get, does not carry cause of disease safely.
Two large classes can be divided at present: a class is natural skin, comprises autologous skin, alloskin and xenogenesis skin in the skin regeneration material of clinical middle use; Another kind of is artificial skin, namely uses the tissue engineering artificial skin that natural polymer (collagen, chitosan, hyaluronic acid etc.) and synthesis macromolecule (silicone rubber, nylon, terylene etc.) are synthesized.Though natural skin has the 26S Proteasome Structure and Function being similar to receptor skin, have that supply source deficiency, preparation flow are loaded down with trivial details, a problem such as immunity rejection, Wound Contraction are serious, viral infection; And although artificial skin can be supplied in a large number, and easily preserve, without communicate illness, without immunological rejection, but also there is various drawback, as Intergra not as autotransplantation skin has elasticity and pliability, and Biobrane may suppress the effect etc. of macrophage.Also have by plantation fibroblast external on artificial skin material and horn cell in addition, and cultivate the artificial skin with tissue-engineered composite being formed and there is corium and epidermal structure, the products such as such as Transcyte, Apligraf, OrCel, compare with traditional material, after transplanted tissue's through engineering approaches artificial skin, Wound healing rate improves, and healing time shortens, but treatment cost increases, and product is prepared loaded down with trivial details consuming time.In addition, in wound healing process, conventional wound dressings without antibiotic effect, very easily in agglutination by extraneous bacteriological infection, cause wound infection, healing slows down.
China, as household that raises a large number of pigs, uses part except daily, at present except Corii Sus domestica is as collagen extraction raw material, small intestinal as outside the extraction raw material of medicament with anticoagulant active heparin sodium, the utilization that all the other compositions all do not improve.
Summary of the invention
For the problems referred to above that prior art exists, the applicant provides the preparation method of the antibacterial skin of a kind of artificial xenogenesis.The present invention not only solves the problem of trade waste process, and solve prepare artificial skin carry out source problem.
Technical scheme of the present invention is as follows:
A preparation method for the antibacterial skin of artificial xenogenesis, comprises the steps:
(1) get the qualified pig of quarantine, asepticly to begin to speak, get thoracic aorta, after medical saline rinsed clean, blunt separation obtains the tunica intima that thickness is 0.3 ~ 0.5mm, then with medical saline cleaning, obtained pig thoracic aorta tunica intima;
(2) the pig thoracic aorta tunica intima of step (1) gained is placed in the sodium chloride solution of 1M, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 24h, use medical saline rinsed clean;
(3) the pig thoracic aorta tunica intima that step (2) obtains is placed in the sodium dodecyl sulfate solution that solubility is 10mg/mL, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 12 ~ 24h, use medical saline rinsed clean;
(4) the pig thoracic aorta tunica intima that step (3) obtains is placed in the trypsin solution that concentration is 2.5mg/mL, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 6 ~ 24h, use medical saline rinsed clean;
(5) the pig thoracic aorta tunica intima that step (4) obtains is placed in the nanometer silver solution that concentration is 100 ~ 150 μ g/mL, under room temperature, soaks 30 DEG C of natural air dryings after 24h;
(6) by the product that step (5) is obtained, need cutting according to clinical, vacuum packaging, preserve under 2 ~ 10 DEG C of environment after electron beam radiation disinfection, obtain the antibacterial skin of the artificial xenogenesis of the present invention.
The monthly age of pig described in step (1) is greater than 5 months.
Solvent in solution described in step (1) ~ step (4) is ultra-pure water.
In step (5), the flux of nanometer silver solution is medical saline, and nanometer silver mean diameter is 5 ~ 50nm.
Medical saline described in step (1) ~ step (5) to be mass concentration be 0.9% sodium chloride solution.
Frequency ultrasonic in described ultrasonic wave concussion process is 40KHz.
The technique effect that the present invention is useful is:
(1) the present invention thoroughly removes endothelial layer and the smooth muscle cell of pig thoracic aorta, makes tunica intima become autologous skin growth support.
(2) the present invention removes endotheliocyte and layer of smooth muscle cells in theca interna by the cleaning of SDS solution and trypsin cleaning step, reduces immunogenicity.
(3) the present invention is soaked by nanometer silver solution, significantly improves the antibacterial activity of finished product, increases the products storage time.
(4) the present invention compares the sterilization methods such as epoxy acetylene, Co 60, after electron beam radiation disinfection product without harmful chemical substance or radiation remaining, ensure that finished product to human body without damaging effect.
(5) the reservation extracellular matrix that the present invention is complete, fibroblast and horn cell can shoot up, and form skin corium and epidermal area, thus avoid causing cicatrization overweight because of skin corium defect; Adhere to closely with wound surface simultaneously, and containing a large amount of angiogenic material, therefore significantly can accelerate the speed of growth of skin revitalization blood vessel, shorten healing time.
(6) preparation process of the present invention is simple, and finished product form is complete, and guarantor's liquid rate, mechanical strength all can meet instructions for use, and Wound Contraction rate is low; Without the need to improving intensity with the toxic reagents such as glutaraldehyde are crosslinked, without the need to increasing the wettability of finished product with wetting agent, compared with existing artificial skin, using chemical drugs few, reducing chemical toxicity during use.
(7) the artificial skin collagen structural integrity of the present invention, is beneficial to the growth of fibroblast and horn cell, is applicable to skin injury or burn, safe and reliable, nontoxic, avoids pathophoresis.
(8) the present invention's artificial skin material abundance, with short production cycle, holding time are long and cheap, have the tremendous potential that can be applicable to Clinical practice.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is specifically described.
Embodiment 1
A preparation method for artificial xenogeneic skin, comprises the following steps:
(1) got for 12 monthly ages to quarantine qualified pig, asepticly to begin to speak, get thoracic aorta, after medical saline rinsed clean, it is 0.3 ~ 0.5mm tunica intima that blunt separation obtains thickness, by pig thoracic aorta tunica intima obtained after medical saline cleaning;
(2) the pig thoracic aorta tunica intima of step (1) gained is placed in the sodium-chloride water solution of 1M, and shake with the rotating speed of 100r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(3) the pig thoracic aorta tunica intima that step (2) obtains is placed in the lauryl sodium sulfate aqueous solution that solubility is 10mg/mL, and shake with the rotating speed of 100r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 12h;
(4) the pig thoracic aorta tunica intima that step (3) obtains is placed in the trypsin solution that concentration is 2.5mg/mL, and shake with the rotating speed of 100r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(the pig thoracic aorta tunica intima that step (4) obtains by (5) is placed in the nanometer silver solution that concentration is 100 μ g/mL, soaks 30 DEG C of natural air dryings after 24h under room temperature;
(6) by the product that step (5) is obtained, need cutting according to clinical, vacuum packaging, preserve under 2 ~ 10 DEG C of environment after electron beam radiation disinfection, obtain the artificial xenogeneic skin of the present invention.
The monthly age of pig described in step (1) is greater than 5 months.
Solvent in solution described in step (1) ~ step (4) is ultra-pure water.
In step (5), the flux of nanometer silver solution is medical saline, and nanometer silver mean diameter is 50nm.
Medical saline described in step (1) ~ step (5) to be mass concentration be 0.9% sodium chloride solution.
Frequency ultrasonic in described ultrasonic wave concussion process is 40KHz.
Embodiment 2
A preparation method for artificial xenogeneic skin, comprises the following steps:
(1) got for 18 monthly ages to quarantine qualified pig, asepticly to begin to speak, get thoracic aorta, after medical saline rinsed clean, it is 0.3 ~ 0.5mm tunica intima that blunt separation obtains thickness, by pig thoracic aorta tunica intima obtained after medical saline cleaning;
(2) the pig thoracic aorta tunica intima of step (1) gained is placed in the sodium-chloride water solution of 1M, and shake with the rotating speed of 150r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(3) the pig thoracic aorta tunica intima that step (2) obtains is placed in the lauryl sodium sulfate aqueous solution that solubility is 10mg/mL, and shake with the rotating speed of 150r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 18h;
(4) the pig thoracic aorta tunica intima that step (3) obtains is placed in the trypsin solution that concentration is 2.5mg/mL, and shake with the rotating speed of 150r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 6h;
(the pig thoracic aorta tunica intima that step (4) obtains by (5) is placed in the nanometer silver solution that concentration is 150 μ g/mL, soaks 30 DEG C of natural air dryings after 24h under room temperature;
(6) by the product that step (5) is obtained, need cutting according to clinical, vacuum packaging, preserve under 2 ~ 10 DEG C of environment after electron beam radiation disinfection, obtain the artificial xenogeneic skin of the present invention.
The monthly age of pig described in step (1) is greater than 5 months.
Solvent in solution described in step (1) ~ step (4) is ultra-pure water.
In step (5), the flux of nanometer silver solution is medical saline, and nanometer silver mean diameter is 50nm.
Medical saline described in step (1) ~ step (5) to be mass concentration be 0.9% sodium chloride solution.
Frequency ultrasonic in described ultrasonic wave concussion process is 40KHz.
Embodiment 3
A preparation method for artificial xenogeneic skin, comprises the following steps:
(1) got for 24 monthly ages to quarantine qualified pig, asepticly to begin to speak, get thoracic aorta, after medical saline rinsed clean, it is 0.3 ~ 0.5mm tunica intima that blunt separation obtains thickness, by pig thoracic aorta tunica intima obtained after medical saline cleaning;
(2) the pig thoracic aorta tunica intima of step (1) gained is placed in the sodium-chloride water solution of 1M, and shake with the rotating speed of 120r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(3) the pig thoracic aorta tunica intima that step (2) obtains is placed in the lauryl sodium sulfate aqueous solution that solubility is 10mg/mL, and shake with the rotating speed of 120r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(4) the pig thoracic aorta tunica intima that step (3) obtains is placed in the trypsin solution that concentration is 2.5mg/mL, and shake with the rotating speed of 120r/min in 37 DEG C of constant-temperature tables, medical saline rinsed clean is used at interval of after 6h ultrasonic wave concussion (frequency is 40KHz) 5min, 24h;
(5) the pig thoracic aorta tunica intima that step (4) obtains is placed in the nanometer silver solution that concentration is 100, under room temperature, soaks 30 DEG C of natural air dryings after 24h;
(6) by the product that step (5) is obtained, need cutting according to clinical, vacuum packaging, preserve under 2 ~ 10 DEG C of environment after electron beam radiation disinfection, obtain the artificial xenogeneic skin of the present invention.
The monthly age of pig described in step (1) is greater than 5 months.
Solvent in solution described in step (1) ~ step (4) is ultra-pure water.
In step (5), the flux of nanometer silver solution is medical saline, and nanometer silver mean diameter is 50nm.
Medical saline described in step (1) ~ step (5) to be mass concentration be 0.9% sodium chloride solution.
Frequency ultrasonic in described ultrasonic wave concussion process is 40KHz.
Test case 1
The antibacterial skin of the artificial xenogenesis embodiment of the present invention 3 obtained carries out antibacterial effect evaluation, in embodiment 3, step (4) gained does not soak the pig thoracic aorta tunica intima of nanometer silver solution as negative control group, with the common staphylococcus aureus of skin surface for test model.
Experimental technique: depletion Staphylococcus aureus strain is recovered in broth bouillon, with the rotating speed of 120r/min concussion overnight incubation in 37 DEG C of constant incubators.Getting normal saline, bacterial suspension to be diluted to 600nm except light absorption value be after about 0.1, is spread evenly across on broth agar culture medium.Cut-off footpath is the both sides that the antibacterial skin of the artificial xenogenesis of embodiment 3 gained of 1cm and negative control group sample are attached to band same bacterio-agar culture medium flat plate, after 37 DEG C of constant temperature culture 24h, inhibition zone size is measured according to GB GB/T20944.1-2007, and combine the average evaluation sample antibacterial effect whether having bacterial growth bottom test sample, parallel testing 5 times.Result is as shown in table 1, and does not soak compared with nano silver material, artificial xenogenesis antibacterial skin anti-Staphylococcus aureus Be very effective.
Table 1 anti-Staphylococcus aureus effect assessment
Test case 2
The antibacterial skin of the artificial xenogenesis embodiment of the present invention 2 obtained carries out antibacterial effect evaluation, in embodiment 2, step (4) gained does not soak the pig thoracic aorta tunica intima of nanometer silver solution as negative control group, with wound infection comparatively common bacteria pseudomonas aeruginosa for test model
Experimental technique: get pseudomonas aeruginosa strain and recover in broth bouillon, with the rotating speed of 120r/min concussion overnight incubation in 37 DEG C of constant incubators.Getting normal saline, bacterial suspension to be diluted to 600nm except light absorption value be after about 0.1, is spread evenly across on broth agar culture medium.Cut-off footpath is the both sides that the antibacterial skin of the artificial xenogenesis of embodiment 2 gained of 1cm and negative control group sample are attached to band same bacterio-agar culture medium flat plate, after 37 DEG C of constant temperature culture 24h, inhibition zone size is measured according to GB GB/T20944.1-2007, and combine the average evaluation sample antibacterial effect whether having bacterial growth bottom test sample, parallel testing 5 times.Result is as shown in table 2, and does not soak compared with nano silver material, the anti-pseudomonas aeruginosa Be very effective of the antibacterial skin of artificial xenogenesis.
The anti-pseudomonas aeruginosa effect of table 2
Test case 3
The antibacterial skin of the artificial xenogenesis embodiment of the present invention 2 obtained carries out the transplantation experiments of SD rat skin defect model, in embodiment 2, step (4) gained does not soak the pig thoracic aorta tunica intima of nanometer silver solution as negative control group, and hospital gauze is as blank group.
Experimental technique: by 15 of the same age, fix with the healthy SD rat anesthesia of build, after rejecting back hair under aseptic condition, cut off skin to mucous layer with surgical scissors, cause diameter to be the wound defect model of 1cm; After being divided into three groups at random, wherein one group of rat applies the obtained antibacterial skin of artificial xenogenesis of the embodiment 2 identical with wound size at random, and one group is applied in wound surface with the negative control group material identical with wound size, remains one group and binds up a wound with hospital gauze.After fixing wrapping, normal diet, observes wound healing situation, record wound healing time.The results are shown in Table shown in 3, compared with hospital gauze, the antibacterial skin of artificial xenogenesis and negative control group anthemorrhagic speed are fast, bond closely, difficult drop-off with wound, can remarkable absorbing wound exudate, significantly promote wound healing; Compared with negative control group, the artificial xenogenesis antibacterial skin inflammatory reaction time is short, and wound is without obviously infecting phenomenon.Illustrate that the present invention significantly can promote wound healing, and have significant antibacterial effect.
Table 3 wound healing

Claims (6)

1. a preparation method for the antibacterial skin of artificial xenogenesis, is characterized in that comprising the steps:
(1) get the qualified pig of quarantine, asepticly to begin to speak, get thoracic aorta, after medical saline rinsed clean, blunt separation obtains the tunica intima that thickness is 0.3 ~ 0.5mm, then with medical saline cleaning, obtained pig thoracic aorta tunica intima;
(2) the pig thoracic aorta tunica intima of step (1) gained is placed in the sodium chloride solution of 1M, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 24h, use medical saline rinsed clean;
(3) the pig thoracic aorta tunica intima that step (2) obtains is placed in the sodium dodecyl sulfate solution that concentration is 10mg/mL, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 12 ~ 24h, use medical saline rinsed clean;
(4) the pig thoracic aorta tunica intima that step (3) obtains is placed in the trypsin solution that concentration is 2.5mg/mL, and shake with the rotating speed of 100 ~ 150r/min in 37 DEG C of constant-temperature tables, at interval of 6h ultrasonic wave concussion 5min, after 6 ~ 24h, use medical saline rinsed clean;
(5) pig thoracic aorta tunica intima obtained for step (4) is placed in the nanometer silver solution that concentration is 100 ~ 150 μ g/mL, under room temperature, soaks 30 DEG C of natural air dryings after 24h;
(6) need cutting according to clinical, vacuum packaging, preserve under 2 ~ 10 DEG C of environment after electron beam sterilization, obtain the antibacterial skin of the artificial xenogenesis of the present invention.
2. method according to claim 1, is characterized in that the monthly age of pig described in step (1) is greater than 5 months.
3. method according to claim 1, is characterized in that the solvent in solution described in step (1) ~ step (4) is ultra-pure water.
4. method according to claim 1, it is characterized in that described in step (5), nanometer silver mean diameter is 5 ~ 50nm, the solvent of nanometer silver solution is medical saline.
5. method according to claim 1, is characterized in that medical saline described in step (1) ~ step (5) to be mass concentration is the sodium chloride solution of 0.9%.
6. method according to claim 1, is characterized in that frequency ultrasonic in described ultrasonic wave concussion process is 40KHz.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049568A2 (en) * 2007-10-17 2009-04-23 Bio-Skin, A.S. Sterile autologous, allogenic or xenogenic implant and the method of its production
CN101623518A (en) * 2009-06-30 2010-01-13 中国人民解放军第二军医大学 Anti-infection bio-derived hernia and body wall repair material, preparation and application thereof
CN102218162A (en) * 2011-05-24 2011-10-19 山西奥瑞生物材料有限公司 Preparation method of homologous acellular dermal matrix
CN103191466A (en) * 2013-04-15 2013-07-10 潘华倩 Method for preparing human body or animal accellular tissues

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049568A2 (en) * 2007-10-17 2009-04-23 Bio-Skin, A.S. Sterile autologous, allogenic or xenogenic implant and the method of its production
CN101623518A (en) * 2009-06-30 2010-01-13 中国人民解放军第二军医大学 Anti-infection bio-derived hernia and body wall repair material, preparation and application thereof
CN102218162A (en) * 2011-05-24 2011-10-19 山西奥瑞生物材料有限公司 Preparation method of homologous acellular dermal matrix
CN103191466A (en) * 2013-04-15 2013-07-10 潘华倩 Method for preparing human body or animal accellular tissues

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