A kind of artificial skin and preparation method thereof
Technical field
The invention belongs to the technical field of biological materials of organizational project, be specifically related to a kind of artificial skin and preparation method thereof.
Background technology
Skin is the vital tissue organ covering and protect body surface.Because the reasons such as inflammation, ulcer, wound, burn, tumor post-operation and congenital abnormality often cause defect and the exception of skin.For this tissue defect method adopting auto-skin grafting at present more, this wound defect not only causing skin donor site new, and be often subject to the restriction for skin source.
In order to repair, substitute the skin histology of defect, various countries scientist is devoted to the research of artificial skin, and only a few countries such as U.S. is grasped this technology and realizes industrialization.Some artificial skin products of external industrialization, comprise Integra, AlloDerm, Biobrane, DermagraftTM etc., also have some products to be in development, as ICX-SKN.But these products are much allosome or xenogenesis skin to be obtained through processing treatment, there is tooling cost high, the problems such as immunological rejection.
In prior art also the synthetic membrane that makes of useful different material as artificial skin.Gelfoam is the hemostasis absorbent dressing used the earliest, but it guides Growth of Cells performance not as collagen.Polyose is as chitose, glycosaminoglycan, the class materials such as hyaluronic acid have the function of Promote cell's growth, such as chitose has anti-inflammation and sterilization, short Keratiocyte growth effect, for good cell growth substrate material, but along with consumption increase, there is the effect being suppressed to fibroblast growth, the elasticity of material is low simultaneously, and matter is crisp.Urethane, terylene, nylon, silicon rubber etc. are bio-inert material, do not degrade, and can not carry out physiological metabolism, can only be used as outer dressing.The biodegradable materials such as second rac-Lactide, degradable, can metabolism, if but its molecular weight is little, insufficient strength, molecular weight disaster is water-soluble, occurs degraded during dissolving, affect the physical strength of material, and the product after its degraded makes its surrounding tissue acidity improve, and occurs aseptic inflammation.Collagen protein is the important component of artificial skin, is the nutraceutical matrix of cytodifferentiation, growth, has the effect increasing reticular tissue reparation and regeneration, but its shortcoming existed be nonelastic, intensity is low.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, provide a kind of product material to originate wide, with short production cycle, the shelf time long, cheap, be suitable for the artificial skin of wide clinical application.
A technical scheme of the present invention is:
A preparation method for artificial skin, comprises and the tendon being rich in collagen protein in animal, corium, cartilage or osseous tissue is made collagen sponge membrane through the treatment process of engineering science or cytobiology.
Preferably, it comprises the steps: tendon or dermal tissue to carry out to clean, cutting, disinfects, skimming treatment, tissue mashing process, acid enzymolysis, to saltout and dialysis treatment, defoaming treatment, crosslinking Treatment, modifier treatment, lyophilize and vacuum-drying; Described animal tendon or dermal tissue are preferably for being rich in tendon or the cartilaginous tissue of collagen in the allosome of people or Mammals; More preferably ox tendon, cartilage, pig heel string, fish-skin, xenogenesis dermal tissue; Preferably wherein said ox tendon tissue is collagen cell extracellular matrix materials.
Preferably, describedly disinfect as adopting broad-spectrum disinfectant liquid to carry out immersion treatment to ox tendon materials at normal temperatures; The processing mode of described thimerosal is preferably: soak 10-60 minute with the mixed aqueous solution of the Peracetic Acid of 0.05-5% and the ethanol of 1-15%, or the neomycin sulphate solution that 0.1-5% sodium hydroxide solution soaks 4-12 hour, 5-20% soaks 15-60 minute.
Preferably, described skimming treatment for adopting under organic solvent or salt normal temperature to ox tendon materials through 1-5 immersion treatment, each immersion 3-12 hour; Preferred described organic solvent or salts solution are the solution of one or more mixing be selected from dehydrated alcohol, methyl alcohol, propyl carbinol, methylene dichloride, trichloromethane, sodium carbonate, and the mass volume ratio of preferred material and organic solvent or salts solution is 1:20-50 (g/ml).
Preferably, described acid enzymolysis is treated to and adopts one or more mixing solutionss in enzyme solution, acid solution or salts solution to carry out immersion treatment; The pepsin solution that preferred described enzyme solution is treated to 0.2-5% soaks 12-48 hour; Described acid solution is treated to 0.2-5mol/L acetum.The disodium ethylene diamine tetra-acetic acid solution that described salts solution is treated to 0.01-0.5M soaks 1-6 hour.The mass volume ratio of preferred material and solution is 1:20-50 (g/ml).
Preferably, collagen fabric is recombinated for adopting physical method by described crosslinking Treatment, is increased purity and the physical strength of material by the process such as nucleation, collegen filament growth, collagen solution system balance of collagen; Detailed process adopts phosphate buffered saline buffer to regulate the pH value of collagen solution system at 5.0-6.5, and collagen solution final concentration is 0.2-1%, is placed in ultrasonator, and adjustment temperature is 20-25 DEG C, and frequency is 20-60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0-7.6 again, be placed in water-bath and leave standstill 2-6 hour, eventually pass high speed centrifugation process.
Preferably, described modifier treatment adopts macromolecular material to carry out mixing of physical agitation mode with collagen material with certain material proportion, preferably described macromolecular material is one or more mixing in starch, poly(lactic acid), polycarbonate, and preferably described material blending ratio is 10-50:1 volume ratio.
Preferably, described collagen solution defoaming treatment method adopts one or both modes in the physics or defoamer immersion vacuumized to combine, and preferably described defoamer process adopts polyethers, organo-siloxane, tributyl phosphate solvent soaking.
Another technical scheme of the present invention is:
A kind of artificial skin, be three layers or multilayer sandwiched supporting structure, extexine is macromolecule membrane, mainly plays physical barriers effect, is equivalent to the epidermal area of human body skin; Sandwich shelf layer is the artificial skin that above-mentioned preparation method is prepared from, and the nutritive ingredient of supply induced cell growth, simultaneously as the support of Growth of Cells; Internal layer is starch film, this kind of material has superabsorbency, and biocompatibility is better, when artificial skin is used for wound art district, the inflammatory infiltrate of wound tissue can be absorbed, strengthen the adhesive power of product simultaneously, adhere to closely with the surface of a wound and there is moisture-keeping functions, avoiding the unconscious activity due to patient to cause product to come off.
Tool of the present invention has the following advantages: preparation technology of the present invention is simple; artificial skin of the present invention both can as the human body temporary wound-surface cover protection surface of a wound; wound repairing can be grown by permanent reservation dermis scaffold again, solve expensive, the shortcomings such as the damage limited, Production Time is long, the shelf time is short, secondary skin-grafting brings for body of originating of existing artificial skin product.Product material source prepared by the present invention is wide, with short production cycle, the shelf time is long, cheap, is suitable for wide clinical application.
Embodiment
Below by embodiment, the present invention is described in further detail, but scope is not limited to described content.
Embodiment 1
Prepare artificial skin, its step comprises:
Get ox tendon materials, clean up, and be cut into fritter, soak 10 minutes with the mixed aqueous solution of the Peracetic Acid of 0.05% and the ethanol of 1%, to adopt under dehydrated alcohol normal temperature to ox tendon materials through 1 immersion treatment, to soak 3 hours at every turn; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 0.2% to soak and carry out immersion treatment in 12 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 30% strong brine to saltout.Centrifugal 4000g, 30min.Precipitation is incorporated in 0.1% acetic acid, be placed in dialysis tubing the 5h that dialyses with the HCL of 1mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 5.0, collagen solution final concentration is 0.2%, is placed in ultrasonator, and adjustment temperature is 20 DEG C, and frequency is 20Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0 again, be placed in water-bath and leave standstill 2 hours, eventually pass high speed centrifugation process.Put it in vacuum defoaming box and carry out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 2
Prepare artificial skin, its step comprises:
Get ox tendon materials, clean up, and be cut into fritter, soak 60 minutes with the mixed aqueous solution of the Peracetic Acid of 5% and the ethanol of 15%, to adopt under sodium carbonate solution normal temperature to ox tendon materials through 5 immersion treatment, to soak 3 hours at every turn; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 5% to soak and carry out immersion treatment in 48 hours, the mass volume ratio of material and solution is 1:50 (g/ml); Add 50% strong brine to saltout.Centrifugal 5000g, 50min.Precipitation is incorporated in 0.2% acetic acid, be placed in dialysis tubing the 15h that dialyses with the HCL of 2mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 6.5, collagen solution final concentration is 1%, is placed in ultrasonator, and adjustment temperature is 25 DEG C, and frequency is 60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.6 again, be placed in water-bath and leave standstill 6 hours, eventually pass high speed centrifugation process.Immersed in organic siloxane solution and carried out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 50:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 3
Prepare artificial skin, its step comprises:
Get cartilage, clean up, and be cut into fritter, soak 4 hours with 0.1% sodium hydroxide solution, to adopt under methyl alcohol normal temperature to cartilage through 1 immersion treatment, to soak 3 hours at every turn; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 0.2% to soak and carry out immersion treatment in 12 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 30% strong brine to saltout.Centrifugal 4000g, 30min.Precipitation is incorporated in 0.1% acetic acid, be placed in dialysis tubing the 5h that dialyses with the HCL of 1mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 5.0, collagen solution final concentration is 0.2%, is placed in ultrasonator, and adjustment temperature is 20 DEG C, and frequency is 20Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0 again, be placed in water-bath and leave standstill 2 hours, eventually pass high speed centrifugation process.Immersed in polyethers and carried out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 4
Prepare artificial skin, its step comprises:
Get cartilage, clean up, and be cut into fritter, soak 12 hours with 5% sodium hydroxide solution, to adopt under propyl carbinol normal temperature to cartilage through 5 immersion treatment, to soak 12 hours at every turn; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 5% to soak and carry out immersion treatment in 48 hours, the mass volume ratio of material and solution is 1:50 (g/ml); Add 50% strong brine to saltout.Centrifugal 5000g, 50min.Precipitation is incorporated in 0.2% acetic acid, be placed in dialysis tubing the 15h that dialyses with the HCL of 2mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 6.5, collagen solution final concentration is 1%, is placed in ultrasonator, and adjustment temperature is 25 DEG C, and frequency is 60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.6 again, be placed in water-bath and leave standstill 6 hours, eventually pass high speed centrifugation process.Immersed in tributyl phosphate solution and carried out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 50:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 5
Prepare artificial skin, its step comprises:
Get pig heel string, clean up, and be cut into fritter, the neomycin sulphate solution with 5% soaks 60 minutes, to adopt under methylene dichloride normal temperature to pig heel string through 1 immersion treatment, soaks 3 hours at every turn; Fully smashed to pieces by material after soaking, adopt 0.2mol/L acetum to soak and carry out immersion treatment in 12 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 30% strong brine to saltout.Centrifugal 4000g, 30min.Precipitation is incorporated in 0.1% acetic acid, be placed in dialysis tubing the 5h that dialyses with the HCL of 1mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 5.0, collagen solution final concentration is 0.2%, is placed in ultrasonator, and adjustment temperature is 20 DEG C, and frequency is 20Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0 again, be placed in water-bath and leave standstill 2 hours, eventually pass high speed centrifugation process.Immersed in organic siloxane solution and carried out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 6
Prepare artificial skin, its step comprises:
Get pig heel string, clean up, and be cut into fritter, soak 12 hours with 5% sodium hydroxide solution, to adopt under trichloromethane normal temperature to pig heel string through 5 immersion treatment, to soak 12 hours at every turn; Fully smashed to pieces by material after soaking, adopt the disodium ethylene diamine tetra-acetic acid solution of 0.01M to soak 6 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 50% strong brine to saltout.Centrifugal 5000g, 50min.Precipitation is incorporated in 0.2% acetic acid, be placed in dialysis tubing the 15h that dialyses with the HCL of 2mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 6.5, collagen solution final concentration is 1%, is placed in ultrasonator, and adjustment temperature is 25 DEG C, and frequency is 60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.6 again, be placed in water-bath and leave standstill 6 hours, eventually pass high speed centrifugation process.Put it in vacuum defoaming box and carry out defoaming treatment, poly(lactic acid) is carried out mixing of physical agitation mode with collagen material with the ratio of 50:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 7
Prepare artificial skin, its step comprises:
Get fish-skin material, clean up, and be cut into fritter, the neomycin sulphate solution with 20% soaks 15 minutes, to adopt under sodium carbonate solution normal temperature to fish-skin material through 5 immersion treatment, soaks 3 hours at every turn; Fully smashed to pieces by material after soaking, adopt the disodium ethylene diamine tetra-acetic acid solution of 0.5M to soak 1 hour, the mass volume ratio of material and solution is 1:50 (g/ml); Add 30% strong brine to saltout.Centrifugal 4000g, 30min.Precipitation is incorporated in 0.1% acetic acid, be placed in dialysis tubing the 5h that dialyses with the HCL of 1mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 5.0, collagen solution final concentration is 0.2%, is placed in ultrasonator, and adjustment temperature is 20 DEG C, and frequency is 20Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0 again, be placed in water-bath and leave standstill 2 hours, eventually pass high speed centrifugation process.Immersed in polyethers and carried out defoaming treatment, polycarbonate is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 8
Prepare artificial skin, its step comprises:
Get fish-skin material, clean up, and be cut into fritter, soak 60 minutes with the mixed aqueous solution of the Peracetic Acid of 5% and the ethanol of 15%, to adopt under dehydrated alcohol normal temperature to fish-skin material through 5 immersion treatment, to soak 12 hours at every turn; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 5% to soak and carry out immersion treatment in 48 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 50% strong brine to saltout.Centrifugal 5000g, 50min.Precipitation is incorporated in 0.2% acetic acid, be placed in dialysis tubing the 15h that dialyses with the HCL of 2mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 6.5, collagen solution final concentration is 1%, is placed in ultrasonator, and adjustment temperature is 25 DEG C, and frequency is 60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.6 again, be placed in water-bath and leave standstill 6 hours, eventually pass high speed centrifugation process.Immersed in organic siloxane solution and carried out defoaming treatment, polycarbonate is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 9
Prepare artificial skin, its step comprises:
Get xenogenesis dermal matrix tissue, clean up, and be cut into fritter, the neomycin sulphate solution with 20% soaks 60 minutes, to adopt under propyl carbinol normal temperature to xenogenesis dermal matrix tissue through 1 immersion treatment, each immersion 12 hours; Fully smashed to pieces by material after soaking, adopt 5mol/L acetum to soak 12 hours, the mass volume ratio of material and solution is 1:20 (g/ml); Add 30% strong brine to saltout.Centrifugal 4000g, 30min.Precipitation is incorporated in 0.1% acetic acid, be placed in dialysis tubing the 5h that dialyses with the HCL of 1mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 5.0, collagen solution final concentration is 0.2%, is placed in ultrasonator, and adjustment temperature is 20 DEG C, and frequency is 20Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.0 again, be placed in water-bath and leave standstill 2 hours, eventually pass high speed centrifugation process.Immersed in organic siloxane solution and carried out defoaming treatment, starch is carried out mixing of physical agitation mode with collagen material with the ratio of 10:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 10
Prepare artificial skin, its step comprises:
Get xenogenesis dermal matrix tissue, clean up, and be cut into fritter, soak 60 minutes with the mixed aqueous solution of the Peracetic Acid of 5% and the ethanol of 15%, to adopt under methylene dichloride normal temperature to xenogenesis dermal matrix tissue through 5 immersion treatment, each immersion 12 hours; Fully smashed to pieces by material after soaking, adopt the pepsin solution of 5% to soak and carry out immersion treatment in 48 hours, the mass volume ratio of material and solution is 1:50 (g/ml); Add 50% strong brine to saltout.Centrifugal 5000g, 50min.Precipitation is incorporated in 0.2% acetic acid, be placed in dialysis tubing the 15h that dialyses with the HCL of 2mol/L.Adopt phosphate buffered saline buffer to regulate the pH value of collagen solution system 6.5, collagen solution final concentration is 1%, is placed in ultrasonator, and adjustment temperature is 25 DEG C, and frequency is 60Hz.Adopt sodium hydroxide solution to regulate potential of hydrogen to obtain between pH value 7.6 again, be placed in water-bath and leave standstill 6 hours, eventually pass high speed centrifugation process.Immersed in tributyl phosphate solution and carried out defoaming treatment, polycarbonate is carried out mixing of physical agitation mode with collagen material with the ratio of 50:1, by product obtained collagen sponge membrane after lyophilize and vacuum-drying, be required artificial skin.
Embodiment 11: artificial skin fundamental property index Test of the present invention
With the product of above-mentioned embodiment of the present invention 1-10 for laboratory sample carries out property indices mensuration, wherein the tensile strength of artificial skin, drag of breaking, Young's modulus and poisson ratio test method are: adopt electronic universal tension testing machine, sample is cut into appropriate size size such as 10 × 1 (cm), carries out Mechanics Performance Testing with the rate of extension of 100mm/min; The density inspect method of artificial skin is: material is cut into square, with after the length and width of vernier callipers Accurate Measurement sample, thickness and the quality of precision weighing sample according to the volume of sample and the density of Mass Calculation final finished; The mean pore size of artificial skin measures through image analysis software according to scanning electron microscope collection of illustrative plates; The thermal denaturation temperature of artificial skin adopts differential calorimetric scan instrument to measure (before measuring sample in PBS damping fluid swelling 48 hours).This experimental results contrasts with the corresponding index of human body skin, and result is as shown in the table:
Artificial skin mechanical performance index test result