CN105597150A - Skin patch for repairing skin burn and preparing method of skin patch - Google Patents
Skin patch for repairing skin burn and preparing method of skin patch Download PDFInfo
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Abstract
The invention relates to a preparing method of a skin patch for repairing a skin burn. The preparing method comprises the following steps that adipose-derived stem cells and an allogenic skin matrix are obtained; the adipose-derived stem cells and hydrogel are mixed and inoculated to the allogenic skin matrix, wherein the adipose-derived stem cells are from the body of a patient suffering from the skin burn, and the hydrogel is prepared by mixing an alginate solution and a gelatin solution. The adipose-derived stem cells are carried in the hydrogel prepared from alginate and gelatin so that bacteria can be blocked, a humid environment is maintained for the adipose-derived stem cells and the skin wound, a good repair environment is provided, and immunological rejection is relieved; in addition, adhesion of the patch and the wound can be prevented when the patch is replaced, and thus the new skin tissue can be protected; the purpose of quickly repairing the skin can be achieved, the pain of the patient can also be relieved, and the phenomena of a scar caused by pigment accumulation at the wound part and the like are not likely to occur.
Description
Technical field
The present invention relates to field of tissue engineering technology, particularly a kind of for repairing the dermal patch of skin burnAnd preparation method thereof.
Background technology
Skin is the barrier that human body contacts with external environment, not only plays protection, secretion, metabolism and sensation etc.Effect, and participate in immune response and maintain the stable of organismic internal environment. The skin of showing most as human body,It is impaired injures large area to scald the unexpected incidence of burn higher. Early stage burn wound covers and can suppressMoisture and electrolytical loss are infected, prevent in bacterial growth, prevention; Auto-skin grafting is treatment burnFirst-selection, but for large-area burns, autologous skin can not satisfy the demands, and the cycle course for the treatment of is longer,In therapeutic process, skin wounds is prone to infection; Allograft skin matrix skin can cause obvious immunity rowScold reaction; And transplanting artificial skin, subcutaneous secretion easily forms blister, and then artificial skin is causedDamage, and artificial skin is more expensive, and manufacture craft is more complicated. Therefore, so far, severe trauma andThe skin source shortage of Patients with Big Area Burn remains a clinical difficult problem urgently to be resolved hurrily.
At present, the research of tissue engineering skin has become one of the focus in life science field, andOn market, also occur for clinical organization engineering skin product, though there is certain clinical effectiveness, notOnly expensive, technical operation is loaded down with trivial details, has certain technical difficulty and cost cost higher.
Summary of the invention
Technical problem to be solved by this invention is to repair skin for available technology adopting allograft skin matrixSkin burn wound easily causes immunological rejection and the defect such as curative effect efficiency is low, provides one can avoid exempting fromEpidemic disease rejection and repairing effect are preferably for repairing dermal patch of skin burn and preparation method thereof.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of and burn for repairing skinThe preparation method of the dermal patch of wound, comprises the following steps:
Obtain respectively fat stem cell and allograft skin matrix;
After being mixed with hydrogel, described fat stem cell is inoculated in described allograft skin matrix;
Wherein, it is autologous that described fat stem cell derives from skin burn patient;
Described hydrogel is mixed by alginate solution and gelatin solution.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described marine algaThe concentration of hydrochlorate is 20～80mg/ml, and described gelatin concentration is 20～80mg/ml.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described water-settingThe preparation process of glue, comprises the following steps:
Get alginate in PBS solution, after alginate dissolves completely, constant temperature places 0.2～0.8Hour, obtain described alginate solution; Get gelatin and be dissolved in deionized water, be placed in 45～55 DEG C, treatAfter gelatin fully dissolves, constant temperature is placed 0.2～0.8 hour, obtains described gelatin solution; By described marine algaAcid salt solution and gelatin solution mix, and stir, and constant temperature is placed to solution gel.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described marine algaHydrochlorate is sodium alginate or calcium alginate.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described fatThe concentration of stem cell is (2～8) × 105Individual/ml.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described fatStem cell is arbitrary fat subsitutes stem cell in 2nd～4 generations.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described allosomeThe acquisition process of scytoblastema matter, comprises the following steps:
Get health pig belly or back skin and carry out degreasing depilation processing, remove the epidermis of 0.2～0.6mm, systemBecome the skin corium skin graft that thickness is 0.2～0.4mm, after cleaning, sterilization 20～40 minutes, cleans repeatedly, inIn digestive juice, digest 2～4 days, repeatedly clean skin graft, then add nonionic surface active agent to hatch 2～4My god, remove completely to cell component, obtain acellular dermal, then use respectively sodium chloride and PBS repeatedlyRinse, obtain allograft skin matrix.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, described digestionIn liquid, include mass percent and be 0.10～0.40% trypsase, 0.01～0.03% ethanedioic acid ethylenediamine and0.01～0.03% NaOH.
Provided by the invention for repairing the preparation method of dermal patch of skin burn, by described fatBefore fat stem cell and hydrogel are inoculated in described allograft skin matrix, also comprise and adopt irradiation to described allosomeScytoblastema matter is carried out the step of sterilization processing.
It is a kind of for repairing the dermal patch of skin burn that the present invention also provides, by above-mentioned for repairing skinThe dermal patch preparation method of burn obtains.
Implement provided by the invention for repairing dermal patch of skin burn and preparation method thereof, Ke YidaTo following beneficial effect: by utilizing the differentiation potential of fat stem cell, and the stimulation of allograft skin matrixInducing action, impels fat stem cell to be divided into epidermal cell; And because fat stem cell derives from burnPatient is autologous, and therefore its antigenicity is lower, is difficult for causing immunological rejection, thereby improves skin repairEffect, reaches the object of quick reparation skin trauma; In addition, fat stem cell is carried on to marine algaIn hydrochlorate-gelatin hydrogel, not only can intercept bacterium, make fat stem cell and skin wound keep oneIndividual wet environment, provides a good repairing environment, and can avoid changing paster time, paster withThereby newborn skin histology is protected in wound adhesion, not only realize the object of repairing fast skin, and canAlleviate patient suffering, be difficult for causing the accumulation of wound site pigment to form scar etc.
Detailed description of the invention
Exist and easily cause that immunological rejection is anti-by allograft skin matrix reparation burned skin for solving in prior artShould and the defect such as remediation efficiency is low, innovative point of the present invention is to provide a kind of load to have skin burn patientDermal patch of autologous fat stem cell and preparation method thereof, by adopting autologous fat stem cell in conjunction with differentBody scytoblastema matter, to reduce the immunological rejection causing because of allograft skin matrix, thereby reduction dermal patchAntigenicity, improves skin repair efficiency.
Particularly, provided by the invention for repairing the dermal patch of skin burn, its preparation method comprisesFollowing steps:
S1, obtain fat stem cell and allograft skin matrix respectively;
Wherein, it is autologous that fat stem cell derives from skin burn patient, to reduce immunological rejection; DifferentBody scytoblastema matter is through de-cell dermal matrix after treatment, can be that alloskin matrix is (except skinMankind's scytoblastema matter beyond fire victim), can be also that heterogenous allosome scytoblastema matter is (as the Animal Skin such as pig, oxMatrix);
S2, after being mixed with hydrogel, fat stem cell is inoculated in allograft skin matrix;
Allograft skin matrix is not only used as the paster of repairing burned skin, and has wherein retained original groupKnit the insoluble components in structure, mainly comprise collagen, elastin laminin, GAG and structural proteinsDeng, for Growth of Cells and skin trauma reparation provide nutrition foundation to provide growth for fat stem cellDesired nutritional material and impel fat stem cell to be divided into the cell factor of epidermal cell, makes fat dry thinBorn of the same parents can maintain its cytoactive, and can be divided into epidermal cell; And because fat stem cell derives fromBurned skin patient body, therefore its antigenicity relatively a little less than, can avoid allograft skin matrix directly to contactThe caused immunological rejection of skin burn part, improves the object of repairing skin efficiency with this. Water-settingGlue is the polymer with cross-linked network structure that a class can absorb and possess large quantity of moisture, has three-dimensionalPore structure, stores abundant water environment, has good biocompatibility, can be that fat is dry thinBorn of the same parents provide apposition growth space, expand the contact area of fat stem cell and moisture, dry thin to improve fatBorn of the same parents' cytoactive. Therefore, after being mixed with fat stem cell, hydrogel is inoculated in allograft skin matrix,Can ensure the cytoactive of fat stem cell, and provide certain solidification to fat stem cell, haveBe beneficial to the stability of paster.
Further, in step S1, the present invention preferentially adopts arbitrary fat subsitutes stem cell in 2nd～4 generations,The acquisition process of fat stem cell, comprises the following steps:
Obtaining of S11A, primary fat stem cell;
Under aseptic condition, get described skin burn patient's adipose tissue, reject in fat macroscopicBlood vessel and pars fibrosa, after PBS solution cleans, shred, and enzymolysis vibration digestion 20～60 minutes, usesComplete medium is ended digestion, then is placed in ice bath piping and druming digestion 10～20 minutes, filters, and removes and does not disappearAfter the adipose tissue and connective tissue of changing, filtrate is carried out centrifugal, remove upper strata oil droplet, take off layer fatCell, PBS obtains primary fat stem cell after cleaning;
Wherein, preferably, complete medium is two for containing (5～10) % hyclone and (0.5～2) %Anti-DMEM in high glucose culture medium.
S12A, the primary fat stem cell obtaining in step S11A is carried out to purifying;
The primary fat stem cell obtaining in washing step S11A, removes red blood cell, uses complete medium weightOutstanding, be then inoculated on the coated culture plate of poly-ornithine, cultivate 2～6 hours, remove after supernatantTrypsinization, removes indigested fibroblast and endothelial cell, obtains the former fat subsitutes after purifyingStem cell.
S13A, to the cultivation of going down to posterity of the primary fat stem cell after purifying;
Get the primary fat stem cell after purifying in step S12A and carry out cell count, with (0.5～2) ×105The density of individual/ml is inoculated in DMEM culture medium carries out former culture, more renews every 2-3 daysDMEM culture medium, after Fusion of Cells degree reaches 70～80%, trypsinization 2～4 minutes, adds etc.PBS solution or the normal saline dilution of volume, piping and druming is cleaned repeatedly, and the then centrifugal supernatant of abandoning is usedAfter DMEM culture medium re-suspended cell, in 1:(3～5) the ratio of going down to posterity cultivate, adopt DMEMCulture medium carries out amplification cultivation first generation fat stem cell, in amplification procedure, changes fresh every 2～3 daysDMEM culture medium, after Fusion of Cells degree reaches 70～80%, so repeats above-mentioned cell dissociation and goes down to posterityStep, completes going down to posterity and amplification cultivation of 2nd～4 fat subsitutes stem cells. In the present invention, preferably,Primary fat stem cell is inoculated in to the cultivation of going down to posterity in cell culture bags, is convenient to post incoulation and is coated onIn allograft skin matrix; In primary fat stem cell incubation, cultivate by slight rolling kinetocyte simultaneouslyBag is more conducive to primary fat stem cell and fully contacts culture medium, to improve fat stem cell activity.
Wherein, (it is main in DMEM culture medium, to be added with the ITS of 2～5% human serums, 5～15ng/mlComposition is insulin, transferrins and selenium), the streptomysin of 50～150U/ml, 50～150U/ml penicillin,EGF (EpidermalGrowthFactor, EGF) and the 5～15ng/ml of 5～35ng/mlLIF (leukemiainhibitoryfactor, LIF ELISA).
Wherein, the insulin in ITS, can reduce protein degradation, increases taking the photograph of amino acid and glucoseEnter, for Growth of Cells and differentiation provide energy, impel fat stem cell in low sugar environment, keep cellActivity, is conducive to fat stem cell and breaks up to epidermal cell; Meanwhile, can promote that cell utilizes glucoseAnd protein, promoting the synthetic of RNA, protein and aliphatic acid, inhibited apoptosis, is extremely importantLiability factor. Transferrins can be intervened the ion metabolism in fat stem cell, promotes fat dryThe generation of cell. And selenium is powerful anti-oxidant element, selenium is eliminated in cell cultivation process by polyphenoilsThe free radical producing, reduces and delays the formation of lipofuscin, to reach the object of anti-cell aging and apoptosis;In addition, selenium, as the composition of glutathione peroxidase, can catalytic reduction type glutathione be oxidized formGlutathione, the biofilm structure of Cell protection and the integrality of function, and Cell protection film function and pre-Anti-meronecrosis and sudden change.
Penicillin can hinder the Cell wall synthesis of bacterium, causes cell to leak dead, thus anti-bacteriaGrowth; And streptomysin can act on the ribosomes of bacterium, impede protein translation, thereby anti-bacteriaGrowth; Under natural environment, do not consider the artificial resistance to the action of a drug producing, to the insensitive overwhelming majority of penicillinMicroorganism is to streptomysin sensitivity, and vice versa; Therefore, most preferably penicillin and streptomysin are takenJoin use and can control almost all common bacteriums, thereby can avoid bacterium to fat stem cell as far as possibleGrowth and the active harmful effect causing.
EGF can promote cell migration, stick, breed and survive, and EGF is can induced lipolysis dryThe propagation of cell, migration, and do not affect its differentiation potential, damaged tissue is had and repaired and defencive function.LIF can Promote cell's growth, propagation with break up, in cell culture medium, add a certain amount of LIF passableReplace trophocyte, maintain the propagation of fat stem cell, improve cell proliferation speed and Long-term Proliferation energyPower, and make fat stem cell can keep undifferentiated state.
Further, the acquisition process of allograft skin matrix is:
S11B, get health pig belly or back skin and carry out degreasing depilation and process, remove the table of 0.2～0.6mmSkin, making thickness is the skin corium skin graft of 0.2～0.4mm, after cleaning, sterilization 20～40 minutes, repeatedly clearWash, in digestive juice, digest 2～4 days, repeatedly clean skin graft, then add nonionic surface active agentHatch 2～4 days, remove completely to cell component, obtain acellular dermal, then use respectively sodium chloride moltenLiquid and PBS buffer solution rinse repeatedly, fully to reduce the antigenicity of scytoblastema matter; Wherein, the matter of sodium chlorideAmount mark is 5～9%, and rinsing the processing time with sodium chloride solution is 8～16 hours, changes liquid every 6 hours;PBS rinses and processes 8～16 hours, changes liquid every 6 hours, obtains allograft skin matrix.
Wherein, preferably, in digestive juice, include mass percent and be 0.10～0.40% trypsase,0.01～0.03% ethanedioic acid ethylenediamine and 0.01～0.03% NaOH; Nonionic surface active agent is(0.4～0.6) %TritonX-100 solution.
In step 2, preferably, hydrogel of the present invention is mixed by alginate and gelatin solutionClose and make. Alginate and gelatin are all natural biologic materials, have cheap, safe and reliable andThe advantages such as biocompatibility is good; But the degradation rate of alginate itself is slow, its hydrogel degraded sideFormula is uncontrollable, and catabolite is difficult to because molecular weight is too high discharge; And the character of easy gel under gelatin normal temperature,Poor controllability, therefore, adopts alginate and gelatin covalent cross-linking to form hydrogel, no in the present inventionOnly there is good biocompatibility, catabolite molecular weight, and have stronger hygroscopicity andSafety in utilization, for making dermal patch and laminating degree in skin trauma position is better, is conducive to wound portionThe fat stem cell acting in conjunction of load in position and hydrogel, to reach the object of reparation skin; Meanwhile,The hydrogel of being made up of alginate and gelatin can intercept bacterium, makes wound keep a moist environment,Can extend the service time of paster, reduce the replacing number of times of paster, can reduce on the one hand and to repair skinCost, thus while paster being changed on the other hand, can not protect newborn skin histology with wound adhesion, reduceThe misery of patient in the time changing paster.
In the present invention, the preparation process of alginate-gelatin hydrogel is:
S21A, get in the PBS solution that alginate is dissolved in pH=7.0～7.7, treat that alginate dissolves completelyAfter, constant temperature is placed 0.2～0.8 hour, the alginate solution that preparation concentration is 20～80mg/ml;
S22A, get gelatin and be dissolved in deionized water, be placed in 45～55 DEG C, after gelatin fully dissolves, perseveranceTemperature is placed 0.2～0.8 hour, the gelatin solution that preparation concentration is 20～80mg/ml;
S23A, by molten the gelatin obtaining in the alginate solution obtaining in step S21A and step S22ALiquid mixes, and fully stirs, and constant temperature is placed to solution gel, obtains alginate-gelatin hydrogel.
Wherein, preferably, alginate is sodium alginate, can certainly be calcium alginate etc.
Further, before inoculation fat stem cell, need carry out sterilization processing to allograft skin matrix, withThe antigenicity that further reduces allograft skin matrix, detailed process is:
Under gnotobasis, get the allograft skin matrix obtaining in step S11B and pack in aseptic aluminium foil film, take out trueEmpty sealing, uses 60Coradiation mould 3～5 hours; Wherein, irradiation dose is 3～5kGy, with notThe physicochemical property that changes allograft skin matrix is advisable.
In addition, before inoculation fat stem cell, also need be in gnotobasis, by the allograft skin after sterilizationMatrix is paved, and allograft skin matrix epithelial surface upward, specifically can allograft skin matrix is fixedly laid in smallerOn the glass plate of allograft skin matrix area, or in tiling and Tissue Culture Dish, unnecessary allosome scytoblastemaMatter infolding, makes the inoculating surfaces of allograft skin matrix in formation state, and then inoculation fat stem cell, andAnd after inoculation fat stem cell, allograft skin matrix need remain formation state so that load has fatThe hydrogel of stem cell is uniformly distributed in allograft skin matrix, avoids fat stem cell to pile up, and affects that it is thinCytoactive and normal growth.
The detailed process of step S2 is:
Get the fat stem cell obtaining in step S13A and carry out cell count, with density (2～8) × 105Individual/ml fully mixes with the alginate-gelatin hydrogel obtaining in step S23A;
Then, the allograft skin matrix upper berth after sterilization processing enters to contain the alginate-Ming of fat stem cellGlue gel, preferably, the height of hydrogel is 1～3mm.
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment,The present invention is further elaborated. Should be appreciated that specific embodiment described herein only in order toExplain the present invention, be not intended to limit the present invention.
Embodiment 1
Provided by the invention a kind of for repairing the dermal patch of skin burn, its preparation method comprises followingStep:
S1a, patient select;
Burn patient inclusion criteria: II～III degree burn wound patient, side by side except following patient:
Conceived women breast-feeding their children; To biologic product allergy sufferers used in treatment; Moderate and severe inhalation andHe damages by important organ, as: hepatic and renal function damage, craniocerebral injury etc.; Serious basic disease is as sugarUrine disease, hypertension, tumour or chronic organ injury etc.; Hinder signature and understand informed consent person.
Before S2a, treatment, patient is carried out to inspection and evaluation;
Evaluation criteria: inspection routine blood test (WBC > 3.0 × 10*9/L, LC > 15%; Hb > 80g/L;PLT > 60 × 10*9/L), hepatic and renal function, hepatitis B three be (surface antigen feminine gender), c-hepatitis antibody feminine gender, Chinese mugwortGrow negative antibody, syphilis antibody feminine gender.
Obtaining of S3a, fat stem cell;
S31a, collection adipose tissue;
Select to reach in step S2a the patient of evaluation criteria, under aseptic condition, extract patient with liposuction machineThe fat contents such as belly, buttocks, thigh are enriched the adipose tissue at position, reject in fat macroscopicBlood vessel and pars fibrosa, clean adipose tissue 3 times with PBS, put into contain dual anti-(penicillin/streptomycin,Be respectively 1% content) buffer solution in, gnotobasis saves backup under 4 DEG C of conditions.
S32a, obtain primary fat stem cell;
Get the adipose tissue obtaining in step S31a, after shredding, add 0.075% I type under aseptic conditionClostridiopetidase A, 37 DEG C of constant temperature also digest 40 minutes with 50rpm vibration velocity vibration shaking table, then add equivalent volumesComplete medium (DMEM in high glucose+8% hyclone+1% is dual anti-) end digestion; Then, will disappearChange liquid and be placed in ice bath, blow and beat Dnase I (the deoxyribose core that adds 10mg/L after 15 times with suction pipeAcid enzyme I) hatch 15 minutes, filter through 100 micron screen, remove indigested adipose tissue and knot and formTissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell below the oil droplet of top layer, trainingSupport base top, remove upper strata oil droplet, floating adipocyte is transferred in new centrifuge tube, add PBSClean, centrifugal 10 minutes of 1000rpm in 4 DEG C of environment, repeats this centrifugal process for several times, obtains primaryFat stem cell.
S33a, fat stem cell purifying;
By the primary fat stem cell obtaining in step S32a with 1% salt water washing 2～3 times with except decontaminationThe red blood cell dying, resuspended with complete medium, be inoculated in the coated culture plate of poly-ornithine, cultivate 4Hour, remove supernatant, with 0.25% trypsinization, remove be difficult to digestion fibroblast, inChrotoplasts etc., obtain the primary fat stem cell after purifying.
The cultivation of going down to posterity of S34a, fat stem cell;
Get the primary fat stem cell obtaining in step S33a, with 1 × 105Individual/cm2Density be inoculated in DMEMIn culture medium, carry out former culture, at 37 DEG C, 5% CO2In incubator, changed fresh every 2-3 daysCulture medium, reach after 70%-80% until Fusion of Cells degree, adopt 1 × TrypLESelect (trypsaseWithout phenol red, derive from American I nvitrogen company) in 37 DEG C of digestion 3 minutes, add isopyknic PBSDilution, piping and druming, cleans up cell repeatedly, then the cell after cleaning is moved in 50mL centrifuge tube,With the centrifugal 5min of 1000-2000rpm rotating speed, abandon supernatant, cell is resuspended in to new DMEM and cultivatesBase, according to the ratio that goes down to posterity of 1:4, goes down to posterity and is inoculated in new cell culture bags, adopts DMEM trainingFoster base was cultivated first generation fat stem cell, and amplification procedure was changed fresh DMEM and cultivated every 2-3 daysBase, reaches after 70%-80% until Fusion of Cells degree, so repeats the above-mentioned cell dissociation process that goes down to posterity, and treatsThird generation Fusion of Cells degree reaches after 70%-80%, and the cultivation that stops going down to posterity, for making paster. Wherein,In DMEM culture medium, be added with the ITS (insulin, transferrins, selenium) of 5% human serum, 10ng/ml,The streptomysin of 100U/ml and penicillin, EGF (EpidermalGrowthFactor, the epidermis of 20ng/mlGrowth factor), the LIF (leukemiainhibitoryfactor, LIF ELISA) of 10ng/ml.
The preparation of S4a, allograft skin matrix;
Whole belly of health pig and back skin are carried out to preliminary degreasing by Mechanical Method, hair is rejected with tweezersTotally, first the hypodermis of pigskin is removed with electric dermatome, then remove epidermis 0.4mm, make thickDegree is the skin corium skin graft of 0.3mm, after physiological saline is cleaned, immerse concentration and be 0.1% medical new clean youGo out in solution and sterilize, after 30 minutes, take out, repeatedly rinse 10 times with SPSS; Then by skin graftBe cut into 1cm × 1cm size, PBS liquid rinses twice, is soaked in 0.25% trypsase, 0.02% is housedIn the vial of ethanedioic acid ethylenediamine (EDTA) and 0.02% NaOH mixture slaking solution, digestion at 4 DEG C3d, rocks left and right every day 5 times. After 4 days, take out skin graft, PBS liquid rinses 2 times, puts into and is equipped withIn the vial of 0.5%TritonX-100 solution, under room temperature, hatch, and sustained oscillation 3d, until cell becomesDivide completely and remove, obtain acellular dermal.
Finally, the PBS liquid that the NaCl solution that is 7% with mass fraction and pH value are 7.4 rinses; Wherein,The NaCl solution-treated time is 12h, and the PBS processing time is 12h, changes liquid every 6h,Repeat 5 times, be stored in 4 DEG C of PBS liquid for subsequent use.
The preparation of S5a, sodium alginate-gelatin mixing water gel;
S51a, get in the PBS solution that sodium alginate is dissolved in pH=7.4, be mixed with the marine alga of 50mg/mlAcid sodium solution, magnetic agitation, until completely dissolved, is placed in 37 DEG C of insulating boxs and places 0.5 hour;
S52a, get gelatin and be dissolved in the gelatin solution that is made into 50mg/ml in deionized water, be first placed on 50 DEG CElectric heating constant-temperature blowing drying box in, until completely dissolved, go in 37 DEG C of insulating boxs and place 0.5 hour.
S53a, get the sodium alginate soln and the gelatin solution that prepare, the two mix and blend is after 15 seconds, straightConnect and pour into or push in preprepared Tissue Culture Plate with double syringe, be placed in 37 DEG C of insulating boxsGel reaction 10 minutes.
The preparation of S6a, dermal patch;
The sterilization processing of S61a, allograft skin matrix;
Take out the allograft skin matrix obtaining in step S4a and between sterile purification, pack aseptic aluminium foil film package bag intoIn, vacuum-pumping and sealing, 60Coradiation sterilizing 4 hours, irradiation dose 4kGy.
S62a, mixing sodium alginate-gelatin hydrogel and fat stem cell;
Get the sodium alginate-gelatin hydrogel obtaining in step S53a and add in centrifuge tube, by step S34aThe third generation fat stem cell of middle acquisition is with 5 × 105Density and the sodium alginate-gelatin hydrogel of individual/ml are fastSpeed is fully mixed, and leaves standstill 30 minutes.
S63a, the sodium alginate-gelatin hydrogel that contains fat stem cell is coated in allograft skin matrix;
Under aseptic condition, get the allograft skin matrix obtaining in step S61a, by containing of obtaining in step S62aThere is the sodium alginate-gelatin hydrogel of fat stem cell to be coated in allograft skin matrix, the height of hydrogel2mm left and right, makes dermal patch.
When use, dermal patch is sticked on the surface of a wound to stitching after alignment, pressure dressing and fixing; And it is rightIn burn severe patient, can adopt multi-point injection fat stem cell to coordinate the treatment of dermal patch method, every some injectionThe volume of the fat stem cell that step S34a obtains is less than 250ml, and the density of fat stem cell is 5 × 105Individual/ml.
Embodiment 2
Be with the difference of embodiment 1, in the present embodiment,
Obtaining of S3b, fat stem cell;
S31b, collection adipose tissue;
Select to reach in step S2a the patient of evaluation criteria, under aseptic condition, extract patient with liposuction machineThe fat contents such as belly, buttocks, thigh are enriched the adipose tissue at position, reject in fat macroscopicBlood vessel and pars fibrosa, clean adipose tissue 3 times with PBS, put into contain dual anti-(penicillin/streptomycin,Be respectively 1% content) buffer solution in, gnotobasis saves backup under 4 DEG C of conditions.
S32b, obtain primary fat stem cell;
Get the adipose tissue obtaining in step S31b, after shredding, add 0.075% I type under aseptic conditionClostridiopetidase A, 37 DEG C of constant temperature also digest 20 minutes with 50rpm vibration velocity vibration shaking table, then add equivalent volumesComplete medium (DMEM in high glucose+5% hyclone+2% is dual anti-) end digestion; Then, will disappearChange liquid and be placed in ice bath, blow and beat Dnbse I (the deoxyribose core that adds 10mg/L after 15 times with suction pipeAcid enzyme I) hatch 20 minutes, filter through 100 micron screen, remove indigested adipose tissue and knot and formTissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell below the oil droplet of top layer, trainingSupport base top, remove upper strata oil droplet, floating adipocyte is transferred in new centrifuge tube, add PBSClean, centrifugal 10 minutes of 1000rpm in 4 DEG C of environment, repeats this centrifugal process for several times, obtains primaryFat stem cell.
S33b, fat stem cell purifying;
The primary fat stem cell obtaining in step S32b is depolluted to remove for 3 times with 1% salt water washingRed blood cell, resuspended with complete medium, be inoculated in the coated culture plate of poly-ornithine, cultivate 2 littleTime, remove supernatant, with 0.25% trypsinization, remove the fibroblast, the endothelium that are difficult to digestionCells etc., obtain the primary fat stem cell after purifying.
The cultivation of going down to posterity of S34b, fat stem cell;
Get the primary fat stem cell obtaining in step S33b, with 0.5 × 105Individual/cm2Density be inoculated inIn DMEM culture medium, carry out former culture, at 37 DEG C, 5% CO2In incubator, every 2-3 daysChange fresh culture medium, reach after 70%-80% until Fusion of Cells degree, adopt 1 × TrypLESelectIn 37 DEG C of digestion 3 minutes, add isopyknic PBS dilution, piping and druming, cleans up cell repeatedly,Then the cell after cleaning is moved in 50mL centrifuge tube, with the centrifugal 5min of 1000-2000rpm rotating speed,Abandon supernatant, cell is resuspended in to new DMEM culture medium, according to the ratio that goes down to posterity of 1:3, go down to posterityBe inoculated in new cell culture bags, adopt DMEM culture medium to cultivate first generation fat stem cell,Amplification procedure was changed fresh DMEM culture medium every 2-3 days, treat that Fusion of Cells degree reaches 70%-80%After, so repeat the above-mentioned cell dissociation process that goes down to posterity, treat that third generation Fusion of Cells degree reaches 70%-80%After, the cultivation that stops going down to posterity, for making paster.
Wherein, the ITS that is added with 3% human serum, 5ng/ml in DMEM culture medium (insulin, turns ironAlbumen, selenium), the streptomysin of 50U/ml and the penicillin of 50U/ml, the EGF (Epidermbl of 5ng/mlGrowthFbctor, EGF), and the LIF of 5ng/ml (leukemibinhibitoryfbctor, whiteThe sick inhibiting factor of blood).
The preparation of S4b, allograft skin matrix;
Whole belly of health pig and back skin are carried out to preliminary degreasing by Mechanical Method, hair is rejected with tweezersTotally, first the hypodermis of pigskin is removed with electric dermatome, then remove epidermis 0.2mm, make thickDegree is the skin corium skin graft of 0.2mm, after physiological saline is cleaned, immerse concentration and be 0.1% medical new clean youGo out in solution and sterilize, after 20 minutes, take out, repeatedly rinse 10 times with SPSS; Then by skin graftBe cut into 1cm × 1cm size, PBS liquid rinses twice, is soaked in 0.10% trypsase, 0.01% is housedIn the vial of ethanedioic acid ethylenediamine (EDTB) and 0.01% NaOH mixture slaking solution, digestion at 4 DEG C3d, rocks left and right every day 5 times. After 4 days, take out skin graft, PBS liquid rinses 2 times, puts into and is equipped withIn the vial of 0.4%TritonX-100 solution, under room temperature, hatch, and sustained oscillation 2d, until cell becomesDivide completely and remove, obtain acellular dermal.
Finally, the PBS liquid that the NaCl solution that is 5% with mass fraction and pH value are 7.4 rinses; Wherein,The NbCl solution-treated time is 8h, and the PBS processing time is 8h, changes liquid every 6h, heavyMultiple 5 times, be stored in 4 DEG C of PBS liquid for subsequent use.
The preparation of S5b, sodium alginate-gelatin mixing water gel;
S51b, get in the PBS solution that sodium alginate is dissolved in pH=7.4, be mixed with the marine alga of 20mg/mlAcid sodium solution, magnetic agitation, until completely dissolved, is placed in 37 DEG C of insulating boxs and places 0.5 hour;
S52b, get gelatin and be dissolved in the gelatin solution that is made into 20mg/ml in deionized water, be first placed on 50 DEG CElectric heating constant-temperature blowing drying box in, until completely dissolved, go in 37 DEG C of insulating boxs and place 0.5 hour.
S53b, get the sodium alginate soln and the gelatin solution that prepare, the two mix and blend is after 30 seconds, straightConnect and pour into or push in preprepared Tissue Culture Plate with double syringe, be placed in 37 DEG C of insulating boxsGel reaction 20 minutes.
The preparation of S6b, dermal patch;
The sterilization processing of S61b, allograft skin matrix;
Take out the allograft skin matrix obtaining in step S4b and between sterile purification, pack aseptic aluminium foil film package bag intoIn, vacuum-pumping and sealing, 60Coradiation sterilizing 3 hours, irradiation dose 3kGy.
S62b, mixing water gel and fat stem cell;
Get the sodium alginate-gelatin hydrogel obtaining in step S53b and add in centrifuge tube, by step S34bThe third generation fat stem cell of middle acquisition is with 2 × 105Density and the sodium alginate-gelatin hydrogel of individual/ml are fastSpeed is fully mixed, and leaves standstill 30 minutes.
S63b, the sodium alginate-gelatin hydrogel that contains fat stem cell is coated in allograft skin matrix;
Under aseptic condition, get the allograft skin matrix obtaining in step S61b, by containing of obtaining in step S62bThere is the sodium alginate-gelatin hydrogel of fat stem cell to be coated in allograft skin matrix, height 3mm left and right,Make dermal patch.
When use, dermal patch is sticked on the surface of a wound to stitching after alignment, pressure dressing and fixing; And it is rightIn burn severe patient, can adopt multi-point injection fat stem cell to coordinate the treatment of dermal patch method, every some injectionThe volume of the third generation fat stem cell that step S34b obtains is less than 250ml, and the density of fat stem cell is2×105Individual/ml.
Embodiment 3
Be with the difference of embodiment 1, in the present embodiment,
Obtaining of S3c, fat stem cell;
S31c, collection adipose tissue;
Select to reach in step S2a the patient of evaluation criteria, under aseptic condition, extract patient with liposuction machineThe adipose tissue of belly, rejects macroscopic blood vessel and pars fibrosa in fat, cleans fatty with PBSOrganize 3 times, put in the buffer solution that contains dual anti-(penicillin/streptomycin is respectively 1% content), asepticEnvironment saves backup under 4 DEG C of conditions.
Wherein, this patient is deepⅱdegreeburn, and arm part burn surface area is 100cm2Left and right.
S32c, obtain primary fat stem cell;
Get the adipose tissue obtaining in step S31c, after shredding, add 0.075% I type under aseptic conditionClostridiopetidase A, 37 DEG C of constant temperature also digest 60 minutes with 50rpm vibration velocity vibration shaking table, then add equivalent volumesComplete medium (DMEM in high glucose+10% hyclone+0.5% is dual anti-) end digestion; Then, willDigestive juice is placed in ice bath, blows and beats the Dncse I (deoxyribose that adds 10mg/L after 15 times with suction pipeNuclease I) hatch 20 minutes, filter through 100 micron screen, remove indigested adipose tissue and knotForm tissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell below the oil droplet of top layer,Culture medium top, removes upper strata oil droplet, and floating adipocyte is transferred in new centrifuge tube, addsPCS cleans, and centrifugal 10 minutes of 1000rpm in 4 DEG C of environment repeats this centrifugal process for several times, obtainsPrimary fat stem cell.
S33c, fat stem cell purifying;
The primary fat stem cell obtaining in step S32c is depolluted to remove for 3 times with 1% salt water washingRed blood cell, resuspended with complete medium, be inoculated in the coated culture plate of poly-ornithine, cultivate 6 littleTime, remove supernatant, with 0.25% trypsinization, remove the fibroblast, the endothelium that are difficult to digestionCells etc., obtain the primary fat stem cell after purifying.
The cultivation of going down to posterity of S34c, fat stem cell;
Get the primary fat stem cell obtaining in step S33c, with 2 × 105Individual/cm2Density be inoculated inIn DMEM culture medium, carry out former culture, at 37 DEG C, 5% CO2In incubator, every 2-3 daysChange fresh culture medium, reach after 70%-80% until Fusion of Cells degree, adopt 1 × TrypLESelectIn 37 DEG C of digestion 3 minutes, add isopyknic PBS dilution, piping and druming, cleans up cell repeatedly,Then the cell after cleaning is moved in 50mL centrifuge tube, with the centrifugal 5min of 1000-2000rpm rotating speed,Abandon supernatant, cell is resuspended in to new DMEM culture medium, according to the ratio that goes down to posterity of 1:5, go down to posterityBe inoculated in new cell culture bags, adopt DMEM culture medium to cultivate first generation fat stem cell,Amplification procedure was changed fresh DMEM culture medium every 2-3 days, treat that Fusion of Cells degree reaches 70%-80%After, so repeat the above-mentioned cell dissociation process that goes down to posterity, treat that third generation Fusion of Cells degree reaches 70%-80%After, the cultivation that stops going down to posterity, for making paster.
Wherein, the ITS that is added with 2% human serum, 15ng/ml in DMEM culture medium (insulin, turnsFerritin, selenium), the streptomysin of 150U/ml and the penicillin of 150U/ml, the EGF of 35ng/ml(EpidermclGrowthFcctor, EGF), the LIF (leukemicinhicitory of 15ng/mlFcctor, LIF ELISA).
The preparation of S4c, allograft skin matrix;
Whole belly of health pig and back skin are carried out to preliminary degreasing by Mechanical Method, hair is rejected with tweezersTotally, first the hypodermis of pigskin is removed with electric dermatome, then remove epidermis 0.6mm, make thickDegree is the skin corium skin graft of 0.4mm, after physiological saline is cleaned, immerse concentration and be 0.1% medical new clean youGo out in solution and sterilize, after 40 minutes, take out, repeatedly rinse 10 times with SPSS; Then by skin graftBe cut into 1cm × 1cm size, PBS liquid rinses twice, is soaked in 0.40% trypsase, 0.03% is housedIn the vial of ethanedioic acid ethylenediamine (EDTC) and 0.03% NaOH mixture slaking solution, digestion at 4 DEG C2d, rocks left and right every day 5 times. After 4 days, take out skin graft, PBS liquid rinses 2 times, puts into and is equipped withIn the vial of 0.6%TritonX-100 solution, under room temperature, hatch, and sustained oscillation 4d, until cell becomesDivide completely and remove, obtain acellular dermal.
Finally, the PBS liquid that the NaCl solution that is 9% with mass fraction and pH value are 7.4 rinses; Wherein,The NaCl solution-treated time is 16h, and the PBS processing time is 16h, changes liquid every 6h,Repeat 5 times, be stored in 4 DEG C of PBS liquid for subsequent use.
The preparation of S5c, sodium alginate-gelatin mixing water gel;
S51c, get in the PBS solution that sodium alginate is dissolved in pH=7.4, be mixed with the marine alga of 80mg/mlAcid sodium solution, magnetic agitation, until completely dissolved, is placed in 37 DEG C of insulating boxs and places 0.5 hour;
S52c, get gelatin and be dissolved in the gelatin solution that is made into 80mg/ml in deionized water, be first placed on 50 DEG CElectric heating constant-temperature blowing drying box in, until completely dissolved, go in 37 DEG C of insulating boxs and place 0.5 hour.
S53c, get the sodium alginate soln and the gelatin solution that prepare, the two mix and blend is after 10 seconds, straightConnect and pour into or push in preprepared Tissue Culture Plate with double syringe, be placed in 37 DEG C of insulating boxsGel reaction 5 minutes.
The preparation of S6c, dermal patch;
The sterilization processing of S61c, allograft skin matrix;
Take out the allograft skin matrix obtaining in step S4c and between sterile purification, pack aseptic aluminium foil film package bag intoIn, vacuum-pumping and sealing, 60Coradiation sterilizing 5 hours, irradiation dose 5kGy.
S62c, mixing sodium alginate-gelatin hydrogel and fat stem cell;
Get the sodium alginate-gelatin hydrogel obtaining in step S53c and add in centrifuge tube, by step S34cThe third generation fat stem cell of middle acquisition is with 8 × 105Density and the sodium alginate-gelatin hydrogel of individual/ml are fastSpeed is fully mixed, and leaves standstill 40 minutes.
S63c, the sodium alginate-gelatin hydrogel that contains fat stem cell is coated in allograft skin matrix;
Under aseptic condition, get the allograft skin matrix obtaining in step S61c, by what obtain in step S62cSodium alginate-the gelatin hydrogel that contains fat stem cell is coated in allograft skin matrix, a height 1mm left sideThe right side, makes dermal patch.
Before patient's burned part paster, the localized burn surface of a wound need be made to conventional debridement treatment, remove dirtyThing and the epithelium that comes off, remove bubble and sterilization, by the dermal patch of preparing in the present embodiment according to burn portionPlane is long-pending to be built with shape, then by 130cm2The dermal patch of left and right, facing to burn wound, makesPaster and the surface of a wound are adjacent to and cover the whole surface of a wound, and paster is greater than approximately 2～3cm of edge of wound. After 6d, open gauze,Observe the surface of a wound, find that surface of a wound exudate is less, the dermal patch more renewing, while changing paster, pain is brightThe aobvious allograft skin paster that does not use autologous fat stem cell to smear that is lighter than. Every 6d, open gauze again, findDamage location starts to occur epithelialization, without infecting, and the paster again more renewing. Until 21d opens yarnCloth, the surface of a wound heals substantially. After healing, the surface of a wound is followed up a case by regular visits to, and healing surface of a wound scar proliferation is few, and pigmentation is few.And available technology adopting allograft skin matrix reparation burned skin needs 30 days even longer reparation phases, phaseLonger for dermal patch required time of the present invention, and because immunological rejection more easily causes inflammation,Easily cause pigment accumulation.
In addition, to adopting the patient of the dermal patch that provides of the present embodiment to detect respectively it before treatment and controllingImmunoglobulin IgA, IgG, IgM content after treating the 7th, 14, in 21d blood, testing result is as followsTable 1:
Table 1
(wherein, control group is the content of Immunoglobulin IgA, IgG and IgM in normal human blood. )
Testing result: by adopting dermal patch treatment skin burn wound provided by the invention, in treatmentThe 7th day, immunoglobulin content obviously raise; In the time treating the 14th day, immunoglobulin content is baseThis recovery is normal, illustrates that thus dermal patch provided by the invention has not only shortened the skin repair time, andAnd repairing effect is better.
Above embodiments of the invention are described, above-mentioned concrete but the present invention is not limited toEmbodiment, above-mentioned detailed description of the invention is only schematically, instead of restrictive, this areaThose of ordinary skill under enlightenment of the present invention, protect not departing from aim of the present invention and claimScope situation under, also can make a lot of forms, within these all belong to protection of the present invention.
Claims (10)
1. for repairing the preparation method of dermal patch for skin burn, it is characterized in that, comprise withLower step:
Obtain respectively fat stem cell and allograft skin matrix;
After being mixed with hydrogel, described fat stem cell is inoculated in described allograft skin matrix;
Wherein, it is autologous that described fat stem cell derives from skin burn patient;
Described hydrogel is mixed by alginate solution and gelatin solution.
2. according to claim 1 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, the concentration of described alginate is 20～80mg/ml, and described gelatin concentration is 20～80mg/ml.
3. according to claim 2 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, the preparation process of described hydrogel, comprises the following steps:
Get alginate in PBS solution, after alginate dissolves completely, constant temperature places 0.2～0.8Hour, obtain described alginate solution; Get gelatin and be dissolved in deionized water, be placed in 45～55 DEG C, treatAfter gelatin fully dissolves, constant temperature is placed 0.2～0.8 hour, obtains described gelatin solution; By described marine algaAcid salt solution and gelatin solution mix, and stir, and constant temperature is placed to solution gel.
4. according to claim 3 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, described alginate is sodium alginate or calcium alginate.
5. according to claim 1 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, the concentration of described fat stem cell is (2～8) × 105Individual/ml.
6. according to claim 1 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, described fat stem cell is arbitrary fat subsitutes stem cell in 2nd～4 generations.
7. according to claim 1 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, the acquisition process of described allograft skin matrix, comprises the following steps:
Get health pig belly or back skin and carry out degreasing depilation processing, remove the epidermis of 0.2～0.6mm, systemBecome the skin corium skin graft that thickness is 0.2～0.4mm, after cleaning, sterilization 20～40 minutes, cleans repeatedly, inIn digestive juice, digest 2～4 days, repeatedly clean skin graft, then add nonionic surface active agent to hatch 2～4My god, remove completely to cell component, obtain acellular dermal, then use respectively sodium chloride and PBS repeatedlyRinse, obtain allograft skin matrix.
8. according to claim 7 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, in described digestive juice, include mass percent and be 0.10～0.40% trypsase,0.01～0.03% ethanedioic acid ethylenediamine and 0.01～0.03% NaOH.
9. according to claim 8 for repairing the preparation method of dermal patch of skin burn, itsBe characterised in that, before described fat stem cell and hydrogel are inoculated in to described allograft skin matrix, also compriseAdopt irradiation described allograft skin matrix to be carried out to the step of sterilization processing.
10. for repairing a dermal patch for skin burn, it is characterized in that, by claim 1-9Described in any one for repairing, the dermal patch preparation method of skin burn obtains.
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| CN106110402A (en) * | 2016-08-31 | 2016-11-16 | 朱家源 | Organization engineering skin that Sodium Alginate Hydrogel Films support builds and preparation method thereof |
| RU2625002C1 (en) * | 2016-07-18 | 2017-07-11 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Волгоградский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Method for adhesiogenesis stimulation in pleural cavity in case of polytraumas with predominant thoracic involvement |
| CN110302426A (en) * | 2019-07-10 | 2019-10-08 | 山东大学 | A kind of stem cell skin adhesive piece and its preparation method and application |
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