CN105013013A - Preparation method of skin ulcer repairing matrix - Google Patents
Preparation method of skin ulcer repairing matrix Download PDFInfo
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- CN105013013A CN105013013A CN201510454579.3A CN201510454579A CN105013013A CN 105013013 A CN105013013 A CN 105013013A CN 201510454579 A CN201510454579 A CN 201510454579A CN 105013013 A CN105013013 A CN 105013013A
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Abstract
The invention provides a preparation method of a skin ulcer repairing matrix. According to the preparation method, a natural extracellular matrix subjected to decellularization, antigen removal and thickening treatment is adopted as a biological scaffold, seed cells are stratified on the surface of the natural extracellular matrix to form a cell membrane with multi-layer cells, and the skin ulcer repairing matrix is obtained after freeze drying treatment. By adopting the preparation method, the effects of surface cell stratifying of the extracellular matrix and partial cell ingrowth in the extracellular matrix are achieved; after the skin ulcer repairing matrix is used, the wound repairing can be effectively protected and promoted, and little scar is formed; the skin ulcer repairing matrix is short in preparation period, simple in process, suitable for industrialization, wide in application range, and good in wound repairing effect; the elasticity and toughness of the healed wound are close to those of the normal skin. By adopting freeze drying sterilization treatment, the product is guaranteed to have the expected use effect, the quality guarantee period is prolonged, and the transportation and storage requirements are reduced greatly.
Description
Technical field
The invention belongs to tissue engineering technique field, relate to the preparation method that a kind of skin ulcer repairs substrate.
Background technology
Skin is the first defensive barrier of human body, has the critical function maintaining body stable state, and therefore, the skin deficiencies damage no matter which kind of reason causes, all needs to treat in time.Small size shallow-layer skin injury can by human body self-regulation self-healing, but wound diameter is greater than 20cm or is deep to skin corium and chronic skin ulcer patient, needs to be treated by other means.
Chronic skin ulcer (chronicskinulcer, CSU) be intractable ulcer again, is a kind of clinical common disease.Diabetic foot and venous ulcer etc. all belong to intractable ulcer.In recent years along with people's living standard improves and social senilization, diabetes (diabctesmellitus, DM) sickness rate rises year by year.Current China diabetics about 9,200 ten thousand, wherein the patient of 15% ~ 25% can suffer from foot ulcers.If do not treated, amputation may be caused.
For the various skin injury for the treatment of, there is many unvanquishable shortcomings in traditional dermatoplastic method, as caused many places wound surface, increases the misery of patient; The demand etc. that large area is implanted can not be met.In addition, as some intractable ulcer wound surface such as diabetic foot and venous ulcer also cannot realize wound healing by simple skin-grafting.Skin ulcer repairs substrate then for these difficult problems provide the approach of solution.
Organization engineering skin is generally prepared from by seed cell and timbering material.The kind of timbering material is more, but limit due to material nature and processing method etc., at present the support of report be not well positioned to meet cell proliferation require or preparation technology loaded down with trivial details.Extracellular matrix (extracellularmatrix, ECM) is the general name of extracellular material in connective tissue, is a kind of well timbering material.The effect that it can play storage, display to somatomedin and discharge.Existing a lot of report proves that extracellular matrix can effectively guide fibrocyte to grow into and promote that new vessels is formed.The somatomedin of the emiocytosis of inoculation has material impact to the wound such as ulcer, burn and scald.Somatomedin directly can promote the reproduction restraint of transplanted cells, also can maintain its biological function; The activity decrease of the wound surface such as ulcer, deep burn tropic growth factors and receptor thereof and the shortage of the relative or absolute of quantity are the pathophysiological basis that wound surface is difficult to heal.
Number of patent application be 200910078357.0 Chinese invention patent (patent right lost efficacy) disclose a kind of with people source epidermis cell for seed cell, the extracellular matrix that people source fibroblasts to secrete is formed is the method that biological support builds organization engineering skin.There is the defect of the high and complex process of incubation time long (being generally 20 ~ 50 days), cost in the method.
Number of patent application be 200810116108.1 Chinese invention patent (patent right lost efficacy) be referred to a kind of with people source fibroblast for seed cell, be inoculated in the method building organization engineering skin in gel biological material.There is the shortcomings such as cultivation cycle long (being generally 15 ~ 20 days), cost are high, complex process equally in the method.
Number of patent application is the Chinese invention patent application of 201510100264.9, disclose a kind of organization engineering skin and application thereof, it is with acellular dermal matrix (Acellular dermal matrix, ADM) as timbering material, umbilical cord mesenchymal stem cells is the organization engineering skin that seed cell builds.Although this technology shortens incubation time, but need at acellular dermal matrix pan coating one deck gelatin in preparation process, then repeatedly inoculate, and cultivate a period of time after bundle hole is carried out on ADM surface, just can reach the effect that cell membrane covers ADM surface.If do not wrap by gelatin, the product that surface has intact cell film cannot be obtained.This kind of preparation method does not only make acellular matrix give full play to self-acting when cell culture, and technique is comparatively loaded down with trivial details, and cost is high, is not suitable for industrialization.And, this preparation method also can only form cell monolayer film on ADM surface, and the somatomedin of emiocytosis can fast degradation in vivo as a kind of protein, thus the amount of the cell membrane secretion somatomedin of monolayer can not meet the effect regulating local wound microenvironment far away, thus makes it limited to the repairing effect of wound surface.In addition, nearly all there is same problem in the organization engineering skin series products reported at present, namely causes the shelf-life short due to product form, to storage condition and the high shortcoming of movement requirement.
Summary of the invention
The object of the invention is to solve that the cultivation cycle that existing organization engineering skin construction method exists is long, cost is high, complex process, be not suitable for industrialization, the effect regulating local wound microenvironment can not be met, thus make that it is limited to the repairing effect of wound surface, the shelf-life is short, to storage condition and the high shortcoming of movement requirement.
For this reason, the invention provides the preparation method that a kind of skin ulcer repairs substrate, mainly adopt de-cell, remove antigen and thicken process after natural extracellular matrix as biological support, compound seed cell forms the cell membrane with multi-layer cellular on natural extracellular matrix surface, namely obtains skin ulcer and repair substrate after lyophilization process;
Described seed cell be autologous or allogeneic source fibroblast or stem cell.
Above-mentioned skin ulcer repairs the preparation method of substrate, specifically comprises the following steps:
1) preparation of step one, extracellular matrix: go the collagem membrane tissue dipping by lye of antigen and inactivation treatment to carry out thickening process by completing de-cell, then with 0.01 ~ 0.05M PBS solution carry out cleaning to pH be 6 ~ 7, and within 1 ~ 6 hour, obtain extracellular matrix with described PBS solution immersion, for subsequent use after then using the sterilizing of 10 ~ 25kGy 60Coradiation;
2) preparation of step 2, culture medium:
2.1), basal liquid is prepared: by the volume ratio of 9:1, commercial for DMEM/F12 culture fluid and hyclone or new-born calf serum are mixed into basal liquid;
2.2), basal medium is prepared: by basal liquid Standard entertion insulin 1 ~ 100ng described in 500ml, transferrins 1 ~ 50mg, sodium selenite 1 ~ 30 μ g, hydrogenation can pine 10 ~ 600 μ g, epithelical cell growth factor 1 ~ 20 μ g, basic fibroblast growth factor 1 ~ 100ng, transforminggrowthfactor-β1 ~ 100ng, vitamin C 1 ~ 100mg, is mixed with basal medium;
2.3), preparation culture medium: add Niu Chuiti extracting solution 10 ~ 30 μ g/ml, adenine 1 ~ 50mg, HRG 50 ~ 300ng/ml and platelet derived growth factor 1 ~ 20ng/ml respectively in described basal medium, be mixed with following four kinds of different culture medium respectively:
Culture medium a: basal medium+Niu Chuiti extracting solution 10 ~ 30 μ g/ml;
Culture medium b: basal medium+adenine 1 ~ 100 μ g/ml;
Culture medium c: basal medium+HRG 50 ~ 300ng/ml;
Culture medium d: basal medium+platelet derived growth factor 1 ~ 20ng/ml;
Wherein, the concentration of described cattle hypophysis extracting solution, adenine, HRG and platelet derived growth factor is final concentration;
3) separation of step 3, seed cell and In vitro culture: adopt and organize adherent method to carry out being separated of seed cell from required tissue, or adopt enzyme digestion from required tissue, carry out the separation of seed cell, or differential attachment method carries out the separation of seed cell from required tissue;
And carry out subculture in vitro separately cultivation with the hyclone of F12 or MEM or DMEM or RPMI-1640 culture medium mixing 5% ~ 20%, cultivation temperature is 25 ~ 38 DEG C, the CO of 3% ~ 7%
2concentration;
4) step 4, inoculating cell and dimensional culture: in an aseptic environment, by 1 × 10
4~ 10 × 10
6individual/cm
2the extracellular matrix prepared in described step one of inoculum density on carry out cell inoculation, thrust described extracellular matrix 0.2 ~ 1mm during inoculation and inoculate, inject 0.5 ~ 1.0ml at every turn, pinhold density is 0.1 ~ 0.5/cm
2, and carry out point-like inoculation on described extracellular matrix surface, then make described seed cell be evenly distributed on described extracellular matrix surface by slight oscillatory;
At 37 DEG C, the CO of 5%
2in incubator, postvaccinal extracellular matrix is immersed in culture medium a completely, cultivate 1 ~ 2 day, replaced medium b afterwards, postvaccinal extracellular matrix described in submergence, cultivates and is replaced by culture medium c after 1 ~ 2 day, culture medium c liquid level is concordant with described postvaccinal extracellular matrix, continue cultivation and be replaced by culture medium d after 1 ~ 2 day, postvaccinal extracellular matrix described in same submergence, cultivate 1 ~ 2 day; Between culture period, every day changes liquid;
5) step 5, lyophilizing packaging and sterilizing: the sample described step 3 cultivated carries out lyophilizing, control lyophilizing program and be down to-80 ~-20 DEG C with the speed of 5 ~ 12 DEG C/min, keep after 1 ~ 4 hour, lyophilization 10 ~ 24 hours, pack afterwards, then repair substrate namely to obtain skin ulcer after the sterilizing of 10 ~ 25kGy 60Coradiation.
Above-mentioned dipping by lye process means at ambient temperature, with 1M aqueous slkali soaking 5 ~ 30min, or soaks 1 ~ 2 hour with the sodium hypochlorite of 1 ‰ ~ 1%, carries out immersion treatment, makes the thickness of described collagem membrane tissue increase by 2 ~ 7 times.
Above-mentioned aqueous slkali is sodium hydroxide aqueous slkali, sodium bicarbonate aqueous slkali, any one in sodium carbonate aqueous slkali and potassium hydroxide aqueous slkali.
The thickness of above-mentioned collagem membrane tissue increases by 3 ~ 5 times.
After in above-mentioned steps one, on the described extracellular matrix of preparation, evenly punching processes, for subsequent use after then using the sterilizing of 10 ~ 25kGy 60Coradiation;
The aperture in described hole is 0.5 ~ 2mm, and the hole density in described hole is 0.2 ~ 1/cm
2, for subsequent use after sterilizing.
Above-mentioned collagem membrane tissue is any one in Skin allograft and amniotic membrane;
Or cattle, the skin in pig source, any one in pericardium and peritoneum.
Above-mentioned stem cell is any one in umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, skin progenitor cell, oral mucosa stem cell and dental pulp mescenchymal stem cell, fat mesenchymal stem cell, mesenchymal stem cells MSCs, hematopoietic stem cell, liver stem cells, neural stem cell.
Compared with prior art, the present invention has the following advantages:
(1) method that the present invention relates to, namely single inoculation realizes the effect forming multi-layer cellular film on extracellular matrix surface, ensures the cytokine profiles can secreting q.s when wound repairing, and induction new vessels is formed, wound healing.In addition the protection of natural extracellular matrix, support, barrier action and three-dimensional space structure guide cell to grow into and the effect of tissue regeneration etc., can promote the reparation of wound surface, the formation of less cicatrix by available protecting.
(2) take off the collagem membrane after cell process with dipping by lye, not only method is simple, and makes the extracellular matrix collagen fiber structure that obtains more loose.The growing multiplication that this loose collagen fiber structure is not only cell provides more suitable three-D space structure, and ensure that the nutritional labeling in culture medium can exchange infiltration fast, facilitates the propagation of cell greatly.Carry out cleaning and dipping by PBS solution afterwards, not removing only residual reagent, and improve the microenvironment of growth and proliferation of cell.Formation cladding cell membrane is sticked on extracellular matrix more closely, cellular portions even can be made to grow in substrate, also extend the time that cytokine plays a role.
(3) adopt the mode of lyophilizing to process, not only ensure that product has the result of use of expection, and can extend the shelf life, the requirement greatly reducing transport and store.
(4) manufacturing cycle is short, and technique is simple, and be applicable to industrialization, the scope of application is wide, and wound repair is effective, reduces cicatrization, the wound surface of healing and the elasticity of normal skin and tough similar temperament.
Accompanying drawing explanation
Figure 1A is the HE picture of the bovine pericardium after dipping by lye process.
Figure 1B is the HE picture of the bovine pericardium after dipping by lye process.
Fig. 2 A is the scanning electron microscope (SEM) photograph of cultivation extracellular matrix superficial cell film after 2 days.
Fig. 2 B is the scanning electron microscope (SEM) photograph of cultivation extracellular matrix superficial cell film after 3 days.
Fig. 2 C is the scanning electron microscope (SEM) photograph of cultivation extracellular matrix superficial cell film after 5 days.
Fig. 3 is the HE coloration result of single inoculation cell culture after 5 days on extracellular matrix.
Fig. 4 A is that rat empyrosis wound surface uses skin ulcer to repair the pictorial diagram of the rear 1w (1 week) of substrate treatment.
Fig. 4 B is the pictorial diagram after treatment of control group rat empyrosis wound surface 1w (1 week).
Fig. 4 C is that rat empyrosis wound surface uses skin ulcer to repair the pictorial diagram of the rear 3w (3 weeks) of substrate treatment.
Fig. 4 D is the pictorial diagram after treatment of control group rat empyrosis wound surface 3w (3 weeks).
Detailed description of the invention
In order to the cultivation cycle solving the existence of existing organization engineering skin construction method is long, cost is high, complex process, be not suitable for industrialization, the effect regulating local wound microenvironment can not be met, thus make it limited to the repairing effect of wound surface, shelf-life is short, to storage condition and the high shortcoming of movement requirement, present embodiments provide the preparation method that a kind of skin ulcer repairs substrate: by seed cell direct inoculation on the extracellular matrix through special handling, achieve the effect that extracellular matrix superficial cell cladding and extracellular matrix interior section cell are grown into, and carried out lyophilizing sterilization treatment, prepared skin ulcer repairs substrate effectively can promote diabetic foot ulcers, venous ulcer, the healing of the chronic wounds such as deep burn, and the formation of cicatrix can be reduced, the wound surface of healing and the elasticity of normal skin and tough similar temperament.
This skin ulcer repair the preparation method of substrate specifically adopt de-cell, remove antigen and thicken process after natural extracellular matrix as biological support, compound seed cell forms the cell membrane (having the cell of 2 ~ 10 confluent monolayer cells films) of multi-layer cellular on natural extracellular matrix surface, namely obtains skin ulcer and repair substrate after lyophilization process; The seed cell wherein related to is autologous or the fibroblast in allogeneic source or embryo or stem cell.
According to preparation process, this skin ulcer repairs the preparation method of substrate, mainly comprises the preparation of extracellular matrix, the preparation of culture medium, the separation of seed cell and In vitro culture, inoculating cell and the step such as dimensional culture, lyophilizing; Concrete preparation process is as follows:
The preparation of step one, extracellular matrix:
De-cell: the collagem membrane for the preparation of extracellular matrix is organized in except after degrease and other foreign material, carry out de-cell and go antigen and inactivation treatment, it is that any one the de-cell recorded in CN201110145229.0, CN201210251134.1, CN201310358048.5, CN201310117569.1 and CN201310109216.7 goes antigen and inactivation treatment mode that concrete processing method can refer to number of patent application.If number of patent application is the method mentioned in CN201310109216.7: the ammonia mixed solution that collagem membrane pre-treatment completed is placed in 2.0M potassium chloride and 0.1M soaks 60 minutes, the purified water of replacing equivalent afterwards jolts 1 hour, alternate cycles process like this 3 times, finally cleans to pH value 6.0 ~ 8.0 by purified water.
The acquisition of extracellular matrix: the collagem membrane tissue (abbreviation collagem membrane) that above-mentioned process is completed 1M aqueous slkali soaking 5 ~ 30min, as sodium hydroxide solution, sodium bicarbonate solution, sodium carbonate liquor, potassium hydroxide solution etc., or soak 1 ~ 2 hour with the sodium hypochlorite of 1 ‰ ~ 1%, making the thickness of collagem membrane increase by 2 ~ 7 times, is preferably thicken 3 ~ 5 times.Then undertaken cleaning to pH by a large amount of 0.01 ~ 0.05M PBS solution to be 6 ~ 7, and to soak by PBS solution and obtain extracellular matrix in 1 ~ 6 hour.This step makes the collagen fiber structure of collagem membrane more loose by the effect of alkali liquor, shown in Figure 1B (compared with Figure 1A, illustrate that base extraction can effectively loosen collagen fiber structure), be convenient to cell and better adhere to and even grow into, make the stuctures and properties of product more stable.
The inner microenvironment of collagem membrane not only can be made more to be applicable to growth and proliferation of cell by PBS solution cleaning and dipping in addition, and other remaining reagent can be removed, ensure that extracellular matrix can not produce any negative effect to the propagation of cell.Then extracellular matrix is cut into certain specification, as circle, rectangle etc., (be divided into and get through and do not get through, aperture 0.5 ~ 2mm) process of punching also can be carried out on extracellular matrix, make hole be evenly distributed on extracellular matrix, hole density is 0.2 ~ 1/cm
2, for subsequent use after the sterilizing of 10 ~ 25kGy 60Coradiation.
Be not difficult to find out, now extracellular matrix is here the de-cell collagen film thickening process through alkali liquor, main component is I, III Collagen Type VI, comprises Skin allograft, amniotic membrane etc., also comprises the tissue film's class such as skin, pericardium, peritoneum as the heterologous source such as cattle, pig.Not only close with the composition of human body skin, and there is certain elasticity and toughness, almost non-immunogenicity.Collagem membrane simultaneously after process has more loose three-dimensional space structure, can guide cell and grow into and tissue regeneration, makes the wound surface of healing have characteristic close to normal skin, can also suppress cicatrization.
Step 2: the preparation of culture medium:
By the volume ratio of 9:1, commercial for DMEM/F12 culture fluid and hyclone or new-born calf serum are mixed into basal liquid; Press 500ml basal liquid Standard entertion insulin 1 ~ 100ng again, transferrins 1 ~ 50mg, sodium selenite 1 ~ 30 μ g, hydrogenation can pine 10 ~ 600 μ g, epithelical cell growth factor 1 ~ 20 μ g, basic fibroblast growth factor 1 ~ 100ng, transforminggrowthfactor-β1 ~ 100ng, vitamin C 1 ~ 100mg, is mixed with basal medium, be mixed with following culture medium with basal medium again, wherein the concentration of additive is final concentration:
Culture medium a: basal medium+Niu Chuiti extracting solution 10 ~ 30 μ g/ml;
Culture medium b: basal medium+adenine 1 ~ 100 μ g/ml;
Culture medium c: basal medium+HRG 50 ~ 300ng/ml;
Culture medium d: basal medium+platelet derived growth factor 1 ~ 20ng/ml;
The Niu Chuiti extracting solution added, adenine, HRG, platelet derived growth factor all have the effect of Promote cell's growth propagation, contribute to realizing cell membrane cladding.
Separation and the In vitro culture of step 3, seed cell: being separated of relevant seed cell and In vitro culture can be carried out with reference to prior art, organize adherent method from required collagem membrane tissue, carry out the separation of seed cell as adopted, or adopt enzyme digestion from required collagem membrane tissue, carry out the separation of seed cell, or differential attachment method carries out the separation of seed cell from required collagem membrane tissue; And carry out subculture in vitro separately cultivation with the hyclone of F12 or MEM or DMEM or RPMI-1640 culture medium mixing 5% ~ 20%, cultivation temperature is 25 ~ 38 DEG C, the CO of 3% ~ 7%
2concentration.
Step 4, inoculating cell and dimensional culture: in an aseptic environment, by 1 × 10
4~ 10 × 10
6individual/cm
2inoculum density on extracellular matrix, carry out cell inoculation.During inoculation with the utensil that top is sharp-pointed thrust extracellular matrix 0.2 ~ 1mm carry out " formula of having an injection " inoculation inject wherein, inject 0.5 ~ 1.0ml, pinhold density is 0.1 ~ 0.5/cm at every turn
2, and carry out on extracellular matrix surface point-like inoculation namely dropwise drop in extracellular matrix on the surface, then make cell be evenly distributed on extracellular matrix surface by slight oscillatory.Condition of culture: 37 DEG C, 5%CO
2, at 37 DEG C, the CO of 5%
2in incubator, postvaccinal extracellular matrix is immersed in culture medium a completely, cultivate 1 ~ 2 day, replaced medium b afterwards, postvaccinal extracellular matrix described in submergence, cultivates and is replaced by culture medium c after 1 ~ 2 day, culture medium c liquid level is concordant with described postvaccinal extracellular matrix, continue cultivation and be replaced by culture medium d after 1 ~ 2 day, postvaccinal extracellular matrix described in same submergence, cultivate 1 ~ 2 day; Between culture period, every day changes liquid.
Here the cell mentioned is seed cell, autologous or allogeneic source fibroblast, stem cell, as medium in umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, skin progenitor cell, oral mucosa stem cell and dental pulp mescenchymal stem cell, fat mesenchymal stem cell, mesenchymal stem cells MSCs, hematopoietic stem cell, liver stem cells and neural stem cell.
More preferably selecting of inoculation inoculates on the matsurface of extracellular matrix, because the collagen fiber structure of matsurface is more loose, and can better for the growing multiplication of cell provides three-dimensional space.In addition the dipping by lye in step one has loosened the structure of collagen fiber (collagem membrane), the extracellular matrix obtained is made to have the solid space of appropriate cell growth propagation more, finally make cell be easy to be adhered tightly on extracellular matrix, even guide cell to grow into wherein.In addition, this inoculation method too increases the adhesiveness making cells on extracellular matrix, and the cell membrane of cladding is formed on its surface, as shown in Fig. 2 A, Fig. 2 B, Fig. 2 C (cell membrane of the extracellular matrix after thin cultivation is cladding) and Fig. 3, extracellular matrix intimate surface after base extraction has adhered to multi-layer cellular, and inner shallow-layer also distributed a certain amount of cell).
Step 5, lyophilizing packaging and sterilizing: the sample cultivated is carried out lyophilizing, control lyophilizing program and be down to-80 ~-20 DEG C with the speed of 5 ~ 12 DEG C/min, keep after 1 ~ 4 hour, lyophilization 10 ~ 24 hours, obtains the sample after lyophilizing.Sample is packed, again obtains skin ulcer after the sterilizing of 10 ~ 25kGy 60Coradiation and repair substrate.
The skin ulcer obtained by the method repairs substrate, first abundant cytokine after transplanting in product plays a role, regulate the microenvironment of the unfavorable biological cells and tissues regeneration of wound surface, remove microcirculation blood capillary spasm, thus promote that epithelium regeneration reparation and skin heart are reinvented.Now, the effect of extracellular matrix mainly protects wound surface, keeps moist environment, the release cells factor etc.After having new vessels and granulation tissue to occur; the effect of extracellular matrix just highlights; its natural 3-D solid structure can effectively guide cell to grow into; wound healing can be protected; and the collagen fiber excessive accumulation of formation can be prevented; the formation of less cicatrix, the wound surface of healing and the elasticity of normal skin and tough similar temperament.
Below in conjunction with example, technical solution of the present invention is described in further detail.
Embodiment 1
Bovine pericardium is carried out after de-cell process cleaned, soak 5min with 1M sodium hydroxide solution at ambient temperature, afterwards with a large amount of 0.01M PBS solution carry out cleaning to pH be about 7.Now bovine pericardium thickness adds 3 ~ 4 times.Then bovine pericardium is immersed in 2h in PBS solution.Bovine pericardium is cut into the circle of 10cm, for subsequent use after the sterilizing of 10kGy 60Coradiation.
By the volume ratio of 9:1, the hyclone of commercial for DMEM/F12 culture fluid and 10% is mixed into basal liquid; Press 500ml basal liquid Standard entertion insulin 50ng again, transferrins 30mg, sodium selenite 10 μ g, hydrogenation can loose 60 μ g, epithelical cell growth factor 20 μ g, basic fibroblast growth factor 40ng, transforming grouth factor beta 2 0ng, adenine 10mg, vitamin C 30mg.
Be laid in by bovine pericardium after sterilizing in the sterile petri dish that diameter is 10cm, pressing makes it be attached to completely bottom culture dish gently.Be taken at neonatal umbilical cord, with the umbilical cord mesenchymal stem cells in Secondary Culture to 2 ~ 6 generation for seed cell, by 5 × 10
5/ cm
2inoculum density inoculate on bovine pericardium matsurface.Asepsis injector first with 2ml during inoculation thrusts bovine pericardium 1mm, injects 0.5ml at every turn, and pinhold density is 0.5/cm
2, multiple point is evenly inoculated, and then Cell sap is dropped in bovine pericardium surface, and vibration makes its uniform fold in bovine pericardium gently.The culture medium a adding appropriate preparation makes its just submergence bovine pericardium, adherent growth 2 hours, and then supplemented medium a makes its liquid level distance bovine pericardium surface about 1cm, puts into 37 DEG C of 5%CO
2carry out shaken cultivation in incubator, cultivate 1 day, replaced medium b, floods bovine pericardium afterwards, and cultivate and be replaced by culture medium c after 2 days, liquid level is concordant with bovine pericardium, continues cultivation and is replaced by culture medium d after 1 day, same submergence bovine pericardium, cultivate 2 days.Between culture period, every day changes liquid.
Repeatedly clean the bovine pericardium cultivated by aseptic PBS solution, then control lyophilizing program and be down to-80 DEG C with the speed of 5 DEG C/min, keep after 1 hour, lyophilization 10 hours.Pack after lyophilizing, again obtain skin ulcer after the sterilizing of 10kGy 60Coradiation and repair substrate.
Embodiment 2
Bovine pericardium is carried out after de-cell process cleaned, soak 2h with 1 ‰ liquor natrii hypochloritises at ambient temperature, afterwards with a large amount of 0.05M PBS solution carry out cleaning to pH be about 7.Now bovine pericardium thickness adds 3 ~ 6 times.Then bovine pericardium is immersed in 6h in PBS solution.Bovine pericardium is cut into the circle of 10cm, for subsequent use after the sterilizing of 25kGy 60Coradiation.
By the volume ratio of 9:1, the hyclone of commercial for DMEM/F12 culture fluid and 10% is mixed into basal liquid; Press 500ml basal liquid Standard entertion insulin 60ng again, transferrins 50mg, sodium selenite 20 μ g, hydrogenation can loose 100 μ g, epithelical cell growth factor 20 μ g, basic fibroblast growth factor 50ng, transforming growth factor β 40ng, Niu Chuiti extracting solution 5mg, vitamin C 50mg.
Be laid in by bovine pericardium after sterilizing in the sterile petri dish that diameter is 10cm, pressing makes it be attached to completely bottom culture dish gently.With by application on human skin through trypsin treatment, be separated and the epidermal stem cells of In vitro culture for seed cell, by 1 × 10
4/ cm
2inoculum density inoculate on bovine pericardium matsurface.Asepsis injector first with 2ml during inoculation thrusts bovine pericardium 0.5mm, injects 0.6ml at every turn, and pinhold density is 0.3/cm
2, multiple point is evenly inoculated, and then Cell sap is dropped in bovine pericardium surface, and vibration makes its uniform fold in bovine pericardium gently.The culture medium a adding appropriate preparation makes its just submergence bovine pericardium, adherent growth 2 hours.And then supplemented medium a makes its liquid level distance bovine pericardium surface about 1cm.Put into 37 DEG C of 5%CO
2shaken cultivation is carried out in incubator.Cultivate 2 days, replaced medium b, floods bovine pericardium afterwards, and cultivate and be replaced by culture medium c after 1 day, liquid level is concordant with bovine pericardium, continues cultivation and is replaced by culture medium d after 2 days, same submergence bovine pericardium, cultivate 2 days.Between culture period, every day changes liquid.
Repeatedly clean the bovine pericardium cultivated by aseptic PBS solution, then control lyophilizing program and be down to-20 DEG C with the speed of 12 DEG C/min, keep after 4 hours, lyophilization 24 hours.Pack after lyophilizing, again obtain skin ulcer after the sterilizing of 25kGy 60Coradiation and repair substrate.
Embodiment 3
Pig peritoneum is carried out after de-cell process cleaned, soak 1h with 1% liquor natrii hypochloritis at ambient temperature, afterwards with a large amount of 0.05M PBS solution carry out cleaning to pH be about 7.Now pig peritoneum thickness adds 4 ~ 7 times.Then pig peritoneum is immersed in 6h in PBS solution.Pig peritoneal scissors is cut into the circle of 10cm, for subsequent use after the sterilizing of 10kGy 60Coradiation.
By the volume ratio of 9:1, the new-born calf serum of commercial for DMEM/F12 culture fluid and 10% is mixed into basal liquid; Press 500ml basal liquid Standard entertion insulin 10ng again, transferrins 1mg, sodium selenite 1 μ g, hydrogenation can loose 600 μ g, epithelical cell growth factor 1 μ g, basic fibroblast growth factor 100ng, transforminggrowthfactor-β1 00ng, HRG15mg, vitamin C 100mg.
Be laid in by pig peritoneum after sterilizing in the sterile petri dish that diameter is 10cm, pressing makes it be attached to completely bottom culture dish gently.With In vitro culture cord blood stem cell for seed cell, by 10 × 10
7/ cm
2inoculum density inoculate on pig peritoneum matsurface.Asepsis injector first with 2ml during inoculation thrusts bovine pericardium 0.2mm, injects 1ml at every turn, and pinhold density is 0.4/cm
2, multiple point is evenly inoculated, and then Cell sap is dropped in pig peritoneal surface, and vibration makes its uniform fold on pig peritoneum gently.The culture medium a adding appropriate preparation makes its just submergence pig peritoneum, adherent growth 2 hours.And then supplemented medium a makes its liquid level distance pig about peritoneal surface 1cm, puts into 37 DEG C of 5%CO
2carry out shaken cultivation in incubator, cultivate 2 days, replaced medium b afterwards, floods pig peritoneum, and cultivate and be replaced by culture medium c after 1 day, liquid level is concordant with pig peritoneum, continues cultivation and is replaced by culture medium d after 2 days, same submergence pig peritoneum, cultivate 1 day.Between culture period, every day changes liquid;
Repeatedly clean the pig peritoneum cultivated by aseptic PBS solution, then control lyophilizing program and be down to-40 DEG C with the speed of 8 DEG C/min, keep after 3 hours, lyophilization 16 hours.Pack after lyophilizing, again obtain skin ulcer after the sterilizing of 25kGy 60Coradiation and repair substrate.
Embodiment 4
Pig dermis is carried out after de-cell process cleaned, soak 1h with 1M potassium hydroxide solution at ambient temperature, afterwards with a large amount of 0.05M PBS solution carry out cleaning to pH be about 7.Now pig dermis thickness adds 2 ~ 4 times.Then pig dermis to be immersed in PBS solution 4 hours.Pig dermis is cut into the circle of 10cm, for subsequent use after the sterilizing of 15kGy 60Coradiation.
By the volume ratio of 9:1, the new-born calf serum of commercial for DMEM culture fluid and 10% is mixed into basal liquid; Press 500ml basal liquid Standard entertion insulin 1ng again, transferrins 10mg, sodium selenite 5 μ g, hydrogenation can loose 80 μ g, epithelical cell growth factor 15 μ g, basic fibroblast growth factor 30ng, transforming growth factor β 70ng, platelet derived growth factor 5mg, vitamin C 80mg.
Be laid in by pig dermis after sterilizing in the sterile petri dish that diameter is 10cm, pressing makes it be attached to completely bottom culture dish gently.With neonatal foreskin through collagenase treatment, be separated the fibroblast that obtains of In vitro culture for seed cell, by 5 × 10
6/ cm
2inoculum density inoculate on pig peritoneum matsurface.Asepsis injector first with 2ml during inoculation thrusts pig dermis 0.3mm, injects 0.1ml at every turn, and pinhold density is 0.1/cm
2, multiple point is evenly inoculated, and then Cell sap is dropped in pig dermis surface, and vibration makes its uniform fold on pig dermis gently.The culture medium a adding appropriate preparation makes its just submergence pig dermis, adherent growth 2 hours, and then supplemented medium a makes its liquid level distance pig dermis surface about 1cm.Put into 37 DEG C of 5%CO
2carry out shaken cultivation in incubator, cultivate 1 day, replaced medium b, floods pig dermis afterwards, and cultivate and be replaced by culture medium c after 1 day, liquid level is concordant with pig dermis, continues cultivation and is replaced by culture medium d after 2 days, same submergence pig dermis, cultivate 1 day.Between culture period, every day changes liquid;
Repeatedly clean the pig dermis cultivated by aseptic PBS solution, then control lyophilizing program and be down to-60 DEG C with the speed of 6 DEG C/min, keep after 3 hours, lyophilization 20 hours.Pack after lyophilizing, again obtain skin ulcer after the sterilizing of 15kGy 60Coradiation and repair substrate.
Embodiment 5
SD rat 10, body weight 200 ~ 250g, male and female half and half, sub-cage rearing 3d (English day writes a Chinese character in simplified form, " my god " the meaning), scald fasted for one day prior, and with pentobarbital sodium (35mg/kg) intraperitoneal injection of anesthesia, hair is shaved at back, dries skin.Take spinal column as center line, manufacture with the hand-held scald apparatus of automatic control the circular wound surface that diameter is 2cm respectively in the both sides, back of rat, scalding temperature is 80 DEG C, and the scald time is 5s.Lumbar injection 10mL ringer's solution shock simultaneously, sub-cage rearing.Learn that scald degree is dark II degree through histopathological examination.Dead skin tissue is removed after scalding 3d, process wound surface, the bovine pericardium (matched group) then the skin ulcer after rehydration being repaired substrate (material group) and non-inoculating cell is cut into applicable size, covers on wound surface respectively, sew up, packing is fixing.Intraperitoneal injection contains normal saline (20000IU/mL) the 0.2mL prevention infection of penicillin, rat sub-cage rearing.
1w (material group, Fig. 4 A; Matched group Fig. 4 B, w are writing a Chinese character in simplified form of the English week in week) and 3w (material picture group 4C; Matched group Fig. 4 D) after take pictures result display:
1w after the transfer, material group can merge preferably with the autologous skin of rat, and along with time lengthening, in graft, the quantity of blood capillary increases gradually, substantially heals after 3w, and cicatrix is less.Matched group does not heal completely, and graft area cicatrization, color is darker.Result shows that material group can wound healing, minimizing cicatrization in burning/wound tissue healing process.
Observe the healing time of wound surface after application combination of materials matched group, Wound healing rate, wound surface inflammatory reaction and with or without situations such as anaphylaxiss.Wherein some computation time of Wound healing rate is chosen postoperative 11 days, 18 days, 25 days and is carried out, and computing formula is:
Wound surface area × 100% before set time Wound healing rate=(before treatment wound surface area-set time wound surface area)/treatment.
Less in the inflammatory reaction in early stage of observation process in which materials group, without anaphylaxis appearance, wound healing time and healing rate result as shown in table 1.Showing to apply the average healing of wound surface after material group is 18 days, and matched group is 25 days.The wound repair successful of application material group is better than matched group.
Table 1. dark II degree of burning/wound tissue healing time and healing rate
Claims (8)
1. the preparation method of a skin ulcer reparation substrate, it is characterized in that: adopt de-cell, remove antigen and thicken process after natural extracellular matrix as biological support, compound seed cell forms the cell membrane with the multi-layer cellular of 2 ~ 10 layers on natural extracellular matrix surface, namely obtains skin ulcer and repair substrate after lyophilization process;
Described seed cell be autologous or allogeneic source fibroblast or stem cell.
2. skin ulcer according to claim 1 repairs the preparation method of substrate, it is characterized in that, comprises the following steps:
1) preparation of step one, extracellular matrix: go the collagem membrane tissue dipping by lye of antigen and inactivation treatment to carry out thickening process by completing de-cell, then with 0.01 ~ 0.05M PBS solution carry out cleaning to pH be 6 ~ 7, and within 1 ~ 6 hour, obtain extracellular matrix with described PBS solution immersion, for subsequent use after then using the sterilizing of 10 ~ 25kGy 60Coradiation;
2) preparation of step 2, culture medium:
2.1), basal liquid is prepared: by the volume ratio of 9:1, commercial for DMEM/F12 culture fluid and hyclone or new-born calf serum are mixed into basal liquid;
2.2), basal medium is prepared: by basal liquid Standard entertion insulin 1 ~ 100ng described in 500ml, transferrins 1 ~ 50mg, sodium selenite 1 ~ 30 μ g, hydrogenation can pine 10 ~ 600 μ g, epithelical cell growth factor 1 ~ 20 μ g, basic fibroblast growth factor 1 ~ 100ng, transforminggrowthfactor-β1 ~ 100ng, vitamin C 1 ~ 100mg, is mixed with basal medium;
2.3), preparation culture medium: add Niu Chuiti extracting solution 10 ~ 30 μ g/ml, adenine 1 ~ 50mg, HRG 50 ~ 300ng/ml and platelet derived growth factor 1 ~ 20ng/ml respectively in described basal medium, be mixed with following four kinds of different culture medium respectively:
Culture medium a: basal medium+Niu Chuiti extracting solution 10 ~ 30 μ g/ml;
Culture medium b: basal medium+adenine 1 ~ 100 μ g/ml;
Culture medium c: basal medium+HRG 50 ~ 300ng/ml;
Culture medium d: basal medium+platelet derived growth factor 1 ~ 20ng/ml;
Wherein, the concentration of described cattle hypophysis extracting solution, adenine, HRG and platelet derived growth factor is final concentration;
3) separation of step 3, seed cell and In vitro culture: adopt and organize adherent method to carry out being separated of seed cell from required tissue, or adopt enzyme digestion from required tissue, carry out the separation of seed cell, or from required tissue, carry out the separation of seed cell with differential attachment method;
And carry out subculture in vitro separately cultivation with the hyclone of F12 or MEM or DMEM or RPMI-1640 culture medium mixing 5% ~ 20%, cultivation temperature is 25 ~ 38 DEG C, the CO of 3% ~ 7%
2concentration;
4) step 4, inoculating cell and dimensional culture: in an aseptic environment, by 1 × 10
4~ 10 × 10
6individual/cm
2the extracellular matrix prepared in described step one of inoculum density on carry out cell inoculation, thrust described extracellular matrix 0.2 ~ 1mm during inoculation and inoculate, inject 0.1 ~ 1.0 ml at every turn, pinhold density is 0.1 ~ 0.5/cm
2, and carry out point-like inoculation on described extracellular matrix surface, then make described seed cell be evenly distributed on described extracellular matrix surface by slight oscillatory;
At 37 DEG C, the CO of 5%
2in incubator, postvaccinal extracellular matrix is immersed in culture medium a completely, cultivate 1 ~ 2 day, replaced medium b afterwards, postvaccinal extracellular matrix described in submergence, cultivates and is replaced by culture medium c after 1 ~ 2 day, culture medium c liquid level is concordant with described postvaccinal extracellular matrix, continue cultivation and be replaced by culture medium d after 1 ~ 2 day, postvaccinal extracellular matrix described in same submergence, cultivate 1 ~ 2 day; Between culture period, every day changes liquid;
5) step 5, lyophilizing packaging and sterilizing: the sample described step 3 cultivated carries out lyophilizing, control lyophilizing program and be down to-80 ~-20 DEG C with the speed of 5 ~ 12 DEG C/min, keep after 1 ~ 4 hour, lyophilization 10 ~ 24 hours, pack afterwards, then repair substrate namely to obtain skin ulcer after the sterilizing of 10 ~ 25kGy 60Coradiation.
3. skin ulcer repairs the preparation method of substrate according to claim 2, it is characterized in that: described dipping by lye process means at ambient temperature, with 1M aqueous slkali soaking 5 ~ 30min, or soak 1 ~ 2 hour with the sodium hypochlorite of 1 ‰ ~ 1%, carry out immersion treatment, make the thickness of described collagem membrane tissue increase by 2 ~ 7 times.
4. skin ulcer repairs the preparation method of substrate according to claim 3, it is characterized in that: described aqueous slkali is sodium hydroxide aqueous slkali, sodium bicarbonate aqueous slkali, any one in sodium carbonate aqueous slkali and potassium hydroxide aqueous slkali.
5. skin ulcer repairs the preparation method of substrate according to claim 3, it is characterized in that: the thickness of described collagem membrane tissue increases by 3 ~ 5 times.
6. skin ulcer according to claim 2 repairs the preparation method of substrate, it is characterized in that: after in described step one, on the described extracellular matrix of preparation, evenly punching processes, for subsequent use after then using the sterilizing of 10 ~ 25kGy 60Coradiation;
The aperture in described hole is 0.5 ~ 2mm, and the hole density in described hole is 0.2 ~ 1/cm
2, for subsequent use after sterilizing.
7. skin ulcer according to claim 2 repairs the preparation method of substrate, it is characterized in that: described collagem membrane tissue is any one in Skin allograft and amniotic membrane;
Or cattle, the skin in pig source, any one in pericardium and peritoneum.
8. skin ulcer according to claim 1 or 2 or 3 or 5 or 6 or 7 repairs the preparation method of substrate, it is characterized in that: described stem cell is any one in umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, skin progenitor cell, oral mucosa stem cell and dental pulp mescenchymal stem cell, fat mesenchymal stem cell, mesenchymal stem cells MSCs, hematopoietic stem cell, liver stem cells, neural stem cell.
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| CN105013013B (en) | 2020-01-21 |
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