CN102038976A - Regeneration material of dermis substitution for tissue engineering skin for loading rhGM-CSF and preparation method thereof - Google Patents
Regeneration material of dermis substitution for tissue engineering skin for loading rhGM-CSF and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a regeneration material of artificial dermis substitution and a preparation method thereof. In the regeneration material of a dermis substitution for tissue engineering skin for loading rhGM-CSF, the regeneration material is formed by a heparinizing collagen-chitosan bracket loading the rhGM-CSF solution. The invention further discloses a preparation method of the regeneration material. The invention provides an artificial dermis substitution with good performance for curing the whole skin coloboma of trauma, burning, chronic skin ulcer, thereby being capable of remarkably quickening the vascularization process of the regeneration material of artificial dermis substitution, decreasing the infection risk, promoting the healing of the surface of a wound and reducing the hyperplasia of scar, and thus easing the pain of patients. The invention can be widely applied for the aspects of trauma, burning, surgery reshaping, and the like. The preparing method is simple, the source of the material is wide, the production efficiency is high, and the invention is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of artificial dermis substitute regrown material and preparation method thereof.
Background technology
Skin mainly is made of epidermal area, skin corium and subcutaneous tissue as the organ of human body maximum, contains abundant blood vessel and nerve simultaneously.In the routine work life because a variety of causes such as wound, burn, chronic disease, operation etc. are easy to cause the damaged of skin.Though skin histology has stronger regeneration capacity, under large-area holostrome skin injury situation, because the disappearance of skin corium, the regeneration capacity of skin obviously weakens, and very easily produces a large amount of cicatrizations or cicatricial contracture.
The key of treatment holostrome skin injury is reconstruction and regeneration dermal tissue, promotes that wound surface seals, avoids microorganism invasion etc. as early as possible.At present, the mode of reply tissue defect mainly contains three kinds, that is: autograft, allosome/heteroplasm transplant, artificial material substitutes.Self-skin transplant remains the effective method of clinical treatment holostrome skin injury, but this method is faced with from the under-supply shortcoming of body skin, and is the way of a kind of " curing the wound with wound "; The restriction that is subjected to immunological rejection is again transplanted by allosome or heteroplasm; Another Therapeutic Method commonly used adopts artificial material skin exactly.Artificial skin has Apligraf, Integra, Transcyte, AlloDerm and Dermagraft etc. on the international market at present.
Collagen-chitin porous support dermal substitute regrown material can effectively be induced migration, propagation and the differentiation of defective tissue place cell, and original position is induced the regeneration of damaged dermal tissue.This dermal substitute regrown material not only can play the good supporting effect in vivo, and the collagen that it adopted and chitosan are the natural macromolecular materials with good biocompatibility, reduced immunogenicity and degradability.
Chinese invention patent (application number: 02112498.1 applying date: 2002-07-10) disclose the preparation method of used in tissue engineering collagen/chitosan porous rack, it is raw material that this method adopts natural biologic material collagen and chitosan, by freezing-lyophilizing, further by vacuum dry heating method, aldehyde compound or Carbodiimides compound crosslink, preparation has the porous support of controlled, suitable degradation rate of micro structure and good biocompatibility again.The good biocompatibility of the collagen/chitosan support of this invention, degradation rate is controlled, and material source is extensive, cost is low, preparation technology's simple possible, good reproducibility, constructed support can be widely used in field of tissue engineering technology, has good potential applicability in clinical practice.
Further, Chinese invention patent (application number: 200510060749.6 applyings date: 2005-09-13) disclose the preparation method that composite vascular generates plain heparinization collagen/chitosan porous rack, this method is mixed with beef tendon collagen, chitosan 0.5 ~ 5% solution respectively with acetic acid solution, mix homogeneously injects mould, chitosan solution content is 5～30%, and lyophilizing obtains collagen/chitosan porous rack in the freezing rearmounted freeze dryer; After the application of vacuum, it is dipped in the 2-N-morpholino ethane sulfonic acid solution that contains heparin sodium soaks, adding 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor then handles, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Be dipped in the angiogenesis cellulose solution.This patent technology is simple, effective, and the influence factor is few, more easily promotes, and the heparinization collagen/chitosan porous rack of preparation has suitable aperture and porosity, is fit to the dermal substitute of skin tissue engineering.
Artificial dermis substitute regrown material vascularization speed is the key that influences its regeneration capacity, material transplanting success.Owing to lack the blood vessel network that is interconnected in the artificial dermis substitute regrown material, be difficult to realize with wound surface basal area blood vessel fast in succession.At transplant early, the needed nutrient substance of cell in the artificial dermis substitute regrown material mainly relies on diffusion to realize, and functions such as the propagation of cell and differentiation are often limited because of the shortage of nutritional labeling, even cause natural death of cerebral cells, thereby influence its reproduction speed and survival rate.The vascularization speed of present artificial dermis substitute reconstituted product is slower, and the complete vascularization of implant generally needs the time in several weeks (2 ~ 4 week), has increased patient's hospital stays and infection risk.Therefore, how promoting the vascularization speed of artificial dermis substitute regrown material, and then shorten the wound surface Ischemia Time, improve the transplanting survival rate, is the key issue that needs to be resolved hurrily during skin regeneration is studied with reparation.
The multinomial rhGM-CSF of studies confirm that has shown tangible positive role in the skin injury treatment that multiple reasons such as ulcer due to deep, the chronic disease cause, rhGM-CSF can pass through from bone marrow mobilization and recruit hematopoietic stem cell and endothelial stem cell, to regulate propagation, differentiation and the survival of hematopoietic cell, also stimulate myeloid progenitor to granulocyte and macrophage differentiation, to transfer immune system specifically.But cytokine exists active unstable, holds degradation-labile defect.
Summary of the invention
Exist active unstable in order to solve cytokine, hold degradation-labile defect, an object of the present invention is to provide the organization engineering skin dermal substitute regrown material of a kind of load rhGM-CSF, this regrown material has quick vascularization, can be widely used in holostrome skin injury reparations such as wound and burn.Another object of the present invention provides the preparation method of the organization engineering skin dermal substitute regrown material of a kind of above-mentioned load rhGM-CSF.
In order to realize above-mentioned first purpose, the present invention has adopted following technical scheme:
The organization engineering skin dermal substitute regrown material of load rhGM-CSF, this regrown material is made of the described rhGM-CSF solution of collagen-chitin support load of heparinization.
As preferably, the concentration of above-mentioned rhGM-CSF solution is 100ng/ml ~ 300 μ g/ml.
As preferably, the collagen-chitin support of above-mentioned heparinization is containing heparin, EDC(1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides by the collagen-chitin support) and NHS(N-maloyl imines) the MES(2-N-morpholino ethane sulfonic acid) cross-linking reaction after the vacuum defoamation in the solution, lyophilization makes then, the mass ratio of the collagen in heparin and the collagen-chitin support is 1:20 ~ 1:4, and the concentration ratio of EDC and NHS is 1:1 ~ 4:1.
Mass ratio as chitosan and collagen in preferred again, the above-mentioned collagen-chitin support is 1:9 ~ 5:5.
In order to realize above-mentioned second purpose, the present invention has adopted following technical scheme:
A kind of method for preparing the organization engineering skin dermal substitute regrown material of above-mentioned load rhGM-CSF, this method comprises the steps:
1) with mass concentration be 0.3% ~ 5% acetic acid solution to prepare mass concentration respectively be 0.05% ~ 5% collagen solution and chitosan solution, chitosan solution is added in the collagen solution, stir, after the vacuum defoamation, obtain the collagen-chitin mixed solution; The collagen-chitin blended liquid is injected mould, after 24 hours, adopt the freeze-dried method in 4 ℃ of abundant down effects, lyophilization under-5 ℃ ~-80 ℃ temperature obtains the collagen-chitin three-dimensional porous rack;
2) with above-mentioned steps 1) the collagen-chitin three-dimensional porous rack of gained is 80 ℃ ~ 180 ℃ xeothermic crosslinking Treatment of following vacuum 24 hours;
3) with heparin in the collagen-chitin support in the mass ratio of collagen be that the ratio of 1:20 ~ 1:4 places the MES solution that contains EDC and NHS, abundant stirring and evenly mixing; Again with above-mentioned steps 2) collagen-chitin after the dry heat treatment of gained props up and is placed in this kind mixed liquor vacuum defoamation, crosslinked 1 ~ 24 hour; After the flushing, lyophilization under-5 ℃ ~-80 ℃ temperature once more makes the collagen-chitin support of described heparinization repeatedly;
4) utilize water for injection or deionized water compound concentration rhGM-CSF solution for 100ng/ml ~ 300 μ g/ml, the collagen-chitin support is immersed in this solution, leave standstill under 4 ℃ and hatch 24 hours, promptly obtain the organization engineering skin dermal substitute regrown material of described load rhGM-CSF.
Certainly, collagen-chitin support of the present invention can adopt the preparation of Chinese invention patent 02112498.1 disclosed method, and the collagen-chitin support of heparinization also can adopt the preparation of 200510060749.6 disclosed methods.
As preferably, above-mentioned EDC and NHS are than being 1:1 ~ 4:1.
As preferably, the concentration 0.05M of above-mentioned MES solution, pH5.4.
The present invention combines cytokine therapy with the method and the technology of organizational project regenerative medicine, selection has the natural macromolecular material of good biocompatibility, reduced immunogenicity and degradability, mutually compound with rhGM-CSF, formation has the bioactive compound organization engineering skin dermal substitute regrown material of rhGM-CSF, realize the controlled lasting release of rhGM-CSF, promote the vascularization of artificial dermis substitute regrown material, the final compound organization engineering skin dermal substitute regrown material that obtains to have high transplanting success and good Regeneration and Repair effect.
The organization engineering skin dermal substitute regrown material of the load rhGM-CSF of the present invention preparation, its outstanding feature are to be primary raw material with biodegradable collagen protein, add chitosan and play and promote crosslinked, enhancing and degradation resistant effect; By control lyophilization condition, obtain the having specific microstructure porous support of (aperture, porosity etc.).By changing rhGM-CSF load capacity, the artificial dermis substitute regrown material that obtains having different blood vessel speed.The result shows that this artificial dermis substitute regrown material can effectively promote migration, propagation and the differentiation of endotheliocyte, promotes fibroblastic immersion, has the favorable tissue compatibility.Vascularization fast (10 days) is found in SD subcutaneous rat heeling-in experiment.This artificial dermis substitute regrown material can be used as the falsework of tissue growth, promotes the vascularization of wound surface, the growth in porous support of granulation tissue, endotheliocyte and fibroblast.Tissue that these are grown into or cell can be secreted corresponding extracellular matrix and then be divided into new skin histology, promote the regeneration of damaged skin.Meanwhile, ectogenic collagen-chitin porous support can be degraded gradually along with the continuous growth of cambium, and is absorbed by body, and what finally obtain is and the almost consistent skin histology of human body self.
The present invention provides a kind of well behaved artificial dermis substitute for the treatment of holostrome skin injury such as wound, burn and chronic skin ulcer, can significantly quicken vascularization process, the reduction infection risk of artificial dermis substitute regrown material, promote the healing of wound surface, reduce the hypertrophy of cicatrix, and then alleviate patient's misery.Can be widely used in aspects such as wound, burn and surgical plastic.Preparation method of the present invention is simple, and material source is extensive, and the production efficiency height is applicable to industrialization production.Compare with external like product, product cost of the present invention aspect tool has great advantage.
Description of drawings
Fig. 1 is scanning electron microscope (SEM) photo of the compound support of collagen-chitin of load rhGM-CSF of the present invention.
Fig. 2 is the operative photographs of the compound support of the collagen-chitin of load rhGM-CSF of the present invention at SD rat in-vivo embed.
Fig. 3 be the compound support of the collagen-chitin of load rhGM-CSF of the present invention at the cardinal principle photo of SD rat in-vivo embed after 7 days, 14 days, dotted line is represented the implant material border.
Fig. 4 be the compound support of the collagen-chitin of load rhGM-CSF of the present invention at the cardinal principle photo of SD rat in-vivo embed after 14 days, dotted line is represented the implant material border.
Fig. 5 is the compound support of the collagen-chitin of load rhGM-CSF of the present invention at the HE figure of SD rat in-vivo embed 7 after everyday, and arrow is represented blood vessel, and m represents implant material, * 200.
Fig. 6 is that the compound support of the collagen-chitin of load rhGM-CSF of the present invention is schemed at the HE of SD rat in-vivo embed after 14 days, and arrow is represented blood vessel, and m represents implant material, * 200.
The specific embodiment
It is 0.5% acetic acid solution that collagen and chitosan are made into mass fraction respectively, after then collagen solution and chitosan solution being mixed according to the volume ratio of 9:1, stir, injecting diameter is the mould of 2cm, make it highly reach 1.5mm, place 4 ℃ after following 24 hours, in-20 ℃ freezing 24 hours, placed the freeze dryer lyophilizing then 16 hours; Freeze dried support was in xeothermic crosslinked 24 hours of 105 ℃ of following vacuum; According to heparin and collagen mass ratio is the ratio of 1:10, heparin is added into the EDC(20mmmol that molar concentration rate is 2:1) and the 0.05M MES(pH5.4 of N-maloyl imines (NHS)) in the solution, again be placed in this mixed liquor crosslinked 24 hours after the above-mentioned lyophilizing, freezing once more-lyophilizing obtains the collagen-chitin support (micro structure is seen Fig. 1) of the heparinization after crosslinked after cleaning repeatedly.
After the collagen-chitin support ethane via epoxyethane sterilization with heparinization, dripping concentration is the rhGM-CSF solution 300 μ l of 100 μ g/ml, and 4 ℃ of following overnight incubation are standby.
Embodiment 2
It is 0.5% acetic acid solution that collagen and chitosan are made into mass fraction respectively, after then collagen solution and chitosan solution being mixed according to the volume ratio of 9:1, stir, injecting diameter is the mould of 2cm, make it highly reach 1.5mm, place 4 ℃ after following 24 hours, in-20 ℃ freezing 24 hours, placed the freeze dryer lyophilizing then 16 hours; Freeze dried support was in xeothermic crosslinked 24 hours of 105 ℃ of following vacuum; According to heparin and collagen mass ratio is the ratio of 1:10, heparin is added into the EDC(20mmmol that molar concentration rate is 2:1) and the 0.05M MES(pH5.4 of N-maloyl imines (NHS)) in the solution, again be placed in this mixed liquor crosslinked 24 hours after the above-mentioned lyophilizing, freezing once more-lyophilizing obtains the collagen-chitin support (micro structure is seen Fig. 1) of the heparinization after crosslinked after cleaning repeatedly.
After the collagen-chitin support ethane via epoxyethane sterilization with heparinization, dripping concentration is the rhGM-CSF solution 200 μ l of 200 μ g/ml, and 4 ℃ of following overnight incubation are standby.
Embodiment 3
It is 0.5% acetic acid solution that collagen and chitosan are made into mass fraction respectively, after then collagen solution and chitosan solution being mixed according to the volume ratio of 9:1, stir, injecting diameter is the mould of 2cm, make it highly reach 1.5mm, place 4 ℃ after following 24 hours, in-20 ℃ freezing 24 hours, placed the freeze dryer lyophilizing then 16 hours; Freeze dried support was in xeothermic crosslinked 24 hours of 105 ℃ of following vacuum; According to heparin and collagen mass ratio is the ratio of 1:10, heparin is added into the EDC(20mmmol that molar concentration rate is 2:1) and the 0.05M MES(pH5.4 of N-maloyl imines (NHS)) in the solution, again be placed in this mixed liquor crosslinked 24 hours after the above-mentioned lyophilizing, freezing once more-lyophilizing obtains the collagen-chitin support (micro structure is seen Fig. 1) of the heparinization after crosslinked after cleaning repeatedly.
After the collagen-chitin support ethane via epoxyethane sterilization with heparinization, dripping concentration is the rhGM-CSF solution 100 μ l of 300 μ g/ml, and 4 ℃ of following overnight incubation are standby.
The test example
Operation the previous day cuts off the Mus hair of SD rat back, loses hair or feathers with depilatory then; During operation, respectively do two " capsule " (see figure 2)s in the spinal column both sides of SD rat, gap 1cm, the compound support of collagen-chitin of the load rhGM-CSF that embodiment 1 is prepared is implanted open and flatly then, sews up.Drew materials in 7 days, 14 days in postoperative, gross examination of skeletal muscle and HE dyeing is observed in vivo biological property of compound rest and (is seen Fig. 3 ~ Fig. 6).See substantially visible a large amount of vascularitys in the surface of the artificial dermis substitute regrown material of implanting with on every side; As seen HE dyeing observed, and the artificial dermis substitute regrown material of implantation is connected closely with surrounding tissue, abundant blood vessel arranged around material and inner formation, the trend that the also oriented embedded material infiltration of surrounding tissue is grown.
Claims (7)
1. the organization engineering skin dermal substitute regrown material of load rhGM-CSF, it is characterized in that: this regrown material is made of the described rhGM-CSF solution of collagen-chitin support load of heparinization.
2. the organization engineering skin dermal substitute regrown material of load rhGM-CSF according to claim 1 is characterized in that: the concentration of rhGM-CSF solution is 100ng/ml ~ 300 μ g/ml.
3. the organization engineering skin dermal substitute regrown material of load rhGM-CSF according to claim 1 and 2, it is characterized in that: the collagen-chitin support of heparinization is by collagen-chitin support cross-linking reaction after the vacuum defoamation in containing the MES solution of heparin, EDC and NHS, lyophilization makes then, the mass ratio of the collagen in heparin and the collagen-chitin support is 1:20 ~ 1:4, and the concentration ratio of EDC and NHS is 1:1 ~ 4:1.
4. the organization engineering skin dermal substitute regrown material of load rhGM-CSF according to claim 3 is characterized in that: the mass ratio of chitosan and collagen is 1:9 ~ 5:5 in the collagen-chitin support.
5. a method for preparing the organization engineering skin dermal substitute regrown material of the described load rhGM-CSF of claim 1 is characterized in that this method comprises the steps:
1) with mass concentration be 0.3% ~ 5% acetic acid solution to prepare mass concentration respectively be 0.05% ~ 5% collagen solution and chitosan solution, chitosan solution is added in the collagen solution, stir, after the vacuum defoamation, obtain the collagen-chitin mixed solution; The collagen-chitin blended liquid is injected mould, after 24 hours, adopt the freeze-dried method in 4 ℃ of abundant down effects, lyophilization under-5 ℃ ~-80 ℃ temperature obtains the collagen-chitin three-dimensional porous rack;
2) with above-mentioned steps 1) the collagen-chitin three-dimensional porous rack of gained is 80 ℃ ~ 180 ℃ xeothermic crosslinking Treatment of following vacuum 24 hours;
3) with heparin in the collagen-chitin support in the mass ratio of collagen be that the ratio of 1:20 ~ 1:4 places the MES solution that contains EDC and NHS, abundant stirring and evenly mixing; Again with above-mentioned steps 2) collagen-chitin after the dry heat treatment of gained props up and is placed in this kind mixed liquor vacuum defoamation, crosslinked 1 ~ 24 hour; After the flushing, lyophilization under-5 ℃ ~-80 ℃ temperature once more makes the collagen-chitin support of described heparinization repeatedly;
4) utilize water for injection or deionized water compound concentration rhGM-CSF solution for 100ng/ml ~ 300 μ g/ml, the collagen-chitin support is immersed in this solution, leave standstill under 4 ℃ and hatch 24 hours, promptly obtain the organization engineering skin dermal substitute regrown material of described load rhGM-CSF.
6. the method for the organization engineering skin dermal substitute regrown material of load rhGM-CSF according to claim 5 is characterized in that: EDC is 1:1 ~ 4:1 with the NHS ratio.
7. the method for the organization engineering skin dermal substitute regrown material of load rhGM-CSF according to claim 5 is characterized in that: the concentration 0.05M of MES solution, pH5.4.
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| CN105561400A (en) * | 2015-12-30 | 2016-05-11 | 广州迈普再生医学科技有限公司 | Artificial dermal scaffold and production method thereof |
| CN105920680A (en) * | 2016-06-03 | 2016-09-07 | 昆明理工大学 | Soft tissue engineering porous scaffold and preparation method thereof |
| CN108721690A (en) * | 2018-06-14 | 2018-11-02 | 福州大学 | A kind of preparation method and products thereof of medicament slow release type antiseptic dressing |
| CN108744014A (en) * | 2018-06-14 | 2018-11-06 | 福州大学 | A kind of preparation method and products thereof with slow releasing function antiseptic dressing |
| CN114344449A (en) * | 2018-07-04 | 2022-04-15 | 杭州彗搏科技有限公司 | Application of histaminin 1 polypeptide in preparation of composite material for promoting repair of large-area skin defect |
| CN116569916B (en) * | 2023-07-13 | 2023-11-28 | 九天览月生物科技(天津)有限公司 | Frozen cells, cell frozen solution, frozen method and application |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105561400A (en) * | 2015-12-30 | 2016-05-11 | 广州迈普再生医学科技有限公司 | Artificial dermal scaffold and production method thereof |
| CN105561400B (en) * | 2015-12-30 | 2019-12-13 | 广州迈普再生医学科技股份有限公司 | Artificial dermis stent and preparation method thereof |
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| CN108721690A (en) * | 2018-06-14 | 2018-11-02 | 福州大学 | A kind of preparation method and products thereof of medicament slow release type antiseptic dressing |
| CN108744014A (en) * | 2018-06-14 | 2018-11-06 | 福州大学 | A kind of preparation method and products thereof with slow releasing function antiseptic dressing |
| CN108744014B (en) * | 2018-06-14 | 2021-04-27 | 福州大学 | Preparation method of antibacterial dressing with slow release effect and product thereof |
| CN114344449A (en) * | 2018-07-04 | 2022-04-15 | 杭州彗搏科技有限公司 | Application of histaminin 1 polypeptide in preparation of composite material for promoting repair of large-area skin defect |
| CN114344449B (en) * | 2018-07-04 | 2024-04-12 | 杭州彗搏科技有限公司 | Application of histamine 1 polypeptide in preparing composite material for promoting repair of large-area skin defect |
| CN116569916B (en) * | 2023-07-13 | 2023-11-28 | 九天览月生物科技(天津)有限公司 | Frozen cells, cell frozen solution, frozen method and application |
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