CN101773687A - Preparation method of composite soft-tissue patch - Google Patents
Preparation method of composite soft-tissue patch Download PDFInfo
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Abstract
The invention discloses a preparation method of a composite soft-tissue patch. Sustained-release microspheres containing human cell culture secretion are mixed with gel solution, then the mixture is compounded with an acellular dermal matrix under vacuum, and after drying and sterilization, the composite soft-tissue patch is obtained. The prepared composite patch can improve the biocompatibility and the bioactivity of the dermal matrix effectively. The experiment shows that compared with the single acellular dermal matrix, the patch can promote cell proliferation and collagen formation effectively and has better biocompatibility and better tissue remolding. The patch can be widely applied to closure of various wound surfaces, repair of various soft-tissue defects and reinforcement of weak places and can be prepared into micro particles to be used for filling various fine and weak or dented places by injection, thus having wider application prospect in clinic.
Description
Technical field
The invention belongs to the tissue engineering technique field of bio-medical material, be specifically related to a kind of preparation method that is used for the complex tissue sticking patch of soft tissue defects reparation.
Background technology
Damaged, the weakness of soft tissue is FAQs clinically, and the reason that causes has birth defect, pathological factor and aging etc., and clinical manifestation is: hernia, fistula, cicatrix and wrinkle etc.Damaged, the weakness of this class soft tissue can't be repaired usually voluntarily, have influence on the function and the outward appearance of corresponding organ tissue greatly.Mainly rely on tissue patch filling reparation at present clinically, therefore the research and development to tissue patch have become one of focus of modern bio-medical material.
The kind of tissue patch is a lot, has by the material classification: nylon sticking patch, polypropylene sticking patch, expanded polytetrafluoroethylsealing sticking patch, biological sticking patch, composite patch etc.Abiotic sticking patch wherein have biocompatibility relatively poor, can not degrade, influence problems such as postoperative life quality.In recent years, the biological sticking patch that comes into the market mostly is allosome/xenogeneic cell products and derived product thereof of taking off, as: take off the cell human dermis, take off cell cattle corium etc.Simple takes off cell material because also removed a large amount of active substances in the process of removing cell, and biological activity reduces greatly; But the improvement by to method for removing cells can effectively improve the biological activity that takes off cell material, in order further to improve the character of taking off cell material, makes it possess better biocompatibility simultaneously, realizes certain physiological function, and composite patch becomes the emphasis of research.
Chinese patent (200610027044.9) discloses allosome/xenogenesis acellular dermal substitute that a kind of human fibroblasts is modified, be with fibroblast and the compound cultivation of acellular dermal matrix, make the acellular dermal surface form the single or multiple lift human fibroblasts, the affinity and the vigor of acellular dermal have been improved, as dermal substitute.The deficiency of this method is: prepared dermal substitute, and its range of application only limits to skin histology; This dermal substitute contains cell, is easy to generate immunologic rejection in clinical practice, therefore is difficult to be applied to the reparation of in-vivo tissue; In addition, celliferous sticking patch complicated process of preparation, the production cost height, difficult quality control, the industrialization bottleneck is not broken through as yet.
Chinese patent (200610153403.5) discloses a kind of surgical patch of biologically active agent, and this sticking patch has the function that discharges antiinflammatory, anti-platelet agents, anticoagulant, fibrinolytic agent, cell cycle inhibitor and/or antiproliferative; But it only limits to vascular patch, and just adopts simple medicine coating process preparation, can't realize the continuous action of medicine.
By retrieval, up to the present still do not have a kind of sticking patch and can satisfy requirements for clinical application fully.
Summary of the invention
At the deficiencies in the prior art, the preparation method that the purpose of this invention is to provide a kind of composite soft-tissue patch, the gained soft-tissue patch is not only had repair the function of filling, also have the ability that promotes cell proliferation and secretion substrate, biocompatibility and tissue reconstruction better effects if in the clinical manifestation.
Composite soft-tissue patch preparation method provided by the present invention, employing has through taking off the dermal matrix that cell is handled, it is characterized in that, be to contain the sustained-release micro-spheres that human body cell is cultivated secretions, be mixed in the gel solution, compound under vacuum condition with acellular dermal matrix again, obtain can be used for the composite soft-tissue patch that human body soft tissue is repaired after the drying sterilization.Described acellular dermal matrix can derive from xenogenesis or allosome, has natural three dimensional structure, and its porosity can reach more than 90%, helps cell and grows into, and is a kind of well behaved biologic bracket material.Described human body cell is cultivated secretions, the cytotrophy factor that constitutes for the human body cell excretory various active factor and stromatin in incubation; Institute's cultured cells is according to sticking patch purposes needs, can be cells all types of in the tissue; Collect the secretions of these cell culture, with the compound sustained-release micro-spheres that contains cell culture secretions that becomes of microsphere; Described microsphere can adopt any or several mixture of glycidyl methacrylate dextran, polylactic acid, polyglycolic acid or gelatin to be prepared from.Prepared tissue patch has higher biological activity, can slowly discharge cell culture secretions in three months at implant into body and promote cell proliferation and substrate secretion, promotes tissue reconstruction, thereby reaches better clinical therapeutic efficacy.
The concrete steps of composite soft-tissue patch preparation method of the present invention comprise:
Step 1, preparation acellular dermal matrix: get the mammiferous fresh skin of health (people, pig, cattle etc.), clean the back and remove subcutaneus adipose tissue and epidermis, keep the thick skin corium skin graft of 0.1mm～2mm, clean with aseptic PBS solution respectively with deionized water, soak abundant swelling in 4 ℃ of deionized waters; Place-80 ℃ freezing more than 30 minutes, melt to organizing to take out after the complete deep colling, can effectively destroy cell, cellular content is come out, be convenient to next step removing, this frozen-thaw process is at least once; Adopting W/V again is 0.1%～0.3% trypsin solution digestion skin graft, removes being connected between collagen protein and other albumen, and the removal of antigen protein is convenient in crumbly texture; Clean skin graft with any or several blended detergent solution that contain TritonX 100/200, NaTDC or sodium lauryl sulphate, remove most antigenic component, still stay part small fragment nucleic acid; The DNA enzymatic solution digestion of reuse 40～150U/ml is removed residual DNA, along with digestion time can be suitably shortened in the increase of temperature; With the NaOH solution ablation corium skin graft 1h of 1M,, guarantee the safe in utilization of prepared dermal matrix to remove the virus in the corium; At last skin graft is cleaned with PBS solution and pure water respectively, after lyophilization, sterilization, 4 ℃ of preservations are standby; Resulting acellular dermal matrix has higher biological activity and biocompatibility, and its porosity is more than 90%.
Step 2, preparation cell culture secretions: the culture fluid under changing in the collecting cell incubation, cell culture processes can be with reference to " tissue engineering philosophy and technique " (publishing house of banket The Fourth Military Medical University published in 2004) or other prior arts; Concentrate (the ultrafiltration post can adopt the 30K aperture) by hyperfiltration process, desalt with washed with de-ionized water; Adopt the protein purification instrument to remove bovine serum albumin, remaining protein ingredient is the cell culture secretions that contains the required cytotrophy factor, preserves after the filtration sterilization;
Step 3, preparation contains the sustained-release micro-spheres of cell culture secretions: with reference to " experimentation of the biological controlled-released promotion paradenlal tissue regeneration of somatomedin " (Chen Faming, thesis for the doctorate) or other prior arts, micro-sphere material can adopt the glycidyl methacrylate dextran, polylactic acid, polyglycolic acid, or the mixing of any or two kinds of gelatin, the particle diameter of prepared microsphere is 10nm～3um, after the lyophilizing sterilization, the method of water solution absorbs under aseptic condition, make the cell culture secretions that contains 100～500mg in the 1g microsphere, after lyophilizing, promptly get the sustained-release micro-spheres that contains cell culture secretions;
Step 4, preparation composite soft-tissue patch: any of employing collagen, cellulose, alginic acid, hyaluronic acid or chitosan or a several mixture are raw material, preparation W/V is 0.2～1.5% gel solution, add the sustained-release micro-spheres that contains cell culture secretions therein, the final concentration that makes cell culture secretions is 50～200mg/ml, evenly cover behind the mixing and be applied to the acellular dermal matrix surface, the gel solution that contains cell culture secretions microsphere fully is penetrated in the hole of acellular dermal, and covering the amount of being coated with is 0.5～1ml/cm
2, be composite soft-tissue patch after the drying.
The preservation of composite soft-tissue patch of the present invention and use: should preserve down at 0～8 ℃; During use sticking patch is cut into required size and places sterile chamber, the normal saline immersion 5～30min with 10～50 times of amounts can be used for the sealing of various wound surface, the repairing of soft tissue defects and the enhancing of weakness; Sticking patch fragment and cutting residue can adopt injection system to be used for the filling of various tiny soft tissue weaknesses or recess after crushed.
The composite soft-tissue patch of the present invention's preparation has kept the good three dimensional structure of acellular dermal matrix, for adhering to of cell provides natural three dimensions; Owing to the use of uniting of methods such as enzyme processing, detergent cleaning and alkali treatment, effectively removed antigenic component, can not cause tangible immunological rejection; The gel that compound tense adopted can promote growing into of cell; Carry a large amount of cell growth factor and extracellular matrix components in the sustained-release micro-spheres and can promote cell proliferation and substrate secretion; Sustained-release micro-spheres is adsorbed onto the release time that can effectively prolong the emiocytosis thing in the hole of acellular dermal, makes the effect of emiocytosis thing more stable lasting.The present invention is not owing to adopt the compound cultivation of cell, makes that making is easier, cost is lower, quality is controlled easily, it is more convenient to use.Experiment shows, compares with simple acellular dermal matrix, and sticking patch of the present invention can effectively promote the secretion of the propagation and the substrate of cell, has better biocompatibility and more excellent tissue reconstruction.Sticking patch of the present invention can be widely used in the sealing of various wound surface, the repairing of soft tissue defects and the enhancing of weakness, also can be made into powder and is used for the filling of various tiny weaknesses or recess by injection, has application prospect widely clinically.
The specific embodiment
Below in conjunction with instantiation technical solution of the present invention is described in further detail.
Embodiment 1,
Step 1, preparation acellular dermal matrix: get healthy fresh Corii Sus domestica skin, remove subcutaneus adipose tissue and epidermis with the bark fetching drum, keep the thick skin corium of 0.1mm, clean with deionized water, the aseptic PBS solution of reuse cleans, after 4 ℃ of deionized waters soak abundant swelling, place-80 ℃ freezing more than 30 minutes, place 37 ℃ of environment to melt 2 times repeatedly to organizing to take out after the complete deep colling; Adopt the trypsin solution of 0.1% (W/V) to digest skin graft 1 day; Use 0.02% (W/V) TritonX, 100/200 solution to clean subsequently 1 day; Adopt the DNA enzymatic solution of 40U/ml to digest 2 hours for 37 ℃; NaOH solution soaking corium skin graft 1h with 1M; After cleaning with PBS solution and pure water respectively, after lyophilization, sterilization, standby in 4 ℃ of preservations; Acellular dermal matrix porosity by this method preparation is 91%;
Step 2, preparation cell culture secretions: be carried out to fibrocyte with reference to " tissue engineering philosophy and technique " (publishing house of banket The Fourth Military Medical University published in 2004) and cultivate, collect the culture fluid under changing; The employing hyperfiltration process concentrates, and washed with de-ionized water desalts, and the ultrafiltration post adopts the 30K aperture; Adopt protein purification instrument (AKTA PRIME) to remove bovine serum albumin, remaining protein ingredient is required people's cell culture secretions, surveys secretion content (in Tot Prot), and concentration transfers to 200mg/ml, preserves after the filtration sterilization;
Step 3, preparation contain the sustained-release micro-spheres of cell culture secretions: with reference to " experimentation of the biological controlled-released promotion paradenlal tissue regeneration of somatomedin " (Chen Faming, thesis for the doctorate) method in prepares glycidyl methacrylate dextran microsphere, the mean diameter of microsphere is 50～100nm, after the lyophilizing sterilization, the aqueous solution that in the 1g microsphere, adds 1ml cell culture secretions under the aseptic condition, placed one day down, must contain the sustained-release micro-spheres of cell culture secretions after the lyophilizing for 4 ℃;
Step 4, preparation composite soft-tissue patch: at W/V is that to add cell culture secretions final concentration in 0.3% the collagen solution be the sustained-release micro-spheres that contains cell culture secretions of 80mg/ml, behind the mixing this gel solution evenly covered and be applied to the acellular dermal matrix surface, covering the amount of being coated with is 0.6ml/cm
2, make under the vacuum condition in its hole that fully is penetrated into acellular dermal, promptly get composite soft-tissue patch after the lyophilization.
The composite soft-tissue patch thickness of this example preparation is 0.1～0.15mm, and fracture tensile strength is greater than 0.1Mpa.Sticking patch is preserved under 4 ℃ of conditions, during use the sticking patch taking-up is cut into suitable size as required and places sterile chamber, and the normal saline that adds 10～20 times of amounts soaks 10min.This sticking patch can be used for the reparation of soft tissue defects in the human body body and the enhancing of soft tissue weakness, as: the sealing of wound surface etc. behind hernia reparation, the tumor operation; Its sticking patch fragment and cutting residue can be used for local the filling after crushed, as injecting reduce wrinkle etc.
Embodiment 2,
Step 1, preparation acellular dermal matrix: get healthy abortive calfskin, clean manual subcutaneus adipose tissue and the epidermis removed in back, keep the thick skin corium of 1mm, clean with deionized water, the aseptic PBS solution of reuse cleans, after 4 ℃ of deionized waters soak abundant swelling, place-80 ℃ freezing more than 30 minutes, place 37 ℃ of environment to melt 3 times repeatedly to organizing to take out after the complete deep colling; Adopt the trypsin solution of 0.25% (W/V) to digest 2 hours, reuse 0.05% sodium dodecyl sulfate solution cleaned 8 hours; Adopt the DNA enzymatic solution of 80U/ml to digest 2 hours for 37 ℃; NaOH solution ablation corium skin graft 1h with 1M; After corium after the processing is cleaned with PBS solution and pure water respectively, after lyophilization, sterilization, standby in 4 ℃ of preservations; Acellular dermal matrix porosity by this method preparation is 93%;
Step 2, preparation cell culture secretions: with reference to the comparative study of spready hide sheet method in " Chinese reconstruction surgery " (2004 06 phases) and lingel method cultivation epidermis cell, carry out epidermis cell and cultivate, collect the culture fluid under changing; Be carried out to fibrocyte by example 1 and cultivate, collect the culture fluid of changing; Adopt hyperfiltration process to concentrate respectively, washed with de-ionized water desalts, and the ultrafiltration post adopts the 30K aperture; Adopt protein purification instrument (AKTA PRIME) to remove bovine serum albumin, obtain two kinds of required cell culture secretions respectively, survey secretion content (in Tot Prot) respectively, preserve after the filtration sterilization;
Step 3, preparation contains the sustained-release micro-spheres of cell culture secretions: with reference to " sustained-release micro-spheres of preparation composite growth factor is also investigated the influence of its pair cell " (yellow sand, banket etc., 2004 the 25th the 4th phases of volume of " Shanghai biomedical engineering " magazine) preparation method of gelatine microsphere in, the preparation mean diameter is the gelatine microsphere of 100～500nm, after the lyophilizing sterilization, in the 2g microsphere, add under the aseptic condition and contain the aqueous solution that the 250mg epidermis cell is cultivated secretions and contained 100mg fibroblast cultivation secretions, placed one day down, promptly get the sustained-release micro-spheres that contains cell culture secretions after the lyophilizing for 4 ℃;
Step 4, preparation composite soft-tissue patch: in containing the mixed gel solution that W/V is 0.15% hyaluronate sodium and 0.15% collagen, add the sustained-release micro-spheres that contains cell culture secretions, the final concentration that makes cell culture secretions is 120mg/ml, behind the mixing this gel solution evenly covered and be applied to the acellular dermal matrix surface, covering the amount of being coated with is 1ml/cm
2, making under the vacuum condition in its hole that fully is penetrated into acellular dermal, lyophilizing promptly gets composite soft-tissue patch.
The composite soft-tissue patch thickness of this example preparation is 1～1.2mm, and fracture tensile strength is greater than 0.2Mpa; Sticking patch is preserved under 4 ℃ of conditions, during use the sticking patch taking-up is cut into suitable size as required and places sterile chamber, and the normal saline that adds 10～20 times of amounts soaks 15min.This sticking patch is applicable to the sealing of human body skin wound surface, can effectively promote the healing of refractory wound surface, chronic ulcer.
Claims (2)
1. the preparation method of a composite soft-tissue patch, employing has through taking off the dermal matrix that cell is handled, it is characterized in that, be to contain the sustained-release micro-spheres that human body cell is cultivated secretions, be mixed in gel solution, down compound with acellular dermal matrix in vacuum condition again, drying obtains composite soft-tissue patch after sterilizing.
2. preparation method according to claim 1 is characterized in that concrete steps comprise:
Step 1, preparation acellular dermal matrix: get after fresh skin cleans, remove subcutaneus adipose tissue and epidermis, keep the thick skin corium skin graft of 0.1mm～2mm, clean with deionized water and aseptic PBS solution respectively, soak abundant swelling in 4 ℃ of deionized waters; Place-80 ℃ freezing more than 30 minutes, melt to organizing to take out after the complete deep colling; After adopting W/V to be 0.1%～0.3% trypsin solution digestion skin graft again, clean skin graft with the de-sludging agent solution; The DNA enzymatic solution digestion of reuse 40～150U/ml with the NaOH solution ablation corium skin graft 1h of 1M, is cleaned skin graft respectively at last with PBS and pure water, after lyophilization, sterilization, that the 4 ℃ of preservations of acellular dermal matrix that obtain are standby;
Step 2, preparation cell culture secretions: the culture fluid under changing in the collecting cell incubation, concentrate by hyperfiltration process, desalt with washed with de-ionized water; Adopt the protein purification instrument to remove bovine serum albumin, remaining protein ingredient is a cell culture secretions, preserves after the filtration sterilization;
Step 3, preparation contain the sustained-release micro-spheres of cell culture secretions: the microsphere that with particle diameter is 10nm～3um is after the lyophilizing sterilization, the method of water solution absorbs under aseptic condition, make the cell culture secretions that contains 100～500mg in the 1g microsphere, after lyophilizing, be the sustained-release micro-spheres that contains cell culture secretions;
Step 4, preparation composite soft-tissue patch: in W/V is 0.2～1.5% gel solution, add the sustained-release micro-spheres that contains cell culture secretions, the final concentration that makes cell culture secretions is 50～200mg/ml, evenly cover behind the mixing and be applied to the acellular dermal matrix surface, covering the amount of being coated with is 0.5～1ml/cm
2, under the vacuum condition it is fully permeated, be composite soft-tissue patch after the drying.
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| CN102172418A (en) * | 2011-02-18 | 2011-09-07 | 上海交通大学医学院附属新华医院 | Acellular matrix material capable of sustainedly releasing growth factors |
| CN105833353A (en) * | 2016-05-09 | 2016-08-10 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation and application of bioengineering decellularized dermal matrix |
| CN105903080A (en) * | 2016-05-23 | 2016-08-31 | 苏州恒瑞迪生医疗科技有限公司 | Breast patch and preparation method thereof |
| CN107376023A (en) * | 2017-08-31 | 2017-11-24 | 上海市第六人民医院 | A kind of preparation method of timbering material suitable for urethra reconstruction |
| CN109529106A (en) * | 2018-11-14 | 2019-03-29 | 山东隽秀生物科技股份有限公司 | A kind of chronic wound repairs the preparation method of matrix |
| CN110960731A (en) * | 2019-12-12 | 2020-04-07 | 成都奇璞生物科技有限公司 | Preparation method of medical surgical biological patch |
| CN113301928A (en) * | 2018-12-28 | 2021-08-24 | 黄玲惠 | Biological scaffolds and methods for making same |
| CN114146221A (en) * | 2021-12-09 | 2022-03-08 | 杭州帕莱拉医疗科技有限公司 | Injectable dextran hydrogel microsphere filling agent and preparation method thereof |
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| WO2007051221A1 (en) * | 2005-11-04 | 2007-05-10 | Craig Mclachlan | Substrate for tissue growth |
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| CN102172418A (en) * | 2011-02-18 | 2011-09-07 | 上海交通大学医学院附属新华医院 | Acellular matrix material capable of sustainedly releasing growth factors |
| CN105833353A (en) * | 2016-05-09 | 2016-08-10 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation and application of bioengineering decellularized dermal matrix |
| CN105833353B (en) * | 2016-05-09 | 2018-04-20 | 拜欧迪赛尔（北京）生物科技有限公司 | A kind of preparation of bioengineering acellular dermal matrix and purposes |
| CN105903080A (en) * | 2016-05-23 | 2016-08-31 | 苏州恒瑞迪生医疗科技有限公司 | Breast patch and preparation method thereof |
| CN107376023A (en) * | 2017-08-31 | 2017-11-24 | 上海市第六人民医院 | A kind of preparation method of timbering material suitable for urethra reconstruction |
| CN109529106A (en) * | 2018-11-14 | 2019-03-29 | 山东隽秀生物科技股份有限公司 | A kind of chronic wound repairs the preparation method of matrix |
| CN109529106B (en) * | 2018-11-14 | 2021-09-10 | 山东隽秀生物科技股份有限公司 | Preparation method of chronic wound repair matrix |
| CN113301928A (en) * | 2018-12-28 | 2021-08-24 | 黄玲惠 | Biological scaffolds and methods for making same |
| CN110960731A (en) * | 2019-12-12 | 2020-04-07 | 成都奇璞生物科技有限公司 | Preparation method of medical surgical biological patch |
| CN114146221A (en) * | 2021-12-09 | 2022-03-08 | 杭州帕莱拉医疗科技有限公司 | Injectable dextran hydrogel microsphere filling agent and preparation method thereof |
| CN115054732A (en) * | 2022-06-07 | 2022-09-16 | 东华大学 | Suture-free multilayer drug-loaded myocardial patch and preparation method thereof |
| CN115054732B (en) * | 2022-06-07 | 2023-10-13 | 东华大学 | Suture-free multi-layer drug-loaded myocardial patch and preparation method thereof |
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