CN106730038A - A kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof - Google Patents
A kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof Download PDFInfo
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- CN106730038A CN106730038A CN201710022057.5A CN201710022057A CN106730038A CN 106730038 A CN106730038 A CN 106730038A CN 201710022057 A CN201710022057 A CN 201710022057A CN 106730038 A CN106730038 A CN 106730038A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
Abstract
The invention discloses a kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof, the invention belongs to biomedical engineering technology field, a kind of described tunica fibrosa for tracheae soft tissue repair is nucleocapsid porous network structure, and the tunica fibrosa for tracheae soft tissue repair is made up of polycaprolactone, NTx, recombinant conversion grouth factor beta 3 (rhTGF β 3) and bovine serum albumin (BSA).This kind of tracheae soft tissue repair tunica fibrosa immunogenicity is low, and biocompatibility and induction repair ability are excellent, and has the effect for significantly promoting healing to being damaged tracheae soft tissue.
Description
Technical field
The invention belongs to biomedical engineering technology field, particularly tracheae soft tissue repair technical field of material,
Disclose a kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof.
Background technology
If tracheal neoplasm, thyroid gland and other region tumors invasion and attack tracheae are clinically met, and tracheae congenital or wound
When wound property reason causes tracheae Serious Stenosis, it is both needed to the excision of promoting the circulation of qi pipe and rebuilds.Due to the special physiological structure of tracheae, determine
The problems such as successful key factor of graft of trachea includes the vascularization of graft, immunological rejection and histiocytic regeneration.Reason
The trachea substitute thought should possess following characteristics：Unrestricted in order to ensure neck activity, tracheae must be easily bent but not
Cause is collapsed；In order to meet ventilatory function and prevent mediastinal infection, tube chamber must be closed air tight；Inner wall smooth, prevents into fiber
Cell and the intrusion of bacterium；Secondly, to be conducive to tracheal mucosa epithelial growth, make it have the function of cleaning air flue；It is more important
Be a little to have good histocompatbility.
Tissue engineering trachea can be repaired because of the trachea defect that wound, tumour, inflammation etc. cause.Regenerated because cartilage lacks
Ability, is difficult to itself and repairs, it is necessary to repaired and rebuild using its hetero-organization or alternative materials after disease damage.In nearly 20 years,
Field of tissue engineering technology has been intended to recover or change damage or has lost tissue and organ.Tissue engineered trachea is needed for structure,
The tissue of machinery and function.The benign or malignant change of tracheae generally requires extensive tracheal reconstruction.For tracheae substitute mode
It has been be reported that, including used the group of autologous tissue, autotransplantation allograft, prosthetic material or these methods
Close.But in these methods, tracheostenosis, fibrosis, bacterium infection and immunological rejection, material failure often occurs, cause
Trachea substitute carries out graft failure.Furthermore the biocompatibility of the tissue engineering trachea of some is low, degradation property difference is also limit
Tissue engineering trachea processed further applying clinically.
Recombinant conversion growth factor-beta 3（rhTGF-β3）, it is cell factor important during embryo's Scarless wound healing reacts.Have
People is found that the Scarless wound healing phenomenon of body early embryo and compares the cell of Scarless wound healing and cicatrization and divide very early
Handset system.Recombinant conversion growth factor-beta 3（rhTGF-β3）The initial form generation in repair process after wound, subsequent is thin
Played an important role in born of the same parents' propagation and final tissue reconstruction.Recombinant conversion growth factor-beta 3（rhTGF-β3）Have passed through big
The polycentric control of amount and prospective clinical trial, it was demonstrated that it can efficiently reduce scar in various surgical operations, it is clinical
Experiment confirms the effect in terms of security, validity and the improvement scar outward appearance of recombined human TGF β 3.
The content of the invention
It is an object of the invention to tracheostenosis, fiber occurs after being directed to existing tracheal tissue's repair materials application
Change, immunological rejection, material failure, cause trachea substitute to carry out graft failure, and its biocompatibility and degradability it is low
Deng not enough, there is provided a kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof, the tracheae soft tissue repair tunica fibrosa
Immunogenicity is low, and material source enriches, with good biocompatibility and degradability, and with induction soft tissue repair
Ability and the appearance that impaired tracheal-fibrosis can be suppressed.
To achieve the above object, the technical scheme is that:A kind of tunica fibrosa for tracheae soft tissue repair
It is core-shell structure copolymer porous network structure, by polycaprolactone, NTx, recombinant conversion growth factor-beta 3（rhTGF-β3）And cow's serum
Albumen（BSA）Composition.
Further, a kind of tunica fibrosa by tracheae soft tissue repair by weight based on by following component group
Into：
10 parts of polycaprolactone；
10 parts of NTx；
Recombinant conversion growth factor-beta 3（rhTGF-β3）0.01-0.05 parts；
Bovine serum albumin（BSA）0.05-0.1 parts.
Preferably, the molecular weight of described polycaprolactone is 8 × 105Da, the molecular weight of described NTx is 1 × 105
Da, described recombinant conversion growth factor-beta 3（rhTGF-β3）Its molecular weight is 25 kDa.
Further, a kind of preparation method for tracheae soft tissue repair, its preparation method passes through following steps
Realize：First, the preparation of fiber film material, two, the spinning of tracheae soft tissue repair tunica fibrosa.
Further, the preparation of the fiber film material is concretely comprised the following steps：
（1）It is prepared by Shell Materials：By polycaprolactone and NTx in mass ratio 4:1 is dissolved in hexafluoroisopropanol stirs 12 hours, matches somebody with somebody
The mixed solution of polycaprolactone/NTx that mass concentration is 12% is made, is then produced during ultrasonically treated 30 min removals stirring
Raw bubble, is obtained Shell Materials mixed solution；
（2）It is prepared by core layer material：
S1：By recombinant conversion growth factor-beta 3（rhTGF-β3）Be added to the h of gentle agitation 1 in phosphate buffer be configured to it is dense
Spend the solution of rhTGF- β 3 for 40 μ g/mL；
S2：By bovine serum albumin（BSA）It is 200 μ g/mL to be added to the h of gentle agitation 1 in phosphate buffer and be configured to concentration
BSA solution；
S3：Then by BSA that the solution of rhTGF- β 3 and concentration obtained in S2 that concentration obtained in S1 is 40 μ g/mL are 200 μ g/mL
Solution mixes in equal volume, is configured to the mixed solution of rhTGF- β 3/BSA；
S4：NTx is dissolved in deionized water and is made into the NTx solution that mass concentration is 20%；
S5：By the mixed solution of obtained rhTGF- β 3/BSA in NTx solution that mass concentration obtained in S4 is 20% and S3
Isometric mixing, is obtained core layer material mixed solution.
Further, the spinning of the tracheae soft tissue repair tunica fibrosa is comprised the following steps that：
S1:The Shell Materials mixed solution and the core layer material mixed solution are added separately in coaxial electrostatic spinning equipment
Shell Materials syringe and core layer material syringe in, stand 30-60 min to draining the Shell Materials syringe and core
The air of layer material syringe；
S2：Distance between adjusting roller receiver and Coaxial nozzle is 15-20 cm, regulation high voltage power supply to 15 ~ 20 kV, regulation
The output speed for being loaded with the micro-injection pump of Shell Materials syringe is adjusted to 1mL/h, and regulation is loaded with core layer material syringe
The output speed of micro-injection pump to 0.2mL/h, the rotating speed of roller receiver is controlled in 200 ~ 350 rpm；
S3:After core layer material is exported completely, terminate spinning, spinning film is removed from collection device, obtain final product a kind of for tracheae
The tunica fibrosa of soft tissue repair.
Preferably, described coaxial electrostatic spinning silk device is by high voltage power supply, Shell Materials syringe, core layer material injection
Device, micro-injection pump, collection device, injector head and roller receiver.
Further, the coaxial injector head internal orifice dimension is 0.5 mm, and the outer aperture of the coaxial injector head is 0.8 mm.
The present invention by with polycaprolactone and NTx as matrix, with recombinant conversion growth factor-beta 3（rhTGF-β3）With
Bovine serum albumin（BSA）It is bioactive ingredients, by coaxial electrostatic spinning technology, prepares a kind of tool core-shell structure copolymer porous web
The tunica fibrosa of shape structure, this kind of repairing fiber film have good tridimensional network and biocompatibility be conducive to cell
Its surface adhesion and growth, and because it has nucleocapsid structure, can effectively solve recombinant conversion growth factor-beta 3（rhTGF-
β3）And bovine serum albumin（BSA）The easy problem of failure during preparation, and can make this kind of repairing fiber film there is slow-release function, can
Recombinant conversion growth factor-beta 3 is gone out with slow sustained release（rhTGF-β3）And bovine serum albumin（BSA）To damaged part, can have
Effect ground promotes the healing of damage location and suppresses the appearance of fibrosis.And the matrix that this kind of repairing fiber film is used is for biology can
Degradable material, harmless small molecule can be degraded into after the completion of tissue repair and be excluded in vitro with human circulation.
Beneficial effects of the present invention are：
（1）The present invention prepares a kind of tunica fibrosa of tool core-shell structure copolymer porous network structure by coaxial electrostatic spinning technology, should
Planting repairing fiber film has good tridimensional network beneficial to cell in its surface adhesion and growth, so as to accelerate newborn group
The growth knitted.And because it has nucleocapsid structure, can effectively solve recombinant conversion growth factor-beta 3（rhTGF-β3）And ox
Haemocyanin（BSA）Easily fail in the preparation and the low problem of load factor, and can have this kind of repairing fiber film and be sustained work(
Can, slowly can go out recombinant conversion growth factor-beta 3 by sustained release（rhTGF-β3）And bovine serum albumin（BSA）To impaired portion
Position, the healing that damage location can be effectively facilitated and the appearance for suppressing fibrosis；
（2）The present invention is by with recombinant conversion growth factor-beta 3（rhTGF-β3）And bovine serum albumin（BSA）It is core layer material,
Assigning obtained repairing fiber film has the effect for promoting damaged part healing.Recombinant conversion growth factor-beta 3（rhTGF-β
3）Can be assembled by reducing inflammatory cell and connective tissue deposition reduces scar, the effect of antagonistic TGF beta 1 and TGF β 2 makes wound
Collage synthesis in mouthful repair process are in poised state with decomposing, and both can guarantee that the healing of wound, again will not scar it is excessive.This
Outward, recombinant conversion growth factor-beta 3（rhTGF-β3）Can promote the differentiation of mescenchymal stem cell, and bovine serum albumin（BSA）
Can be outside the growth and differentiation raising nutrition of mescenchymal stem cell, moreover it is possible to as protective agent protection recombinant conversion growth factor-beta 3
（rhTGF-β3）Extend its half-life period in human body.
Brief description of the drawings
Fig. 1 is the release in vitro evaluation experimental result figures of rhTGF- β 3 in embodiment 4；
Fig. 2 is in embodiment 5 and mescenchymal stem cell（MSCS）Cytotoxic evaluation result figure after cultivating 1 day and 7 days.
Specific embodiment
Technical scheme is described further with reference to embodiment.
Embodiment 1
A kind of tunica fibrosa for tracheae soft tissue repair, the tunica fibrosa for tracheae soft tissue repair is that core-shell structure copolymer is porous
Network structure, by polycaprolactone, NTx, recombinant conversion growth factor-beta 3（rhTGF-β3）And bovine serum albumin（BSA）Group
In groups into；The tunica fibrosa by tracheae soft tissue repair by weight based on by 10 parts of polycaprolactone, 10 parts of NTx,
Recombinant conversion growth factor-beta 3（rhTGF-β3）0.01 part and bovine serum albumin（BSA）0.05 part of composition；Described polycaprolactone
Molecular weight be 8 × 105Da, the molecular weight of described NTx is 1 × 105 Da, described recombinant conversion growth factor-beta 3
（rhTGF-β3）Molecular weight be 25 kDa.
The above-mentioned tunica fibrosa for tracheae soft tissue repair, its preparation method is comprised the following steps that：
First, the preparation of fiber film material:
（1）It is prepared by Shell Materials：
By polycaprolactone and NTx in mass ratio 4:1 is dissolved in hexafluoroisopropanol stirs 12 hours, and being configured to mass concentration is
The mixed solution of 12% polycaprolactone/NTx, the bubble for then being produced during ultrasonically treated 30 min removals stirring, is obtained shell
Layer material mixed solution；
（2）It is prepared by core layer material：
S1：By recombinant conversion growth factor-beta 3（rhTGF-β3）Be added to the h of gentle agitation 1 in phosphate buffer be configured to it is dense
Spend the solution of rhTGF- β 3 for 40 μ g/mL；
S2：By bovine serum albumin（BSA）It is 200 μ g/mL to be added to the h of gentle agitation 1 in phosphate buffer and be configured to concentration
BSA solution;
S3：Then by BSA that the solution of rhTGF- β 3 and concentration obtained in S2 that concentration obtained in S1 is 40 μ g/mL are 200 μ g/mL
Solution mixes in equal volume, is configured to the mixed solution of rhTGF- β 3/BSA；
S4：NTx is dissolved in deionized water and is made into the NTx solution that mass concentration is 20%；
S5：Will be obtained in NTx solution that mass concentration obtained in S4 is 20% and S3
The mixed solution of rhTGF- β 3/BSA mixes in equal volume, and core layer material mixed solution is obtained.
2nd, the spinning of tracheae soft tissue repair tunica fibrosa:
S1:The Shell Materials mixed solution and the core layer material mixed solution are added separately in coaxial electrostatic spinning equipment
Shell Materials syringe and core layer material syringe in, stand 30-60 min to draining the Shell Materials syringe and core
The air of layer material syringe；
S2:The electric wire between roller receiver and Coaxial nozzle is connected, the distance between adjusting roller receiver and Coaxial nozzle is
15cm；High voltage power supply is connected, regulation high voltage power supply to 15kV starts micro-injection pump, and regulation is loaded with Shell Materials syringe
The output speed of micro-injection pump is adjusted to 1mL/h, and regulation is loaded with the output speed of the micro-injection pump of core layer material syringe
Regulation starts roller receiver and receives spinning to 0.2mL/h, and the rotating speed of roller receiver is controlled in 200 rpm；
S3:After core layer material is exported completely, terminate spinning, close high voltage power supply, spinning film is removed from collection device, i.e.,
Obtain a kind of tunica fibrosa for tracheae soft tissue repair.
Described coaxial electrostatic spinning silk device includes high voltage power supply, and Shell Materials syringe, core layer material syringe is micro
Syringe pump, collection device, Coaxial nozzle and roller receiver.
The coaxial injector head internal orifice dimension is 0.5 mm, and the outer aperture of the coaxial injector head is 0.8 mm.
Embodiment 2
A kind of tunica fibrosa for tracheae soft tissue repair, the tunica fibrosa for tracheae soft tissue repair is that core-shell structure copolymer is porous
Network structure, by polycaprolactone, NTx, recombinant conversion growth factor-beta 3（rhTGF-β3）And bovine serum albumin（BSA）Group
In groups into；The tunica fibrosa by tracheae soft tissue repair by weight based on by 10 parts of polycaprolactone, 10 parts of NTx,
Recombinant conversion growth factor-beta 3（rhTGF-β3）0.025 part and bovine serum albumin（BSA）0.075 part of composition；Described gathers in oneself
The molecular weight of ester is 8 × 105Da, the molecular weight of described NTx is 1 × 105 Da, described recombinant conversion growth factor-beta
3（rhTGF-β3）Molecular weight be 25 kDa.
The above-mentioned tunica fibrosa for tracheae soft tissue repair, its preparation method is comprised the following steps that：
First, the preparation of fiber film material:
（1）It is prepared by Shell Materials：
By polycaprolactone and NTx in mass ratio 4:1 is dissolved in hexafluoroisopropanol stirs 12 hours, and being configured to mass concentration is
The mixed solution of 12% polycaprolactone/NTx, the bubble for then being produced during ultrasonically treated 30 min removals stirring, is obtained shell
Layer material mixed solution；
（2）It is prepared by core layer material：
S1：By recombinant conversion growth factor-beta 3（rhTGF-β3）Be added to the h of gentle agitation 1 in phosphate buffer be configured to it is dense
Spend the solution of rhTGF- β 3 for 40 μ g/mL；
S2：By bovine serum albumin（BSA）It is 200 μ g/mL to be added to the h of gentle agitation 1 in phosphate buffer and be configured to concentration
BSA solution;
S3：Then by BSA that the solution of rhTGF- β 3 and concentration obtained in S2 that concentration obtained in S1 is 40 μ g/mL are 200 μ g/mL
Solution mixes in equal volume, is configured to the mixed solution of rhTGF- β 3/BSA；
S4：NTx is dissolved in deionized water and is made into the NTx solution that mass concentration is 20%；
S5：Will be obtained in NTx solution that mass concentration obtained in S4 is 20% and S3
The mixed solution of rhTGF- β 3/BSA mixes in equal volume, and core layer material mixed solution is obtained.
2nd, the spinning of tracheae soft tissue repair tunica fibrosa:
S1:The Shell Materials mixed solution and the core layer material mixed solution are added separately in coaxial electrostatic spinning equipment
Shell Materials syringe and core layer material syringe in, stand 30-60 min to draining the Shell Materials syringe and core
The air of layer material syringe；
S2:The electric wire between roller receiver and Coaxial nozzle is connected, the distance between adjusting roller receiver and Coaxial nozzle is
17cm；High voltage power supply is connected, regulation high voltage power supply to 17kV starts micro-injection pump, and regulation is loaded with Shell Materials syringe
The output speed of micro-injection pump is adjusted to 1mL/h, and regulation is loaded with the output speed of the micro-injection pump of core layer material syringe
Regulation starts roller receiver and receives spinning to 0.2mL/h, and the rotating speed of roller receiver is controlled in 300 rpm；
S3:After core layer material is exported completely, terminate spinning, close high voltage power supply, spinning film is removed from collection device, i.e.,
Obtain a kind of tunica fibrosa for tracheae soft tissue repair.Described coaxial electrostatic spinning silk device includes high voltage power supply, Shell Materials
Syringe, core layer material syringe, micro-injection pump, collection device, Coaxial nozzle and roller receiver.
The coaxial injector head internal orifice dimension is 0.5 mm, and the outer aperture of the coaxial injector head is 0.8 mm.
Embodiment 3
A kind of tunica fibrosa for tracheae soft tissue repair, the tunica fibrosa for tracheae soft tissue repair is that core-shell structure copolymer is porous
Network structure, by polycaprolactone, NTx, recombinant conversion growth factor-beta 3（rhTGF-β3）And bovine serum albumin（BSA）Group
In groups into；The tunica fibrosa by tracheae soft tissue repair by weight based on by 10 parts of polycaprolactone, 10 parts of NTx,
Recombinant conversion growth factor-beta 3（rhTGF-β3）0.05 part and bovine serum albumin（BSA）0.1 part of composition；Described polycaprolactone
Molecular weight be 8 × 105Da, the molecular weight of described NTx is 1 × 105 Da, described recombinant conversion growth factor-beta 3
（rhTGF-β3）Molecular weight be 25 kDa.
The above-mentioned tunica fibrosa for tracheae soft tissue repair, its preparation method is comprised the following steps that：
First, the preparation of fiber film material:
（1）It is prepared by Shell Materials：
By polycaprolactone and NTx in mass ratio 4:1 is dissolved in hexafluoroisopropanol stirs 12 hours, and being configured to mass concentration is
The mixed solution of 12% polycaprolactone/NTx, the bubble for then being produced during ultrasonically treated 30 min removals stirring, is obtained shell
Layer material mixed solution；
（2）It is prepared by core layer material：
S1：By recombinant conversion growth factor-beta 3（rhTGF-β3）Be added to the h of gentle agitation 1 in phosphate buffer be configured to it is dense
Spend the solution of rhTGF- β 3 for 40 μ g/mL；
S2：By bovine serum albumin（BSA）It is 200 μ g/mL to be added to the h of gentle agitation 1 in phosphate buffer and be configured to concentration
BSA solution;
S3：Then by BSA that the solution of rhTGF- β 3 and concentration obtained in S2 that concentration obtained in S1 is 40 μ g/mL are 200 μ g/mL
Solution mixes in equal volume, is configured to the mixed solution of rhTGF- β 3/BSA；
S4：NTx is dissolved in deionized water and is made into the NTx solution that mass concentration is 20%；
S5：By the mixed solution of obtained rhTGF- β 3/BSA in NTx solution that mass concentration obtained in S4 is 20% and S3
Isometric mixing, is obtained core layer material mixed solution.
2nd, the spinning of tracheae soft tissue repair tunica fibrosa:
S1:The Shell Materials mixed solution and the core layer material mixed solution are added separately in coaxial electrostatic spinning equipment
Shell Materials syringe and core layer material syringe in, stand 30-60 min to draining the Shell Materials syringe and core
The air of layer material syringe；
S2:The electric wire between roller receiver and Coaxial nozzle is connected, the distance between adjusting roller receiver and Coaxial nozzle is
20cm；High voltage power supply is connected, regulation high voltage power supply to 20kV starts micro-injection pump, and regulation is loaded with Shell Materials syringe
The output speed of micro-injection pump is adjusted to 1mL/h, and regulation is loaded with the output speed of the micro-injection pump of core layer material syringe
Regulation starts roller receiver and receives spinning to 0.2mL/h, and the rotating speed of roller receiver is controlled in 350 rpm；
S3:After core layer material is exported completely, terminate spinning, close high voltage power supply, spinning film is removed from collection device, i.e.,
Obtain a kind of tunica fibrosa for tracheae soft tissue repair.Described coaxial electrostatic spinning silk device includes high voltage power supply, Shell Materials
Syringe, core layer material syringe, micro-injection pump, collection device, Coaxial nozzle and roller receiver.
The coaxial injector head internal orifice dimension is 0.5 mm, and the outer aperture of the coaxial injector head is 0.8 mm.
Embodiment 4
Comparative example：To have loaded the Xenogenic acellular dermal matrix of rhTGF- β 3.
Xenogenic acellular dermal matrix is with reference to a kind of good different of the biocompatibility disclosed in CN201510549616.9
Plant the preparation method of acellular dermal base.Load rhTGF- β's 3 concretely comprises the following steps：RhTGF- β 3 are dissolved in deionized water, are matched somebody with somebody
Into the solution of rhTGF- β 3 that concentration is 40 μ g/mL, then will be stood in the solution of Xenogenic acellular dermal matrix immersion rhTGF- β 3
12 h, are subsequently placed in -80 DEG C of freeze-dryings in freeze drier, obtain final product the xenogenesis acellular dermal base for having loaded rhTGF- β 3
Matter.
Experimental group 1 ~ 3：It is for tracheae soft tissue repair tunica fibrosa obtained by embodiment 1~3.
By obtained in a kind of tunica fibrosa for tracheae soft tissue repair prepared by above-described embodiment 1~3 and comparative example
Having loaded the Xenogenic acellular dermal matrix of rhTGF- β 3 carries out the release in vitro evaluation experimentals of rhTGF- β 3, and comparative example 1 ~ 3
With the releasing effects of rhTGF- β 3 of comparative example.Experimental result such as Fig. 1 shows.
By above-mentioned release evaluation experimental, it will be seen from figure 1 that being used for gas by one kind that preparation method of the present invention is obtained
The tunica fibrosa of pipe soft tissue repair has slow-release function, slowly can constantly discharge recombinant conversion growth factor-beta 3（rhTGF-
β3）.
Embodiment 5
Comparative example：Xenogenic acellular dermal matrix（A kind of biocompatibility with reference to disclosed in CN201510549616.9 is good
Xenogenesis acellular dermal base preparation method obtained by）
Experimental group 1 ~ 3：It is the obtained tunica fibrosa for tracheae soft tissue repair of embodiment 1~3.
The tunica fibrosa for tracheae soft tissue repair prepared by above-described embodiment 1~3 is carried out into cell toxicant with comparative example
Property evaluation experimental（Tested by GB GB/T 16886.5-2003）.Experimental result is as shown in Figure 2.
Cytotoxicity testing result show embodiment 1 ~ 3 with mescenchymal stem cell（MSCS）Co-culture 1 day and 7 days after its
With respect to proliferation rate more than 95%, cytotoxicity is rated 0 grade to corresponding cell, it was demonstrated that it has good describing property of cell,
And comparative example and mescenchymal stem cell（MSCS）Its correspondence cell is respectively less than 80% with respect to proliferation rate after co-culturing 1 day and 7 days, carefully
Cellular toxicity is rated 3 grades, with cytotoxicity higher.Additionally, the relative proliferation rate of the extension embodiment 1 ~ 3 of the time of co-cultivation
It is significantly improved, more negative group obvious with respect to proliferation rate of its cell after co-culturing 7 days of embodiment 1 ~ 3 will height（It is above
110%）, it was demonstrated that had using the tunica fibrosa for tracheae soft tissue repair obtained by preparation method disclosed in this invention and promoted
Enter mescenchymal stem cell（MSCS）Growth effect, be conducive to the reparation of damaged part.
Embodiment 6
Experimental group 1 ~ 3：The tunica fibrosa for tracheae soft tissue repair obtained by embodiment 1~3.
By the tunica fibrosa for tracheae soft tissue repair prepared by above-described embodiment 1~3 carry out acute toxicity evaluation and
Hemolysis in vitro evaluation is evaluating its biological safety.
Method of testing：
Acute toxicity evaluation method：By GB/T 16886.11-2011《The part of BiologicalEvaluationofMedicalDevice the 11st：Whole body poison
Property experiment》In acute toxicity detection method detected.
Hemolysis in vitro evaluation method：2 mL blood are centrifuged 5min in 1000 × g, supernatant liquor is removed, appropriate PBS is added
Solution（pH=7.4）, slowly shake up, 5 min are centrifuged with 1000 × g again after red blood cell is cleaned into three times, remove layer red blood cell and hang
Liquid PBS solution is configured to 16% red blood cell suspension.Take the 50 above-mentioned red blood cell suspensions of μ L, be separately added into 1 mL distilled water,
In the sample extraction liquor of PBS solution and 1mg/mL, positive controls, negative control group and test group are set to, gently
1 h is incubated after mixing at 37 DEG C, by each group sample in 1000 × g be centrifuged 5 min, take the μ L of supernatant 200 add 96 orifice plates in,
Every group of 3 Duplicate Samples, test its absorbance under 540nm wavelength, repeat experiment 3 times.According to the absorbance for measuring, by formula
（1）Calculate the hemolysis rate of each experiment（Y）.Y(%) = （A-A0）/(A100-A0) ×100
Skin irritatin evaluation method：By GB/T 16886.10-2005《The part of BiologicalEvaluationofMedicalDevice the 10th：Stimulate with
Delayed allergy is tested》Middle intracutaneous stimulates detection method to be detected.
Evaluation result is as shown in table 1：
The acute toxicity evaluation of table 1 and hemolysis in vitro evaluation result
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Acute toxicity is evaluated | Normally, it is asymptomatic | Normally, it is asymptomatic | Normally, it is asymptomatic |
| Hemolysis in vitro is evaluated | 2.33% | 1.25% | 3.11% |
Knowable to upper table result, the skin regeneration material with bioactivity obtained by embodiment 1 ~ 3 is without acute toxicity, body
Outer haemolysis meets hemolysis rate criterion of acceptability, it was demonstrated that the tunica fibrosa for tracheae soft tissue repair prepared by the present invention has good
Good biological safety.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not right
The restriction of embodiments of the present invention；For those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, there is no need and unable to be exhaustive to all of implementation method；It is all this
Any modification, equivalent and improvement made within the spirit and principle of invention etc., should be included in the claims in the present invention
Protection domain within.
Claims (8)
1. a kind of tunica fibrosa for tracheae soft tissue repair, it is characterised in that described one kind is used for tracheae soft tissue repair
Tunica fibrosa be core-shell structure copolymer porous network structure, the tunica fibrosa for tracheae soft tissue repair by polycaprolactone, NTx,
Recombinant conversion growth factor-beta 3（rhTGF-β3）And bovine serum albumin（BSA）Composition.
2. a kind of tunica fibrosa for tracheae soft tissue repair according to claim 1, it is characterised in that described for gas
The tunica fibrosa of pipe soft tissue repair is counted consist of the following composition by weight:
10 parts of polycaprolactone;
10 parts of NTx;
Recombinant conversion growth factor-beta 3（rhTGF-β3）0.01-0.05 parts;
Bovine serum albumin（BSA）0.05-0.1 parts.
3. a kind of tunica fibrosa for tracheae soft tissue repair according to claim 1, it is characterised in that described gathers oneself
The molecular weight of lactone is 8 × 105Da, the molecular weight of described NTx is 1 × 105 Da, described recombinant conversion growth because
Son-β 3（rhTGF-β3）Molecular weight be 25 kDa.
4. the preparation method of a kind of tunica fibrosa for tracheae soft tissue repair according to any one of claim 1 ~ 3, its
It is characterised by, described preparation method is realized by following steps：First, the preparation of fiber film material;2nd, tracheae soft tissue repair
The spinning of tunica fibrosa.
5. the preparation method of a kind of tunica fibrosa for tracheae soft tissue repair according to claim 4, it is characterised in that
The preparation of the fiber film material is concretely comprised the following steps：
（1）It is prepared by Shell Materials：
By polycaprolactone and NTx in mass ratio 4:1 is dissolved in hexafluoroisopropanol stirs 12 hours, and being configured to mass concentration is
The mixed solution of 12% polycaprolactone/NTx, the bubble for then being produced during ultrasonically treated 30 min removals stirring, is obtained shell
Layer material mixed solution；
（2）It is prepared by core layer material：
S1：By recombinant conversion growth factor-beta 3（rhTGF-β3）Be added to the h of gentle agitation 1 in phosphate buffer be configured to it is dense
Spend the solution of rhTGF- β 3 for 40 μ g/mL；
S2：By bovine serum albumin（BSA）It is 200 μ g/mL to be added to the h of gentle agitation 1 in phosphate buffer and be configured to concentration
BSA solution;
S3：Then by BSA that the solution of rhTGF- β 3 and concentration obtained in S2 that concentration obtained in S1 is 40 μ g/mL are 200 μ g/mL
Solution mixes in equal volume, is configured to the mixed solution of rhTGF- β 3/BSA；
S4：NTx is dissolved in deionized water and is made into the NTx solution that mass concentration is 20%；
S5：By the mixed solution of obtained rhTGF- β 3/BSA in NTx solution that mass concentration obtained in S4 is 20% and S3
Isometric mixing, is obtained core layer material mixed solution.
6. the preparation method of a kind of tunica fibrosa for tracheae soft tissue repair according to claim 4, it is characterised in that
The spinning of the tracheae soft tissue repair tunica fibrosa is concretely comprised the following steps：
S1:The Shell Materials mixed solution and the core layer material mixed solution are added separately in coaxial electrostatic spinning equipment
Shell Materials syringe and core layer material syringe in, stand 30-60 min to draining the Shell Materials syringe and core
The air of layer material syringe；
S2：Distance between adjusting roller receiver and Coaxial nozzle is 15-20 cm, regulation high voltage power supply to 15 ~ 20 kV, regulation
The output speed for being loaded with the micro-injection pump of Shell Materials syringe is adjusted to 1mL/h, and regulation is loaded with core layer material syringe
The output speed of micro-injection pump to 0.2mL/h, the rotating speed of roller receiver is controlled in 200 ~ 350 rpm；
S4:After core layer material is exported completely, terminate spinning, spinning film is removed from collection device, obtain final product a kind of for tracheae
The tunica fibrosa of soft tissue repair.
7. the preparation method of a kind of tunica fibrosa for tracheae soft tissue repair according to claim 6, it is characterised in that
Described coaxial electrostatic spinning silk device include high voltage power supply, Shell Materials syringe, core layer material syringe, micro-injection pump,
Collection device, Coaxial nozzle and roller receiver.
8. the preparation method of a kind of tunica fibrosa for tracheae soft tissue repair according to claim 7, it is characterised in that
The coaxial injector head internal orifice dimension is 0.5 mm, and the outer aperture of the coaxial injector head is 0.8 mm.
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