CN105903077A - Preparation method of biological matrix gel material for promoting regeneration and repair of cartilage, gel performance determination method and application - Google Patents
Preparation method of biological matrix gel material for promoting regeneration and repair of cartilage, gel performance determination method and application Download PDFInfo
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- CN105903077A CN105903077A CN201610230098.9A CN201610230098A CN105903077A CN 105903077 A CN105903077 A CN 105903077A CN 201610230098 A CN201610230098 A CN 201610230098A CN 105903077 A CN105903077 A CN 105903077A
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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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
- A61L27/20—Polysaccharides
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
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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
- A61L27/52—Hydrogels or hydrocolloids
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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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
- A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
Abstract
The invention provides a preparation method of a biological matrix gel material for promoting regeneration and repair of cartilage, a gel performance determination method and application. The steps include: adding a GP solution into a mixed solution of a human amniotic membrane-PEI system and a mixed solution of a chitosan solution to conduct blending treatment to obtain a chitosan/human amniotic membrane-PEI/GP system mixed solution with the biological matrix gel material as the matrix. The gel formed by the chitosan solution and the GP solution maintains the activity of cells in the mixed solution of the human amniotic membrane-PEI system, promotes the regeneration and repair of cartilage, and artificial joint replacement is no longer carried out for treatment, therefore, the non-surgical treatment effect of severe osteoarthritis formed by further development of cartilage injury is improved.
Description
One, technical field
The present invention relates to the preparation method of a kind of bio-matrix gel rubber material, a kind of for promoting what regenerating bone or cartilage was repaired
The preparation method of bio-matrix gel rubber material, gelling performance assay method and application.
Two, background technology
Owing to there is not blood vessel and neutral net, knee cartilage, almost without self repair ability, particularly defines big
During damage location, the forfeiture of arthralgia and function of joint can be caused, often progress to osteoarthritis.Further, since aged or fortune
The dynamic excessive use causing joint, knee articular cartilage defect patient gets more and more, has had a strong impact on the quality of life of patient, therefore
For promoting that the preparation method of the bio-matrix material of regenerating bone or cartilage reparation is a kind of important biological method, existing for
Promote that in the preparation method of bio-matrix material of regenerating bone or cartilage reparation, the treatment of osteoarthritis is mainly focused on and eliminates affected part
Pain or inflammation, by being to give nonsteroidal antiinflammatory disease medicine to realize patient positions, reduce gerontal patient's renal function, from
From the point of view of safety, it is difficult to continuous oral gives nonsteroidal antiinflammatory disease medicine；Can also be by the composition of cartilage joint liquid
One of hyaluronic acid make product that preparation obtains by giving in articular cavity, have and improve the lubricating function in joint and press down
The effect of pain processed, but the severe osteoarthritic that progress further for cartilage injury is formed, it is necessary to carry out prosthetic replacement
Treat, currently there are no a kind of preparation method of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair, gel
Performance measurement method and application.
Three, summary of the invention
The object of the present invention is the preparation method of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair,
The object of the present invention is the gelling performance mensuration side of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair
Method,
The object of the present invention is the application of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair.
In order to overcome above-mentioned technical disadvantages, it is an object of the invention to provide a kind of biology for promoting regenerating bone or cartilage to repair
The preparation method of matrix gel material, gelling performance assay method and application, therefore improve cartilage injury and be in progress shape further
The non-operative treatment effect of the severe osteoarthritic become.
For reaching above-mentioned purpose, the present invention adopts the technical scheme that: a kind of biology for promoting regenerating bone or cartilage to repair
The preparation method of matrix gel material, molten with addition GP in the mixed liquor of chitosan solution at the mixed liquor of people's amniotic membrane-PEI system
Liquid carries out reconciliation process, and the chitosan/people's amniotic membrane-PEI/GP system obtaining the matrix as bio-matrix gel rubber material mixes
Liquid.
Owing to devising the mixed liquor of people's amniotic membrane-PEI system, chitosan solution and GP solution, by chitosan solution and
In the gel that GP solution is formed, maintain the activity of cell in the mixed liquor of people's amniotic membrane-PEI system, promote regenerating bone or cartilage
Repair, no longer carry out prosthetic replacement and treat, therefore improve cartilage injury further be in progress formed severe bone close
The non-operative treatment effect that joint is scorching.
The present invention devises, and mixes with the combination of the mixed liquor of chitosan solution according to the mixed liquor of people's amniotic membrane-PEI system
Close liquid obtaining final bio-matrix gel rubber material by the reaction of GP solution is technical essential.
The present invention devises, and the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 1.8-2.2cm × 2.8-3.2cm people's amniotic membrane, is mixed by shaking mode after adding 0.02% EDTA solution
Conjunction obtains middle mixing liquid I, by centrifugation, centre mixing liquid is separated, and after reject supernatant, adds 0.23-
0.27 mol acetum, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, sets
Parameter is rotating speed 3800-4200rpm, middle mixed liquor body II is centrifuged processing by time 28-32min, takes supernatant
Liquid, extract supernatant through excess pressure be 65.55-69.55KPa, temperature be 110.6-119.6 DEG C and time 8-12min
Disinfect dress and obtain middle mixing liquid III, centre mixing liquid III-18 to-22 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 0.8-1.2%, regulate PEI by NaOH weak solution
The pH value of solution is 4-6, add in the middle of mixing liquid III make PEI concentration be set to 0.4-0.6% to obtain people's amniotic membrane-PEI system
Mixed liquor, lyophilization.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree, more than 90% and chitosan powder that weight is 0.18-0.22g is dissolved into the 28-32ml that concentration is 0.5M
Acetum in, to being completely dissolved by the way of magnetic agitation, be then settled to 88-102ml with acetum, obtain dense
Degree is the chitosan solution of 2mg/ml, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in
In the autoclave of 120-122 DEG C sterilize 9-11 minute, subject to sterilization after chitosan solution temperature be down to room temperature after put into 3-5 DEG C of ice
Case preserves；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 54-58%, is removed by the membrane filtration of 0.22 μm
Obtain GP solution after bacterium, be 3-5 DEG C of preservation in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 18-22:1
Ratio add in sterile beaker, beaker is put into the supersound process carried out 0.9-1.1 hour on ice and obtains chitosan people's amniotic membrane
Solution, is put into the chitosan people's amniotic membrane solution in beaker and is stirred on ice and (b) step in second step is obtained
To GP solution be the most progressively added dropwise in chitosan people's amniotic membrane solution, during instillation observe chitosan people's amniotic membrane solution be
The obvious flocculent substance of no existence occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixed
The ultimate density closing the GP in liquid IV is 580-620mg/ml and obtains to 6.9-7.2 with saturated sodium dihydrogen phosphate regulation pH value
To chitosan/people's amniotic membrane-PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to chitosan people's amniotic membrane
The process of solution.
The present invention devises, and the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 2.2cm × 3.2cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.24 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 4100rpm, time 31min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 68.0KPa, temperature is 111.0 DEG C and time 9min obtains middle mixing liquid III, middle
Mixing liquid III is-21.5 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1.1%, regulate PEI solution by NaOH weak solution
PH value be 5.8, add in the middle of mixing liquid III makes PEI concentration be set to 0.48% mixed liquor obtaining people's amniotic membrane-PEI system,
Lyophilization.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree 95% and chitosan powder that weight is 0.19g are dissolved into the acetum of the 28ml that concentration is 0.5M
In, to being completely dissolved by the way of magnetic agitation, then it is settled to 99ml with acetum, obtains the shell that concentration is 2mg/ml
Polysaccharide solution, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in the autoclave of 120 DEG C
Sterilize 9 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 4.5 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 55%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 4.5 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 21.4:1's
Ratio adds in sterile beaker, beaker is put into the supersound process carried out on ice 0.9 hour and obtains chitosan people's amniotic membrane solution,
Chitosan people's amniotic membrane solution in beaker is put into and is stirred on ice and molten for the GP that (b) step in second step is obtained
Liquid is the most progressively added dropwise in chitosan people's amniotic membrane solution, observes whether chitosan people's amniotic membrane solution exists bright during instillation
Aobvious flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixing liquid IV
In the ultimate density of GP be 589mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 7.0 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
The present invention devises, the survey of the gelling performance of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair
Determine method, the steps include:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 35-39 DEG C of water-bath, every
Within 0.8-1.2 minute, it is inverted and observes mobility, take inversion 28-32 second chitosan/people's amniotic membrane-PEI/GP system mixed liquor immobilising
Time is designated as gel time.
The present invention devises, the application of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair, and shell is gathered
Inject the cartilage defect of articular cavity after sugar/people's amniotic membrane-PEI/GP system mixed liquor mixing umbilical cord blood mesenchymal stem cells, pass through
Heat lamp is heated, and seed cell is fixed on performance Regeneration and Repair effect at defect, makes colloidal sol one cell complexes undergo phase transition
To bio-matrix gel rubber material.
In the technical program, GP solution refers to phosphoglycerol two sodium solution.
The method have technical effect that:
Compared with the support of other forms, safely and effectively, this support contains the most natural I-type collagen and is adhered egg
In vain, growth and the mesenchymal stem cells into chondrocytes induction differentiation of chondrocyte it are especially suitable for.It addition, support is modified it is
Cell provides communicating with each other between good cell and extracellular matrix.Thus, can farthest promote that mesenchyme is done
Cell breaks up to chondrocyte growth, and then beneficially cartilage injury repairs.The timbering material that this invention uses is n cell
Epimatrix composition, degradation in vivo product totally nontoxic is harmless, internal uses safety to have no side effect, it is possible to soft with around host
Osseous tissue has preferable biocompatibility.Cheap.Compared with prior art, this invention greatly reduces cartilage injury's reparation
Required expense.Prior art many uses somatomedin carrys out inducing mesenchymal stem cell to be broken up to chondrocyte direction, promotes soft
Bone injury is repaired.Somatomedin large usage quantity, price is much more expensive.And the somatomedin concentration of present invention use is low, valency
Lattice are compared the biggest advantage；The gel stent additionally used is drawn materials simply, and processing technology is also than existing rack making technology
Simply.Therefore, it is possible to greatly reduce the cost that cartilage injury repairs.Use simple, reliable.Existing support vivo applications side
Method, needs to use open surgical techniques, is implanted by timbering material, and wound is more greatly and costly.The ECM that this invention uses
Material modified and hyaluronate sodium mixed gel can be injected at internal cartilage defect, then under the irradiation of ultraviolet point source
Gel in-situ, simple, convenient, wound is little and spends cheap, is especially suitable for the application that clinical cartilage injury repairs, significantly carries
High clinical replicability.Chitosan biological superior performance, has natural antibiotic property, biodegradable, good tissue
The compatibility, and structurally to the hyaluronic acid in glycosaminoglycans and articular cartilage, there is similar structure, so by extensively
It is applied to cartilage defect repair.But, individually use chitosan ossification is limited and mechanical property is poor.In amnion stroma
Containing compositions such as substantial amounts of collagen, fibronectin and laminin，LNs, it it is a kind of cheap and easy to get, tissue of good mechanical performance
Engineering material.Wherein there is special cell recognition signal (specific amino acid sequence) on type i collagen fiber and laminins surface,
Can promote that cell breaks up, the substrate of composition is conducive to keeping the form of chondrocyte, and the lower osteocyte of induction is to Chondrocyte Differentiation
Propagation.Material amnion stroma good for natural biological property is effectively physical crosslinking by the present invention with chitosan, maximum limit
The respective natural quality of holding of degree, the advantage playing respective material, the shortcoming making up the other side, build one and can play multiple merit
The compound injection aquagel timbering material of the intelligence of effect.This invention material operation is simple, convenient, mechanical strength is big, gel
Time is short, and wound is little and spends cheap, is especially suitable for the application that clinical cartilage injury repairs, and substantially increasing clinic can promote
Property.In the actual reproduction medicinal of cartilaginous lesion site, from the affinity of cell and body, easily use degree, control
Therapeutic effect angularly considers to be not appropriate for actual application.Osteoarthritis is easily to produce a kind of disease that morbid state deteriorates.Therefore, right
In the medicine of osteoarthritis, demand has the cartilage protecting the effect of cartilage, suppression abrasion or inflammation to cause in abrasion concurrently
The effect of the plyabilities such as the effect of the effect of degenerative change, the effect of reparation cartilaginous lesion site, suppression inflammation or pain.Logical
Cross and the amnion stroma containing type i collagen fiber and laminins is carried out cationic polymer modification, make modified amniotic membrane base
Matter is dissolved in chitosan gel rubber system and forms new injectable type bio-matrix material.Needed for this material easily combines repair of cartilage
Other materials such as stem cell, cell inducible factors, hyaluronic acid etc., the compositions collectively constituting tool mobility is applied to cartilage damage
Position, traumatic part, it is not necessary to excessive surgical method, can promote regenerating bone or cartilage by easy method.The compositions of the present invention is in embedding
When having Placenta Hominis source mescenchymal stem cell, it is thus achieved that the most excellent regenerating bone or cartilage.Also find, when not embedding these cells, this
The compositions of invention also obtains good hyaline cartilage regeneration.It addition, the compositions contained is applied to osteoarthritis
Cartilaginous lesion site, finds to suppress cartilage degenerative change, obtain the effect protecting cartilage.
In the technical program, mixed liquor, chitosan solution and the GP solution of people's amniotic membrane-PEI system is that important technology is special
Levy, in the preparation method of bio-matrix gel rubber material, gelling performance assay method and application for promoting regenerating bone or cartilage to repair
Technical field in, there is novelty, creativeness and practicality, the term in the technical program is all can to lead by this technology
Patent documentation in territory explains and understands.
Four, detailed description of the invention
According to guidelines for examination, to used in the present invention such as " having ", " comprising " and " including " term should be understood to not
Allot other element one or more or the existence of a combination thereof or interpolation.
In describing the invention, it should be noted that term " " center ", " on ", D score, "left", "right", " vertically ",
Orientation or the position relationship of the instruction such as " level ", " interior ", " outward " are orientation or position relationship, are for only for ease of the description present invention
Describe rather than indicate or imply that the device of indication or element must have specific orientation, with specific orientation structure with simplifying
Make and operate, be therefore not considered as limiting the invention.Additionally, term " first ", " second ", " the 3rd " are only used for describing
Purpose, and it is not intended that indicate or hint relative importance.
In describing the invention, it should be noted that unless otherwise clearly defined and limited, term " is installed ", " phase
Even ", " connection " should be interpreted broadly, for example, it may be fixing connection, it is also possible to be to removably connect, or be integrally connected；Can
To be mechanical connection, it is also possible to be electrical connection；Can be to be joined directly together, it is also possible to be indirectly connected to by intermediary, Ke Yishi
The connection of two element internals.For the ordinary skill in the art, can understand that above-mentioned term is at this with concrete condition
Concrete meaning in invention.
If additionally, the most non-structure of technical characteristic involved in invention described below difference embodiment
Become conflict just can be combined with each other.
Below in conjunction with embodiment, further describing the present invention, following example are intended to illustrate rather than this
The further restriction of invention.
A kind of preparation method of the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair,
First embodiment, the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 1.8cm × 2.8cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.23 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 3800rpm, time 28min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 65.55KPa, temperature is 110.6 DEG C and time 8min obtains middle mixing liquid III, middle
Mixing liquid III is-18 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 0.8%, regulate PEI solution by NaOH weak solution
PH value be 4, add in the middle of mixing liquid III makes PEI concentration be set to 0.4% mixed liquor obtaining people's amniotic membrane-PEI system, cold
Lyophilizing is dry.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree more than 90% and chitosan powder that weight is 0.18g to be dissolved into the acetic acid of the 28ml that concentration is 0.5M molten
In liquid, to being completely dissolved by the way of magnetic agitation, being then settled to 88ml with acetum, obtaining concentration is 2mg/ml's
Chitosan solution, preserves in room temperature, and the chitosan solution kraft paper stirred sealing is placed in the autoclave of 120 DEG C
Middle sterilization 9 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 3 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 54%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 3 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by the ratio of 18:1
Example adds in sterile beaker, beaker is put into the supersound process carried out on ice 0.9 hour and obtains chitosan people's amniotic membrane solution, burning
Chitosan people's amniotic membrane solution in Bei is put into and is stirred on ice and the GP solution that (b) step in second step is obtained
Slowly progressively it is added dropwise in chitosan people's amniotic membrane solution, during instillation, observes whether chitosan people's amniotic membrane solution exists substantially
Flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes in middle mixing liquid IV
The ultimate density of GP be 580mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 6.9 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
Second embodiment, the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 2.2cm × 3.2cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.27 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 4200rpm, time 32min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 69.55KPa, temperature is 119.6 DEG C and time 12min obtains middle mixing liquid III, in
Between mix liquid III-22 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1.2%, regulate PEI solution by NaOH weak solution
PH value be 6, add in the middle of mixing liquid III makes PEI concentration be set to 0.6% mixed liquor obtaining people's amniotic membrane-PEI system, cold
Lyophilizing is dry.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree 91% and chitosan powder that weight is 0.22g are dissolved into the acetum of the 32ml that concentration is 0.5M
In, to being completely dissolved by the way of magnetic agitation, then it is settled to 102ml with acetum, obtaining concentration is 2mg/ml's
Chitosan solution, preserves in room temperature, and the chitosan solution kraft paper stirred sealing is placed in the autoclave of 122 DEG C
Middle sterilization 11 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 5 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 58%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 5 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by the ratio of 22:1
Example adds in sterile beaker, beaker is put into the supersound process carried out on ice 1.1 hours and obtains chitosan people's amniotic membrane solution, burning
Chitosan people's amniotic membrane solution in Bei is put into and is stirred on ice and the GP solution that (b) step in second step is obtained
Slowly progressively it is added dropwise in chitosan people's amniotic membrane solution, during instillation, observes whether chitosan people's amniotic membrane solution exists substantially
Flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes in middle mixing liquid IV
The ultimate density of GP be 620mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 7.2 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
3rd embodiment, the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 1.8-2.2cm × 2.8-3.2cm people's amniotic membrane, is mixed by shaking mode after adding 0.02% EDTA solution
Conjunction obtains middle mixing liquid I, by centrifugation, centre mixing liquid is separated, and after reject supernatant, adds 0.23-
0.27 mol acetum, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, sets
Parameter is rotating speed 3800-4200rpm, middle mixed liquor body II is centrifuged processing by time 28-32min, takes supernatant
Liquid, extract supernatant through excess pressure be 65.55-69.55KPa, temperature be 110.6-119.6 DEG C and time 8-12min
Disinfect dress and obtain middle mixing liquid III, centre mixing liquid III-18 to-22 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 0.8-1.2%, regulate PEI by NaOH weak solution
The pH value of solution is 4-6, add in the middle of mixing liquid III make PEI concentration be set to 0.4-0.6% to obtain people's amniotic membrane-PEI system
Mixed liquor, lyophilization.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree, more than 90% and chitosan powder that weight is 0.18-0.22g is dissolved into the 28-32ml that concentration is 0.5M
Acetum in, to being completely dissolved by the way of magnetic agitation, be then settled to 88-102ml with acetum, obtain dense
Degree is the chitosan solution of 2mg/ml, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in
In the autoclave of 120-122 DEG C sterilize 9-11 minute, subject to sterilization after chitosan solution temperature be down to room temperature after put into 3-5 DEG C of ice
Case preserves；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 54-58%, is removed by the membrane filtration of 0.22 μm
Obtain GP solution after bacterium, be 3-5 DEG C of preservation in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 18-22:1
Ratio add in sterile beaker, beaker is put into the supersound process carried out 0.9-1.1 hour on ice and obtains chitosan people's amniotic membrane
Solution, is put into the chitosan people's amniotic membrane solution in beaker and is stirred on ice and (b) step in second step is obtained
To GP solution be the most progressively added dropwise in chitosan people's amniotic membrane solution, during instillation observe chitosan people's amniotic membrane solution be
The obvious flocculent substance of no existence occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixed
The ultimate density closing the GP in liquid IV is 580-620mg/ml and obtains to 6.9-7.2 with saturated sodium dihydrogen phosphate regulation pH value
To chitosan/people's amniotic membrane-PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to chitosan people's amniotic membrane
The process of solution.
4th embodiment, the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 2.0cm × 3.0cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.25 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 4000rpm, time 30min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 67.55KPa, temperature is 114.6 DEG C and time 10min obtains middle mixing liquid III, in
Between mix liquid III-20 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1.0%, regulate PEI solution by NaOH weak solution
PH value be 5, add in the middle of mixing liquid III makes PEI concentration be set to 0.5% mixed liquor obtaining people's amniotic membrane-PEI system, cold
Lyophilizing is dry.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree 93% and chitosan powder that weight is 0.20g are dissolved into the acetum of the 30ml that concentration is 0.5M
In, to being completely dissolved by the way of magnetic agitation, then it is settled to 92ml with acetum, obtains the shell that concentration is 2mg/ml
Polysaccharide solution, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in the autoclave of 121 DEG C
Sterilize 10 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 4 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 56%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 4 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by the ratio of 20:1
Example adds in sterile beaker, beaker is put into the supersound process carried out on ice 1.0 hours and obtains chitosan people's amniotic membrane solution, burning
Chitosan people's amniotic membrane solution in Bei is put into and is stirred on ice and the GP solution that (b) step in second step is obtained
Slowly progressively it is added dropwise in chitosan people's amniotic membrane solution, during instillation, observes whether chitosan people's amniotic membrane solution exists substantially
Flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes in middle mixing liquid IV
The ultimate density of GP be 600mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 7.0 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
5th embodiment, the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 2.2cm × 3.2cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.24 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 4100rpm, time 31min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 68.0KPa, temperature is 111.0 DEG C and time 9min obtains middle mixing liquid III, middle
Mixing liquid III is-21.5 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1.1%, regulate PEI solution by NaOH weak solution
PH value be 5.8, add in the middle of mixing liquid III makes PEI concentration be set to 0.48% mixed liquor obtaining people's amniotic membrane-PEI system,
Lyophilization.
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree 95% and chitosan powder that weight is 0.19g are dissolved into the acetum of the 28ml that concentration is 0.5M
In, to being completely dissolved by the way of magnetic agitation, then it is settled to 99ml with acetum, obtains the shell that concentration is 2mg/ml
Polysaccharide solution, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in the autoclave of 120 DEG C
Sterilize 9 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 4.5 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 55%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 4.5 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 21.4:1's
Ratio adds in sterile beaker, beaker is put into the supersound process carried out on ice 0.9 hour and obtains chitosan people's amniotic membrane solution,
Chitosan people's amniotic membrane solution in beaker is put into and is stirred on ice and molten for the GP that (b) step in second step is obtained
Liquid is the most progressively added dropwise in chitosan people's amniotic membrane solution, observes whether chitosan people's amniotic membrane solution exists bright during instillation
Aobvious flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixing liquid IV
In the ultimate density of GP be 589mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 7.0 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
A kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair the assay method of gelling performance,
First embodiment, the steps include:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 35 DEG C of water-baths, every 0.8
Minute be inverted observe mobility, take inversion 28 seconds chitosan/people the amniotic membrane-PEI/GP system mixed liquor immobilising time be designated as coagulating
The glue time.
Anastrophe is used to measure its gel time: gel time is defined as being changed into the time needed for gel by colloidal sol, when
System is defined as dissolved colloidal state when being in working fluid state, is defined as gel state when system is in and does not flows semisolid.Result
Showing with this understanding, the final gelling temp of material of the present invention can shorten to 2.5 minutes.
The detection of the biocompatibility of chitosan/people's amniotic membrane-PEI/GP system mixed liquor:
Take healthy adult SD rat 12, with the pentobarbital sodium intraperitoneal injection of anesthesia of 2%, at back, rat spine both sides skin
Under, spaced for 1.0cm, the chitosan disinfected/people amniotic membrane-PEI/GP system mixed liquor 1.0mL is injected separately into rat
Subcutaneous.At room temperature feed.
Within postoperative 2,4,6,8,12 weeks, injection site cuts, and observes the relation of implant and surrounding tissue.
After result of the test shows to apply the new bio matrix gel material of the present invention to carry out skin grafing and mending 12 weeks, body
Formula microscope and om observation, implant is tightly combined with autologous tissue, shows that cartilage repairing scaffold material has good life
The thing compatibility, is the material of a kind of effective treatment articular cartilage defect.
Concrete detection data refer to table 1
Biocompatibility after table 1 crosslinking
| Time | Inflammatory reaction | Fibrosis | Hemorrhagic necrosis | Lymphocyte number | Neutrophilic granulocyte number | Phagocyte number |
| 2 weeks | Nothing | Gently | Nothing | 53 | 62 | 3 |
| 4 weeks | Weak | Gently | Nothing | ～200 | Nothing | 12 |
| 6 weeks | Nothing | Gently | Nothing | Nothing | Nothing | Nothing |
| 8 weeks | Nothing | Gently | Nothing | Nothing | Nothing | 2 |
| 12 weeks | Nothing | Gently | Nothing | Nothing | Nothing | 3 |
The interior animal experiment of chitosan/people's amniotic membrane-PEI/GP system mixed liquor:
The chitosan of invention/people amniotic membrane-PEI/GP system mixed liquor 0.2g is dissolved in 5ml normal saline solution, fully mixes,
10 minutes are stood at 4 DEG C.After Umbilical cord blood mesenchymal stem cells counting, add 3 × 106Cell fully mixes.Matched group injection glass
Glass acid sodium.
Take Adult New Zealand White Rabbit, under aseptic condition, on new zealand white rabbit right hindlimb patellofemoral joint face, use ophthalmology
Trepan gets out the cartilage defect of the deep about 2mm of diameter about 3.0mm, and layering is sewed up a wound and made cartilage injury's model.Postoperative 5d is every
The anti-infection of intramuscular injection antibiotic.Postoperative 4 weeks row arthrocentesiSs, it was demonstrated that after thrusting articular cavity, inject 0.5ml chitosan/people
Amniotic membrane-PEI/GP system mixed liquor composite, is repeated 1 times for 4 weeks.Matched group intraarticular injection 0.5ml hyaluronic acid sodium is combined
Thing.
Placing heat riser (such as heat lamp) above damaged part at 10-15cm makes the temperature of defect area keep 37
Under the conditions of DEG C 6 minutes.Within postoperative 12 weeks, put to death rabbit to draw materials, carry out gross examination of skeletal muscle and histological observation.
Gross examination of skeletal muscle is visible: test group articular surface is more smooth, and repair tissue and surrounding cartilage are integrated good, and color and luster is close；
Blank group: cave at defect, sharpness of border, there is a small amount of reparation at defect edge.
Histological observation is visible: test group is hyaline cartilage sample reparation, repair tissue surfacing, integrates with cartilage around
Closely, thickness close, cell density is slightly larger；Blank group: gained repair tissue is fibroid tissue, and surface is crude, cell
Epimatrix arrangement disorder.
Concrete results of comparison such as table 2
Table 2. cartilage defect repair effectiveness comparison table
| Synergy | Cartilage state around | Reparation degree | Matrix fiber arranges | |
| Matched group | Difference, interface is uneven | Without significant degradation | Central part little area reparation | Arrangement disorder |
| Test group | Well | Without degenerating | All repair | Similar normal cartilage |
Second embodiment, the steps include:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 39 DEG C of water-baths, every 1.2
Minute be inverted observe mobility, take inversion 32 seconds chitosan/people the amniotic membrane-PEI/GP system mixed liquor immobilising time be designated as coagulating
The glue time.
3rd embodiment, the steps include:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 35-39 DEG C of water-bath, every
Within 0.8-1.2 minute, it is inverted and observes mobility, take inversion 28-32 second chitosan/people's amniotic membrane-PEI/GP system mixed liquor immobilising
Time is designated as gel time.
4th embodiment, the steps include:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 37 DEG C of water-baths, every 1.0
Minute be inverted observe mobility, take inversion 30 seconds chitosan/people the amniotic membrane-PEI/GP system mixed liquor immobilising time be designated as coagulating
The glue time.
The application of a kind of bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair, chitosan/people amniotic membrane-PEI/
Inject the cartilage defect of articular cavity after GP system mixed liquor mixing umbilical cord blood mesenchymal stem cells, heated by heat lamp, will plant
Daughter cell is fixed on performance Regeneration and Repair effect at defect, makes colloidal sol one cell complexes undergo phase transition and obtains bio-matrix gel
Material.
Bio-matrix gel rubber material has the advantages such as easy to use, simple to operate, therapeutic process not damaged, determined curative effect.
A kind of enforcement of the alternate manner of the preparation method of the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair
Example:
The preparation of people's amniotic membrane-PEI system
Prepared by full solution-off cell people's amniotic membrane: after obtaining the informed consent of cesarean patient, it is thus achieved that its postcesarean people's amniotic membrane, uses
PBS removes bloodstain, and repeatedly cleans.People's amniotic membrane is cut into 2cm × 3cm, shakes after adding 0.02% EDTA solution.Centrifugal
After abandon supernatant, slowly shake in 90 DEG C of water after adding 0.25 mol acetum, until being completely dissolved.Due to viscosity relatively
Greatly, it is difficult to Entkeimung.Having processed in acetic acid, 4000rpm is centrifuged 30min and draws supernatant, through 67.55KPa(115.6 degree)
Subpackage after autoclaving 10min ,-20 DEG C of preservations.
The preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1%, during with NaOH weak solution regulation pH value 4-6
Add a certain amount of complete molten people's amniotic membrane solution to make PEI concentration be maintained at 0.5% i.e. to obtain mixed system, lyophilization.
The preparation of embodiment 1 chitosan/people's amniotic membrane-PEI/GP system
1) preparation of chitosan solution and sterilization
Balance accurately weigh 0.2g take off second phthalein degree more than 90% chitosan powder be scattered in the acetum of 30ml 0.5mol/L,
Magnetic agitation, to being completely dissolved, is then settled to 100ml with acetum, obtains 2mg/ml chitosan solution, room temperature preservation.Will
The chitosan solution kraft paper sealing stirred is placed in the autoclave sterilization pot of 121 DEG C sterilizes 10 minutes, subject to sterilization after
Chitosan solution temperature be down to room temperature after to put into 4 DEG C of Refrigerator stores standby.
2) preparation of phosphoglycerol disodium (GP) and sterilization
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 56%, and the membrane filtration of 0.22 μm is degerming, 4 DEG C of guarantors
Deposit.
3) prepared by chitosan/people amniotic membrane-PEI/GP
In super-clean bench, measure the aseptic chitosan solution prepared with the people's amniotic membrane-PEI solution prepared in ratio add nothing
In bacterium beaker, standby in the most ultrasonic 1 hour.Stir on ice, the aseptic GP solution previously prepared the most progressively is dripped
Enter in the chitosan people's amniotic membrane solution prepared, observe solution during instillation and occur without obvious flocculent substance, otherwise regard
For failure.In the chitosan obtained/people's amniotic membrane-PEI/GP system, the ultimate density of GP is 600mg/ml, uses saturated biphosphate
Sodium regulation pH value is between 6.9-7.2.
Embodiment 2 chitosans/people's amniotic membrane-PEI/GP system gelling performance measures
Anastrophe is used to measure its gel time: gel time is defined as being changed into the time needed for gel by colloidal sol, works as system
It is defined as dissolved colloidal state when being in working fluid state, is defined as gel state when system is in and does not flows semisolid.Take 2.0ml molten
Glue is put in ampere bottle, is placed in 37 DEG C of water-baths, was inverted every 1 minute and observes mobility, takes inversion time within 30 seconds, colloidal sol is immobilising
Between be designated as gel time.Result shows with this understanding, and the final gelling temp of material of the present invention can shorten to 2.5 minutes.
The detection of embodiment 3 biocompatibility
1) healthy adult SD rat 12 is taken, with the pentobarbital sodium intraperitoneal injection of anesthesia of 2%, at back, rat spine both sides
Subcutaneous, spaced for 1.0cm, the gel 1.0mL disinfected is injected separately into subcutaneous rat.At room temperature feed.
2) postoperative 2,4,6,8,12 weeks injection sites cut, and observe the relation of implant and surrounding tissue.
After result of the test shows to apply the new bio matrix gel material of the present invention to carry out skin grafing and mending 12 weeks, body
Formula microscope and om observation, implant is tightly combined with autologous tissue, shows that cartilage repairing scaffold material has good life
The thing compatibility, is the material of a kind of effective treatment articular cartilage defect.
Concrete detection data refer to table 1
Biocompatibility after table 1 crosslinking
| Time | Inflammatory reaction | Fibrosis | Hemorrhagic necrosis | Lymphocyte number | Neutrophilic granulocyte number | Phagocyte number |
| 2 weeks | Nothing | Gently | Nothing | 53 | 62 | 3 |
| 4 weeks | Weak | Gently | Nothing | ～200 | Nothing | 12 |
| 6 weeks | Nothing | Gently | Nothing | Nothing | Nothing | Nothing |
| 8 weeks | Nothing | Gently | Nothing | Nothing | Nothing | 2 |
| 12 weeks | Nothing | Gently | Nothing | Nothing | Nothing | 3 |
Embodiment 5. interior animal experiment
1) the gel 0.2g of invention is dissolved in 5ml normal saline solution, fully mix, at 4 DEG C, stand 10 minutes.Between people's umbilical blood
After mesenchymal stem cells counting, add 3 × 106Cell fully mixes.Matched group injection hyaluronic acid sodium.
2) take Adult New Zealand White Rabbit, under aseptic condition, on new zealand white rabbit right hindlimb patellofemoral joint face, use eye
Section's trepan gets out the cartilage defect of the deep about 2mm of diameter about 3.0mm, and layering is sewed up a wound and made cartilage injury's model.Postoperative 5d
Every anti-infection of intramuscular injection antibiotic.Postoperative 4 weeks row arthrocentesiSs, it was demonstrated that after thrusting articular cavity, inject 0.5ml composite wood
Material, is repeated 1 times for 4 weeks.Matched group intraarticular injection 0.5ml hyaluronic acid sodium complex.
3) placing heat riser (such as heat lamp) above damaged part at 10-15cm makes the temperature of defect area keep 37 DEG C
Under the conditions of 6 minutes.Within postoperative 12 weeks, put to death rabbit to draw materials, carry out gross examination of skeletal muscle and histological observation.
Gross examination of skeletal muscle is visible: test group articular surface is more smooth, and repair tissue and surrounding cartilage are integrated good, and color and luster is close；
Blank group: cave at defect, sharpness of border, there is a small amount of reparation at defect edge.
Histological observation is visible: test group is hyaline cartilage sample reparation, repair tissue surfacing, integrates with cartilage around
Closely, thickness close, cell density is slightly larger；Blank group: gained repair tissue is fibroid tissue, and surface is crude, cell
Epimatrix arrangement disorder.
Concrete results of comparison such as table 2
Table 2. cartilage defect repair effectiveness comparison table
| Synergy | Cartilage state around | Reparation degree | Matrix fiber arranges | |
| Matched group | Difference, interface is uneven | Without significant degradation | Central part little area reparation | Arrangement disorder |
| Test group | Well | Without degenerating | All repair | Similar normal cartilage |
The injectable cartilage regeneration biological host material mixing umbilical cord blood mesenchymal stem cells utilizing the present invention to prepare passes through articular cavity
Inject cartilage defect, heated by heat lamp and make colloidal sol one cell complexes undergo phase transition, thus seed cell is fixed on
Playing Regeneration and Repair effect at defect, this material has easy to use, simple to operate, therapeutic process not damaged, determined curative effect etc.
Advantage
The present invention has a lower feature:
1, owing to devising the mixed liquor of people's amniotic membrane-PEI system, chitosan solution and GP solution, by chitosan solution and GP
In the gel that solution is formed, maintain the activity of cell in the mixed liquor of people's amniotic membrane-PEI system, promote regenerating bone or cartilage and repair
Multiple, no longer carry out prosthetic replacement and treat, therefore improve cartilage injury and be in progress the severe osteoarthrosis formed further
Scorching non-operative treatment effect.
2, owing to devising chitosan solution and GP solution, the gel of formation does not produces physiological stimulation to patient.
3, owing to devising the mixed liquor of people's amniotic membrane-PEI system, the activity of the regenerative cell of holding is good.
4, owing to devising acetic acid, the infringement to people's amniotic membrane is little.
5, owing to devising NaOH weak solution and saturated sodium dihydrogen phosphate, carry out substep and carry out pH value regulation, meet system
The requirement of standby technique, improves the activity of bio-matrix gel rubber material.
6, planform has been carried out the restriction of numerical range owing to devising, the technical side making numerical range be the present invention
Technical characteristic in case, calculates or is tested by limited number of time the technical characteristic drawn not by formula, and test shows this numerical value
The technical characteristic of scope achieves good technique effect.
7, owing to devising the technical characteristic of the present invention, in the effect of the set individually and each other of technical characteristic,
Being shown by test, the property indices of the present invention is at least 1.7 times of existing property indices, is had by assessment
There is good market value.
Also have other with the mixed liquor of people's amniotic membrane-PEI system, chitosan solution and GP solution same or like as
Technical characteristic is all one of embodiments of the invention, and each technical characteristic of embodiment described above can carry out arbitrary group
Close, for meeting the requirement of Patent Law, patent regulation and guidelines for examination, no longer to each technical characteristic in above-described embodiment
The embodiment of all possible combination is all described.
Above-described embodiment is the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair provided by the present invention
A kind of way of realization of preparation method, gelling performance assay method and application, other according to scheme provided by the present invention become
Shape, increases composition therein or step, or the present invention is used for other the technical field close with the present invention, belongs to this
The protection domain of invention.
Claims (6)
1. it is used for a preparation method for the bio-matrix gel rubber material promoting regenerating bone or cartilage to repair, it is characterized in that: its step:
The mixed liquor of people's amniotic membrane-PEI system carries out reconciliation process with addition GP solution in the mixed liquor of chitosan solution, obtains as life
The chitosan of the matrix of thing matrix gel material/people's amniotic membrane-PEI/GP system mixed liquor.
The preparation method of the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair the most according to claim 1, its
Feature is: pass through GP solution according to the mixed liquor of people's amniotic membrane-PEI system with the combined hybrid liquid of the mixed liquor of chitosan solution
Reaction to obtain final bio-matrix gel rubber material be technical essential.
The preparation method of the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair the most according to claim 1, its
Feature is: the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 1.8-2.2cm × 2.8-3.2cm people's amniotic membrane, is mixed by shaking mode after adding 0.02% EDTA solution
Conjunction obtains middle mixing liquid I, by centrifugation, centre mixing liquid is separated, and after reject supernatant, adds 0.23-
0.27 mol acetum, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, sets
Parameter is rotating speed 3800-4200rpm, middle mixed liquor body II is centrifuged processing by time 28-32min, takes supernatant
Liquid, extract supernatant through excess pressure be 65.55-69.55KPa, temperature be 110.6-119.6 DEG C and time 8-12min
Disinfect dress and obtain middle mixing liquid III, centre mixing liquid III-18 to-22 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 0.8-1.2%, regulate PEI by NaOH weak solution
The pH value of solution is 4-6, add in the middle of mixing liquid III make PEI concentration be set to 0.4-0.6% to obtain people's amniotic membrane-PEI system
Mixed liquor, lyophilization,
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree, more than 90% and chitosan powder that weight is 0.18-0.22g is dissolved into the 28-32ml that concentration is 0.5M
Acetum in, to being completely dissolved by the way of magnetic agitation, be then settled to 88-102ml with acetum, obtain dense
Degree is the chitosan solution of 2mg/ml, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in
In the autoclave of 120-122 DEG C sterilize 9-11 minute, subject to sterilization after chitosan solution temperature be down to room temperature after put into 3-5 DEG C of ice
Case preserves；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 54-58%, is removed by the membrane filtration of 0.22 μm
Obtain GP solution after bacterium, be 3-5 DEG C of preservation in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 18-22:1
Ratio add in sterile beaker, beaker is put into the supersound process carried out 0.9-1.1 hour on ice and obtains chitosan people's amniotic membrane
Solution, is put into the chitosan people's amniotic membrane solution in beaker and is stirred on ice and (b) step in second step is obtained
To GP solution be the most progressively added dropwise in chitosan people's amniotic membrane solution, during instillation observe chitosan people's amniotic membrane solution be
The obvious flocculent substance of no existence occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixed
The ultimate density closing the GP in liquid IV is 580-620mg/ml and obtains to 6.9-7.2 with saturated sodium dihydrogen phosphate regulation pH value
To chitosan/people's amniotic membrane-PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to chitosan people's amniotic membrane
The process of solution.
The preparation method of the bio-matrix gel rubber material for promoting regenerating bone or cartilage to repair the most according to claim 3, its
Feature is: the steps include:
First step, the preparation of people's amniotic membrane-PEI system:
A prepared by (), full solution-off cell people's amniotic membrane: people amniotic membrane PBS is carried out bloodstain removing process, then carries out instead with normal saline
Multiple cleaning, is cut into 2.2cm × 3.2cm people's amniotic membrane, in being mixed to get by shaking mode after adding 0.02% EDTA solution
Between mix liquid I, by centrifugation, centre mixing liquid is separated, after reject supernatant, adds 0.24 mol acetic acid
Solution, slowly shakes in the water that temperature is 90 DEG C, until being completely dissolved middle mixing liquid, setup parameter is rotating speed
Middle mixed liquor body II is centrifuged processing by 4100rpm, time 31min, takes supernatant, the supernatant warp extracted
The dress of disinfecting that excess pressure is 68.0KPa, temperature is 111.0 DEG C and time 9min obtains middle mixing liquid III, middle
Mixing liquid III is-21.5 DEG C of preservations；
B (), the preparation of people's amniotic membrane-PEI system: PEI solution is diluted to concentration 1.1%, regulate PEI solution by NaOH weak solution
PH value be 5.8, add in the middle of mixing liquid III makes PEI concentration be set to 0.48% mixed liquor obtaining people's amniotic membrane-PEI system,
Lyophilization,
The preparation of second step, chitosan/people's amniotic membrane-PEI/GP system:
The preparation of (a), chitosan solution
De-second phthalein degree 95% and chitosan powder that weight is 0.19g are dissolved into the acetum of the 28ml that concentration is 0.5M
In, to being completely dissolved by the way of magnetic agitation, then it is settled to 99ml with acetum, obtains the shell that concentration is 2mg/ml
Polysaccharide solution, preserves in room temperature, the chitosan solution kraft paper stirred sealing is placed in the autoclave of 120 DEG C
Sterilize 9 minutes, subject to sterilization after chitosan solution temperature be down to room temperature after put into 4.5 DEG C of Refrigerator stores；
(b), the preparation of phosphoglycerol disodium i.e. GP solution
GP powder is dissolved in tri-distilled water, it is thus achieved that mass concentration is the solution of 55%, by the membrane filtration of 0.22 μm degerming after
Obtain GP solution, be 4.5 DEG C of preservations in temperature；
Prepared by (c), chitosan/people amniotic membrane-PEI/GP
In super-clean bench, the people's amniotic membrane-PEI solution measuring the aseptic chitosan solution prepared with preparing, second step
In the chitosan solution that obtains of (a) step and first step in the mixed liquor of people's amniotic membrane-PEI system that obtains by 21.4:1's
Ratio adds in sterile beaker, beaker is put into the supersound process carried out on ice 0.9 hour and obtains chitosan people's amniotic membrane solution,
Chitosan people's amniotic membrane solution in beaker is put into and is stirred on ice and molten for the GP that (b) step in second step is obtained
Liquid is the most progressively added dropwise in chitosan people's amniotic membrane solution, observes whether chitosan people's amniotic membrane solution exists bright during instillation
Aobvious flocculent substance occurs and mixing liquid IV in the middle of not having obvious flocculent substance to occur to obtain, and makes middle mixing liquid IV
In the ultimate density of GP be 589mg/ml and with saturated sodium dihydrogen phosphate regulation pH value to 7.0 obtain chitosan/people's amniotic membrane-
PEI/GP system mixed liquor, when flocculent substance occurs that just terminating GP solution is added dropwise to the process of chitosan people's amniotic membrane solution.
5. it is used for an assay method for the gelling performance of the bio-matrix gel rubber material promoting regenerating bone or cartilage to repair, its step
It is:
Take 1.82.2ml chitosan/people's amniotic membrane-PEI/GP system mixed liquor to be put in ampere bottle, be placed in 35-39 DEG C of water-bath, every
Within 0.8-1.2 minute, it is inverted and observes mobility, take inversion 28-32 second chitosan/people's amniotic membrane-PEI/GP system mixed liquor immobilising
Time is designated as gel time.
6. it is used for an application for the bio-matrix gel rubber material promoting regenerating bone or cartilage to repair, chitosan/people's amniotic membrane-PEI/GP body
It is the cartilage defect injecting articular cavity after mixed liquor mixing umbilical cord blood mesenchymal stem cells, is heated by heat lamp, by seed cell
It is fixed on performance Regeneration and Repair effect at defect, makes colloidal sol one cell complexes undergo phase transition and obtain bio-matrix gel rubber material.
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| 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 |
| CN113707247A (en) * | 2020-09-02 | 2021-11-26 | 成都域时信息科技有限公司 | Method for quantitatively predicting cartilage repair rate after mesenchymal stem cell therapy based on machine learning of block chain |
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| 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 |
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| CN113707247A (en) * | 2020-09-02 | 2021-11-26 | 成都域时信息科技有限公司 | Method for quantitatively predicting cartilage repair rate after mesenchymal stem cell therapy based on machine learning of block chain |
| CN113707247B (en) * | 2020-09-02 | 2023-09-22 | 上海深鱼赛跑科技有限公司 | Method for quantitatively predicting cartilage repair rate after mesenchymal stem cell therapy based on machine learning of blockchain |
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