CN108159499A - A kind of plural gel and its preparation method and application - Google Patents
A kind of plural gel and its preparation method and application Download PDFInfo
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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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
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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
- A61L27/50—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
-
- 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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention discloses a kind of plural gel and its preparation method and application, the plural gel includes enrichment mesenchymal stem cell and collagen-based materials, and enrichment mesenchymal stem cell with collagen-based materials is mixed and dissolves to obtain the plural gel.The preparation method includes the following steps：It is dissolved by the pretreatment of collagen hydrogel raw material and using acid solution, and adjusts using aqueous slkali that its pH obtains liquid or the hydrogel of semisolid is spare；By the BMSCs of acquisition under 4 DEG C of environment with gained hydrogel according to abundant mixing to get the plural gel product.The plural gel can be applied to the reparation of patient's meniscus tear.
Description
Technical field
The invention belongs to bio-medical material or Biocomposite material field more particularly to a kind of plural gel and its systems
Preparation Method and application.
Background technology
The incidence of meniscus injury is higher and higher, and meniscus is mainly made of fibrocartilage, and clinically it is lacked
Effective therapy, and meniscus plays extremely important undertake in kneed continuous movement and transmits load, buffers and bear
The effects that lotus, stabilization and lubricating joint, but 2/3rds region of meniscus is no blood supply area, and this results in meniscus
The place of no blood supply once be damaged cannot self-healing, traditional treatment means are often to the meniscus of damage
The means such as cut off, corrected and shaped, although the slight illness of releasing patient that can be of short duration, the corresponding function of meniscus also with
And disappear, and eventually lead to the generation of knee joint endoprosthesis inflammation.At present, mainly there is artificial prosthesis for the method for meniscal repairs
Implantation repair, three kinds of methods of allograft and organizational project, but above-mentioned three kinds of methods there are it is certain the defects of, effect
Difference, and easily fail after a period of time is undergone.
Mesenchymal stem cell (BMSCs) is bone marrow stroma stem cell, and BMSCs initially has found in marrow, because of its tool
There is the features such as multi-lineage potential, hematopoiesis support and promotion stem cell implantation, immunoregulation and self-replacation and be increasingly subject to people
Concern.BMSCs is in vivo or in vitro under specific inductive condition, can be divided into fat, bone, cartilage, muscle, tendon, tough
The Various Tissues cells such as band, nerve, liver, cardiac muscle, endothelium are still dived with Multidirectional Differentiation after continuous passage culture and freezen protective
Can, there can be potentially clinical answer as ideal seed cell for injuries of tissues and organs reparation caused by aging and lesion
With value.
The paper that Qin Jing, Zhao Zhenlin were delivered in 2010 on Immunology Today magazine about《Mesenchymal stem cell
Immunogenic characteristics and its immunoregulation capability》Middle confirmation mesenchymal stem cell has extremely low immunogenicity, no
Allosome rejection can occur, undifferentiated mesenchymal stem cell non-immunogenicity breaks up between the marrow of skeletonization or cartilage
The immunogenicity of mesenchymal stem cells is still relatively low.Lei Rong, sieve dare to delivering on Chinese clinician's magazine (electronic edition) for 2013
Paper《Bone Marrow Mesenchymal Stem Cells Transplantation enters the progress of survival rate after heart》Middle confirmation mesenchymal stem cell exists
Generation immunological rejection is showed no in allogeneic and heterograft.
Invention content
In order to solve the above-mentioned technical problem, one of the objects of the present invention is to provide it is a kind of prepare conveniently, it is easy to use and
The plural gel of the meniscal repairs of good, the curative effect period length to meniscus tear repairing effect.
One of the objects of the present invention is to provide a kind of preparation method of above-mentioned meniscus complex repairation gel, method letters
It is single, of low cost.
To achieve the above object, another technical solution of the invention is：A kind of preparation method of plural gel, the method
Include the following steps,
(1) spongiform collagen-based materials are torn up, and is sterilized using physical method；
(2) the acid solution acetic acid of 0.5M will be used with 10-20mg/mL by obtained collagen-based materials after step (1) processing
Concentration dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that it is all dissolved, and aqueous slkali is used by its PH at 4 DEG C
Value is adjusted to 7-7.2, and the hydrogel for obtaining liquid or semisolid is spare；
(4) it is enriched with to obtain enrichment medulla mesenchyma to human marrow mesenchymal stem cell using cell enrichment technology to do carefully
Born of the same parents are with spare；Wherein human marrow mesenchymal stem cell from directly buying on the market；
(5) under 4 DEG C of environment by hydrogel obtained by step (3) according to 15-25：1 volume ratio is added drop-wise to obtained by step (4)
Enrichment mesenchymal stem cell above, by its abundant mixing to get the plural gel product.
The advantageous effect of above-mentioned technical proposal is：Its method is simple, and preparation condition is mild, the plural gel of generation
Material solidification effect is good, can be good at carrying out good combination at the defect with meniscus.
Method for disinfection in step described in above-mentioned technical proposal (1) is sterilized using ultraviolet light irradiation.
The advantageous effect of above-mentioned technical proposal is：Its bactericidal effect is good, and collagen-based materials will not be generated any bad
It influences.
Acid solution described in above-mentioned technical proposal is the acetum of a concentration of 0.5M.
The advantageous effect of above-mentioned technical proposal is：The acetum is acid mild and collagen hydrogel will not be generated
Undesirable influence.
Aqueous slkali described in above-mentioned technical proposal is the NaOH solution of a concentration of 0.5-1.5M.
The advantageous effect of above-mentioned technical proposal is：It is convenient to prepare.
Collagen-based materials described in above-mentioned technical proposal are collagen hydrogel.
The advantageous effect of above-mentioned technical proposal is：Collagen hydrogel has the characteristic of rapid shaping, can be lacked in meniscus
It is solidified at damage, meniscal tissue main component is a kind of collagen, and the collagen hydrogel after solidifying is formed and Cartilage tissue constructs
It is much like, it can be grown for BMSCs and microenvironment is provided, contribute to the reparation of meniscus injury.
The second object of the present invention is to protect one kind by the preparation-obtained plural gel of the above method.
The advantageous effect of above-mentioned technical proposal is：And collagen-based materials can supplement half a month by rapid shaping at meniscus defect
At plate defect, the BMSCs of enrichment is solved when being treated with autogenous bone marrow blood merely, because of BMSCs in unit volume marrow blood
Amount reduces curative effect less.Simultaneously using enrichment BMSCs avoid adopt Stem cells cultured in vitro amplification patient need it is longer
The disadvantage of stand-by period；And avoid the risk of the various microbial contaminations of in vitro culture.Therefore, the BMSCs of high concentration is enriched with, then
A kind of new approach is likely to provide, promotes cell therapy in clinical application.
The third object of the present invention is to provide one kind by above-mentioned plural gel or the obtained plural gel of the above method
Application in the reparation of patient's meniscus tear.
Description of the drawings
Fig. 1 is each experimental group modeling meniscus HE coloration results of experiment in vivo of the present invention；
Fig. 2 is each experimental group sarranine-O coloration results of experiment in vitro of the present invention；
Fig. 3 is each experimental group modeling meniscus HE coloration results of experiment in vivo of the present invention；
Fig. 4 is each experimental group modeling meniscus sarranine-O coloration results of experiment in vivo of the present invention.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
An embodiment of the present invention provides a kind of preparation methods of meniscal repairs plural gel, and the method includes walking as follows
Suddenly,
(1) spongiform solid state collagen material is sterilized using the method for ultraviolet light irradiation；
(2) acetum of a concentration of 0.5M will be used with 10- by obtained collagen-based materials after step (1) processing
The concentration of 20mg/mL is dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that it is all dissolved, and in 4 DEG C of a concentration of 0.5- of use
For the NaOH solution of 1.5M by its pH value adjustment to 7-7.2, the hydrogel for obtaining liquid or semisolid is spare；
(4) above-mentioned human marrow mesenchymal stem cell purchased in market is enriched with using cell enrichment technology to obtain enrichment marrow
Mescenchymal stem cell is with spare；
(5) under 4 DEG C of environment by hydrogel obtained by step (3) according to 15-25：1 volume ratio is added drop-wise to obtained by step (4)
Enrichment mesenchymal stem cell above, by its abundant mixing to get the plural gel product.
Wherein, the collagen-based materials are collagen hydrogel, it is preferred that the collagen-based materials are the collagen water of calf-skin extraction
Gel.
Heretofore described human marrow mesenchymal stem cell from directly buying on the market；For example, purchased from plug industry biotechnology
The mesenchymal stem cells in adult human with combined density gradient centrifugation of Co., Ltd, article No. HUXMA-01001 or Beijing Ke Ruisibo biotechnologies are limited
The human marrow mesenchymal stem cell of company, product identification are CE9987 etc..In order to better illustrate the technology of present invention effect
Fruit, in present invention below embodiment by taking the mesenchymal stem cells in adult human with combined density gradient centrifugation of Sai Ye bio tech ltd as an example；It needs
Illustrate, the human marrow mesenchymal stem cell in other sources can also realize identical technique effect.
Wherein, the complete medium of mesenchymal stem cell described in each embodiment is also from directly buying on the market, with
To be purchased from the mesenchymal stem cells in adult human with combined density gradient centrifugation complete medium of Sai Ye bio tech ltd in the lower embodiment of the present invention
For.
Embodiment 1
Wherein collagen-based materials are mainly carried with the collagen hydrogel of calf-skin extraction, the collagen hydrogel of pigskin extraction and horse skin
The collagen hydrogel taken is tested；
Acid solution is mainly tested using hydrochloric acid solution, phosphoric acid solution and acetum；
Aqueous slkali mainly uses NaOH solution or KOH solution；
Pass through the collagen hydrogel, three kinds of different acid solutions and two different alkali solubles of above-mentioned three kinds of different Animal Skins
Liquid random combine prepares hydrogel in accordance with the following steps,
(1) spongiform collagen hydrogel is torn up, and is sterilized using the method for ultraviolet irradiation；
(2) 0.5M acetums will be used with 10-20mg/mL's by obtained collagen hydrogel after step (1) processing
Concentration is dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that it is all dissolved, and aqueous slkali is used by its PH at 4 DEG C
Value is adjusted to 7-7.2, and the hydrogel for obtaining liquid or semisolid is spare；18 kinds of different hydrogels are respectively obtained, pass through comparison
It was found that collagen hydrogel, acetic acid and the NaOH of calf-skin extraction are obtained respectively as collagen-based materials, acid solution and aqueous slkali
Hydrogel forming effect is best.
Embodiment 2
Experiment in vitro：Experiment is divided into four groups：1) BMSCs beads (about 107A cell/every)；2) it is enriched with BMSCs
(2mL)；3) BMSCs (about 107A cell) mixing collagen hydrogel；4) enrichment BMSCs (2mL) mixes collagen hydrogel.To
Respectively according to 1 in above-mentioned four groups of experiments：50 volume adds in mesenchymal stem cells in adult human with combined density gradient centrifugation complete medium, in 5vol%
CO2, cultivate respectively 7 days, 14 days, 21 days under 37 DEG C of environment；Compare the expression of fibrocartilage related gene and albumen；To sample
The detection of this progress cell Proliferation, HE dyeing, sarranine-O dyeing, RT-PCR detections fibrocartilage related gene (COL1A1,
COL2A1, COLX, SOX9, Aggrecan), WB detections fibrocartilage GAP-associated protein GAP (COL1A1, COL2A1, COLX, SOX9,
Aggrecan)。
Embodiment 3
The preparation method of BMSCs and enrichment BMSCs mixing collagen hydrogels is as follows wherein in embodiment 2：
(1) spongiform collagen hydrogel is sterilized by the method that ultraviolet light irradiates；
(2) acetum of a concentration of 0.5M will be used with 15mg/ by obtained collagen hydrogel after step (1) processing
The concentration of mL is dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that its is all dissolved, and 4 DEG C of a concentration of 1M's of use
For NaOH solution by its pH value adjustment to 7.0, the hydrogel for obtaining liquid or semisolid is spare；
(4) under 4 DEG C of environment by hydrogel obtained by step (3) according to 20：1 volume ratio is added drop-wise to BMSCs (about 107
A cell) and enrichment BMSCs (2mL) is above.
Embodiment 4
The preparation method of BMSCs and enrichment BMSCs mixing collagen hydrogels is as follows wherein in embodiment 2：
(1) spongiform solid collagen hydrogel is sterilized by the method that ultraviolet light irradiates；
(2) acetum of a concentration of 0.5M will be used with 15mg/ by obtained collagen hydrogel after step (1) processing
The concentration of mL is dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that its is all dissolved, and 4 DEG C of a concentration of 0.2M's of use
For NaOH solution by its pH value adjustment to 7.0, the hydrogel for obtaining liquid or semisolid is spare；
(4) under 4 DEG C of environment by hydrogel obtained by step (3) according to 20：1 volume ratio is added drop-wise to BMSCs (about 107
A cell) and enrichment BMSCs (2mL) is above, and respectively by its abundant mixing to get the plural gel product.
Embodiment 5
The preparation method of BMSCs and enrichment BMSCs mixing collagen hydrogels is as follows wherein in embodiment 2：
(1) spongiform solid state collagen hydrogel is sterilized by the method that ultraviolet light irradiates；
(2) acetum of a concentration of 0.5M will be used with 15mg/ by obtained collagen hydrogel after step (1) processing
The concentration of mL is dissolved；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that its is all dissolved, and 4 DEG C of a concentration of 1M's of use
For NaOH solution by its pH value adjustment to 7.0, the hydrogel for obtaining liquid or semisolid is spare；
(4) under 4 DEG C of environment by hydrogel obtained by step (3) according to 20：1 volume ratio is added drop-wise to BMSCs (about 107
A cell) and enrichment BMSCs (2mL) is above, and respectively by its abundant mixing to get the plural gel product.
Analysis：
Using the embodiment 3-5 BMSCs collagen hydrogels prepared and enrichment BMSCs collagen hydrogels according to embodiment 2
Method is tested, and experimental result is shown, BMSCss collagen hydrogels can preferably be grown, and has higher activity, and energy
Constantly rised in value；HE coloration results show that collagen hydrogel mixing enrichment BMSCss collagen hydrogels have more fibrocartilages to give birth to
Into specifically as shown in Figure 1；Under the action of sarranine-O coloration results display enrichment BMSCss collagen hydrogel mixing collagen hydrogels
More Collagen Type VI tissue can be formed, collagen hydrogel, which can induce the relevant P-glycoprotein expression of cartilage, to be increased, and is specifically shown in Fig. 2.
Animal experiment in vivo：
Operating procedure：The right above-knee meniscus injury model of new zealand white rabbit is made, experiment in vivo is divided into five groups：1)BMSCs
Bead (about 107A cell/every)；2) enrichment BMSCs (2mL)；3) BMSCs (about 107A cell) epoxy glue raw water coagulates
Glue；4) enrichment BMSCs (2mL) mixes collagen hydrogel；5) control group, wherein, 3) BMSCs (about 107A cell) epoxy glue
Raw water gel and 4) enrichment BMSCs (2mL) mix collagen hydrogel and are prepared according to the method for embodiment 3, and control group is according to implementation
The method of example 3 prepares but BMSCs is added without in step (4) and is enriched with the blank group that BMSCs is formed, by grouping by above-mentioned intervention
Object is injected at the meniscus injury of one and a half months dash-board injury model, closes suture articular cavity and the surface of a wound, and injection in continuous three days is anti-
Raw element.It takes out corresponding meniscus postoperative June out of big white mouse body, observes gross specimen, and make HE dyeing, safranin O dyeing.
Experimental result is shown：
1. gross examination of skeletal muscle：Corresponding enrichment BMSCs (2mL) mixes meniscus notch in collagen hydrogel group and has obscured,
There is apparent cambium, repair degree and be significantly better than other groups.
2. HE coloration results are shown：Enrichment BMSCs mixing collagen hydrogel group meniscus defects have more newborn soft
Ostosis is specifically shown in Fig. 3；
3. sarranine-O coloration results are shown：It is enriched with the visible more similar Collagen Type VI group of BMSCs mixing collagen hydrogel groups
It knits, collagen hydrogel, which can induce the relevant P-glycoprotein expression of cartilage, to be increased, and is specifically shown in Fig. 4.
Wherein, overall effect is ordered as enrichment BMSCs (2mL) and mixes collagen hydrogel ＞ BMSCs (about 107A cell/
Every) mixing collagen hydrogel ＞ enrichment BMSCs (2mL) ＞ BMSCs (about 107A cell/every) ＞ blank groups.
Pathological experiment：
The patient of 10 meniscus tears is chosen, is prepared by the method injected at its meniscus tear corresponding to embodiment 3
Enrichment BMSCs (2mL), close the suture articular cavity and surface of a wound, continuous three days injection of antibiotics, then postoperative three in patient
Month, six months and 1 year conditions of patients recovery situation is observed using nuclear magnetic resonance in real time, three months whens, are seen by Nuclear Magnetic Resonance
It examines patient's meniscus to have started to heal, at six months, obscured at meniscus tear, and meniscus tear place has been after 1 year
There is no trace, all equal rehabilitations of patient are good.
It these results suggest that collagen hydrogel load enrichment BMSCs can be conducive to fibrocartilaginous generation, and then promote
The reparation of meniscus injury.The enrichment BMSCs of collagen hydrogel load opens up for meniscus injury reparation for meniscus treatment
New road.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of preparation method of plural gel, which is characterized in that described method includes following steps,
(1) collagen-based materials are crushed, and is sterilized using physical method；
(2) by through step (1) processing after obtained collagen-based materials acid solution is used to be dissolved with the concentration of 10-20mg/mL；
(3) solution described in step (2) is placed in 4 DEG C of environment and treats that it is all dissolved, and aqueous slkali is used by its pH value tune at 4 DEG C
For section to 7-7.2, the hydrogel for obtaining liquid or semisolid is spare；
(4) use cell enrichment technology human marrow mesenchymal stem cell is enriched with to obtain enrichment mesenchymal stem cell with
It is spare；
(5) under 4 DEG C of environment by hydrogel obtained by step (3) according to 15-25：1 volume ratio is added drop-wise to the richness obtained by step (4)
Collect above mesenchymal stem cell, by its abundant mixing to get the plural gel product.
2. the preparation method of plural gel according to claim 1, which is characterized in that the sterilization side in the step (1)
Method is sterilized using ultraviolet light irradiation.
3. the preparation method of plural gel according to claim 1, which is characterized in that the acid solution is acetum.
4. the preparation method of plural gel according to claim 3, which is characterized in that the acetum it is a concentration of
0.5M。
5. the preparation method of plural gel according to claim 1, which is characterized in that the aqueous slkali is NaOH solution.
6. the preparation method of plural gel according to claim 5, which is characterized in that the NaOH solution it is a concentration of
0.5-1.5M。
7. the preparation method of plural gel according to claim 1, which is characterized in that the collagen-based materials are collagen water-setting
Glue.
8. a kind of plural gel prepared by method as described in any one of claim 1-7.
It is 9. a kind of such as any one of the claim 1-7 obtained plural gels of the method or as claimed in claim 8 multiple
Close application of the gel in the reparation of patient's meniscus tear.
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