CN115487358B - Gel composite scaffold for cartilage tissue repair and preparation method thereof - Google Patents
Gel composite scaffold for cartilage tissue repair and preparation method thereof Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/30756—Cartilage endoprostheses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/3094—Designing or manufacturing processes
- A61F2/30942—Designing or manufacturing processes for designing or making customized prostheses, e.g. using templates, CT or NMR scans, finite-element analysis or CAD-CAM techniques
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- A—HUMAN NECESSITIES
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- 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
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/30756—Cartilage endoprostheses
- A61F2002/30766—Scaffolds for cartilage ingrowth and regeneration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures
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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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention provides a preparation method of a gel composite scaffold for cartilage tissue repair, which comprises the following steps: (1) Loading KGN into PELA electrospun fibers by using an electrostatic spinning method to obtain KGN-PELA fibers; loading GDF-5 into the PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the length of both electrospun fibers to be not more than 100 mu m; (2) Adding the aldehyde hyaluronic acid and succinylated chitosan into PBS solution, adding KGN-PELA fiber and GDF-5-PCL fiber, and obtaining the gel composite scaffold after the reaction is completed. The gel composite scaffold has good mechanical properties and can be injected; meanwhile, KGN and GDF-5 in the invention have synergistic effect in inducing cartilage differentiation of mesenchymal stem cells; the invention has good application prospect in the aspect of treating cartilage defect repair.
Description
Technical Field
The invention belongs to the technical field of biological materials, relates to the field of cartilage tissue repair biological materials, and in particular relates to a gel composite scaffold for cartilage tissue repair and a preparation method thereof.
Background
Articular cartilage tissue is free of vascular, neural and lymphoid tissue, has a low cell content, and is supplied with nutrients derived from the moist of the surrounding joint synovial fluid, so that it cannot or is difficult to repair itself after injury. The main methods for clinically treating the articular cartilage injury at present have a plurality of risks and defects, such as fibrocartilage repair, difficult integration of transplanted cartilage and a recipient self cartilage matrix, immune rejection, related cells not arranged according to a tissue structure, wound bacterial infection and the like, and finally lead to cartilage repair failure and even lesions.
At present, the tissue engineering technology is considered as one of ideal methods for repairing cartilage defects, and the tissue engineering scaffold not only can simulate the extracellular matrix of chondrocytes so as to promote the growth and differentiation of the chondrocytes, but also can provide physical signals for the chondrocytes and enhance the adhesion and migration capacity of the chondrocytes. In practical applications, injectable stents have advantages over surgical implantable stents in that injectable hydrogel stents are based on in situ polymerization, filling irregularly shaped defects, and reducing the complexity of the surgical procedure. Wang [1] The four-arm star-shaped polyethylene glycol hydrogel modified by vinyl sulfone is prepared by the et al, so that the cartilage defect is treated by injection filling. The gel is in a porous structure after crosslinking, which is favorable for gas and substance exchange, promotes cell migration adhesion and promotes the tight combination of the nascent cartilage and surrounding cartilage tissues. Therefore, the injection type hydrogel provides a new treatment idea for cartilage tissue repair, not only can be used for in-situ filling treatment, but also can reduce the risk of operation and secondary injury.
Improving the mechanical strength of hydrogels to achieve the same viscoelastic and mechanical magnitudes as normal cartilage is a key issue in repairing cartilage defects. The common gel crosslinking modes at present are responsive crosslinking and chemical crosslinking. The response type crosslinking is related to factors such as temperature, pH, light intensity, enzyme substrate and the like, the mechanical property of the crosslinking is easily influenced by energy and substrate, the responsiveness is poor, and the problems of toxic and side effects of an initiator, uncontrollable exogenous energy and the like occur. Chemical crosslinking mainly reacts by collisions of chemical groups. Mechanical strength and chemical transformation of gelThe density of the chemical groups and the gel concentration are related, and the method has good controllability. Tan (Tan) [2] And the prepared aldehyde Hyaluronic Acid (HA) is crosslinked with chitosan to form gel by utilizing Schiff base reaction. The gel has good viscoelasticity, is beneficial to cell adhesion, and can shorten the hydrogel crosslinking time by adjusting the reaction proportion of aldehyde groups and amino groups. However, the gel has a narrow regulation range of mechanical properties and few optimization conditions, and cannot realize accurate regulation of ideal stent parameters. In order to solve the problems, the regulation and control range of the mechanical properties of the gel is enlarged by adopting methods of gel double cross-linking, gel and rigid nano particle cross-linking, and a composite bracket formed by the gel and a solid porous bracket. Supansa [3] The silk fibroin-hydrogel composite material similar to a reinforced concrete combination is designed by taking silk fibroin fibers and silk fibroin hydrogel as raw materials, and the silk fibroin fibers penetrate through the internal structure of the gel, so that the mechanical properties of the silk fibroin-hydrogel composite material are remarkably improved, but the massive composite material can only be implanted into a defect part through operation, and can easily cause secondary damage to a body.
To solve the problems in stent implantation, one has designed to dope injectable materials in combination with gels, hong [4] The collagen-coated polylactic acid microcarrier and chitosan are adopted by the et al to crosslink and prepare the injectable hydrogel, so that the mechanical property of a composite system is remarkably improved, and the physiological function of chondrocytes is regulated. However, nanofibers of the same size are larger than the specific surface of the microsphere; from the aspect of morphology, the contact area between the microspheres is smaller; the steric hindrance of the microspheres is large and the voids formed after aggregation are smaller, which may reduce the porosity of the gel. Wei (Wei) [5] The mechanical strength of the gel is improved by doping short fibers, and KGN is loaded to induce MSC (mesenchymal stem cells) to differentiate into chondrocytes, so that the repairing effect of cartilage defects is obtained.
However, loading the inducer into the electrospun fibers suffers from the problems of small drug loading and difficulty in achieving good induction of chondrocyte differentiation. That is, although short fibers have a remarkable advantage in terms of improving gel strength, they are limited in the effect of inducing differentiation of MSCs into chondrocytes due to the drug loading problem. In order to solve this problem, a more inducible agent is sought, or an agent that can act synergistically with the existing agent is sought for co-use.
KGN (Kartogenin) as a small molecule cartilage differentiation inducer, it was discovered for the first time in 2012 that KGN is dose dependent to induce MSC to differentiate into chondrocytes, and the induction effect is ideal. Currently, there are very limited studies on agents that can produce a synergistic effect with KGN in inducing differentiation of cells of interest into chondrocytes, and only a few reports are made. Such as Yanhong Zhao [6] The inventors found that KGN and TGF-beta 3 have a synergistic effect in inducing the differentiation of human umbilical cord mesenchymal stem cells into chondrocytes; likewise, wenshuai Fan [7] The KGN and TGF-beta 3 also have been found by the et al to have a synergistic effect in inducing differentiation of endogenous mesenchymal stem cells; in addition, zhaofen Jia [8] KGN and TGF-. Beta.3 were also found to have a synergistic effect on promoting differentiation of SF-MSCs chondrocytes.
To the best of the inventors' knowledge, current reports of active substances that can produce synergistic effects with KGN in promoting chondrocyte differentiation have focused mainly on TGF- β3. This therefore places a great limitation on the loading of KGN into staple fibers and the preparation of corresponding injectable gels to effect repair of cartilage defects.
In view of the above, there is still a need in the art to find substances that can act synergistically with KGN in inducing MSC differentiation into chondrocytes as active ingredients in preparing injectable gels with better mechanical properties to achieve cartilage defect repair.
Reference is made to:
[1]J Wang,F Zhang,W P Tsang et al.Fabrication of injectable high strength hydrogel based on 4-arm star PEG for cartilage tissue engineering[J].Biomaterials,2017,120:11-21.
[2]H Tan,C R Chu,K A Payne et al.Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering[J].Biomaterials,2009,30:2499-2506.
[3]S Yodmuang,S L McNamara,A B Nover et al.Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair[J].Acta Biomaterialia,2016,11:27-36.
[4]Y Hong,Y Gong,C Gao et al.Collagen-coated polylactide microcarriers/chitosan hydrogel composite:Injectable scaffold for cartilage regeneration[J].Part A:Journal of Biomedical Materials Research,2008,85A:628-637.
[5]Wei J,Ran P,Li Q,et al.Hierarchically structured injectable hydrogels with loaded cell spheroids for cartilage repairing and osteoarthritis treatment[J].Chemical Engineering Journal,2022,430:132211-.
[6]Synergistic Effects of Kartogenin and Transforming Growth Factor-β3on Chondrogenesis of Human Umbilical Cord Mesenchymal Stem Cells InVitro[J].Orthopaedic Surgery,2020,12(3).
[7]Fan W,Yuan L,Li J,et al.Injectable double-crosslinked hydrogels with kartogenin-conjugated polyurethane nano-particles and transforming growth factorβ3for in-situ cartilage regeneration[J].Materials Science and Engineering:C,2020,110:110705-.
[8]Jia Z,Wang S,Liang Y,et al.Combination of kartogenin and transforming growth factor-β3supports synovial fluid-derived mesenchymal stem cell-based cartilage regeneration[J].American Journal ofTranslational Research,2019,11(4).
disclosure of Invention
Aiming at the defects of the prior art, the core aim of the invention is to find out other substances which can have synergistic effect with KGN in inducing MSC to differentiate into chondrocytes besides TGF-beta 3 so as to overcome the defect that KGN has insufficient effect of repairing cartilage defects in injectable hydrogel preparations; on the basis, the invention also aims to provide a composite gel bracket similar to reinforced concrete, so that the mechanical property of the bracket is improved, and the application value in the aspect of cartilage defect repair is further improved.
Aiming at the purposes, the invention provides the following technical scheme:
a method for preparing a gel composite scaffold for cartilage tissue repair, the method comprising the steps of:
(1) Loading KGN into PELA electrospun fibers by using an electrostatic spinning method to obtain KGN-PELA fibers; loading GDF-5 into the PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the length of both electrospun fibers to be not more than 100 mu m;
(2) Adding aldehyde hyaluronic acid and succinylated chitosan into PBS solution, adding KGN-PELA fiber and GDF-5-PCL fiber, and obtaining the gel composite scaffold after the reaction is finished;
wherein the aldehyde hyaluronic acid is obtained by reacting hyaluronic acid with sodium periodate; succinylated chitosan is obtained by reacting chitosan with succinic anhydride;
in the step (2), the weight ratio of KGN to GDF-5 in KGN-PELA fiber and GDF-5-PCL fiber is 1:2.
GAGs and Col II are secretions specific to chondrocytes and can be used as evidence of the effect of cartilage differentiation. As shown in the related examples and experimental examples of the present invention, the gel composite scaffold of the present invention can significantly increase the secretion amount of GAG and Col II, while the secretion amount of GAG and Col II in the comparative example is smaller. This shows that KGN and GDF-5 have a synergistic effect in promoting cartilage differentiation in the gel composite scaffolds of the present invention.
The inventors have appreciated that GDF-5 has also been used to induce cartilage differentiation, but there are very few substances that act synergistically with it in terms of cartilage production. For example B.Appel [9] The et al found that GDF-5 has a synergistic effect with insulin in cartilage formation; s. Pat. No. Nia fontTellado [10] TGF-b2 and GDF5 have been found to increase expression of cartilage markers and type II collagen; wang Jianyang it was found that GDF-5 [11] The combination of icaritin can induce the cartilage differentiation of rat BMSCs, wherein the icaritin plays a role in promoting. Likewise, GDF-5 has limited application in the repair of cartilage defects due to its lack of materials that act synergistically in inducing cartilage differentiation.
It will be appreciated that the present invention has found that in addition to TGF-. Beta.3, another substance that can act synergistically with KGN in promoting cartilage differentiation. Meanwhile, the gel composite scaffold is injectable gel, so that the gel composite scaffold can be better used for repairing cartilage defects.
As a preferable technical scheme of the invention, when KGN-PELA fibers are prepared, KGN is dissolved in acetone, PELA is dissolved in a mixed solvent of acetone and DMF with the volume ratio of 6:1, and then KGN solution and PELA polymer solution are uniformly mixed to obtain KGN-PELA spinning solution; when preparing the GDF-5-PCL fiber, dissolving GDF-5 in acetone, dissolving PCL in a mixed solvent of chloroform and acetone in a volume ratio of 1:1, and uniformly mixing the GDF-5 solution and the PCL polymer solution to obtain a GDF-5-PCL spinning solution;
the spinning conditions for electrostatic spinning of KGN-PELA spinning solution and GDF-5-PCL spinning solution are as follows: the pushing speed is 1.6mL/h, the distance between the injector nozzle and the receiver is 15cm, the voltage is 20kV, the solid roller is used as a receiving device, and the rotating speed is 2500r/min.
As a preferable technical scheme of the invention, in the KGN-PELA fiber, the drug loading rate of KGN is 0.1wt%; in the GDF-5-PCL fiber, the drug loading of GDF-5 is 0.2wt%.
As a preferred technical scheme of the invention, the preparation method of the hydroformylation hyaluronic acid comprises the following steps: adjusting the pH value of deionized water to 6.0 by dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150ml of deionized water with the pH value of 6.0; magnetically stirring for 24 hours to fully dissolve the materials; then 16.5ml of 0.25mol/L sodium periodate solution is added, and after magnetic stirring for 3 hours at 40 ℃, 30ml of glycol is added to terminate the reaction; and then dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.
As a preferred technical scheme of the invention, the preparation method of succinylated chitosan comprises the following steps: 1.0g of chitosan is dissolved in dilute hydrochloric acid with the volume concentration of 0.37 percent, and glucosamine is added to ensure that the concentration is 6.20mmol/L, so as to obtain chitosan solution; succinic anhydride 0.63g was dissolved in 5ml of pyridine to obtain succinic anhydride solution. Dropwise adding succinic anhydride solution into chitosan solution at room temperature (25-30 ℃) in a fume hood under the stirring condition; adjusting the pH value to 7.0, reacting for 4 hours, and adding 20w/v% NaCl solution to terminate the reaction; adding ethanol, filtering, soaking the precipitate in diethyl ether, and drying.
As a preferable technical scheme of the invention, the length of the KGN-PELA fiber and the GDF-5-PCL fiber is 50 μm.
As a preferred embodiment of the present invention, in the step (2), the reaction is carried out at 37℃for 12 hours.
In the present invention, the gel composite scaffold for cartilage tissue repair is injectable in vivo and reacts at body temperature.
Another object of the present invention is to provide a gel composite scaffold for cartilage tissue repair prepared by the above preparation method.
It is still another object of the present invention to provide the use of the gel composite scaffold described above for the preparation of a formulation for cartilage tissue repair.
The invention has the beneficial effects that:
the gel composite scaffold for cartilage tissue repair provided by the invention has good mechanical properties and can be injected; meanwhile, KGN and GDF-5 in the invention have synergistic effect in inducing cartilage differentiation of bone marrow mesenchymal stem cells, and overcome the defect of lower cartilage differentiation effect in the prior art; the invention has good application prospect in the aspect of treating cartilage defect repair.
Reference is made to:
[9]Appel B,Baumer J,Eyrich D,et al.Synergistic effects of growth and differentiation factor-5(GDF-5)and insulin on expanded chondrocytes in a 3-D environment[J].Osteoarthritis&Cartilage,2009,17(11):1503-1512.
[10]Tellado,Sonia,Font,et al.Heparin functionalization increases retention of TGF-beta 2and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering[J].Acta Biomaterialia,2018.
[11] wang Jianyang research on the differentiation and mechanism of BMSCs into chondroid cells by GDF-5 in combination with icaritin [ D ]. University of Nanchang.
Drawings
FIG. 1 shows the results of swelling and compression properties of gel composite scaffolds doped with different concentrations of short fibers (part a in FIG. 1), and the rheology profile (part b in FIG. 1);
FIG. 2 is an SEM image of a gel composite scaffold obtained in example 1 of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will occur to those skilled in the art in light of the foregoing disclosure.
The meanings of the abbreviations in the present invention are as follows, unless otherwise indicated:
KGN: kartogenin,2- ([ 1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid;
PELA: polylactic acid-polyethylene glycol copolymer;
PCL: polycaprolactone;
GDF-5-PCL fiber: PCL fibers loaded with GDF-5;
KGN-PELA fiber: KGN-loaded pila fibers;
BMSC: bone marrow mesenchymal stem cells mesenchymal stem cells; example 1
1. Experimental materials
PELA (mw=50 kda, mw/mn=1.23), given to the college of capital medical science; PCL (mw=80 kDa), purchased from shanghai-derived leaf technologies limited; hyaluronic acid (mw=38 kDa), purchased from rad Hua Xifu rayda biomedical company; KGN (Kartogenin) and GDF-5 were purchased from Aba Ding Gongsi; chitosan (degree of deacetylation 96.0%), purchased from shandong Hua Xifu rayleigh biological medicine company; other reagents are available from the laboratory.
2. Preparation of drug-loaded short fiber
(1) KGN with different concentrations (the concentration range is 0.1mg-1mg, the gradient is 0.05 mg) is dissolved in 2mL of acetone, 500mg of PELA is dissolved in 3.0mL of mixed solvent of acetone and DMF with the volume ratio of 6:1, and then KGN solution and PELA polymer solution are uniformly mixed to obtain KGN-PELA spinning solution; carrying out electrostatic spinning on KGN-PELA spinning solution, wherein the spinning conditions of the electrostatic spinning are as follows: the pushing speed is 1.6mL/h, the distance between the injector nozzle and the receiver is 15cm, the voltage is 20kV, the solid roller is used as a receiving device, and the rotating speed is 2500r/min. And finally, KGN-PELA fiber with the drug loading of 0.1 weight percent is selected.
The drug-loaded oriented fiber membrane was immersed in distilled water and folded at 1cm intervals along the direction perpendicular to the direction of oriented fiber arrangement, and frozen at-70℃for 5min. The microtome was set to a cut thickness of 50 μm. And finally, cleaning, centrifugally collecting short fibers, freeze-drying, and storing in a dry and cool place for later use.
(2) GDF-5 with different concentrations (the concentration range is 0.1mg-1mg, the gradient is 0.1 mg) is dissolved in 2mL of acetone, 450mg of PCL is dissolved in a mixed solvent of chloroform and acetone with the volume ratio of 1:1, and then the GDF-5 solution and the PCL polymer solution are uniformly mixed to obtain GDF-5-PCL spinning solution; carrying out electrostatic spinning on the GDF-5-PCL spinning solution, wherein the spinning conditions of the electrostatic spinning are as follows: the pushing speed is 1.6mL/h, the distance between the injector nozzle and the receiver is 15cm, the voltage is 20kV, the solid roller is used as a receiving device, and the rotating speed is 2500r/min. GDF-5-PCL fiber was finally selected with a drug loading of 0.2wt%.
The drug-loaded oriented fiber membrane was immersed in distilled water and folded at 1cm intervals along the direction perpendicular to the direction of oriented fiber arrangement, and frozen at-70℃for 5min. The microtome was set to a cut thickness of 50 μm. And finally, cleaning, centrifugally collecting short fibers, freeze-drying, and storing in a dry and cool place for later use.
3. Preparation of hydroformylation hyaluronic acid
Adjusting the pH value of deionized water to 6.0 by dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150ml of deionized water with the pH value of 6.0; magnetically stirring for 24 hours to fully dissolve the materials; then 16.5ml of 0.25mol/L sodium periodate solution is added, and after magnetic stirring for 3 hours at 40 ℃, 30ml of glycol is added to terminate the reaction; and then dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.
4. Preparation of succinylated chitosan
Chitosan is dissolved in acetic acid with the concentration of 1v/v percent, slowly added into 20ml of acetone solution dissolved with succinic anhydride in a dropwise manner, and stirred in a water bath for reaction for 4 hours; adding excessive acetone for precipitation, filtering to obtain a solid phase, washing with acetone, and drying for 4 hours to obtain solid particles, namely succinylated chitosan. .
5. Preparation of composite gel scaffold
Adding the same amount of aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the adding amount of 0.2 g/ml; adding KGN-PELA fiber and GDF-5-PCL fiber in an equal weight; reacting for 12 hours at 37 ℃ to obtain hydrogel; regulating the total weight of KGN-PELA fiber and GDF-5-PCL fiber in the obtained gel scaffold to be 1.0% of the total dry weight of the gel scaffold; and the gel concentration is regulated to be 3%, and the gel concentration is the total dry weight (w/v) of the aldehyde group hyaluronic acid and the succinylated chitosan contained in each unit solution volume.
Comparative example 1
The procedure of example 1 was followed except that only KGN-PELA fibers were added during the preparation of the composite scaffold. Namely, the gel scaffold method of the comparative example is as follows:
adding the same amount of aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the adding amount of 0.2 g/ml; KGN-PELA fiber is added and reacts for 12 hours at 37 ℃ to obtain hydrogel; in the gel scaffold obtained by regulation, the total weight of KGN-PELA fibers accounts for 1.0 percent of the weight of the gel scaffold.
Comparative example 2
The procedure of example 1 was followed except that only GDF-5-PCL fiber was added in the preparation of the composite scaffold. Namely, the gel scaffold method of the comparative example is as follows:
adding the same amount of aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the adding amount of 0.2 g/ml; adding GDF-5-PCL fiber; reacting for 12 hours at 37 ℃ to obtain hydrogel; in the gel scaffold obtained, the total weight of GDF-5-PCL fibers accounts for 1.0% of the weight of the gel scaffold.
Experimental example 1
1. Culture of Stem cell spheres
Reference (Wang W, li B, li Y, et al in vivo restoration of full-thickness cartilage defects by poly(lactide-co-glycolide)sponges filled with fibrin gel,bone marrow mesenchymal stem cells and DNA complexes[J]Biomaterials.2010, 31:5953-5965) BMSC stem cell spheres, namely: extracting bone marrow from rabbits, and separating BMSC from the bone marrow by a density gradient centrifugation method; rabbits (12 kg,1 month old) were subjected to general anesthesia (50 mg/kg dose) with 3g/L sodium pentobarbital, dehairing treatment at ilium on a lateral operating table, disinfection with iodophor, and local anesthesia at joints with 2g/L lidocaine. 1mL of heparin sodium (3000 kU/L) was withdrawn in a 10mL syringe, and then the syringe was connected to a 16-gauge bone marrow puncture needle, and the needle was inserted into the bone marrow cavity to withdraw 34mL of bone marrow fluid. The bone marrow fluid was then added to a serum-free medium containing lymphocyte isolates and centrifuged at 2000rpm for 30min to remove the supernatant containing monocytes. Cells were resuspended in 5mL of serum-free medium, centrifuged at 1500rpm for 5min, the supernatant discarded, and the cells were washed again. The pelleted cells were resuspended in low glucose DMEM medium containing 10% foetal calf serum, 100mg/mL penicillin and 100U/mL streptomycin and placed at 37℃with 5% CO 2 Is changed once every 2 days, and is amplified after the cells are fused to more than 90 percent.
BMSC differentiation function test
The effect of the composite gel scaffold injection procedure obtained in example 1, comparative example 1 and comparative example 2 on BMSC cell spheres was examined. Taking example 1 as an example, firstly, uniformly dispersing the KGN-PELA fiber and the GDF-5-PCL fiber with equal weight in a PBS solution containing the aldehyde group hyaluronic acid, dispersing BMSC cell spheres in the PBS solution containing the succinylated chitosan, rapidly and uniformly mixing the two solutions (after mixing, ensuring that the aldehyde group hyaluronic acid and the succinylated chitosan are both 0.2G/ml, and the KGN-PELA fiber and the GDF-5-PCL fiber account for 1w/w% of gel obtained except the BMSC cell spheres), injecting the gel into a 48-hole plate through a 16G needle syringe, adding a culture medium for culturing after the gel is completely crosslinked, and changing the liquid once every two days. Comparative examples 1 and 2 were carried out with reference to the above-described method.
Functional test of BMSC cell spheres wrapped in composite gel scaffold: the secretion of GAGs was measured by the dimethylmethylene blue (DMMB) method; the content of Col II was determined by means of a Col II ELISA kit. The test results are shown in Table 1.
TABLE 1 results of GAG and Col II secretion
Experimental example 2
Determination of aldehyde group content in the hydroformylation hyaluronic acid obtained in example 1: 0.2g of aldehyde hyaluronic acid is weighed and placed in a 100mL beaker, 10mL of deionized water is added to dissolve the aldehyde hyaluronic acid completely, then the pH is adjusted to 5.0, 8mL of hydroxylamine hydrochloride reagent (0.05 g/mL, pH=5.0) is accurately added into the solution, stirring is carried out for 4 hours under the water bath condition of 40 ℃, finally the solution is titrated to pH=5.0 by using 0.01mol/L of NaOH standard solution, and the volume of the consumed NaOH solution is recorded. And weighing hyaluronic acid with the same quality for blank experiment comparison.
Aldehyde group content = C (V1-V2)/m/376 x 100%
Wherein, C, naOH standard solution concentration mol/L; v1 is the volume number (L) of NaOH consumed by the sample specimen; v2 is the volume number (L) of NaOH consumed by the hyaluronic acid blank experiment; m is the mass (g) of the sample specimen.
The aldehyde group content of the hydroformylation hyaluronic acid in example 1 was calculated to be 62%.
Experimental example 3
The degree of acylation of succinylated chitosan was examined according to the method of "preparation of succinylated chitosan film and its pH-sensitive study" ([ 1] Li Zuowei, zhang Liyan, zeng Qingxiao. Preparation of succinylated chitosan film and its pH-sensitive study [ J ]. Food industry science and technology, 2009 (1): 3 ]), and the degree of acylation of succinylated chitosan of example 1 was examined to be 12.1%.
Experimental example 4
The gel composite scaffolds obtained in example 1 were tested for swelling rate, compression resistance, storage modulus G' and loss modulus G″. As shown in FIG. 1, the gel composite scaffold obtained in example 1 has a good swelling rate and good mechanical properties.
Claims (10)
1. A method for preparing a gel composite scaffold for cartilage tissue repair, which is characterized by comprising the following steps:
(1) Loading KGN into PELA electrospun fibers by using an electrostatic spinning method to obtain KGN-PELA fibers; loading GDF-5 into the PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the length of both electrospun fibers to be not longer than 100 mu m;
(2) Adding aldehyde hyaluronic acid and succinylated chitosan into PBS solution, adding KGN-PELA fiber and GDF-5-PCL fiber, and obtaining the gel composite scaffold after the reaction is finished;
wherein the aldehyde hyaluronic acid is obtained by reacting hyaluronic acid with sodium periodate; succinylated chitosan is obtained by reacting chitosan with succinic anhydride;
in the step (2), the weight ratio of KGN to GDF-5 in KGN-PELA fiber and GDF-5-PCL fiber is 1:2.
2. The preparation method of the KGN-PELA fiber according to claim 1, wherein KGN is dissolved in acetone, PELA is dissolved in a mixed solvent of acetone and DMF in a volume ratio of 6:1, and KGN solution and PELA polymer solution are uniformly mixed to obtain KGN-PELA spinning solution; when preparing the GDF-5-PCL fiber, dissolving GDF-5 in acetone, dissolving PCL in a mixed solvent of chloroform and acetone in a volume ratio of 1:1, and uniformly mixing the GDF-5 solution and the PCL polymer solution to obtain a GDF-5-PCL spinning solution;
the spinning conditions for electrostatic spinning of KGN-PELA spinning solution and GDF-5-PCL spinning solution are as follows: the pushing speed is 1.6mL/h, the distance between the injector nozzle and the receiver is 15cm, the voltage is 20kV, the solid roller is used as a receiving device, and the rotating speed is 2500r/min.
3. The method according to claim 1, wherein the KGN-PELA fiber has a KGN drug loading of 0.1wt%; in the GDF-5-PCL fiber, the drug loading rate of GDF-5 is 0.2wt%; the total weight of KGN-PELA fibers and GDF-5-PCL fibers accounts for 1.0% of the total dry weight of the gel scaffold.
4. The method of claim 1, wherein the method of preparing the aldehyde-based hyaluronic acid comprises: adjusting the pH value of deionized water to 6.0 by dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150mL of deionized water with the pH value of 6.0; magnetically stirring for 24 hours to fully dissolve the materials; then 16.5mL of 0.25mol/L sodium periodate solution is added, and after magnetic stirring for 3 hours at 40 ℃, 30mL of ethylene glycol is added to terminate the reaction; and then dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.
5. The preparation method of the succinylated chitosan according to claim 1, wherein the preparation method of the succinylated chitosan comprises the following steps: chitosan is dissolved in acetic acid with the concentration of 1v/v percent, slowly added into 20mL of acetone solution dissolved with succinic anhydride in a dropwise manner, and stirred in a water bath for reaction for 4 hours; adding excessive acetone for precipitation, filtering to obtain a solid phase, washing with acetone, and drying for 4 hours to obtain solid particles, namely succinylated chitosan.
6. The method according to claim 1, wherein the KGN-PELA fiber and GDF-5-PCL fiber have a length of 50 μm.
7. The process according to claim 1, wherein in step (2), the reaction is carried out at 37℃for a period of 12 hours.
8. The method of claim 1, wherein the gel composite scaffold for cartilage tissue repair is injectable in vivo and reacts at body temperature.
9. A gel composite scaffold for cartilage tissue repair, characterized in that the gel composite scaffold is prepared by the preparation method of any one of claims 1 to 8.
10. Use of the gel composite scaffold of claim 9 for the preparation of a formulation for cartilage tissue repair.
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