CN115869464A - Joint paint for repairing and regenerating non-full-thickness cartilage defects - Google Patents
Joint paint for repairing and regenerating non-full-thickness cartilage defects Download PDFInfo
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- CN115869464A CN115869464A CN202111132669.2A CN202111132669A CN115869464A CN 115869464 A CN115869464 A CN 115869464A CN 202111132669 A CN202111132669 A CN 202111132669A CN 115869464 A CN115869464 A CN 115869464A
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- chondroitin sulfate
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- aldehyde
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- hyaluronic acid
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Images
Abstract
The invention provides joint paint for repairing and regenerating non-full-thickness cartilage defects, which is characterized in that chondroitin sulfate and gelatin are controllably modified, and the aldehyde chondroitin sulfate is creatively used as a bridge for connecting cartilage tissues and hydrogel, the aldehyde chondroitin sulfate is fully contacted with the surfaces of the cartilage tissues, firm covalent bonds are formed through Schiff base reaction, and the aldehyde chondroitin sulfate bridge is prevented from being solidified in advance by using the aldehyde chondroitin sulfate bridge; the aldehyde chondroitin sulfate can be further crosslinked with methacrylic anhydride gelatin to further improve the adhesion, so that the joint paint has better tissue adhesion, can be formed on the surface of the cartilage in situ, and meets the requirement of non-full-layer cartilage defect repair; the components closer to natural cartilage ECM are adopted, so that the cartilage repair liquid has better biological adhesion, better cell adhesion effect and higher cell activity when used for repairing non-full-thickness cartilage defects, can better maintain the phenotype of chondrocytes, and has better repair effect.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to joint paint for repairing and regenerating non-full-thickness cartilage defects.
Background
Osteoarthritis is a high-incidence, disabling-rate joint disease, with about 3000 million patients in the united states alone. The incidence of the disease gradually increases with the age, and China is facing the progress of the population aging society, so that osteoarthritis seriously affects the life quality of people in China and loads the people and the society. The mechanism of formation of osteoarthritis is complex, with cartilage defects at the articular surface being an important cause. Therefore, repair of damaged articular cartilage is an important approach for the treatment of osteoarthritis. Cartilage is a special tissue, free of blood vessels and lymph, and has poor self-repairing ability after defects. Cartilage defects are generally classified into three types according to the depth of the defect site: non-full-thickness cartilage defects, and osteochondral defects (see fig. 1). Among them, non-full-thickness cartilage defects in the articular surface are most common in osteoarthritis and most troublesome for the clinician. Compared with the full-layer defect and the osteochondral defect, the non-full-layer cartilage defect does not damage subchondral bone, does not have blood supply and bone marrow exudation, and a nearby microenvironment is not suitable for stem cell adhesion, so that the self-repairing capability is completely lost. In addition, early superficial defects also fail to heal themselves, eventually leading to the vicious circle of osteoarthritis. At present, no mature clinical treatment strategy exists for non-full-thickness cartilage defects in osteoarthritis, and development of new treatment means is urgently needed.
With the development of tissue engineering and regenerative medicine, hydrogel materials have been developed like spring bamboo shoots after rain in the research of cartilage defect repair due to the water content similar to natural tissues, excellent biocompatibility and convenient operability. Although the research at home and abroad has made some progress on the application of hydrogel in cartilage defect repair, the non-full-layer cartilage defect repair model is difficult to establish, most of the research on cartilage defect repair selects full-layer defects and osteochondral defects as models, and the research on non-full-layer cartilage defects is relatively lacked.
The non-full-thickness cartilage defect is shallow, such as the surface of knee cartilage, and the hydrogel material for repair is easy to fall off due to friction if the adhesion force is insufficient during the movement process, thereby seriously affecting the repair effect. The research group applied a patent of 'a three-component biological glue and preparation and application thereof' in 2017 (CN 201710353560.7), but the biological glue disclosed in the application is more suitable for the condition of deeper cartilage damage degree and is not suitable for the repair of non-full-layer cartilage defect.
Therefore, a hydrogel material which has stronger biological tissue adhesion, better cell adhesion effect and cell activity and can better maintain the phenotype of chondrocytes is urgently needed to be prepared aiming at the non-full-thickness cartilage defect, and the repair effect can be further improved.
Disclosure of Invention
In order to solve the problems, the invention provides joint paint for repairing and regenerating non-full-thickness cartilage defects, wherein aldehyde chondroitin sulfate is creatively used as a bridge for connecting cartilage tissues and hydrogel, and the adhesion points and the cross-linking points of the joint paint are increased in a Schiff base mode; by utilizing the bionic components of the cartilage and controllably modifying chondroitin sulfate and gelatin, a hydrogel material, namely 'joint paint', which can be formed on the surface of the cartilage in situ, has tissue adhesion, maintains the phenotype of chondrocytes, protects the cartilage from further degradation and promotes the regeneration of the cartilage is constructed.
The invention is inspired by automobile paint-spraying renovation, and designs 'joint paint' for the incomplete cartilage defect, which can bring new growth to the cartilage with the affected scar like paint-spraying renovation to old automobiles (see figure 2).
The natural cartilage ECM (extracellular matrix) mainly consists of type II collagen, chondroitin sulfate and hyaluronic acid, but the type II collagen has higher price, is difficult to store and relatively difficult to clinically transform, and can be replaced by more mature, low-cost and easily transformed gelatin.
Since the defect degree of the non-full-thickness cartilage is shallow, such as the surface of the knee cartilage, the hydrogel material for repair is very easy to fall off due to friction if the adhesion force is not enough during the movement process, so that the repair effect is seriously influenced, and the requirement on the tissue adhesion force of the hydrogel material is very high.
The joint paint is prepared from bionic components of natural cartilage, chondroitin sulfate and gelatin are modified, the conventional use method of hydrogel is changed, and the biological tissue adhesion is greatly improved by coating the adhesive and then coating the hydrogel, so that the repair effect of non-full-layer cartilage defect is obviously improved.
In one aspect, the present invention provides an articular paint comprising a binder and a photocurable hydrogel formulation, the binder and the photocurable hydrogel formulation being packaged separately; wherein the binder is an aldehydized chondroitin sulfate, and the photocurable hydrogel formulation comprises a methacrylated gelatin and hyaluronic acid, which does not require any modification.
The invention modifies chondroitin sulfate to prepare aldehyde chondroitin sulfate, which improves the adhesiveness to the cartilage surface. The modified aldehyde chondroitin sulfate can respectively perform Schiff base reaction on amino on the surface of the cartilage and amino on the surface of hydrogel through aldehyde groups to form a dynamic covalent bond, and then is subjected to photocuring, so that a film (joint paint) is formed at the defect part of the superficial cartilage, and the film can be firmly adhered to the cartilage.
The application of the binder prior to the application of the photocurable hydrogel formulation has the following advantages: 1. the aldehyde chondroitin sulfate is coated firstly, so that the aldehyde chondroitin sulfate is fully contacted with the surface of the cartilage to form firmer adhesion, the coated joint paint cannot fall off from the surface of the cartilage, and the repair effect of the cartilage defect on the superficial layer is further ensured; 2. if the adhesive and the light-cured hydrogel preparation are mixed and then directly used, advanced curing can be caused, the curing effect with controllable paint can not be achieved, the aldehyde chondroitin sulfate is coated firstly, so that the aldehyde chondroitin sulfate is fully contacted with the surface of the cartilage to form firmer adhesive force, and then the light-cured hydrogel preparation is coated and then light-cured, so that the adhesive force with the surface of the cartilage can be obviously improved, and the repair effect of superficial cartilage defects can be improved.
The adhesive and the light-cured hydrogel preparation provided by the invention need to be separately used, so that the adhesive and the light-cured hydrogel preparation need to be separately and independently packaged, and the separate and independent packaging of the adhesive and the light-cured hydrogel preparation is more favorable for storage and transportation and has longer shelf life.
The hyaluronic acid has the functions of resisting inflammation, promoting tissue repair and the like, has an important effect on maintaining the phenotype of cartilage cells, can obviously induce the expression of high COL2A1 genes, but can change the molecular structure of the modified hyaluronic acid, and can easily cause the components to lose the original activity even if the components are excessively modified, so that the repair effect is influenced.
Furthermore, the structural formula of the aldehyde chondroitin sulfate is shown as a formula I, and the structural formula of the methacrylated gelatin is shown as a formula II.
Gelatin reacts with methacrylic anhydride in PBS environment, and a structure containing carbon-carbon double bonds is covalently connected to gelatin molecules by amidation reaction of acid anhydride and amino groups on the gelatin, so that the modified gelatin molecule methacrylic anhydride gelatin capable of undergoing polymerization reaction is obtained. The research proves that the substitution degree of amino in the gelatin by methacrylic anhydride can be controlled by controlling the addition of the methacrylic anhydride, so that the gelatin can retain a certain proportion of amino, the gelatin can react with the formylated chondroitin sulfate by Schiff base to form a firm covalent bond, and the formylated chondroitin sulfate can further crosslink with the methacrylic anhydride gelatin to further improve the adhesion.
Research proves that when 40% of amino groups in methacrylic anhydride gelatin are reserved (as shown in formula II, the lower two amino groups in the structural formula of the methacrylic anhydride gelatin are not substituted) by controlling the addition of the methacrylic anhydride, the prepared joint paint is most suitable, the photocuring effect of the methacrylic anhydride gelatin is good, the reaction with the aldehyde chondroitin sulfate is sufficient, the biological adhesion force when the methacrylic anhydride gelatin is coated on the surface of cartilage is very good, the adhesion strength is higher than the gel strength, the adhesion bursting force and the shear strength are several times of those of the current clinical adhesives, the chondrocyte phenotype can be maintained for a long time, and the repair of superficial layer cartilage defects is promoted.
Furthermore, the dosage of the aldehyde chondroitin sulfate is 5-15%, the dosage of the methacrylated gelatin is 10-20%, and the dosage of the hyaluronic acid is 0-0.5%.
Further, the dosage of the aldehyde chondroitin sulfate is 12.5%, the dosage of the methacrylated gelatin is 10%, and the dosage of the hyaluronic acid is 0.5%.
The joint paint provided by the invention takes the aldehyde chondroitin sulfate as a bridge for connecting the cartilage tissue and the hydrogel, adopts the components closer to the natural cartilage ECM, has better biological adhesion, better cell adhesion effect and higher cell activity when being used for repairing the non-full-thickness cartilage defect, and can better maintain the phenotype of the cartilage cells.
In another aspect, the invention provides a preparation method of joint paint, which mainly comprises the following steps:
(1) Synthesizing formylated chondroitin sulfate and methacrylated gelatin;
(2) Packing the aldehyde chondroitin sulfate independently; and independently packaging a mixed solution of the methacrylated gelatin and the hyaluronic acid.
Further, the reaction formula for synthesizing the aldehyde chondroitin sulfate in the step (1) is shown as a formula III;
the reaction formula for synthesizing the methacrylated gelatin is shown as a formula IV;
further, the method for synthesizing the aldehyde chondroitin sulfate in the step (1) comprises the following steps: 3g chondroitin sulfate powder is weighed and dissolved in 50mL deionized water, 1g NaIO is added 4 Reacting for 6h in a dark environment at 25 ℃, adding 0.55mL of ethylene glycol to stop the reaction, adding 200mL of deionized water after 1h to dilute the reaction solution to 250mL, stirring and dialyzing.
Further, the method for synthesizing the methacrylated gelatin in the step (1) comprises the following steps: weighing 10g of gelatin, dissolving in 100mL of phosphate buffer solution, stirring and dissolving at 50 ℃, slowly dropwise adding 1mL of methacrylic anhydride, continuously reacting for 2h at 50 ℃, adding 100mL of methacrylic anhydride, preheating to 50 ℃, stirring, and dialyzing.
Further, the step (2) comprises the steps of:
a. dissolving the methacrylated gelatin in a PBS buffer solution to obtain 20% (w/v) of the methacrylated gelatin, dissolving hyaluronic acid in deionized water to obtain 1% (w/v) of hyaluronic acid, and dissolving the formylated chondroitin sulfate in the deionized water to obtain 25% (w/v) of formylated chondroitin sulfate;
b. 10-20% (w/v) of methacrylated gelatin, 0-1% (w/v) of hyaluronic acid and 5-25% (w/v) of formylated chondroitin sulfate are sterilized by a 0.22 mu m filter membrane;
c. independently packaging the aldehyde chondroitin sulfate solution;
d. the methacrylated gelatin and hyaluronic acid are mixed according to the proportion of 1.
In another aspect, the invention provides a method for using the joint paint, wherein the method comprises the steps of coating the aldehyde chondroitin sulfate solution on the cartilage defect, adding a mixed solution of methacrylated gelatin and hyaluronic acid after 1-5 minutes, adding LAP, and carrying out photocuring.
In a further aspect, the present invention provides the use of an articular paint as described above for the repair of a non-full thickness cartilage defect, said articular paint requiring the application of a binder followed by the application of a light-curable hydrogel formulation.
Further, the defect depth of the non-full-thickness cartilage defect is not more than 0.5mm.
Further, the defect depth of the non-full-thickness cartilage defect is generally about 0.2 mm.
The joint paint provided by the invention can be formed on the surface of cartilage in situ, which is also a place difficult to realize by the existing cartilage repair hydrogel. The hydrogel provided by the invention has certain shape maintaining capability before photocuring by using the aldehyde chondroitin sulfate as a bridge, and the condition that the shape is changed by random flowing before curing can not occur, so that the cartilage surface can be repaired more beautifully as new after curing.
In the prior art, in order to increase the adhesive force, all components in the hydrogel are usually modified, such as hyaluronic acid needs to be further modified, the invention only needs to controllably modify chondroitin sulfate and gelatin, so that the excellent tissue adhesive force can be achieved, the requirements of the incomplete cartilage defect repair are completely met, the hyaluronic acid does not need to be further modified at all, the normal hyaluronic acid has the functions of resisting inflammation, promoting the tissue repair and the like, but the molecular structure of the normal hyaluronic acid is changed after the modification, the component loses the original activity even is easily caused by the excessive modification, and the repair effect is influenced.
The joint paint for repairing and regenerating the non-full-thickness cartilage defect provided by the invention has the following beneficial effects:
1. by controllably modifying chondroitin sulfate and gelatin and creatively taking the aldehyde chondroitin sulfate as a bridge for connecting the cartilage tissue and the hydrogel, the aldehyde chondroitin sulfate is fully contacted with the surface of the cartilage tissue, and a firm covalent bond is formed by Schiff base reaction to prevent advanced curing; the aldehyde chondroitin sulfate can be further crosslinked with methacrylic anhydride gelatin to further improve the adhesion, so that the joint paint has better tissue adhesion, can be formed on the surface of the cartilage in situ, and meets the requirement of non-full-layer cartilage defect repair;
2. adopts the components closer to the natural cartilage ECM without modifying hyaluronic acid, has better biological adhesion when being used for repairing the incomplete cartilage defect, and the cell adhesion effect is better, the cell activity is higher, the phenotype of the chondrocyte can be better maintained, and the repair effect is better.
Drawings
FIG. 1 is a schematic diagram of cartilage defects classified according to depth of defect sites, from left to right, including non-full-thickness cartilage defects, full-thickness cartilage defects and osteochondral defects in sequence;
FIG. 2 is a schematic illustration of "joint painting" as it is done to paint and refurbish old cars, bringing new growth to the cartilage with scar involvement;
FIG. 3 is a graph showing the results of Fourier transform Infrared Spectroscopy (FTIR) analysis of the molecular structure of the aldehyde-substituted chondroitin sulfate of example 1;
FIG. 4 shows NMR of methacryloylated gelatin molecular structure of example 1: ( 1 H-NMR) analysis result chart;
FIG. 5 is a graph showing the results of analyzing the amino group content in methacrylated gelatin products of different degrees of substitution measured by the TNBS method in example 2;
FIG. 6 is a graph showing the results of the adhesion burst test in example 3;
FIG. 7 is a graph showing the results of the lap shear strength test in example 3;
FIG. 8 is a graph showing the results of comparing the residence time of the joint paint of example 3 and the existing hydrogel on the cartilage surface;
FIG. 9 is an image of stained cells imaged by confocal laser microscopy for the effect of adhesion of joint paint to hASCD in example 4;
FIG. 10 is a graph showing the effect of the vital stain method of example 4 on the activity of hADSCs in joint paint;
FIG. 11 is a graph showing the results of the joint paint of example 5 on the maintenance of chondrocyte phenotype;
FIG. 12 is a graph showing the results of the gross observation and histological analysis evaluation of the articular paint in example 6 on cartilage repair in white rabbits.
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the invention without limiting it in any way. Advantages and features of the present invention will become apparent from the following description and from the claims. It is to be noted that the drawings are in a very simplified form and are not to precise scale, which is merely for the purpose of facilitating and distinctly claiming the embodiments of the present invention.
EXAMPLE 1 preparation of Joint paint
The preparation of the joint paint provided by the embodiment comprises the following steps:
(1) Synthesizing aldehyde chondroitin sulfate, methacrylic acylated gelatin and a photoinitiator;
(1) synthesis of formylated chondroitin sulfate
Weighing 3g of chondroitin sulfate powder, dissolving in 50mL of deionized water, adding 1g of sodium periodate, reacting for 6h in a dark environment at 25 ℃, adding 0.55mL of ethylene glycol to stop the reaction, adding 200mL of deionized water after 1h to dilute the reaction solution to 250mL, stirring for 10min, filling into a dialysis bag (MWCO 3500), dialyzing in the deionized water at normal temperature, changing water 3 times a day, and continuing for 3 days to remove unreacted substances and ions. After dialysis, the product was stored by lyophilizationThe molecular structure of the product is analyzed by Fourier transform infrared spectroscopy (FTIR), and the sample is 400-4000 cm -1 Performing attenuated total reflection infrared spectroscopy. Each sample is measured at 4cm -1 The background test is performed before the sample test, and is automatically subtracted from the sample spectrum, and the analysis result is shown in fig. 3, thereby ensuring that the molecular structure is correct. FIG. 3 shows that at 1731cm -1 An obvious absorption peak appears at the position, which is a characteristic peak of the aldehyde group, and proves that the aldehyde chondroitin sulfate is successfully synthesized.
(2) Synthesis of methacrylated gelatin
Weighing 10g of gelatin, dissolving in 100mL of Phosphate Buffer Solution (PBS), stirring at 50 ℃ for dissolving, slowly dropwise adding 1mL of methacrylic anhydride, continuously reacting at 50 ℃ for 2h, adding 100mL of PBS preheated to 50 ℃, continuously stirring for 10min, filling into a dialysis bag (MWCO 8000-14000), dialyzing in deionized water at 40 ℃, changing water 2 times a day, continuing for 7 days, and removing unreacted substances. After dialysis, the product is freeze-dried and stored, and the molecular structure of the product adopts nuclear magnetic resonance (A) 1 H-NMR), the analysis results are shown in fig. 4, the molecular structure is ensured to be correct, and the amino group content is measured by the TNBS method, and the amino group substitution degree is determined to be 60%. FIG. 4 is a schematic view of 1 The H-NMR results confirmed the presence of carbon-carbon double bonds on the synthesized molecules.
(3) Synthesis of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP)
3.2g (0.018 mol) of 2,4, 6-trimethylbenzoyl chloride are added dropwise to an equimolar amount of dimethylphenylphosphite (3.0 g) at room temperature under an argon atmosphere and the reaction is continued with stirring for 18 hours; a 4-fold excess of lithium bromide (6.1 g) was mixed into 100ml 2-butanone, added to the reaction mixture, and heated to 50 ℃ to form a solid precipitate after about 10 minutes; cooling the solid precipitate to room temperature, standing for 4 hours, and then filtering; washing the solid precipitate with 2-butanone multiple times to remove unreacted lithium bromide; finally the excess solvent was removed by vacuum drying.
(2) Coating the aldehyde chondroitin sulfate solution on the cartilage defect, adding a mixed solution of methacrylated gelatin, hyaluronic acid and a photoinitiator after 1-5 minutes, and carrying out photocuring. The method comprises the following specific steps:
(1) dissolving methacrylated gelatin in PBS buffer solution to obtain 20% (w/v) methacrylated gelatin, dissolving hyaluronic acid in deionized water to obtain 1% (w/v) hyaluronic acid, and dissolving aldehyde chondroitin sulfate in deionized water to obtain 25% (w/v) aldehyde chondroitin sulfate;
(2) sterilizing 20% (w/v) of methacrylated gelatin, 1% (w/v) of hyaluronic acid, and 25% (w/v) of aldehyde-modified chondroitin sulfate through a 0.22 μm filter;
(3) GelMA and hyaluronic acid were mixed in a ratio of 1, and 8.5mM of photoinitiator LAP was added;
(4) when in use, the aldehyde chondroitin sulfate is firstly acted on the cartilage for 1-5 minutes, then the mixed solution of GelMA and HA is added, and the light curing is carried out for 10-30 seconds.
Example 2 Effect of the degree of substitution of amino groups in gelatin by methacrylic anhydride on the preparation of an articular paint
In this example, three groups of methacrylated gelatins with different degrees of substitution were synthesized by the method for synthesizing methacrylated gelatins as provided in example 1: 10g of gelatin was weighed and dissolved in 100mL of Phosphate Buffer Solution (PBS), stirred and dissolved at 50 ℃, 0mL (first group), 1mL (second group), and 8mL (third group) of methacrylic anhydride were slowly added dropwise to prepare methacrylated gelatin, and the amino group content in the product was measured by the TNBS method to calculate the degree of substitution of the amino group, and the results are shown in FIG. 5. As can be seen from fig. 5, when 0mL (first group) was used, all of the amino groups on the gelatin remained; when 1mL of methacrylic anhydride (second group) was used, about 40% of amino groups remained on the prepared gelatin; whereas when 8mL of methacrylic anhydride (third group) was used, the amino group on the gelatin was almost completely substituted with methacrylic acid.
Gelatin can be photocured after being modified by methacrylic anhydride, so that the more amino groups are replaced by methacrylic anhydride, the higher the photocuring strength is, but a certain proportion of amino groups must be reserved in the gelatin, so that the gelatin can react with the aldehyde chondroitin sulfate by Schiff base to form a firm covalent bond, and the aldehyde chondroitin sulfate can be further crosslinked with the methacrylic anhydride gelatin. Research proves that when 40% of amino groups are reserved in the methacrylic anhydride gelatin, namely the substitution degree of the amino groups by the methacrylic anhydride is 60%, the photocuring effect of the methacrylic anhydride gelatin is still good, the methacrylic anhydride gelatin can fully react with the aldehyde chondroitin sulfate to generate very strong adhesion, and therefore the methacrylic anhydride gelatin can be used for clinically repairing non-full-thickness cartilage defects.
Example 3 adhesion of joint paints
This example compares the bioadhesion using the following groups, respectively:
first Group (GHC): adopting an aldehyde chondroitin sulfate binder to directly mix with a photo-curing hydrogel preparation (methacryloylated gelatin, hyaluronic acid and LAP), coating a protein casing for one time, and carrying out photo-curing;
second group (Joint Paint): the joint paint prepared in example 1 is adopted, and a protein casing is adhered in vitro in a mode of coating an aldehyde chondroitin sulfate binder and then coating a photo-curing hydrogel preparation (methacryloylated gelatin, hyaluronic acid and LAP), and is photo-cured;
third group (CA): coating a protein casing by using a clinically common biological adhesive Cyanoacrylate (CA) and curing;
fourth group (Suriflo): coating a protein casing with a clinically common biological adhesive Suriflo, and curing;
fifth group (fibre glue): coating a protein casing with a clinically common biological adhesive Fibrin glue (Fibrin glue), and curing;
according to American Society for Testing and Materials (ASTM) biomedical adhesive standard, the adhesion burst strength test is performed according to a modified ASTM standard (F2392-04) to perform lap shear strength and adhesion burst strength tests, wherein the results of the adhesion burst strength test are shown in FIG. 6, the results of the lap shear strength test are shown in FIG. 7, and the results are shown in groups I, II, III, IV and V from left to right.
As is apparent from FIGS. 6 and 7, the joint paint prepared by the joint paint provided by the invention (second group) and by coating the aldehyde chondroitin sulfate binder and then coating the photo-curing hydrogel preparation has the adhesive bursting power of 34.3 +/-7.3 kPa, the shear strength of 63.9 +/-12.0 kPa, and the failure type is hydrogel center fracture, which means that the adhesive strength is higher than the gel strength and is obviously higher than the adhesive bursting power (17.6 +/-3.2 kPa) and the shear strength (37.3 +/-12.4 kPa) of the first group when the first group is mixed for use, and is also obviously higher than fibrin glue (the adhesive bursting power is 6.9 +/-1.4 kPa, and the shear strength is 7.1 +/-2.9 kPa), cyanoacrylate (the adhesive bursting power is 3.1 +/-0.6 kPa), and Suriflo (the adhesive bursting power is 0.8 +/-0.3 kPa).
The visible joint paint (second group) adopts a mode of firstly coating the aldehyde chondroitin sulfate binder and then coating the light-cured hydrogel preparation, so that the tissue adhesion can be greatly improved, and the adhesion bursting force (34.3 +/-7.3 kPa) and the shear strength (63.9 +/-12.0 kPa) are obviously improved, mainly because the aldehyde chondroitin sulfate is firstly coated, the aldehyde chondroitin sulfate is fully contacted with the cartilage surface to form firmer adhesion, the coated joint paint cannot fall off from the cartilage surface, and the use mode of the aldehyde chondroitin sulfate bridge prevents the advanced curing of gelatin, thereby further ensuring the repair effect of the superficial cartilage defect. The first group of the aldehyde chondroitin sulfate and the photo-curing hydrogel preparation are mixed for use, so that the aldehyde chondroitin sulfate cannot be in full contact with the surface of the cartilage, and can be cured in advance, the adhesive force of the hydrogel material is influenced, the product is not favorable for storage and use, and the method is not suitable for repairing non-full-thickness cartilage defects.
To further examine the adhesion stability of the joint paint (second group) to cartilage, this example further measured the residence time of the "joint paint" on the pig cartilage. First, after creating a surface defect on the pig cartilage, the bridging molecule was spread on the defect site for 5 minutes and then removed with gauze. After the photocurable hydrogel formulation was painted, it was exposed to ultraviolet light (50 mW/cm) 2 30 s), forming elastic hydrogel with smooth surface. It can be seen that the curvature of the cartilage was restored after the joint painting treatment. The cartilage containing the material was then removed in its entirety for further study. Strong adhesion of 'joint paint' to cartilage was observed using Field Emission Scanning Electron Microscopy (FESEM)And (4) sex. Scanning electron microscope shows that the gel film is tightly combined with cartilage without cracks. We believe that one reason for this strong binding is the schiff base reaction between the aldehyde groups of the bridging molecule and the amino groups of the gel membrane and cartilage. To measure the residence time, we soaked the removed cartilage in PBS along with the material, shaken it at 4 ℃ and observed the binding of the material to the cartilage every day. The results show that the mean residence time of the "joint paint" is 136 days, which is 2 times the retention time of the first group (fig. 8). Notably, the gel membrane remained elastic, smooth in surface, and completely attached to the cartilage over the 4 month evaluation period.
Example 4 Effect of Joint paint on cell adhesion
For the treatment of osteoarthritis, hADSC is a very promising cell type, and this example uses human adipose derived stromal cells (hADSC) to study the cell adhesion capacity of cartilage and "joint paint". Freezing-stored human adipose-derived mesenchymal stem cells (hADSCs) at 37 deg.C, 5% 2 The cells were digested with 0.25% trypsin and subcultured. Pig cartilage was coated with the joint paint prepared in example 1 and placed in 48-well plates. hADSCs were pretreated with 2 μ M DiI (Beyotime, institute of Biotechnology). We suspended 100. Mu.l DiI treated hADSCs (2X 10) 4 /ml) were seeded onto the surface of cartilage and hydrogel, and then continued to be cultured at 37 ℃ and 5% in a CO2 environment. After 6 hours of culture, the state of cell adhesion was observed using an inverted fluorescence microscope, and the number of adhered cells was calculated using Image J. In addition, we stained the cytoskeleton, as follows: cells were fixed with 4% (w/v) paraformaldehyde for 30 minutes, washed 3 times with PBS; rupture of membranes for 5 minutes with 0.1% (w/v) Triton X-100 (Sigma-Aldrich) followed by blocking for 30 minutes with 1% (w/v) bovine serum albumin (BSA, sigma-Aldrich); staining with phalloidin (Cytoskeleton, inc.) for 30 min, washing 3 times with PBS; cell nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole) for 10min, washed with PBS 3 times, and the stained cells were imaged with a laser confocal microscope (Zeiss LSM 880). The results are shown in FIG. 9, the number of cells adhering to the "Joint paint" surface compared to the cartilage surfaceThe amount is significantly increased and this enhanced cell adhesion property can be attributed to methacrylated gelatin and hyaluronic acid.
To further investigate the cytocompatibility of "joint paints", this example was performed by coating "joint paints" on cell culture plates and detecting the activity of hADSCs by the live-dead staining method (FIG. 10). After 1d and 4d of culture, cells grew well on normal cell culture plates with fewer dead cells. These results indicate that the joint coating has a high degree of cell compatibility and is capable of promoting stem cell adhesion.
Example 5 Effect of Joint paints on maintaining chondrocyte phenotype
Maintenance of chondrocyte phenotype is critical for cartilage regeneration. To evaluate the effect of joint paint on chondrocyte phenotype, this example cultures human chondrocytes on the surface of a material and detects their gene expression by real-time quantitative polymerase chain reaction. This example quantitatively analyzes Marker genes of chondrocytes such as COL2A1, SOX 9. And the effect of hyaluronic acid concentration on chondrocyte phenotype was investigated.
Human chondrocytes were cultured in F12/DMEM medium (Gibco) containing 10% (v/v) fetal bovine serum (Gibco), 1% (v/v) antibiotic/antifungal (Gibco) and 1% (v/v) L-glutamine (Gibco). Coating of 24-well plates with "Joint paint" procedure 500. Mu.L of chondrocyte suspension (2.4X 10) was applied as described above 4 Per ml) were inoculated onto such "joint paint" coated 24-well plates at 37 ℃ and 5% CO 2 The culture is continued under the environment of (2). The cell culture fluid was changed every two days. RNA of 5 subgroups (Control, GH, GH-6.25% C, GH-12.5% C, and GH-25% C) was extracted from these chondrocytes using RNAlso-Plus (Takara) after 7 days and 14 days of culture. cDNA was synthesized from 2. Mu.gRNA using ReverTra-Ace-qPCR-RT-Master Mix (TOYOBO). Real-time quantitative polymerase chain reaction (qPCR) was performed on Light Cycler appaatus (Roche 480 II) using SYBR Green qPCR Master Mix (Takara). By 2 -△△Ct Calculated by methods, normalized to GAPDH expression. Table 1 shows all primers used in this example.
TABLE 1 primers for qPCR
All data are expressed as mean ± SD, differences between values were assessed using One-way ANOVA, and p <0.05 was considered to be significantly different.
The results showed that the hydrogel membrane composed of methacrylated gelatin (GelMA), hyaluronic Acid (HA) was able to maintain the phenotype of chondrocytes (fig. 11), which had a dose-dependent level of COL2A1 expression at 2 weeks with increasing HA concentration. The highest concentration of HA (0.5%) used in this study showed the best ability to significantly induce high COL2A1 gene expression in chondrocytes. Hyaluronic Acid (HA) is not effective in promoting SOX9 and ACAN expression. Meanwhile, the hyaluronic acid in the joint paint plays an important role in maintaining the phenotype of cartilage cells, can obviously induce the expression of high COL2A1 genes, does not need to modify the hyaluronic acid, prevents the hyaluronic acid from losing the original activity caused by excessive modification, and preferably adopts bionic components closer to natural softness to prepare the joint paint, thereby further improving the repair effect on non-full-thickness cartilage defects.
Next, we investigated the effect of aldehyde Chondroitin Sulfate (CSA) on chondrocyte phenotype. The levels of gene expression of ACAN and SOX9 increased significantly after 7 days of chondrocyte culture on "joint paint" containing 6.25% and 12.5% CSA, indicating that CSA played a positive role in maintaining chondrocyte phenotype (figure 11). After 14 days, the chondrocyte gene expression level of the three marker genes shows a trend of approaching the level of a control group, which shows that the chondrocyte phenotype can still keep normal after the material is cultured for a long time. The results show that HA HAs a significant influence on the COL2A1 gene expression level of chondrocytes, while CSA HAs no significant influence on the COL2A1 gene expression level, but HAs an effect of improving the gene expression of two other marker genes, namely ACAN and SOX 9. Thus, we found that GelMA/HA and CSA combined "joint paint" can promote the expression of genes associated with cartilage regeneration in chondrocytes and maintain their cartilage phenotype in long-term in vitro cell culture. Thus the most favorable components for cartilage regeneration repair were 10% GelMA,0.5% HA and 12.5% CSA.
EXAMPLE 6 repair Effect of Joint paint
To investigate the in vivo cartilage defect repair effect of "joint paint", we used new zealand white rabbits as an animal model for studies. "Joint paint" was applied to the defect (2 mm x 6mm,0.2mm deep) in the trochlear groove and cured in situ on the cartilage surface by photopolymerization. Animals were harvested at 6 and 12 weeks of surgery, their joints collected, and evaluated by gross observation and histological analysis (fig. 12). First, a non-full-thickness cartilage defect treated with "joint paint" filled most of the defect with newly formed tissue, and the cartilage was generally observed to be healthy, similar to the sham group. In contrast to the control group, which did not receive any treatment, further degeneration of cartilage was observed following surface cartilage loss. Histologically, after H & E staining, the chondrocyte distribution of the untreated control group changed significantly after 6 and 12 weeks, and it can be seen that the normal chondrocyte distribution of the sham operated group was columnar, indicating that the cartilage of the control group may have been in a pathological state. To further investigate the development of post-operative damaged cartilage with or without "joint paint" treatment, tissue sections were stained with Safranin-O to assess glycosaminoglycan (GAG) distribution. Safranin-O staining showed that the cartilage surface contour was similar to that of the sham operated group under "joint paint" protection. After 6 weeks of surgery, the "joint paint" group formed a new GAG-rich tissue filling the defect site. The new tissue binds well to autologous cartilage and remains on the cartilage surface after 12 weeks. In contrast, GAG content of the control group that was not "joint paint" treated gradually degraded in the cartilage matrix 6 weeks and 12 weeks post-operatively, indicating further degradation and degeneration of cartilage. In addition, the ICRS score and MODS score were also higher in the "joint paint" treatment group, indicating improved cartilage repair.
The invention is not the best known technology. Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected by one skilled in the art without departing from the spirit and scope of the invention, as defined in the appended claims.
Claims (10)
1. An articular paint, which is characterized by comprising a binder and a light-cured hydrogel preparation, wherein the binder and the light-cured hydrogel preparation are separately and independently packaged; wherein the binder is an aldehydized chondroitin sulfate, and the photocurable hydrogel formulation comprises a methacrylated gelatin and hyaluronic acid, which does not require any modification.
2. The reagent of claim 2, wherein the aldehyde chondroitin sulfate has a formula shown in formula i, and the methacrylated gelatin has a formula shown in formula ii.
3. The reagent according to claim 3, wherein the amount of the aldehyde-substituted chondroitin sulfate is 5 to 25%, the amount of the methacrylated gelatin is 10 to 20%, and the amount of the hyaluronic acid is 0 to 1%.
4. The reagent according to claim 4, wherein the amount of the aldehyde chondroitin sulfate is 12.5%, the amount of the methacrylated gelatin is 10%, and the amount of the hyaluronic acid is 0.5%.
5. A preparation method of joint paint is characterized by comprising the following steps:
(1) Synthesizing aldehyde chondroitin sulfate and methacrylated gelatin;
(2) Packing the aldehyde chondroitin sulfate independently; and independently packaging a mixed solution of the methacrylated gelatin and the hyaluronic acid.
6. The method of claim 5, wherein the reaction formula for the synthesis of the formylated chondroitin sulfate in step (1) is shown in formula III;
the reaction formula for synthesizing the methacrylated gelatin is shown as a formula IV;
7. the method of claim 6, wherein the step (1) of synthesizing the aldehyde-based chondroitin sulfate comprises: 3g chondroitin sulfate powder is weighed and dissolved in 50mL deionized water, 1g NaIO is added 4 Reacting for 6 hours in a dark environment at 25 ℃, adding 0.55mL of ethylene glycol to stop the reaction, adding 200mL of deionized water after 1 hour to dilute the reaction solution to 250mL, stirring and dialyzing;
the method for synthesizing the methacrylated gelatin in the step (1) comprises the following steps: weighing 10g of gelatin, dissolving in 100mL of phosphate buffer solution, stirring and dissolving at 50 ℃, slowly dropwise adding 1mL of methacrylic anhydride, continuously reacting for 2h at 50 ℃, adding 100mL of methacrylic anhydride, preheating to 50 ℃, stirring, and dialyzing.
8. The method of claim 5, wherein the step (2) comprises the steps of:
a. dissolving methacrylated gelatin in PBS buffer solution to obtain 20% (w/v) methacrylated gelatin, dissolving hyaluronic acid in deionized water to obtain 1% (w/v) hyaluronic acid, and dissolving aldehyde chondroitin sulfate in deionized water to obtain 25% (w/v) aldehyde chondroitin sulfate;
b. 10-20% (w/v) of methacrylated gelatin, 0-1% (w/v) of hyaluronic acid and 5-25% (w/v) of formylated chondroitin sulfate are sterilized by a 0.22 mu m filter membrane;
c. independently packaging the formylated chondroitin sulfate solution;
d. the methacrylated gelatin and the hyaluronic acid are mixed according to the proportion of 1.
9. A method for using joint paint is characterized in that aldehyde chondroitin sulfate solution is coated on cartilage defect positions for 1-5 minutes, then mixed liquid of methacrylated gelatin and hyaluronic acid is added, LAP is added, and light curing is carried out.
10. Use of an articular paint according to any one of claims 1 to 5 or of an articular paint prepared by a method according to any one of claims 6 to 7 for the repair of non-full-thickness cartilage defects, characterized in that the defect depth of said non-full-thickness cartilage defects does not exceed 0.5mm; the joint paint is used by coating the binder and then coating the light-cured hydrogel preparation.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106220874A (en) * | 2016-08-17 | 2016-12-14 | 广东工业大学 | The preparation method of composite aquogel and application, composite aquogel repair materials and preparation method thereof |
| US20190224132A1 (en) * | 2016-10-04 | 2019-07-25 | University Of Florida Research Foundation, Inc. | Chondroprotective nanoparticles for the treatment of osteoarthritis |
| CN112245395A (en) * | 2020-11-20 | 2021-01-22 | 佳木斯大学 | Medical cartilage repairing agent and preparation method thereof |
| CN113087819A (en) * | 2021-03-12 | 2021-07-09 | 深圳市光韵达增材制造研究院 | Double-modified compound and preparation method and application thereof |
-
2021
- 2021-09-26 CN CN202111132669.2A patent/CN115869464A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106220874A (en) * | 2016-08-17 | 2016-12-14 | 广东工业大学 | The preparation method of composite aquogel and application, composite aquogel repair materials and preparation method thereof |
| US20190224132A1 (en) * | 2016-10-04 | 2019-07-25 | University Of Florida Research Foundation, Inc. | Chondroprotective nanoparticles for the treatment of osteoarthritis |
| CN112245395A (en) * | 2020-11-20 | 2021-01-22 | 佳木斯大学 | Medical cartilage repairing agent and preparation method thereof |
| CN113087819A (en) * | 2021-03-12 | 2021-07-09 | 深圳市光韵达增材制造研究院 | Double-modified compound and preparation method and application thereof |
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