CN111330075B - Preparation method and application of squid type II gelatin double-network hydrogel - Google Patents
Preparation method and application of squid type II gelatin double-network hydrogel Download PDFInfo
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Abstract
The invention provides a preparation method and application of squid type II gelatin double-network hydrogel, which comprises the following steps: (1) dissolving HAMA in a biocompatible medium, and sequentially adding photosensitizer I2959 and squid type II gelatin to obtain a mixed solution A; (2) dissolving 4-Arm PEG-SS in a biocompatible medium to obtain a PEG solution; (3) mixing the PEG solution with the mixed solution A, and finishing the cross-linking reaction of the first network in the double-network hydrogel at room temperature; (4) and (3) finishing crosslinking of the second network of the hydrogel by illumination to obtain the squid II type gelatin double-network hydrogel. The double-network hydrogel prepared by the invention has high compression strength close to that of costal cartilage of a human body, and is beneficial to avoiding the collapse of the thorax; the cartilage stem/precursor cells from costal cartilage membranes are induced to be differentiated into chondrocytes under the condition of not adding a chondrogenic inducer, the expression level of cartilage matrixes is improved, the regeneration and repair of the defect costal cartilage are promoted, and the repair of costal cartilage defects caused by various reasons such as trauma, plastic surgery and the like is realized.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a squid II type gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel material for repairing costal cartilage defects and a preparation method thereof.
Background
The autologous costal cartilage is frequently used for repairing deformed ears, nose reshaping and beautifying, reconstruction of temporomandibular joints and the like clinically. Generally, there is no corresponding repair treatment for the defect of the costal cartilage in the donor area clinically after the costal cartilage transplantation is performed. The regeneration capacity of cartilage is poor, the costal cartilage is difficult to repair by self after being damaged, complications such as thoracic cavity collapse and deformity can be caused, the body appearance is not only influenced, but also the thoracic cavity loses the capacity of protecting internal organs to a certain extent, and the serious patient can also cause discomfort of the patient. Therefore, the problem of repairing costal cartilage in the supply area of autologous costal cartilage transplantation should be sufficiently considered.
At present, in the case report or clinical research, the treatment measures for the supply area costal cartilage defect part include: the remaining costal cartilage membrane is sutured, with or without the application of fat, autologous costal cartilage debris, or irradiated cadaver costal cartilage to fill the defect area. However, none of the above methods can really realize the regeneration of costal cartilage, and the method of filling the costal cartilage with autologous costal cartilage fragments or irradiated corpse costal cartilage has certain potential safety hazard. Therefore, there is a need to find a more ideal and safer repair material, which can meet the requirement of costal cartilage as much as possible in terms of mechanical properties (studies show that the maximum stress that can be borne by human costal cartilage can reach about 15MPa [ zhanggui bi, normal human rib morphology and mechanical characteristics and two fixing ways of biomechanical studies, master, north-river medical university, 2013 ]), and can maintain the thoracic form and protect internal organs; meanwhile, the material can also have certain biological activity to induce the regeneration and repair of costal cartilage (such as the chondrogenic differentiation of cartilage stem/precursor cells in costal cartilage membrane).
The hydrogel is high in water content and viscoelastic, can simulate cartilage to a certain extent, and is often used for research on repair of articular cartilage defects, but no report on the use of hydrogel for repair of supply region costal cartilage defects exists at present; more importantly, the requirements on the mechanical property and the biological property of the material for repairing the cartilage defect and the costal cartilage defect in the joint area are different. The traditional hydrogel is mainly single-network hydrogel formed by crosslinking based on physical or chemical principles, the formed hydrogel has poor mechanical properties, particularly, the hydrogel prepared by adopting natural biomaterials such as collagen, gelatin, sodium alginate and the like has low compressive strength, and although the hydrogel can be used for the repair research of articular cartilage surface defects, the mechanical property requirements of repairing and replacing costal cartilage are usually difficult to meet. The appearance of double network hydrogel (DN hydrogel) well solves the problem of poor hydrogel strength. DN hydrogel is a special polymer interpenetrating network formed by two polymers with strong structural asymmetry, and compared with single polymer network hydrogel, the mechanical strength and toughness of the hydrogel are greatly improved. Jianping Gong reported that poly (N, N-dimethylacrylamide) (PDMAAm) can form a high-strength DN hydrogel with poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS), wherein the compressive strength of PAMPS-1-4/PAAm-2-0.1 can be up to 17.2 MPa. However, the raw materials of these hydrogels are chemically synthesized polymers, which have the risks of long degradation time, and the degradation products may cause inflammatory reactions in the body. The swimming bladder I-type collagen is used for replacing PAMPS and also can form DN hydrogel with PDMAAm, 90% of compressive stress of the hydrogel is about 1.87-6.81 MPa, and the hydrogel preliminarily shows a certain repairing effect in a osteochondral defect model of a rabbit knee joint. However, the strength of the hydrogel is not suitable for the repair of costal cartilage, and type I collagen is not a main component of hyaline cartilage, and cartilage involved in the repair is often fibrocartilage rather than hyaline cartilage (costal cartilage is hyaline cartilage), and thus, the hydrogel is also not suitable for the repair of defects of costal cartilage.
Chinese patent CN107281550A discloses DN hydrogel for promoting cartilage damage repair, which is a hydrogel scaffold with GelMA-GelMA and GelMA-HSNGLPL co-crosslinking double-network structure formed by photo-crosslinking methacrylate modified gelatin (GelMA) and HSNGLPL polypeptide under the action of a photoinitiator 659. The hydrogel autonomously combines endogenous TGF-beta 1 through HSNGLPL, regulates and controls cartilage matrix secretion of normal chondrocytes around damaged tissues, promotes cartilage differentiation of mesenchymal stem cells, and thus promotes generation of new cartilage. However, it is not clear whether the mechanical properties of the material and the inducing effect on the differentiation of costal perichondrial derived cartilage stem/precursor cells, particularly the chondrogenic effect can be exerted under the complicated factors such as surgical trauma and implantation of exogenous materials.
In conclusion, the DN hydrogel reported in the current research has the following defects and is not suitable for repairing costal cartilage defects: firstly, most of the preparation raw materials are chemically synthesized polymers, so that the risks of slow degradation, body inflammatory reaction caused by degradation products and the like exist; almost no DN hydrogel completely prepared from natural biological materials exists at present, but the hydrogel prepared from the natural biological materials reported in the prior art has relatively poor mechanical properties, and particularly the compression strength can not meet the requirement of repairing costal cartilage; and thirdly, whether the hydrogel has cartilage repair effect is not researched, and the effect is just an important concern for repairing costal cartilage defects in a donor area.
Therefore, it is desirable to provide a double-network hydrogel with excellent mechanical properties and biocompatibility, and a certain chondrogenic effect, to solve the above problems.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of squid type II gelatin double-network hydrogel which has the performance of inducing cartilage stem/precursor cells derived from costal cartilage membranes to be chondrogenic differentiation, and has high compressive strength and good biological activity.
The second purpose of the invention is to provide the squid II type gelatin double-network hydrogel which has the performance of inducing cartilage stem/precursor cells derived from costal cartilage membranes to be chondrogenic differentiation, and has high compressive strength and good bioactivity.
The third purpose of the invention is to provide the application of the squid type II gelatin double-network hydrogel in preparing a repairing material for costal cartilage defects.
In order to realize the first object of the invention, the invention provides a preparation method of squid type II gelatin double-network hydrogel, which is characterized by comprising the following steps:
(1) dissolving methacrylate modified hyaluronic acid HAMA in a biocompatible medium, sequentially adding a photosensitizer I2959 and squid type II gelatin into the biocompatible medium, and adjusting the pH value to be alkalescent by using an alkaline solution to obtain a mixed solution A, wherein the concentration of the HAMA in the mixed solution A is 0.6-6% (w/w), the concentration of the photosensitizer I2959 is 0.06-0.6% (w/w), and the concentration of the squid type II gelatin is 22.5-45% (w/w);
(2) dissolving four-Arm polyethylene glycol succinimidyl ester 4-Arm PEG-SS in a biocompatible medium to obtain a PEG solution, wherein the concentration of the 4-Arm PEG-SS in the PEG solution is 9-15% (w/w);
(3) mixing the PEG solution obtained in the step (2) with the mixed solution A obtained in the step (1) to obtain a first network in the double-network hydrogel, wherein in the obtained product, the final concentration of the squid type II gelatin is 15-30% (w/w), the final concentration of the 4-Arm PEG-SS is 3-5% (w/w), the final concentration of the HAMA is 0.4-4% (w/w), and the final concentration of the I2959 is 0.04-0.4% (w/w);
(4) and (3) irradiating the hydrogel obtained in the step (3) by using ultraviolet light with the wavelength of 365nm to complete cross-linking of a second network of the hydrogel, so as to obtain the squid II type gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel.
As a preferred scheme, the preparation method of the squid type II gelatin in the step (1) comprises the steps of freeze-drying squid cartilage, crushing, adding an EDTA solution at room temperature, slowly stirring, centrifuging, washing the precipitate with deionized water, adding an NaOH solution, slowly stirring, centrifuging, washing the precipitate, sequentially adding an acetic acid solution and pepsin, slowly stirring at 37 ℃ for hydrolysis, heating to 50 ℃ for a period of time, filtering, dialyzing, and then carrying out vacuum freeze-drying on a glue solution to obtain the squid type II gelatin.
Preferably, the concentration of HAMA in the mixed solution A in the step (1) is 1.5% (w/w); the concentration of photosensitizer I2959 was 0.15% (w/w); the concentration of the squid II type gelatin is 30 to 45 percent (w/w).
As a preferable scheme, in the first network of the hydrogel obtained in the step (3), the final concentration of the squid type II gelatin is 20% -30% (w/w), and the final concentration of the 4-Arm PEG-SS is 5% (w/w); in the second network of the hydrogel obtained in the step (4), the final concentration of HAMA is 1% (w/w), and the final concentration of I2959 is 0.1% (w/w).
As a preferable embodiment, the biocompatible medium is selected from one or more of distilled water, physiological saline, buffer solution and cell culture medium solution.
Preferably, the volume ratio of the mixed solution A to the PEG solution is 2: 1.
Preferably, the wavelength of the light source used for illumination in the step (4) is 365 nm; the illumination time is 2-5 minutes; the distance between the light source and the hydrogel was 3 cm.
In order to realize the second object of the invention, the invention provides the squid type II gelatin double-network hydrogel prepared by the preparation method.
In order to realize the third purpose of the invention, the invention provides the application of the squid type II gelatin double-network hydrogel in preparing a repairing material for costal cartilage defects.
Methacrylate-modified hyaluronic acid (HAMA) was prepared by a conventional method: dissolving hyaluronic acid in deionized water, cooling to 0-4 ℃, adding methacrylic anhydride, slowly dropwise adding NaOH, reacting for 24 hours, pouring the reaction solution into a dialysis bag, dialyzing for 2-3 days with deionized water, and freeze-drying to obtain the photosensitive hyaluronic acid derivative HAMA. The reaction formula for preparing HAMA principle is as formula 1:
photosensitizer I2959 refers to 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (I2959).
The alkaline solution for adjusting the pH of the mixed solution a may be a sodium hydroxide solution, a calcium hydroxide solution, a sodium bicarbonate solution, a calcium bicarbonate solution, or the like, the pH may be adjusted to 7 to 12, and preferably the pH is adjusted to 8 to 9 by using a 1M sodium hydroxide solution.
Wherein the gelatin component can be selected from squid type II gelatin, and other various types of gelatin derived from other animals, such as pig, cattle or fish type I or type II gelatin, can also be prepared into double-network hydrogel. However, the gelatin of the present invention is preferably squid type II gelatin in consideration of the following factors: first, type II gelatin is a hydrolysate of type II collagen, which is a characteristic constituent of cartilage tissue and is more advantageous for chondrocyte to secrete cartilage matrix than type I collagen; secondly, marine organism collagen or gelatin can effectively avoid the risk of spreading animal-derived infectious diseases such as mad cow disease, avian influenza and the like in the presence of the marine organism collagen or gelatin; thirdly, the earlier studies of the invention have confirmed that the type II collagen extracted from the cartilage of the marine organism squid has no immunogenicity and higher biological safety compared with the type II collagen of the terrestrial animal cattle; in addition, the squid cartilage II collagen can effectively inhibit chemotactic enrichment of macrophages, reduce the level of inflammatory mediators released by inflammatory macrophages, induce conversion of unactivated macrophages to anti-inflammatory macrophages capable of participating in tissue repair, further promote chondrocytes to secrete cartilage matrixes, and achieve the effect of inducing immunity to form cartilage. Thus, the invention contemplates the use of a hydrolysate of squid type II collagen, type II gelatin, to aid in the repair of costal cartilage: in one aspect, type II gelatin is the same composition as type II collagen and has a similar biological response; on the other hand, collagen or gelatin is formed into hydrogel by reacting amino groups contained therein with various groups to form cross-links, but compared with collagen, the collagen has less folding of peptide chains and a large number of amino groups contained therein are exposed, which is advantageous for obtaining more sufficient cross-linking reaction, increasing the degree of cross-linking and increasing the mechanical strength of hydrogel.
Polyethylene glycol (PEG) is a good hydrogel material, and the derivative thereof, namely four-Arm polyethylene glycol succinimide ester (4-Arm PEG-SS), contains a large number of carboxyl groups, can react with amino groups in gelatin, and spontaneously forms amide bond crosslinking at room temperature. PEG has better biological safety, and various PEGylated medicines are approved by FDA at present. Therefore, the squid II type gelatin and the 4-Arm PEG-SS react to form a first network of DN hydrogel, so that the biological safety of the squid can be ensured while the gelling reaction is good, and the effect of cartilage induction is exerted. In designing the second network of the hydrogel, the invention carries out methacrylate modification on hyaluronic acid which is another natural component in cartilage tissue so as to form photosensitive hyaluronic acid, and then uses light to initiate double bond polymerization reaction to form gel. Thus, DN hydrogel not only simulates important components (type II collagen and hyaluronic acid) in cartilage tissue, but also ensures sufficient crosslinking and gelling reactions, and is an ideal costal cartilage repair hydrogel material.
The invention has the advantages that:
(1) the squid II type gelatin double-network hydrogel is synthesized based on natural biological materials and has a double-network structure, the compression strength of the hydrogel can reach 11MPa, the hydrogel is obviously higher than that of the hydrogel material prepared by taking the natural biological materials as the raw materials, the mechanical properties of costal cartilage are basically met, the collapse of the thorax is prevented, and the internal organs are protected.
(2) The squid II type gelatin double-network hydrogel disclosed by the invention has the advantages of high mechanical strength, good biocompatibility and certain bioactivity, and can actively induce cartilage stem/precursor cells derived from costal cartilage membranes to differentiate into chondrocytes, so that the regeneration of cartilage is realized without the help of seed cells or chondroblast inducers.
(3) The squid II type gelatin double-network hydrogel not only can play an effective chondrogenic induction effect in vitro, but also can better realize the effect of repairing costal cartilage defect in an animal body under the condition of not additionally adding growth factors or seed cells, and simultaneously avoids the risk caused by implanting exogenous cytokines or seed cells into an organism.
Drawings
FIG. 1 is an appearance diagram of squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel.
FIG. 2 shows the mechanical strength and degradation rate of squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel.
FIG. 3 shows the effect of squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel in inducing chondrogenic differentiation of costal perichondrial derived cartilage stem/precursor cells.
FIG. 4 shows the schematic diagram of squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel used for repairing costal cartilage defect of rabbit and the repairing effect after 8 weeks of implantation.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
Example 1 preparation of Squid type II gelatin- [4-Arm PEG-SS ] -HAMA Dual network hydrogel.
Removing attached muscle and flesh from squid cartilage, carrying out vacuum freeze drying, crushing into cartilage powder, adding an EDTA solution at room temperature, slowly stirring for 24 hours, centrifuging, cleaning the precipitate with deionized water, adding an NaOH solution, slowly stirring for 24 hours, centrifuging, completely cleaning the precipitate with deionized water, adding a 0.5M acetic acid solution and pepsin, slowly stirring at 37 ℃ for hydrolysis, heating to 50 ℃, keeping for about half an hour, filtering, filling into a dialysis bag with the cut-off molecular weight of 10kDa, dialyzing for 3 days at room temperature by using pure water as an external dialysate, and carrying out vacuum freeze drying on the dialyzed glue solution to obtain the squid type II gelatin.
Dissolving 1g of hyaluronic acid (48kDa) in 100mL of deionized water, cooling to 0-4 ℃, adding 4mL of methacrylic anhydride, slowly dropwise adding 2mL of 5M NaOH, reacting for 24 hours, pouring the reaction solution into a dialysis bag (MWCO 3500), dialyzing with deionized water for 2-3 days, freeze-drying to obtain a photosensitive hyaluronic acid derivative HAMA (0.92g), and calculating the content of double bonds to be about 54% according to a nuclear magnetic hydrogen spectrogram.
15mg of HAMA was weighed out on an electronic analytical balance, and dissolved sufficiently in 1mL of a phosphate buffer solution (pH 7), and then 1.5mg of I2959 was added thereto to dissolve sufficiently to prepare a mother liquor containing 1.5% (w/w) of HAMA and 0.15% (w/w) of I2959. According to table 1, 225mg, 300mg or 450mg of squid type II gelatin is added to the mother liquor and dissolved sufficiently at 37 ℃ to form a mixed solution a containing 22.5%, 30% or 45% (w/w) of squid type II gelatin, respectively. The pH of mixture A was adjusted to 8.0 with 1M NaOH solution.
TABLE 1
90mg or 150mg of 4-Arm PEG-SS (senopont, xiamen) was weighed on an electronic analytical balance and dissolved in 1mL of phosphate buffer (pH 7) to obtain a PEG solution with a concentration of 9% or 15% (w/w).
And (3) sucking 100 mu L of PEG solution, quickly adding the PEG solution into 200 mu L of mixed solution A, uniformly mixing by using a vortex oscillator, quickly sucking out the PEG solution, putting the PEG solution into a polytetrafluoroethylene mold, and standing for 2-4 minutes to preliminarily complete the first-time crosslinking reaction of the hydrogel. Irradiating with 365nm ultraviolet light for 3 min, and keeping the irradiation distance at 3 cm. In each group of the final double-network hydrogels obtained in this example, the second network had a composition of 1% HAMA-0.1% I2959.
From FIG. 1, it can be seen that the obtained squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel is basically transparent.
Example 2 mechanical Strength test of Squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel
And the mechanical property analysis adopts a GT-TCS-2000 high-molecular tensile machine, and the prepared hydrogel sample is placed on a test platform for testing. This example investigated the effect of squid type II gelatin and PEG concentration on hydrogel mechanical strength. FIG. 2A shows the compressive strength of the hydrogels prepared according to the formulations in Table 1. It can be seen that when the final concentration of gelatin is 20% or 30% and the final concentration of PEG is 5%, the strength of the prepared squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel is the best, and when the final concentration of gelatin is 15%, the mechanical strength of the obtained hydrogel is still poor even if the final concentration of PEG reaches 5%.
Example 3 degradation detection of Squid type II gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel
Preparing 20% gelatin-5% PEG hydrogel and 30% gelatin-5% PEG hydrogel according to the method in the embodiment 1, sterilizing squid type II gelatin by adopting an irradiation method before preparing the hydrogel, keeping aseptic operation in the hydrogel preparation process, and respectively carrying out suction filtration and sterilization treatment on HAMA/12959 mixed solution and PEG solution by using a 0.22-micrometer filter to finally obtain the aseptic hydrogel. Hydrogel samples were weighed, soaked in sterile PBS, sealed and placed in a 37 ℃ environment, and tested for residual hydrogel rates after 2, 4 and 8 weeks, respectively. As can be seen from fig. 2B, the degradation rate of the hydrogel containing 30% gelatin was higher than that of the hydrogel containing 20% gelatin (p < 0.01), which is probably because the ratio of amino groups and carboxyl groups in the 20% gelatin-5% PEG-1% HAMA-0.1% I2959 hydrogel was matched to form a sufficient crosslinking reaction; in the 30% gelatin-5% PEG-1% HAMA-0.1% I2959 hydrogel, the gelatin component is relatively supersaturated, so that uncrosslinked groups exist in the hydrogel, and the hydrogel is relatively insufficient in crosslinking and is easy to degrade.
Residual ratio (%) ═ m 1 -m 0 )/m 0 ×100％
In this experiment, m 1 Refers to the weight, m, of the sterile hydrogel after 2, 4, or 8 weeks of immersion in sterile PBS 0 Refers to the weight of the hydrogel prior to soaking in PBS.
Example 4 Squid type II gelatin- [4-Arm PEG-SS ] -HAMA double network hydrogel induces cartilage differentiation Effect of costal perichondrium-derived cartilage stem/precursor cells in vitro
References [ Xue K, Zhang X, Qi L, et al, Isolation, identification, and compliance of vehicle step promoter cells from autologous vehicle and perenniam, Am J Transl Res, 2016; 8(2):732-41], cartilage stem/precursor cells in mouse costal chondral periosteum were extracted and cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. Inoculating the cartilage stem/precursor cells with good growth state to the surface of squid type II gelatin double-network hydrogel (the formula of the hydrogel is 20% gelatin-5% PEG-1% HAMA-0.1% I2959), culturing for 3 days, 7 days and 10 days conventionally, extracting cell mRNA to perform PCR experiment, and detecting the expression change of chondrocyte differentiation marker genes (Col2 alpha 1, Acan and Sox-9) and chondrocyte dedifferentiation marker genes (Col1 alpha 1 and ColX). As a result, as shown in fig. 3, the hydrogel significantly promoted the expression of the chondrocyte differentiation marker gene in the perichondrial/precursor cells (×) and inhibited the expression of the chondrocyte dedifferentiation marker gene (×) and demonstrated that the hydrogel promoted chondrogenic differentiation of the perichondrial-derived perichondrial/precursor cells. The reason for this is presumed to be that the squid type II gelatin double-network hydrogel contains type II collagen hydrolysate, namely type II gelatin, which promotes the maintenance of the phenotype of chondrocytes, and the hydrogel may release certain amino acid components beneficial to the growth and differentiation of the chondrocytes while being gradually degraded.
TABLE 2
Example 5 in vivo Effect of Squid type II gelatin- [4-Arm PEG-SS ] -HAMA double network hydrogel on promoting repair of costal cartilage defects in rabbits
New Zealand white rabbits were used, and the weights of the rabbits were randomly divided into nine groups before the experiment, and according to the schematic diagram of FIG. 4A, costal periosteum and costal cartilage were carefully separated from the 5 th costal cartilage and the 6 th costal cartilage on the left and right sides of the rabbit, and then 2cm costal cartilage was cut off from the middle. Placing prepared sterile costal cartilage substitute hydrogel at the 5 th and 6 th costal cartilage defect parts on the right side of the rabbit, namely, preparing squid II type gelatin double-network hydrogel with the shape of costal cartilage according to a formula of 20% gelatin-5% PEG-1% HAMA-0.1% I2959 (the specific preparation method comprises the steps of cutting the costal cartilage of the rabbit in advance, removing connective tissues and a perichondrium around the costal cartilage, turning the cut costal cartilage into a female die by using Polytetrafluoroethylene (PTFE), carrying out sterilization treatment on the female die by adopting an autoclave sterilization method, rapidly placing uncured liquid state hydrogel into a die when preparing the hydrogel, taking out the hydrogel after curing is finished, namely, obtaining a costal cartilage substitute repair material used in animal experiments), and suturing the costal cartilage film to serve as a hydrogel repair group; the 5 th and 6 th costal cartilage defects on the left side of the rabbit were not repaired, and the perichondrium was directly sutured to serve as a control group. Performing CT scanning on thoracic cage parts at 2 weeks, 4 weeks and 8 weeks respectively, performing three-dimensional reconstruction, performing excessive anesthesia to kill the rabbits, taking materials from rib defect parts of the hydrogel repair group and the control group, fixing, cutting a small part of normal costal cartilage on two sides of the defect, making tissue sections, and performing safranin O-fast green staining. Fig. 4B shows a CT scan three-dimensional reconstruction of the costal cartilage of a rabbit at 8 weeks after surgery, fig. 4C shows safranin O-fast green staining of the tissue section of the corresponding costal cartilage defect site, both of which suggest: after 8 weeks of operation molding, the cartilage defect at the side not filled with hydrogel is not naturally healed and repaired, but is occupied by granulation tissues and the like; after 8 weeks of filling the defect with hydrogel, the hydrogel was substantially completely degraded and the defect was not visibly infiltrated with inflammatory cells, but rather was filled with cartilage tissue. In addition, safranin O-fast green staining showed a clearly deep red color, demonstrating that the defect site essentially produced new cartilage, presumably cartilage stem/precursor cells present in the remaining costal cartilage membrane were induced by the hydrogel, gradually differentiated, and new cartilage tissue was generated; on the control side, the costal cartilage membrane was also retained, but cartilage differentiation was not achieved due to lack of a suitable differentiation environment. The results show that the squid type II gelatin double-network hydrogel has good biocompatibility and has good repairing effect on the defects of costal cartilage.
The invention firstly makes amino in gelatin react with activated carboxyl in 4-Arm PEG-SS to initially form a first network of hydrogel, then uses illumination to initiate photosensitive HAMA in the hydrogel to generate double bond polymerization reaction, and continuously forms a second network on the basis of the double bond polymerization reaction, further strengthens crosslinking, has the compressive strength as high as 11MPa, and basically meets the requirement of mechanical properties of costal cartilage. The hydrogel has degradability. In vitro, the cartilage stem/precursor cell has good compatibility with cells of cartilage stem/precursor cells derived from mouse costal cartilage membranes, can directly induce cartilage differentiation of cartilage stem/precursor cells, and promotes cartilage repair. In vivo, the squid type II gelatin double-network hydrogel can obviously promote the regeneration and repair of the defective costal cartilage along with the gradual occurrence of degradation under the condition that neither seed cells (chondrocytes or stem cells) are wrapped nor cartilage induction factors are added.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai university of traffic medical college affiliated ninth people hospital
East China University of Technology
<120> preparation method and application of squid type II gelatin double-network hydrogel
<130> /
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<170> PatentIn version 3.5
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Claims (8)
1. A preparation method of squid type II gelatin double-network hydrogel is characterized by comprising the following steps:
(1) dissolving methacrylate modified hyaluronic acid HAMA in a biocompatible medium, sequentially adding a photosensitizer I2959 and squid type II gelatin into the biocompatible medium, and adjusting the pH value to be alkalescent by using an alkaline solution to obtain a mixed solution A, wherein the concentration of the methacrylate modified hyaluronic acid in the mixed solution A is 0.6-6% (w/w), the concentration of the photosensitizer I2959 is 0.06-0.6% (w/w), and the concentration of the squid type II gelatin is 22.5-45% (w/w);
(2) dissolving four-Arm polyethylene glycol succinimidyl ester 4-Arm PEG-SS in a biocompatible medium to obtain a PEG solution, wherein the concentration of the 4-Arm PEG-SS in the PEG solution is 9-15% (w/w);
(3) mixing the PEG solution obtained in the step (2) with the mixed solution A obtained in the step (1), wherein the amino group of the squid type II gelatin in the mixed solution A reacts with the carboxyl group of the 4-Arm PEG-SS to form amide bond crosslinking, so as to obtain a first network in the double-network hydrogel, and in the obtained product, the final concentration of the squid type II gelatin is 15% -30% (w/w), the final concentration of the 4-Arm PEG-SS is 3% -5% (w/w), the final concentration of HAMA is 0.4% -4% (w/w), and the final concentration of I2959 is 0.04% -0.4% (w/w);
(4) and (3) irradiating the hydrogel obtained in the step (3) by using 365nm ultraviolet light, and polymerizing double bonds of the methacrylate modified hyaluronic acid to form gel by light initiation, so that a second network of the hydrogel is crosslinked, and the squid II type gelatin- [4-Arm PEG-SS ] -HAMA double-network hydrogel for preparing the costal cartilage defect repair material is obtained.
2. The preparation method of the squid type II gelatin dual-network hydrogel according to claim 1, wherein the squid type II gelatin in the step (1) is prepared by freeze-drying squid cartilage, crushing, adding EDTA solution at room temperature for slow stirring, washing and precipitating with deionized water after centrifugation, adding NaOH solution for slow stirring, centrifuging, washing and precipitating, sequentially adding acetic acid solution and pepsin, slowly stirring and hydrolyzing at 37 ℃, heating to 50 ℃ for a period of time, filtering, dialyzing, and vacuum freeze-drying a glue solution to obtain the squid type II gelatin.
3. The preparation method of the squid type II gelatin double-network hydrogel according to claim 1, wherein the concentration of HAMA in the mixed solution A in the step (1) is 1.5% (w/w); the concentration of photosensitizer I2959 was 0.15% (w/w); the concentration of the squid type II gelatin is 30 to 45 percent (w/w).
4. The preparation method of the squid type II gelatin double-network hydrogel according to claim 1, wherein in the first network of the hydrogel obtained in the step (3), the final concentration of the squid type II gelatin is 20-30% (w/w), and the final concentration of the 4-Arm PEG-SS is 5% (w/w); in the second network of the hydrogel obtained in the step (4), the final concentration of HAMA is 1% (w/w), and the final concentration of I2959 is 0.1% (w/w).
5. The method for preparing a squid type II gelatin double-network hydrogel as claimed in claim 1, wherein the biocompatible medium is selected from one or more of distilled water, normal saline, buffer solution and cell culture medium solution.
6. The preparation method of the squid type II gelatin double-network hydrogel according to claim 1, wherein the volume ratio of the mixed solution A to the PEG solution is 2: 1.
7. The preparation method of the squid type II gelatin double-network hydrogel according to claim 1, wherein the wavelength of the light source used for illumination in the step (4) is 365 nm; the illumination time is 2-5 minutes; the distance between the light source and the hydrogel was 3 cm.
8. Use of the squid type II gelatin double-network hydrogel prepared by the preparation method of any one of claims 1 to 7 in preparation of costal cartilage defect repair materials.
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