Preparation method of shape-adaptive hydrogel material for tissue repair

Abstract

The invention provides a preparation method of a shape-adaptive hydrogel material for tissue repair. The method comprises the following steps: S1, preparing HPC (Hydroxypropyl Chitosan); s2, preparation of phenylboronic acid functional chitosan PHPC (Polyhydroxyalkanoate); s3, preparing a PHPC solution; s4, preparing a polyvinyl alcohol (PVA) solution; and S5, preparing the dynamic covalent bond hydrogel. According to the invention, a dynamic covalent bond-boric acid ester bond with stimulation response capability is introduced on the basis of common dual-network CS-PVA hydrogel; on the premise of not changing the biocompatibility and biodegradability of the original dual-network hydrogel, the hydrogel is endowed with response capability under physical and chemical stimulation, and the diversity of application scenes is expanded; the dynamic mechanical behavior changes of the PCA hydrogel at four different strain rates of 0.05 mm / s, 0.5 mm / s, 5 mm / s and 50 mm / s are explored, and the passive shape adaptability of the PCA hydrogel along with tissue growth is studied; meanwhile, changes of hydrogel creep deformation and stress relaxation caused by addition of dynamic covalent bonds are researched.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of modification of high molecular materials and preparation of double network hydrogel, and particularly relates to a preparation method of shape-adaptable hydrogel material for tissue repair. BACKGROUND

[0002] For a long time, solution processing of natural polymer has been a difficult problem. In 2004, the team of Academician Zhang Lina first proposed a green dissolving formula based on NaOH and urea, which realized the complete dissolution of difficultly soluble cellulose under normal atmospheric pressure. Reference: Cai J, Zhang L, Zhou J, et al. Novel Fibers Prepared from Cellulose in NaOH / Urea Aqueous Solution [J]. Macromolecular Rapid Communications, 2004, 25(17): 1558-1562. This research result was later applied to the solvent development of various natural polysaccharide polymers including chitosan. Liu et al. reported the gelation mechanism of chitosan in alkali / urea solution, revealed the gelation process of chitosan alkali hydrogel and explored its physicochemical properties, reference: Liu S, Li L. Unique gelation of chitosan in an alkali / urea aqueous solution [J]. Polymer, 2018, 141: 124-131.

[0003] The exploration of hydrogel materials in tissue engineering applications has lasted for a long time, but at present, most of the implementation schemes of hydrogel materials for tissue repair are irreversible fixed cross-linked single network or double network hydrogel materials. For example, Luca et al. (Luca, Coluccino L, Gottardi R, Ayadi F, et al. Porous Poly(vinyl alcohol)-Based Hydrogel for Knee Meniscus Functional Repair [J]. ACS Biomaterials Science & Engineering, 2018: acsbiomaterials.7b00879) prepared a single network porous PVA-based hydrogel material for knee meniscus functional repair scheme, first added sodium bicarbonate powder with different particle sizes into PVA aqueous solution for freeze-thaw cycle, then used hydrochloric acid to clear sodium bicarbonate powder, and finally obtained a stable and biocompatible PVA hydrogel material. In the scheme of double network hydrogel material, for example, Shichao Bi et al. (Bi S, Wang P, Hu S, et al. Construction of physical-crosslink chitosan / PVA double-network hydrogel with surface mineralization for bone repair [J]. Carbohydrate Polymers, 2019, 224: 115176) invented a technology scheme of flexible CS-PVA double network hydrogel based on alkali solution system and hydrogen bond, which first dissolved chitosan in alkali, urea solution through freeze-thaw cycle to form a solution, then added PVA aqueous solution to form a mixed solution, and formed a PVA crystalline network after several freeze-thaw cycles. Then the solution was heated at 45°C for 1.5 hours to form a CS second network. Finally, the CS-PVA double network hydrogel was formed by dialysis.

[0004] Dynamic covalent hydrogel is a material composed of hydrophilic or amphiphilic polymer network cross-linked by dynamic covalent bonds. Compared with hydrogen bond and ionic cross-linking, dynamic covalent cross-linking has stronger stability, and it also has unique characteristics such as self-healing, injectable and responsiveness, which makes it have wide application prospect in the field of biomedicine. Dynamic covalent bonds, such as imine bond, borate ester bond and disulfide bond, endow hydrogel with stimulus responsiveness. These chemical bonds can be broken and recombined under specific physiological stimuli, such as pH change, mechanical stimulus, temperature change, redox condition or enzyme action, so as to realize the dynamic change of materials.

[0005] Hard materials have long been the first choice for tissue repair materials, especially in the repair of hard tissues such as bone tissue with high self-stiffness. For example, external plaster supports, implanted alloy supports, bone cement, etc. Countless patients have benefited from the application of such hard repair materials. At the same time, in the repair of bone defects in children, especially in the treatment of bone defects caused by pathogenic factors such as osteosarcoma common in children, there is a difficult problem to choose, that is, the lack of "growth" of widely used hard materials makes it impossible to match the growth and development process of children. This directly leads to many patients becoming disabled or having to replace the matching implanted material through multiple surgeries. Such problems also occur in the repair process of adult muscles, nerves and skin. After many tissues are damaged, they often appear an acute swelling period and then slowly subside. If hard materials are selected for repair during this process, the material may cause pressure on the tissue during the swelling period and cannot return to the original position after the swelling subsides.

[0006] During the growth and development of the human body or after the soft tissue is damaged, the shape of the tissue changes. When the traditional treatment material faces such a situation, because it does not have the ability to change shape, it will limit tissue growth and repair or squeeze the edematous injured tissue, which will lead to the failure of the repair process.

[0007] For example, the announcement number: CN202310430277 discloses a hydrogel for wound repair and its preparation method and application. The hydrogel for wound repair is formed by a plurality of reversible bonds between polyvinyl alcohol, borax, oligomeric proanthocyanidin and trivalent iron ions to form a hydrogel with shape adaptability, self-healing and strong adhesion, and can be quickly removed on demand; the hydrogel for wound repair of the present application can be used for damage repair at active tissue sites. The hydrogel for wound repair has strong antioxidant, rapid antibacterial and hemostatic functions, as well as photothermal effect, which can quickly eliminate infection, reduce oxidative stress, regulate inflammation, promote angiogenesis, and ultimately accelerate wound healing under the assistance of near-infrared irradiation. The hydrogel material discloses that the hydrogel utilizes the mutual crosslinking action of hydrogen bonds formed between polyvinyl alcohol and oligomeric proanthocyanidin, borate ester bonds formed between polyvinyl alcohol and / or oligomeric proanthocyanidin and sodium tetraborate, and metal chelate bonds formed between polyvinyl alcohol and / or oligomeric proanthocyanidin and trivalent iron ions to realize gelation.

[0008] The invention applies three different crosslinking methods, dynamic covalent bond, ion, hydrogen bond, which are all classified as dynamic crosslinking bond. The hydrogel obtained by this pure dynamic crosslinking method usually does not have good shape stability and weak resistance to external environment changes. It is considered that the rapid degradation in water is conducive to the rapid removal of the hydrogel dressing, which also means that the crosslinking structure of the hydrogel is not stable enough and is prone to structural collapse under external environmental changes.

[0009] In addition, the hydrogel material prepared according to the existing synthesis method is prone to spatial non-uniformity, that is, uneven crosslinking. This will lead to the mixing of dense and loose crosslinking domains in the network. Such non-uniformity significantly affects the macroscopic performance of the hydrogel. According to the preparation method described in the patent application, the inventors do not graft boronic acid groups to the polymer chain, but directly add stirring, which increases the randomness of borate ester bond crosslinking and increases the possibility of uneven crosslinking, which may affect the macroscopic mechanical properties of the hydrogel material to some extent. SUMMARY

[0010] In order to solve the problems of hard materials in the process of tissue repair, including the change of tissue morphology that may occur during the growth and development of the human body or after the soft tissue is damaged, the traditional treatment materials do not have the ability to change the shape when they face such a situation, so they will limit the growth and repair of the tissue or squeeze the edema of the injured tissue. This will lead to the failure of the repair process. Therefore, the present application provides a preparation method of a shape-adaptive hydrogel material for tissue repair.

[0011] A preparation method of a shape-adaptive hydrogel material for tissue repair, the method comprising the following steps:

[0012] S1, preparation of hydroxypropyl chitosan HPC

[0013] Lithium hydroxide, potassium hydroxide, urea and deionized water are configured into an alkali / urea solution of 200 mL in a ratio of 4.5:7:8:80.5, and the prepared solution is placed in a-20℃ refrigerator for pre-cooling for 4h; take out the pre-cooled liquid, weigh 4g of chitosan CS powder and add it to the alkali / urea solution and stir for 2h, then observe the liquid for no agglomeration of insoluble substances, then place the stirred mixed solution in a-20℃ refrigerator and freeze overnight; take out the frozen liquid and thaw at room temperature for 4h, then quickly stir for 2h after all the solid material is thawed; repeat the freezing-thawing cycle 3 times, and observe that the mixed solution is clear and has no obvious insoluble substances, and then the CS / alkali / urea solution is obtained;

[0014] To the CS / base / urea solution, 7 g of 3-chloro-1-propanol was added and stirred at room temperature for 24 h; at the same time, prepare enough 0.1 mol / L hydrochloric acid solution; after the reaction is completed, slowly add 0.1 mol / L hydrochloric acid solution to the reaction liquid to neutralize the residual basic substances in the solvent until the reaction liquid is neutral; the neutralized liquid is loaded into a 8 kDa dialysis bag prepared in advance, and the dialysis bag is placed in 2 L of deionized water, which is replaced every 6 h, and dialysis is performed for 7 days;

[0015] S2, Preparation of phenylboronic acid functionalized chitosan PHPC

[0016] Take 4 g of HPC and dissolve it in 400 mL of 2-(N-morpholino) ethanesulfonic acid MES, 0.05 M buffer, stir at room temperature for 4 h, and observe that the hydroxypropyl chitosan is completely dissolved in the MES buffer; 4.7 g of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC, 3.4 g of N-hydroxysuccinimide NHS, and 4 g of 4-carboxyphenylboronic acid are dissolved in 50 mL of MES, 0.05 M buffer, and stirred in the dark for 30 min; after stirring is completed, slowly add the EDC / NHS / 4-carboxyphenylboronic acid solution to the stirring HPC solution, continue stirring for 48 h after the addition is completed, and the process should be operated in a dark environment; after the reaction is completed, the solution is divided into dialysis bags prepared in advance, and the dialysis bags are placed in 5 L of deionized water, which is replaced every 6 h, and dialysis is performed for 7 days; stop dialysis, transfer the dialysate to a glass dish, seal with a film, and freeze in a -20℃ refrigerator until the liquid is completely frozen;

[0017] S3, Preparation of PHPC solution

[0018] Add 1 g of PHPC to a beaker containing 99 mL of deionized water, heat in a water bath at 60℃ and stir for 1 h, take out the solution and observe that there is no insoluble material, and the solution is clear and transparent, i.e. 1 wt% PHPC solution is obtained;

[0019] S4, Preparation of polyvinyl alcohol PVA solution

[0020] Add 15 g of PVA powder to a beaker containing 85 g of deionized water, heat in a water bath at 90℃ and stir for 6 h, take out the solution and observe that there is no insoluble material, and the solution is clear and transparent, i.e. 15 wt% PVA solution is obtained;

[0021] S5, Preparation of dynamic covalent hydrogel

[0022] Take 4g of PHPC solution and 6g of PVA solution respectively, slowly drop the PVA solution into the PHPC solution under stirring; after the dropping is completed, continue to stir for 2h, and observe that there is no insoluble flocculation in the solution; after the stirring is completed, transfer the solution into a mold, seal and transfer into a-20℃ refrigerator for freezing for 12h; after taking out, a dynamic covalent hydrogel material is obtained.

[0023] Preferably, in step S1, the dialysis is stopped, the dialysate is transferred into a glass dish, sealed with a film, and placed in a-20℃ refrigerator for freezing until the liquid is completely frozen; the pre-frozen liquid is taken out, placed in a freeze dryer, and freeze-dried for 48h to obtain dried HPC.

[0024] Preferably, in step S2, the pre-frozen liquid is taken out, placed in a freeze dryer, and freeze-dried for 48h to obtain dried PHPC.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] 1. The present application introduces a dynamic covalent bond-boronate ester bond with a stimulus response ability on the basis of ordinary double network CS-PVA hydrogel; without changing the biocompatibility and biodegradability of the original double network hydrogel, the response ability under physical and chemical stimulation is given, and the diversity of application scenarios is expanded.

[0027] 2. The present application explores the dynamic mechanical behavior change of PCA hydrogel under four different strain rates of 0.05mm / s, 0.5mm / s, 5mm / s and 50mm / s, studies the passive shape adaptability with tissue growth, and studies the change of hydrogel creep and stress relaxation caused by the addition of dynamic covalent bond.

[0028] 3. The present application explores the morphological change of PCA hydrogel after being stimulated by ROS, and further verifies the possibility of realizing the active shape adaptation process through artificial active intervention in the in-vivo application of the PCA hydrogel. DETAILED DESCRIPTION

[0029] Figure 1 It is a schematic diagram of the molecular mechanism of the dynamic change of the boronate ester bond of the present application; (a) reversible change of boronate ester bond, (b) volume change of hydrogel and (c) molecular mechanism of double stimulation;

[0030] Figure 2 It is the PCA hydrogel of the present application (a) FT-IR diagram; (b) data diagram of grafting rate test by indophenol color reaction;

[0031] Figure 3 It is the SEM diagram of the dynamic covalent bond double network hydrogel of the present application;

[0032] Figure 4Mechanical behavior analysis graph of the present application under high and low strain loading; (a) stress-strain test digital photo and (b) high and low strain loading stress-strain curve, (c) elastic modulus analysis data, balloon simulation tissue growth;

[0033] Figure 5 PCA hydrogel in vitro ROS stimulation experiment graph of the present application; (a) PCA hydrogel digital photo during the experiment process, (b) contour processing stacked image and (c) data analysis, (d) PCA hydrogel rapid degradation after high concentration ROS stimulation and (e) SEM image of microstructure change after ROS treatment;

[0034] Figure 6 In vivo ROS stimulation response experiment graph of the present application; (a) experiment process schematic diagram and (b) experiment process digital photo, (c) IVIS live imaging picture, (d) contour processing stacked image and (e) data analysis graph;

[0035] Figure 7 Histology graph of SD rat organs and skin after different treatments for 7 days of the present application; (a) SD rat heart, liver, spleen, lung, kidney H&E staining picture and (b) contact skin H&E, Masson staining picture. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] A preparation method of a shape-adaptive hydrogel material for tissue repair, the method comprising the following steps:

[0038] S1, preparation of hydroxypropyl chitosan HPC

[0039] Lithium hydroxide, potassium hydroxide, urea and deionized water are configured into an alkali / urea solution of 200 mL in a ratio of 4.5:7:8:80.5, and the prepared solution is placed in a-20℃ refrigerator for pre-cooling for 4h; the pre-cooled liquid is taken out, 4g of chitosan CS powder is weighed and added to the alkali / urea solution and stirred for 2h, after observing that there is no insoluble substance agglomerated into lumps in the liquid, the stirred mixed solution is placed in a-20℃ refrigerator for freezing overnight; the frozen liquid is taken out and thawed at room temperature for 4h, and after all the solid substances are thawed, it is quickly stirred for 2h; the freezing-thawing cycle is repeated for 3 times, and the mixed solution is observed to be clear without obvious insoluble substances, and the CS / alkali / urea solution is obtained;

[0040] To the CS / base / urea solution, 7g of 3-chloro-1-propanol was added and stirred at room temperature for 24h; at the same time, prepare enough 0.1mol / L hydrochloric acid solution; after the reaction is completed, slowly add 0.1mol / L hydrochloric acid solution to the reaction liquid to neutralize the residual basic substances in the solvent until the reaction liquid is neutral; the neutralized liquid is loaded into a 8kDa dialysis bag prepared in advance, and the dialysis bag is placed in 2L of deionized water, which is replaced every 6h, and dialysis is carried out for 7 days;

[0041] S2, Preparation of phenylboronic acid functionalized chitosan PHPC

[0042] Take 4g of HPC and dissolve it in 400mL of 2-(N-morpholino)ethanesulfonic acid MES, 0.05M buffer, stir at room temperature for 4h, and observe that the hydroxypropyl chitosan is completely dissolved in the MES buffer; 4.7g of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC, 3.4g of N-hydroxysuccinimide NHS and 4g of 4-carboxyphenylboronic acid are dissolved in 50mL of MES, 0.05M buffer, and stirred in the dark for 30min; after stirring is completed, slowly add the EDC / NHS / 4-carboxyphenylboronic acid solution to the stirring HPC solution, continue stirring for 48h after the addition is completed, and the process should be operated in the dark; after the reaction is completed, the solution is divided into dialysis bags prepared in advance, and the dialysis bags are placed in 5L of deionized water, which is replaced every 6h, and dialysis is carried out for 7 days; stop dialysis, transfer the dialysate to a glass dish, seal with film, and freeze in a-20℃ refrigerator until the liquid is completely frozen;

[0043] S3, Preparation of PHPC solution

[0044] Add 1g of PHPC to a beaker containing 99mL of deionized water, heat in a water bath at 60℃ and stir for 1h, take out the solution and observe that there is no insoluble material, and the solution is clear and transparent, i.e. 1wt% PHPC solution is obtained;

[0045] S4, Preparation of polyvinyl alcohol PVA solution

[0046] Add 15g of PVA powder to a beaker containing 85g of deionized water, heat in a water bath at 90℃ and stir for 6h, take out the solution and observe that there is no insoluble material, and the solution is clear and transparent, i.e. 15wt% PVA solution is obtained;

[0047] S5, Preparation of dynamic covalent hydrogel

[0048] Take 4 g of PHPC solution and 6 g of PVA solution respectively, slowly add the PVA solution into the PHPC solution under stirring; after the addition is completed, continue to stir for 2 h, and observe that there is no insoluble flocculation in the solution; after the stirring is completed, transfer the solution into a mold, seal and transfer into a-20℃ refrigerator for freezing for 12 h; after taking out, a dynamic covalent hydrogel material is obtained.

[0049] In this embodiment, in step S1, dialysis is stopped, the dialysate is transferred into a glass dish, sealed with a film, and placed in a-20℃ refrigerator to freeze until the liquid is completely frozen; the pre-frozen liquid is taken out and placed in a freeze dryer for freeze-drying for 48 h to obtain dried HPC.

[0050] In this embodiment, in step S2, the pre-frozen liquid is taken out and placed in a freeze dryer for freeze-drying for 48 h to obtain dried PHPC.

[0051] The method content includes "preparation of double network hydrogel with dynamic bond (PCA)", "dynamic mechanical behavior of PCA under high and low strain rate loading", and "morphology study of PCA hydrogel under ROS stimulation", as shown in Figure 1 .

[0052] FT-IR analysis of PCA hydrogel

[0053] The FT-IR results of the dynamic covalent double network hydrogel are shown in Figure 2 , the C-H stretching vibration peak at 2880 cm -1 and the C=O stretching vibration peak of amide I band at 1660 cm -1 are the FT-IR characteristic absorption peaks of CS. After modification by 3-chloro-1-propanol, the N-H stretching vibration peak at 1596 cm -1 appears on the FT-IR spectrum of HPC. After functional modification by phenylboronic acid, the stretching vibration peak of benzene ring C=C (skeleton) appears at 1540 cm -1 , which indicates that 4-carboxyl phenylboronic acid has been successfully grafted onto HPC.

[0054] The characteristic absorption peak of PVA is the C-O stretching vibration peak at 1090 cm -1 and the -OH stretching vibration peak at 3334 cm -1 . In the FT-IR spectrum of the dynamic covalent double network hydrogel, the C=C stretching vibration peak at 1540 cm -1 from PHPC and the C-O stretching vibration peak at 1090 cm -1 and the -OH stretching vibration peak at 3334 cm -1the -OH stretching vibration peak. The above results show that the synthesis of the dynamic covalent bond double network hydrogel is successful. At the same time, the standard curve of CS raw material is drawn by the indantrione standard colorimetric method. The PHPC OD570 of the experimental group is 0.386, and the grafting rate of -NH in PHPC is about 13.65% calculated by the standard curve. This is similar to the grafting rate reported in the literature by modifying CS using a similar method.

[0055] Micro-morphology analysis of PCA hydrogel

[0056] In order to understand the micro-morphology of the PCA dynamic covalent bond double network hydrogel prepared in the experiment, the brittle fracture surface of the freeze-dried hydrogel was observed by SEM, and the results are shown in Figure 3 The image shows that the fracture surface of the PCA hydrogel presents a three-dimensional network porous structure uniformly dispersed, which is consistent with the typical micro-morphology characteristics of hydrogel materials. This proves the successful preparation of the dynamic covalent bond double network hydrogel.

[0057] Dynamic mechanical property analysis of PCA hydrogel

[0058] The prepared PCA hydrogel was clamped in a universal material testing machine according to Figure 4 (a) to perform high and low strain rate tension, and the obtained data were analyzed and processed to obtain the results shown in Figure 4 (b). At an extremely slow tension rate of 0.05 mm / s, the stress of the PCA hydrogel fluctuates within a very small range. With the increase of the tension rate, the stress of the PCA hydrogel also increases synchronously, showing more and more strong elastic mechanical behavior.

[0059] When the tension rate reaches 50 mm / s, the stress of the material rapidly increases with the tension. Through the extraction and analysis of the test data, the elastic modulus results shown in Figure 4 (c) are obtained. At a tension rate of 0.05 mm / s, the elastic modulus of the PCA hydrogel is only about 2 kPa, while at a tension rate of 50 mm / s, the elastic modulus increases to about 28 kPa, showing a significant statistical difference.

[0060] In vitro ROS stimulation analysis of PCA hydrogel

[0061] In the previous experiment, we found that the shape of the PCA hydrogel changes under the stimulation of ROS, and through this experiment we quantified this change process. The results of the change of the PCA hydrogel under the stimulation of ROS are shown in Figure 5(a) shown. On day 0, the size of each group of hydrogels was basically the same. On day 1, the size of each group of hydrogels increased to a certain extent, which might be due to the swelling of the hydrogels in the PBS solution. At the same time, we observed that the size change of the group with ROS was significantly higher than that of the group without ROS stimulation, and the higher the concentration of ROS, the more obvious the size change. Using ImageJ software to process the images, the approximate outline of each group of PCA hydrogels was extracted and stacked.

[0062] The results are shown in Figure 5 (b) shown, the size change of the 0 mM ROS stimulation group within 7 days was very small, the size change of the 0.2 mM ROS stimulation group within 7 days changed to a certain extent, and the size change of the 0.7 mM ROS stimulation group within 7 days was the most obvious. Stacking the outline of each group of PCA hydrogels at day 7, we can see that the size of each group of PCA hydrogels has a relatively obvious difference, and this change has a clear ROS concentration dependence.

[0063] The quantitative analysis data are shown in Figure 5 (c) shown, in which the size change of the 0 mM ROS stimulation group was 6.95%, the size change of the 0.2 mM ROS stimulation group was 29.90%, and the size change of the 0.7 mM ROS stimulation group was 80.02%. This ROS concentration regulated material size change ability means that we can achieve subjective control of the morphology of the implanted material in the body by simply injecting a relatively high ROS environment locally. For children patients in the rapid growth and development period or tissues with a large deformation during the repair process, this material has the ability to adjust the morphology, which has a significant advantage compared to traditional fixed shape materials.

[0064] To further verify the effect of high concentration ROS on PCA hydrogels, high concentration (30wt% hydrogen peroxide) was added to the culture dish and waited for 5min, and it was observed that the PCA hydrogels changed from gel state to solution state, and could be easily poured out from the culture dish. This result shows that the gel network in the PCA hydrogel is completely destroyed under the stimulation of high concentration ROS. In other words, the degradation behavior of the PCA hydrogel is artificially intervened by controlling the concentration of ROS. Figure 5 (d) shown.

[0065] The PCA hydrogel samples after ROS treatment were placed in the SEM to observe the obvious difference in micro-morphology from the untreated samples. As shown in Figure 5(e) shows that the cross-section of the hydrogel sample is flat and the pore size distribution is uniform after ROS treatment. In the enlarged view, the clear hole structure and the closely arranged hole wall structure can be seen. The surface of the sample treated by ROS is no longer flat, and obvious "gullies" appear. In the enlarged view, the hole structure is destroyed, forming a more loose structure. This gradual destruction of the structure allows cells in the repair period to migrate to the interior of the PCA hydrogel, gradually eroding the hydrogel matrix. Finally, the hydrogel is completely degraded, and the newly formed tissue replaces the completely repaired defect tissue.

[0066] In vivo ROS stimulation analysis of PCA hydrogel

[0067] Creation of subcutaneous implant model Figure 6 (b) and IVIS live imaging according to the experimental plan Figure 6 (a). The experimental results are shown in Figure 6 (c). Under the excitation light of 640 nm, the implanted hydrogel material in the body of each group of animal models shows red. By statistical analysis of the area and stacking the contours of the photographed photos of the experimental groups, the results shown in Figure 6 (d) are obtained. Under the action of local high ROS environment stimulation, the size of the hydrogel increases significantly. After statistical analysis of the areas of the experimental and control groups, it can be seen that the area of the pure PVA hydrogel has almost no change within 7 days. The area of the PCA without ROS stimulation group increases slightly, which may be caused by a small amount of ROS generated at the damage site. The PCA and ROS stimulation group shows a significant increase in area compared to the previous two groups, with an increase of about 22% in 7 days. From the above experimental phenomena, it can be known that even if the in vivo environment is more complex and variable than the in vitro experimental environment, the PCA hydrogel can still maintain the response ability to ROS stimulation.

[0068] Tissue compatibility analysis of PCA hydrogel

[0069] To evaluate the treatment safety of PCA hydrogel, the main organs and skin tissue samples in close contact with PCA hydrogel of SD rats were examined by H&E and Masson staining after the end of the experiment. The results are shown in Figure 7 (a). The H&E staining results of the main organ tissue sections of the experimental group SD rats, such as heart, liver, spleen, lung, kidney, etc., show that the main organ structure of SD rats is basically normal, and no obvious pathological tissue is found, which has no obvious difference with the control group.

[0070] Figure 7 (b) shows the H&E and Masson staining results of the skin tissue sections in the contact part with PCA hydrogel. The skin tissue structure is basically normal, the stratum corneum is not obviously thickened, the collagen fibers are arranged in order, and there is no obvious inflammatory cell infiltration in the tissue, and there is no obvious difference with the control group.

[0071] In summary, according to the experimental results above, it can be explained that the PCA hydrogel does not produce obvious toxic side effects in the whole treatment process, and has good biological safety.

[0072] It should be noted that, in this document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0073] The above merely provides preferred embodiments of the present application and does not in any way limit the present application. Although the embodiments of the present application have been shown and described, it is understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a shape-adaptable hydrogel material for tissue repair, characterized by, The method comprises the following steps: S1, preparation of hydroxypropyl chitosan HPC Lithium hydroxide, potassium hydroxide, urea and deionized water are configured in a ratio of 4.5:7:8:80.5 into a 200 mL alkali / urea solution, and the prepared solution is placed in a-20℃ refrigerator for pre-cooling for 4h; take out the pre-cooled liquid, weigh 4g chitosan CS powder into the alkali / urea solution and stir for 2h, and then observe that there is no agglomerated insoluble material in the liquid; after that, the mixed solution is placed in a-20℃ refrigerator for freezing overnight; take out the frozen liquid and thaw at room temperature for 4h; after all the solid material is thawed, quickly stir for 2h; repeat the freezing-thawing cycle for 3 times; observe that the mixed solution is clear and there is no obvious insoluble material, and then the CS / alkali / urea solution is obtained; 7g 3-chloro-1-propanol is added into the CS / alkali / urea solution, and stirred at room temperature for 24h; at the same time, a sufficient amount of 0.1mol / L hydrochloric acid solution is configured; after the reaction is completed, 0.1mol / L hydrochloric acid solution is slowly added into the reaction liquid to neutralize the residual alkaline substances in the solvent until the reaction liquid is neutral; the neutralized liquid is loaded into a prepared 8kDa dialysis bag, and the dialysis bag is placed in 2L deionized water, which is replaced every 6h, and dialysis is carried out for 7 days; S2, preparation of phenylboronic acid functionalized chitosan PHPC 4g HPC is dissolved in 400mL 2-(N-morpholino)ethanesulfonic acid MES, 0.05M buffer, and stirred at room temperature for 4h; after that, it is observed that the hydroxypropyl chitosan is completely dissolved in the MES buffer; 4.7g N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC, 3.4g N-hydroxysuccinimide NHS and 4g 4-carboxyphenylboronic acid are dissolved in 50mL MES, 0.05M buffer, and stirred in the dark for 30min; after the stirring is completed, the EDC / NHS / 4-carboxyphenylboronic acid solution is slowly added into the stirring HPC solution, and after the addition is completed, continuous stirring is carried out for 48h, and the process should be operated in the dark environment; after the reaction is completed, the solution is divided into dialysis bags prepared in advance, and the dialysis bags are placed in 5L deionized water, which is replaced every 6h, and dialysis is carried out for 7 days; stop dialysis, transfer the dialysate to a glass dish, seal with film, and freeze in a-20℃ refrigerator until the liquid is completely frozen; S3, preparation of PHPC solution 1g PHPC is added into a beaker containing 99mL deionized water, heated and stirred in a water bath at 60℃ for 1h, and then the solution is taken out and observed, in which there is no insoluble material and the solution is clear and transparent, and thus 1wt% PHPC solution is obtained; S4, preparation of polyvinyl alcohol PVA solution 15g PVA powder is added into a beaker containing 85g deionized water, heated and stirred in a water bath at 90℃ for 6h, and then the solution is taken out and observed, in which there is no insoluble material and the solution is clear and transparent, and thus 15wt% PVA solution is obtained; S5, preparation of dynamic covalent hydrogel Take 4g PHPC solution and 6g PVA solution respectively, slowly drop the PVA solution into the PHPC solution under stirring; after the dropping is completed, continue to stir for 2h, observe that there is no insoluble flocculent in the solution; after the stirring is completed, transfer the solution into a mold, seal and transfer into a-20℃ refrigerator to freeze for 12h; after taking out, a dynamic covalent hydrogel material is obtained.

2. The method of claim 1, wherein the shape-adaptable hydrogel material for tissue repair is prepared by the steps of: In step S1, stop dialysis, transfer the dialysate into a glass dish, seal with a film, and put into a-20℃ refrigerator to freeze until the liquid is completely frozen; take out the pre-frozen liquid, put it into a freeze dryer to freeze dry for 48h, and obtain dried HPC.

3. The method of claim 1, wherein the shape-adaptable hydrogel material for tissue repair is prepared by the steps of: In step S2, take out the pre-frozen liquid, put it into a freeze dryer to freeze dry for 48h, and obtain dried PHPC.

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