A gradient structure hernia repair patch with ultra-low swelling rate and its preparation method and application

Abstract

The present invention relates to the field of medical materials, specifically a hernia repair material and a preparation method thereof and a hernia patch. The preparation method provided by the present invention adopts layered casting so that different layers can be used as relatively independent functional modules, realizes the functionalization of the patch material, and reduces the swelling rate of the material to the maximum extent by adjusting the thickness of the dense layer and aging treatment; the swelling rate is close to 0 in the optimal case, which helps to achieve tension-free repair; and a gel is generated in situ by phase separation, and a transition zone is generated in situ between layers, and the microstructure is interlocked to ensure the mechanical reliability of the patch material as a whole; through the PVA-DMSO-H2O dissolving system, the pore size of the loose layer is maintained at tens of microns, and the hydrophilicity of the material is improved at the same time, promoting cell growth and tissue adhesion; through the PVA-soluble small molecule filler / DMSO dissolving system, the pore size of the dense layer is maintained at the submicron level, and the hydrophobicity of the material is improved at the same time, preventing cell and tissue adhesion.

Description

Technical Field

[0001] The present invention relates to the field of medical materials, and specifically to a hernia repair material and a preparation method thereof, and a hernia repair sheet. Background Art

[0002] Hernia patches have been widely used in tension-free herniorrhaphy. According to the manufacturing method, hernia patches can be divided into woven and non-woven types. Woven hernia patches include polypropylene (PP) patches, expanded polytetrafluoroethylene (e-PTFE) patches, polyester (PE) patches, etc., which have excellent mechanical properties and biocompatibility; however, their macroscopic morphology presents a large porous structure, and problems such as adhesion of the inner and outer walls, local infection and inflammation cannot be avoided in clinical hernia repair. Non-woven hernia patches are an emerging concept in recent years, such as hydrogel materials, film materials, etc.; ideally, they should not only have an ultra-low swelling rate to achieve tension-free repair, but also have sufficient mechanical properties.

[0003] In fact, there are few research cases that can achieve ultra-low swelling rate and mechanical property guarantee at the same time. For example, researchers such as Sun from Nanjing University published an article entitled "Designing a bi-layer multifunctional hydrogel patch based on polyvinyl alcohol, quaternized chitosan and gallic acid for abdominal wall defect repair" (https: / / doi.org / 10.1016 / j.ijbiomac.2024.130291). PVA, chitosan, and gallic acid were simply blended to form a gel, and then layered casting and salting out were used to prepare a tension-free repair material for abdominal wall hernia with a double-layer structure, antibacterial, and tissue healing. Through modular design, this work achieved anti-tissue adhesion effects in the dense area and antibacterial and tissue healing effects in the porous area. However, its dense layer has no obvious porous structure, and the porous layer is full of interconnected pores with a pore size of hundreds of microns. The dense layer and the porous layer are only connected by PVA, so the mechanical properties of the interface between the layers cannot be fully guaranteed. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a hernia repair material and a preparation method thereof and a hernia patch. The preparation method provided by the present invention can prepare a multi-layer hernia patch, in which different layers have independent functions and high mechanical properties.

[0005] The present invention provides a method for preparing a hernia repair material, comprising the following steps:

[0006] S1) placing the loose layer precursor solution on the dense layer precursor solution;

[0007] The dense layer precursor solution includes PVA and DMSO;

[0008] The loose layer precursor solution includes PVA, DMSO and water; the volume fraction of DMSO in the loose layer precursor solution is 10% to 40%;

[0009] S2) freezing the product obtained in step S1) into a gel to obtain a hernia repair material.

[0010] The present invention first allows the loose layer precursor to stand on the dense layer precursor; specifically, the dense layer precursor is first injected into the mold, and then the loose layer precursor is injected into the mold, so that the loose layer precursor is maintained above the dense layer precursor and stands with the dense layer precursor. In certain embodiments of the present invention, the dense layer precursor is first injected into the mold, and the loose layer precursor is slowly poured into the mold after constant room temperature, so that the loose layer precursor is maintained above the dense layer precursor and stands with the dense layer precursor until the liquid surface is stable and there is no refractive index change in the liquid area under naked eye observation. The present invention adopts layered casting, so that different layers can be used as relatively independent functional modules to realize the functionalization of the patch material; the gel is generated in situ by phase separation, and the transition zone is generated in situ between the layers, and the microstructure is interlocked to ensure the overall mechanical reliability of the patch material.

[0011] The dense layer precursor solution of the present invention includes polyvinyl alcohol (PVA) and dimethyl sulfoxide (DMSO), wherein PVA is a solute and DMSO is a solvent; the loose layer precursor solution includes PVA, DMSO and water, wherein PVA is a solute and DMSO and water are solvents. The mass percentage of PVA in the dense layer precursor solution of the present invention is 9wt% to 11wt%, preferably 10wt%; the mass percentage of PVA in the loose layer precursor solution is 4wt% to 6wt%, preferably 5wt%.

[0012] The volume fraction of the organic solvent in the loose layer precursor solution of the present invention is 10% to 40%, preferably 10% to 30%, more preferably 15% to 25%, even more preferably 18% to 22%, and most preferably 20%. In some embodiments of the present invention, the volume ratio of the dense layer precursor solution and the loose layer precursor solution of the present invention is 4.5: (1 to 2), preferably 4.5: 1.5. The dense layer precursor solution of the present invention is ultimately formed into a dense layer in the hernia repair material, and the loose layer precursor solution is ultimately formed into a loose layer in the hernia repair material. By reasonably setting the thickness ratio of the dense layer to the loose layer, the swelling rate of the material is maintained at a relatively low level, basically reaching within ±0.6%, and the material after immersion in PBS buffer solution for 30 days is even close to 0%.

[0013] The dense layer precursor solution of the present invention is obtained by mixing PVA and DMSO, and the loose layer precursor solution is obtained by mixing PVA, DMSO and water. Specifically, the PVA and DMSO are mixed according to the mass percentage of PVA and the volume fraction of DMSO in the dense layer precursor solution to obtain the dense layer precursor solution, and the PVA, DMSO and water are mixed according to the mass percentage of PVA and the volume fraction of DMSO in the loose layer precursor solution to obtain the loose layer precursor solution.

[0014] The dense layer precursor solution of the present invention also includes a soluble small molecule filler; the mass ratio of the soluble small molecule filler to the PVA in the dense layer precursor solution is (0.8-1.2):1; the soluble small molecule filler is selected from at least one of PEG, ethylene glycol, glycerol, polyglycerol, low molecular weight chitosan and low molecular weight hyaluronic acid. The PVA and PEG described in the present invention are any one or more of all polymerization degrees and alcoholysis degrees. In some embodiments of the present invention, the soluble small molecule filler is selected from PEG200-PEG20000, preferably PEG200-PEG2000, and more preferably PEG1500-PEG2000. The present invention prepares precursor solutions with different functional structural designs and realizes molding by layered casting. Interlayer diffusion forms DMSO-water blend systems with different gradients in the thickness direction of the patch material. The PVA macromolecules dissolved in the system will achieve different degrees of phase separation with the concentration gradient of DMSO, that is, the solubility of the solute in the dominant solvent and the inferior solvent is different, and then different cross-linking degrees are realized in situ. At the same time, a soluble small molecule filler is added to the dense pore layer as a small molecule filler between the PVA macromolecular chains to block the PVA pores, and at the same time, the mechanical strength of the PVA network is improved, thereby enhancing the mechanical strength of the dense layer of the hernia patch.

[0015] According to the present invention, after the loose layer precursor solution is allowed to stand on the dense layer precursor solution, the obtained product is frozen into gel to obtain a hernia repair material; the freezing gel temperature is below 4°C, preferably -25°C to 0°C, more preferably -20°C, and the freezing gel time is 10h to 14h, preferably 12h.

[0016] In certain embodiments of the present invention, after freezing the product obtained by layered pouring into gel, the present invention also includes washing, freeze-drying and rehydrating the gelled product to obtain a hernia repair material. The hernia repair material obtained by the present invention is a hydrogel close to the physiological state. The washing of the present invention specifically involves washing the gelled product with water at a speed of 100 to 200 rpm for 25 to 35 minutes, repeating the washing 5 to 7 times, preferably washing at a speed of 150 rpm for 30 minutes, and repeating the washing 6 times; the washing of the present invention can play a role in removing residual DMSO in the gel. The freeze-drying of the present invention is specifically to pre-freeze the material at -15℃~-25℃ for 1.5h~5h, then freeze the material at -75℃~-85℃ for 1.5h~5h, and then freeze-dry for 20h~25h; preferably, the material is pre-frozen at -15℃~-25℃ for 1.5h~2.5h, then freeze the material at -75℃~-85℃ for 1.5h~2.5h, and then freeze-dry for 20h~25h; most preferably, the material is pre-frozen at -20℃ for 2h, then freeze the material at -80℃ for 2h, and then freeze-dry for 24h. The rehydration of the present invention is specifically to immerse the freeze-dried material in a PBS buffer solution with a concentration of 0.01mol / L~0.02mol / L for hydration for 20h~25h, preferably, the freeze-dried material is immersed in a PBS buffer solution with a concentration of 0.01mol / L for hydration for 24h.

[0017] The present invention can also carry out aging treatment or not carry out aging treatment on the product after gelation before washing, freeze-drying and rehydration. If aging treatment is carried out, the temperature of the aging treatment is room temperature, specifically 20°C to 30°C, and the time of the aging treatment is 3 to 4 days. If the present invention first carries out aging treatment on the product after gelation, and then carries out freeze-drying and rehydration, the obtained hernia repair material can have a lower swelling rate, because the PVA macromolecular chain shows relaxation phenomenon during the phase separation process, and the aging treatment can deepen the PVA phase separation process until it is completely carried out. Therefore, the aging phenomenon makes the PVA network inside the hernia patch material coarsened, and shows good shape-keeping performance during the swelling process. In addition, carrying out aging treatment first, and then freeze-drying and rehydration are also conducive to obtaining better mechanical properties. If the present invention does not carry out aging treatment on the product after gelation, and directly carries out freeze-drying and rehydration, it can also have the functions of promoting cell adhesion and blocking tissue adhesion at the same time.

[0018] The preparation method provided by the present invention is to prepare a precursor solution with different functional structural designs, realize molding by a layered casting method, and adjust the thickness of the dense layer and perform aging treatment to minimize the swelling rate of the material; and interlayer diffusion will form a DMSO-water blend system with different gradients in the thickness direction of the hernia repair material, and the PVA macromolecules dissolved in the system will achieve different degrees of phase separation with the concentration gradient of DMSO, and then realize different crosslinking degrees in situ, and generate a transition zone in situ between the layers, and the microstructure is interlocked to ensure the mechanical reliability of the patch material as a whole; at the same time, a soluble small molecule filler is added between the PVA macromolecular chains of the dense pore layer, blocking the PVA pore formation, and at the same time enhancing the mechanical strength of the dense layer of the hernia patch. In addition, the pore size of the dense layer formed by the PVA-small molecule filler / DMSO dissolution system will be maintained at the submicron level, while improving the hydrophobicity of the material, preventing cell and tissue adhesion; the pore size of the loose layer formed by the PVA-DMSO-H2O dissolution system will be maintained at tens of microns, while improving the hydrophilicity of the material, promoting cell growth and tissue adhesion.

[0019] The present invention also provides a hernia repair material obtained by the preparation method described in any of the above technical solutions. The hernia repair material of the present invention presents the evolution of the material microstructure in the longitudinal section along the thickness direction, and the pore size ranges from 3μm to 5μm in the loose pore area to the submicron level in the dense pore area; the mechanical tensile strength and maximum elongation at break are high, far satisfying the repair strength requirements of general hernia patches; the results of in vivo and in vitro biocompatibility tests show no significant biological toxicity. The hernia repair material of the present invention has a thin thickness and a low swelling rate, which is conducive to achieving tension-free hernia repair. If the hernia repair material of the present invention is not subjected to aging treatment, it can also have the function of promoting cell adhesion and blocking tissue adhesion. The loose pore area can effectively achieve cell adhesion and tissue repair, and the dense pore area can achieve the effect of blocking tissue adhesion inside and outside the hernia orifice.

[0020] The present invention also provides a hernia patch, which is obtained from the hernia patch material obtained by the preparation method described in any of the above technical solutions. The hernia patch processed by the above hernia patch material inherits the performance of the above hernia patch material, is not only suitable for tension-free hernia repair, but also has mechanical tensile strength and maximum elongation at break that far meet the repair strength requirements of general hernia patches, and has no significant biological toxicity; and can also have the function of promoting cell adhesion and blocking tissue adhesion.

[0021] The present invention provides a gradient structure hernia repair patch with ultra-low swelling rate and its preparation method and application. The preparation method provided by the present invention adopts layered casting so that different layers can be used as relatively independent functional modules to realize the functionalization of patch materials. By adjusting the thickness of the dense layer and aging treatment, the swelling rate of the material is reduced to the maximum extent, basically reaching within ±0.6%, and the material after immersion in PBS for 30 days is even close to 0%; and a gel is generated in situ through phase separation, and a transition zone is generated in situ between layers, and the microstructure is interlocked to ensure the overall mechanical reliability of the patch material; through the PVA-DMSO-H2O dissolution system, the pore size of the loose layer is maintained at tens of microns, and the hydrophilicity of the material is improved, promoting cell growth and tissue adhesion; through the PVA-soluble small molecule filler / DMSO dissolution system, the pore size of the dense layer is maintained at the submicron level, and the hydrophobicity of the material is improved, preventing cell and tissue adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a flow chart of the preparation process of the hernia patch material;

[0023] Figure 2 The present invention is a schematic diagram of the process of preparing the hernia patch material;

[0024] Figure 3 This is a SEM image of the loose porous layer of the hydrogel material obtained in Example 1 of the present invention;

[0025] Figure 4 This is a SEM image of the transition region of the hydrogel material obtained in Example 1 of the present invention;

[0026] Figure 5 This is a SEM image of the dense pore layer of the hydrogel material obtained in Example 1 of the present invention;

[0027] Figure 6 This is a SEM image of the transition region of the hydrogel material obtained in Comparative Example 1 of the present invention;

[0028] Figure 7 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 1 of the present invention;

[0029] Figure 8 This is a SEM image of the loose pore layer of the hydrogel material obtained in Comparative Example 2 of the present invention;

[0030] Fig. 9 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 2 of the present invention;

[0031] Fig.10 This is a SEM image of the loose pore layer of the hydrogel material obtained in Comparative Example 3 of the present invention;

[0032] Fig.11 This is a SEM image of the transition region of the hydrogel material obtained in Comparative Example 3 of the present invention;

[0033] Fig.12 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 3 of the present invention;

[0034] Fig.13 This is a longitudinal cross-sectional microscopic morphology of the hydrogel material obtained in Example 2 of the present invention;

[0035] Fig.14 The swelling ratio measurement results of the hydrogel materials obtained in Examples 2 and 4 of the present invention are shown in FIG.

[0036] Fig.15 This is a graph of cell viability in a cytotoxicity test of the hydrogel material of Example 2 of the present invention;

[0037] Fig.16 This is a staining diagram of the cytotoxicity test of the hydrogel material of Example 2 of the present invention;

[0038] Fig.17 The cell adhesion test result diagram of the hydrogel materials of Examples 3, 4 and 5 of the present invention;

[0039] Fig.18 This is an average result diagram of mechanical property testing of three hydrogel materials selected from each of Examples 2, 3, 4 and 5 of the present invention;

[0040] Fig.19 This is a test chart of the swelling rate of the hydrogel material obtained in Example 6 of the present invention at different days. DETAILED DESCRIPTION

[0041] The present invention discloses a hernia repair material and a preparation method thereof and a hernia patch. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The present invention is based on Figure 1 The process flow shown is used to prepare hernia patch materials. Figure 1 This is a process flow chart for preparing the hernia patch material of the present invention. First, raw materials are selected, and then a dense layer precursor solution and a loose layer precursor solution are prepared respectively. Then, layered casting, in-situ gelation, washing, freeze-drying and rehydration are carried out in sequence to obtain a hydrogel close to the physiological state, i.e., the hernia patch material of the present invention.

[0043] In this process, after the precursor liquid is poured in layers, that is, after injection molding, it is left standing at room temperature for 1 hour to allow the precursor liquid to diffuse between layers to produce phase separation, and then placed at -20°C for 12 hours to complete gelation, and finally placed at room temperature for 4 days to complete phase separation, and then washed with deionized water for 3 hours in the washing step, and then the washed patch material is pre-frozen at 20°C for 2 hours, and then freeze-dried for 48 hours, and then the freeze-dried patch material is soaked in PBS buffer for 24 hours for rehydration, and a hydrogel close to the physiological state is obtained. Figure 2 As shown, Figure 2 The present invention is a schematic diagram of the process of preparing the hernia patch material.

[0044] The PBS buffer solution of the present invention is prepared from PBS dry powder of solarbio, with a pH value of 7.2-7.4, and main components are sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride.

[0045] The present invention will be further described below in conjunction with embodiments:

[0046] Example 1

[0047] Step A: Selecting raw materials: polyvinyl alcohol (PVA) and polyethylene glycol (PEG); and deionized water (H2O);

[0048] Step B: Preparation of dense layer precursor solution: 10 g PVA and 10 g PEG2000 were fully dissolved in 100 mL DMSO to obtain PVA-PEG / DMSO solution;

[0049] Step C: Preparation of loose layer precursor solution: 5 g PVA was fully dissolved in 40 mL DMSO and 60 mL water (DMSO aqueous solution with a volume fraction of 40%) to obtain a PVA / DMSO-water solution;

[0050] Step D: Layered pouring: Pour 1.5 mL of the PVA / DMSO solution obtained in step C into a 6 cm flat dish mold, and slowly pour 4.5 mL of the PVA-PEG / DMSO solution obtained in step B after the solution is kept constant at room temperature, and let stand until the liquid surface is stable and there is no refractive index change in the liquid area under naked eye observation;

[0051] Step E: In-situ gelation: placing the mold after layered casting at -20°C for 12 hours to complete the in-situ gelation and obtain the gel;

[0052] Step F: The gel was placed at room temperature for 4 days for aging treatment, and then the aging-treated gel was washed. In order to remove as much DMSO as possible from the gel, the material should be fully washed with triple distilled water. The washing method was to place the gel in a container, soak it in triple distilled water at room temperature, and then place it on a shaker with a shaking speed of 150 rpm for 30 minutes to complete one wash, and a total of 6 washes;

[0053] Step G: freeze-drying the gel washed in step F. Specifically, after washing, pre-freeze the gel at -20°C for 2 hours, then freeze-freeze it at -80°C for 2 hours, and quickly transfer it to a freeze dryer for freeze drying until it is completely dry.

[0054] Step H: rehydration, i.e., soaking the freeze-dried gel in 0.01M PBS buffer solution for 24 hours to obtain a hydrogel material close to the physiological state.

[0055] The microstructure of the hydrogel material obtained above was characterized. Figure 3 to Figure 5 As shown, Figure 3 This is a SEM image of the loose pore layer of the hydrogel material obtained in Example 1 of the present invention. Figure 4 This is a SEM image of the transition region of the hydrogel material obtained in Example 1 of the present invention. Figure 5 This is a SEM image of the dense pore layer of the hydrogel material obtained in Example 1 of the present invention.

[0056] Comparative Example 1

[0057] Compared with Example 1, the difference is:

[0058] Step C: Preparation of loose layer precursor solution: 5 g PVA was fully dissolved in 60 mL DMSO and 40 mL water (DMSO aqueous solution with a volume fraction of 60%) to obtain a PVA / DMSO-water solution;

[0059] The hydrogel material obtained above has poor formability. The microstructure of the hydrogel material obtained above was characterized, and only a transition region and a dense pore layer were found. The results are as follows Figure 6-7 As shown, Figure 6 This is a SEM image of the transition region of the hydrogel material obtained in Comparative Example 1 of the present invention. Figure 7 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 1 of the present invention.

[0060] Comparative Example 2

[0061] Compared with Example 1, the difference is:

[0062] Step C: Preparation of loose layer precursor solution: 5 g PVA was fully dissolved in 80 mL DMSO and 20 mL water (DMSO aqueous solution with a volume fraction of 80%) to obtain a PVA / DMSO-water solution;

[0063] The hydrogel material obtained above has poor formability. The microstructure of the hydrogel material obtained above is characterized, and only loose pore layers and dense pore layers are found. The results are as follows Figure 8-Figure 9 As shown, Figure 8 This is a SEM image of the loose pore layer of the hydrogel material obtained in Comparative Example 2 of the present invention. Fig. 9 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 2 of the present invention.

[0064] Comparative Example 3

[0065] Compared with Example 1, the difference is:

[0066] Step C: Preparation of loose layer precursor solution: fully dissolve 5 g of PVA in 100 mL of water to obtain a PVA aqueous solution;

[0067] The microstructure of the hydrogel material obtained above was characterized. Figure 10 to Figure 12 As shown, Fig.10 This is a SEM image of the loose pore layer of the hydrogel material obtained in Comparative Example 3 of the present invention. Fig.11 This is a SEM image of the transition region of the hydrogel material obtained in Comparative Example 3 of the present invention. Fig.12 This is a SEM image of the dense pore layer of the hydrogel material obtained in Comparative Example 3 of the present invention. Figure 10 to Figure 12 It can be seen that although the hydrogel material obtained in Comparative Example 3 of the present invention can capture the microstructure images of three regions, the molding performance is poor from the perspective of microstructure morphology.

[0068] Comparative Example 4

[0069] Step A: Selecting raw materials: polyvinyl alcohol (PVA) and polyethylene glycol (PEG); and deionized water (H2O);

[0070] Step B: Preparation of precursor solution: 10 g PVA and 10 g PEG2000 were fully dissolved in 100 mL DMSO to obtain PVA-PEG / DMSO solution;

[0071] Step C: Pour 1L of pure water into a 3L beaker, carefully place the plate from step B into the water and place it at the bottom of the beaker. Then use a glass rod to drain and pour another 2L of pure water along the wall of the beaker, and avoid turbulence during this period to disturb the PVA-PEG / DMSO solution in the plate from step B. After standing for 24 hours, no hydrogel material could be obtained.

[0072] Example 2

[0073] Step A: Selecting raw materials: polyvinyl alcohol (PVA) and polyethylene glycol (PEG); and deionized water (H2O);

[0074] Step B: Preparation of dense layer precursor solution: 10 g PVA and 10 g PEG2000 were fully dissolved in 100 mL DMSO to obtain PVA-PEG / DMSO solution;

[0075] Step C: Preparation of loose layer precursor solution: 5 g of PVA was fully dissolved in 20 mL of DMSO and 80 mL of water (DMSO aqueous solution with a volume fraction of 20%) to obtain a PVA / DMSO-water solution;

[0076] Step D: Layered pouring: Pour 1.5 mL of the PVA / DMSO solution obtained in step C into a 6 cm flat dish mold, and slowly pour 4.5 mL of the PVA-PEG / DMSO solution obtained in step B after the solution is kept constant at room temperature, and let stand until the liquid surface is stable and there is no refractive index change in the liquid area under naked eye observation;

[0077] Step E: In-situ gelation: placing the mold after layered casting at -20°C for 12 hours to complete the in-situ gelation and obtain the gel;

[0078] Step F: The gel was placed at room temperature for 4 days for aging treatment, and then the aging-treated gel was washed. In order to remove as much DMSO as possible from the gel, the material should be fully washed with triple distilled water. The washing method was to place the gel in a container, soak it in triple distilled water at room temperature, and then place it on a shaker with a shaking speed of 150 rpm for 30 minutes to complete one wash, and a total of 6 washes;

[0079] Step G: freeze-drying the gel washed in step F. Specifically, after washing, pre-freeze the gel at -20°C for 2 hours, then freeze-freeze it at -80°C for 2 hours, and quickly transfer it to a freeze dryer for freeze drying until it is completely dry.

[0080] Step H: rehydration, i.e., soaking the freeze-dried gel in 0.01M PBS buffer solution for 24 hours to obtain a hydrogel material close to the physiological state.

[0081] The microstructure of the hydrogel material obtained above was characterized. Fig.13 As shown, Fig.13 This is a longitudinal cross-sectional microscopic morphology of the hydrogel material obtained in Example 2 of the present invention.

[0082] Example 3

[0083] Compared with Example 2, the difference is that the aging treatment in step F is not performed, that is, the gel obtained in step E is directly washed and then steps G and H are performed to obtain a hydrogel material.

[0084] Example 4

[0085] Compared with Example 2, the difference is that step G and step H are not performed, that is, the gel obtained in step E is directly subjected to step F to obtain the hydrogel material.

[0086] Example 5

[0087] Compared with Example 2, the difference is that the aging treatment of step F and step G and step H are not performed, that is, the gel obtained in step E is fully washed with triple distilled water, and the washing method is to place the gel in a container, soak it in triple distilled water at room temperature, and then place it on a shaker with a shaking speed of 150 rpm for 30 minutes, completing one washing, and washing a total of 6 times to obtain a hydrogel material.

[0088] Swelling rate determination

[0089] The swelling rate of the hydrogel materials obtained in the above-mentioned Examples 2 and 4 was measured, wherein Example 2 was named as the hydrogel group and Example 4 was named as the exogel group. The specific measurement steps were as follows:

[0090] 1. Sample preparation: Three hydrogel materials were selected from each of the exogel group and the hydrogel group, and the selected materials were cut into small discs with a diameter of 1 cm at the center of the circle.

[0091] 2. Soaking: The cut discs of the exogel group and the hydrogel group were immersed in 50 mL of 0.01 M PBS solution, and the diameters were measured at 0 days, 1 day, 2 days, 3 days, 7 days, and 30 days.

[0092] 3. Measurement: Use a vernier caliper to measure, test each small disc three times, take the average value as the measurement value, and then take the average value of the diameters of the three small discs as a measurement result. The results are as follows: Fig.14 As shown, Fig.14 This is a diagram showing the swelling rate measurement results of the hydrogel materials obtained in Examples 2 and 4 of the present invention.

[0093] 4. Analysis of results: The exogel group began to show a gradual shrinkage trend at 7 days. This was because the inorganic salt components in PBS caused the salting out of the PVA macromolecular chains and further deepened the phase separation process of PVA, resulting in size shrinkage of the sample. For the hydrogel sample, due to the freeze-drying-rehydration process, compared with the exogel sample, the PVA macromolecular chains inside were firmly entangled and the crystal areas were larger and more numerous, so it had better shape retention properties, and not only would it not swell excessively, but also would not shrink excessively.

[0094] Cytotoxicity test

[0095] The hydrogel material obtained in Example 2 was subjected to a cytotoxicity test, and the test process was as follows:

[0096] 1. Sterilization: The hydrogel material obtained in Example 2 was placed in a clean bench, immersed in sterile 0.01 M PBS, and sterilized under ultraviolet light. The material was then turned over once every 6 hours and sterilized under ultraviolet light for another 6 hours.

[0097] 2. Preparation of extract: According to ISO 10993-12:2021, take 1.5g of gel material, soak it in 15mL complete culture medium (84% alpha-MEM basal culture medium + 15% fetal bovine serum + 1% penicillin-streptomycin solution) at a ratio of 0.1g / mL, keep it in a 37°C water bath for 72h, filter the supernatant through a 0.22 micron filter membrane to obtain a 100% concentration extract. Subsequently, the 100% extract is prepared with the aforementioned complete culture medium to obtain extracts with concentrations of 75%, 50% and 25%; and according to the aforementioned complete culture medium and steps, but without adding gel material, a 0% concentration extract is prepared.

[0098] 3. Cell co-culture: Mouse MC3T3 cells were inoculated in 96-well plates (initial inoculation density 5000 cells / well) and co-cultured with the above-mentioned extracts of different concentrations. The cell incubation time was 24h, 48h, and 72h, respectively.

[0099] Cytotoxicity test results Figures 15-16 As shown. Fig.15 This is a cell survival rate graph of the cytotoxicity test of the hydrogel material of Example 2 of the present invention, Fig.16 This is a staining diagram of the cytotoxicity test of the hydrogel material of Example 2 of the present invention.

[0100] Cell adhesion assay

[0101] The hydrogel materials of Examples 3, 4 and 5 were subjected to cell adhesion tests, and the cell growth at the bottom of a common 96-well plate was used as a control. The results are as follows: Fig.17 As shown, Fig.17Graph showing the cell adhesion test results of the hydrogel materials of Examples 3, 4 and 5 of the present invention.

[0102] Depend on Fig.17 It can be seen that the hydrogel materials of Examples 3 and 5 can respectively achieve the effects of promoting tissue healing and anti-tissue adhesion on the inner and outer sides, while the hydrogel material of Example 4 has a reduced cell adhesion effect. This may be because the aging treatment causes the phase separation of PVA to deepen and the PVA network to become coarser, so the pore size of the porous structure composed of the PVA network becomes smaller, which is not conducive to cell adhesion.

[0103] Mechanical properties test

[0104] Three hydrogel materials were selected from each of Example 2, Example 3, Example 4 and Example 5 for mechanical property testing, and the results were averaged. Fig.18 As shown, Fig.18 This is an average result diagram of mechanical property testing of three hydrogel materials selected from Example 3, Example 4 and Example 5 of the present invention.

[0105] Depend on Fig.18 It can be seen that even if the hydrogel material of the present invention has not been freeze-dried and rehydrated, its mechanical properties can still meet the basic requirements of the repair of extraperitoneal hernia; and the hydrogel material that has been freeze-dried and rehydrated, regardless of whether it has been aged for 4 days, the mechanical properties of the patch can be improved by one order of magnitude compared with the hydrogel material that has not been freeze-dried and rehydrated, but the mechanical properties will be better after 4 days of aging. At the same time, it can be noted that for hydrogel materials that have not been freeze-dried and rehydrated, 4 days of aging can effectively improve both strength and toughness, while for hydrogel materials that have been freeze-dried and rehydrated, 4 days of aging can effectively improve mechanical properties, but has no significant effect on toughness.

[0106] Example 6

[0107] Step A: Selecting raw materials: polyvinyl alcohol (PVA) and polyethylene glycol (PEG); and deionized water (H2O);

[0108] Step B: Preparation of dense layer precursor solution: 20 g PVA and 20 g PEG2000 were fully dissolved in 100 mL DMSO to obtain PVA-PEG / DMSO solution;

[0109] Step C: Preparation of loose layer precursor solution: 10 g PVA was fully dissolved in 10 mL DMSO and 90 mL water (10% by volume DMSO aqueous solution) to obtain a PVA / DMSO-water solution;

[0110] Step D: Layered pouring: Pour 1.5 mL of the PVA / DMSO solution obtained in step C into a 6 cm flat dish mold, and slowly pour 4.5 mL of the PVA-PEG / DMSO solution obtained in step B after the solution is kept constant at room temperature, and let stand until the liquid surface is stable and there is no refractive index change in the liquid area under naked eye observation;

[0111] Step E: In-situ gelation: placing the mold after layered casting at -20°C for 12 hours to complete the in-situ gelation and obtain the gel;

[0112] Step F: The gel was thoroughly washed with triple distilled water. The washing method was to place the gel in a container, soak it in triple distilled water at room temperature, and then place it on a shaker with a shaking speed of 150 rpm for 30 minutes to complete one wash, and a total of 6 washes were performed.

[0113] The swelling rate of the hydrogel material obtained in Example 6 was measured in the same way as above, except that the time points were changed to 0d, 4d, 8d and 12d. The results are as follows: Fig.19 As shown, Fig.19 This is a test chart of the swelling rate of the hydrogel material obtained in Example 6 of the present invention at different days.

[0114] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a hernia repair material, characterized in that: The following steps are involved: S1) placing the loose layer precursor solution on the dense layer precursor solution; The dense layer precursor solution comprises PVA and DMSO, and the mass percentage of PVA in the dense layer precursor solution is 9wt% to 11wt%; The loose layer precursor solution comprises PVA, DMSO and water; the volume fraction of DMSO in the loose layer precursor solution is 10% to 40%, and the mass percentage of PVA in the loose layer precursor solution is 4wt% to 6wt%; S2) freezing the product obtained in step S1) into a gel to obtain a hernia repair material.

2. The preparation method according to claim 1, characterized in that: In step S1), the volume ratio of the dense layer precursor solution to the loose layer precursor solution is 4.5:(1-2).

3. The preparation method according to claim 1, characterized in that: In step S1), the dense layer precursor solution also includes a soluble small molecule filler; The mass ratio of the soluble small molecule filler to the PVA in the dense layer precursor solution is (0.8-1.2):

1.

4. The preparation method according to claim 3, characterized in that: The soluble small molecule filler is selected from at least one of PEG, ethylene glycol, glycerol, polyglycerol, low molecular weight chitosan and low molecular weight hyaluronic acid.

5. The preparation method according to claim 1, characterized in that: In step S2), the freezing gelling temperature is below 4°C, and the freezing gelling time is 10 hours to 14 hours.

6. The preparation method according to any one of claims 1 to 5, characterized in that: In step S2), after the product obtained in step S1) is frozen into gel, the method further includes washing, freeze-drying and rehydrating the frozen gel product.

7. The preparation method according to claim 6, characterized in that: Before washing, freeze-drying and rehydrating the frozen gel product, the frozen gel product is subjected to an aging treatment, the aging treatment temperature is 20° C. to 30° C., and the aging treatment time is 3 to 4 days.

8. A hernia repair material obtained by the preparation method according to any one of claims 1 to 7.

9. A hernia patch, characterized in that: The hernia repair material is obtained by the preparation method described in any one of claims 1 to 8.

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