A method for preparing an abdominal wall defect repair material

Abstract

The application aims to provide a preparation method of abdominal wall defect repair material, and belongs to the technical field of biomedical materials.The pure polyvinyl alcohol outer layer hydrogel is prepared by film forming method combined with salting-out method; the inner layer hydrogel is prepared by 3D printing means from sodium alginate / polyvinyl alcohol / chitosan quaternary ammonium salt.The application is inspired by the abdominal wall, and a double-layer asymmetric hydrogel is prepared to contact the abdominal wall and internal organs respectively.The tensile strength of the outer layer PVA film is increased by ten times through freeze-thaw crosslinking combined with salting-out method.Compared with the simple freeze-thaw crosslinking method, the method can conveniently adjust the mechanical properties of the outer layer hydrogel to better match the requirements of the abdominal wall, and the minimum suture tensile strength can reach 0.12 MPa, which is 0.02 MPa higher than the maximum abdominal pressure.Secondly, the method can effectively reduce the attachment of fibroblasts on the material, prevent the adhesion between the damaged abdominal wall and internal organs, and better promote the repair of the damaged abdominal wall in cooperation with the antibacterial material of the inner layer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a material for repairing abdominal wall defects. Background Technology

[0002] Internal soft tissue defects of the abdominal wall, chest wall, and dura mater are often caused by surgical trauma, resection of primary or secondary tumors, traumatic injury, and soft tissue infection. Globally, at least 400,000 surgeries are performed annually, resulting in over $10 billion in medical costs. In recent years, the repair of internal soft tissue defects that damage the serosal and muscle layers has received considerable attention due to surgical challenges and unsatisfactory treatment outcomes. Muscle layer damage is difficult to suture, and serosal injury can cause significant organ adhesions. Over the past few decades, tension-free repair surgery has been recommended as the standard treatment for soft tissue defects such as abdominal wall defects, with various types of patches being widely used.

[0003] Traditional synthetic patches (e.g., polypropylene (PP) and polyester mesh) have advantages such as high strength, light weight, and resistance to deformation, and have been widely used for tension-free repair of soft tissue defects. However, these patches can lead to severe visceral adhesions and poor wound healing due to significant foreign body reactions, possibly because they lack the microstructures necessary for cell migration and growth. Summary of the Invention

[0004] To match the mechanical strength of the abdominal wall to be repaired and to prevent adhesion, this invention, inspired by the peritoneum, provides a method for preparing an abdominal wall defect repair material. This method achieves mechanical matching between the material and the repair site, solves the problem of anti-adhesion during the repair process, and also provides good anti-Staphylococcus aureus properties, which can effectively reduce the risk of intraoperative infection.

[0005] The present invention adopts the following technical solution: A method for preparing an abdominal wall defect repair material involves using Solidworks to construct a composite material inner scaffold model, and then combining a 3D-printed sodium alginate (SA) / polyvinyl alcohol (PVA) / chitosan quaternary ammonium salt (HACC) inner scaffold with a PVA outer scaffold prepared by membrane fabrication and salting-out methods to form an asymmetric abdominal wall repair material.

[0006] Specifically, the steps include the following: The first step involves preparing the outer repair material using a combination of membrane fabrication and salting-out methods: (1) Pour 14.4g of polyvinyl alcohol into a three-necked flask, add 80ml of deionized water, set the temperature to 90℃, set the speed to 400r / min, and stir continuously for two hours to obtain a viscous and transparent polyvinyl alcohol liquid. (2) Place the polyvinyl alcohol liquid in a magnetic ultrasonic cleaner and sonicate for 10 minutes to remove the air bubbles and obtain the liquid membrane manufacturing precursor; (3) Place the membrane fabrication precursor in a 10ml syringe and slowly inject it into the mold; (4) The material was subjected to three freeze-thaw cycles: freezing at -20℃ for 12 hours and thawing at -4℃ for 12 hours; (5) Prepare a series of sodium sulfate solutions with different concentration gradients: 0.1, 0.3, 0.5, 0.7, and 0.9 mol / L, 20 ml of each; (6) Salting out the polyvinyl alcohol film after freezing and thawing of the mold by immersing it in sodium sulfate solution of different concentrations for 24 hours and deionized water for 12 hours. The second step involves fabricating the hydrogel scaffold using 3D printing. 3D printing bio-ink was placed in a 5ml syringe, and a printing needle was connected. Under the following conditions: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, retraction 0.25mm, elevation 0.2mm, size 10*10 / 15*15mm, line spacing 1.2mm, layer height 0.2mm, and scaffold height 0.4mm, the printing time lasted for 3 minutes to obtain a hydrogel scaffold. After chemical cross-linking with 4wt% calcium chloride solution, the hydrogel scaffold was frozen at -20℃ for 12h and thawed at -4℃ for 12h, and then subjected to repeated freeze-thaw physical cross-linking three times. The third step involves bonding the outer membrane prepared by the membrane fabrication method combined with the salting-out method to a 3D-printed hydrogel scaffold using 0.5ml~1ml of 3D-printed bio-ink, ultimately obtaining a material for repairing abdominal wall defects.

[0007] The outer layer prepared by combining membrane fabrication and salting-out methods has a dense structure on its surface after film formation, with a water contact angle of 20° and a water content of 60% (salting-out concentration of 0.7 mol / L).

[0008] The preparation method of the 3D printing bio-ink is as follows: (1) Weigh out sodium alginate, polyvinyl alcohol and chitosan quaternary ammonium salt powder in a mass ratio of 5:6:1 for later use; (2) Polyvinyl alcohol was continuously stirred in a magnetic stirring pot at 90°C and 600 r / min for 2 hours; (3) Then cool to room temperature, add sodium alginate powder, and mechanically stir at room temperature for 30 minutes at a speed of 400 r / min until the sodium alginate powder is completely dissolved; (4) Slowly add 0.2g of chitosan quaternary ammonium salt powder to the dissolved solution and continue stirring for 30min under the same conditions to obtain 3D printing bio-ink.

[0009] The beneficial effects of this invention are as follows: This invention presents a material for repairing abdominal wall defects with an asymmetric structure, prepared using a membrane fabrication method combined with salting-out and 3D printing technology. Pure polyvinyl alcohol is cross-linked via freeze-thaw cross-linking using the membrane fabrication method. After improving mechanical properties through salting-out, it can well match the mechanical properties of the abdominal wall. Simultaneously, it can replicate the density of the peritoneum to achieve an anti-adhesion effect. The outer layer of the repair material mainly serves a protective and supportive role, providing necessary mechanical strength and preventing soft tissue ingrowth and adhesion during the repair process. For abdominal wall repair, this can alleviate the burden on patients with complications such as intestinal obstruction. The porous inner layer (3D-printed hydrogel) is prepared from 5 wt% sodium alginate, 6 wt% polyvinyl alcohol (PVA), and 1 wt% chitosan quaternary ammonium salt (HACC). The inner layer mainly serves an antibacterial function, and its porous structure provides a suitable structure and microenvironment for cell migration. Ultimately, a material for repairing damaged abdominal walls is obtained.

[0010] This invention exhibits excellent biocompatibility, low toxicity, and low immunogenicity. At the site of abdominal wall injury, the outer layer effectively prevents adhesion and avoids other complications, while the inner layer effectively controls infection and promotes skin regeneration. It also facilitates cell adhesion and growth, thereby improving the quality and rate of wound healing. Attached Figure Description

[0011] Figure 1 The images shown are macroscopic views of the outer abdominal wall defect repair materials prepared in Examples 1-6.

[0012] Figure 2 Microscopic images of the outer abdominal wall defect repair materials prepared for Examples 1-6.

[0013] Figure 3 Examples 1-6 show the tensile stress-strain diagrams of the outer abdominal wall defect repair materials.

[0014] Figure 4 Examples 1-6 show the suture tensile stress-strain diagrams of the prepared outer abdominal wall defect repair materials.

[0015] Figure 5 Examples 1-6 show cell anti-adhesion diagrams of the prepared outer abdominal wall defect repair materials.

[0016] Figure 6 Examples 1-6 show the antibacterial properties of the prepared inner hydrogel scaffold materials.

[0017] Figure 7 Examples 1-6 show the blood compatibility of the prepared inner hydrogel scaffold materials.

[0018] Figure 8CCK-8 diagrams of the abdominal wall defect repair materials prepared for Examples 1 and 5. Detailed Implementation

[0019] The present invention will be further illustrated by the following embodiments, but is not limited to the following embodiments.

[0020] Example 1 Preparation of the inner hydrogel scaffold by 3D printing: Dissolve 1.2g of polyvinyl alcohol (PVA) in 20ml of deionized water, cover with tin foil (to reduce the evaporation of deionized water), adjust the temperature to 90℃ and the stirring speed to 400r / min, and stir continuously for 2h until the polyvinyl alcohol (PVA) is completely dissolved; after the polyvinyl alcohol (PVA) solution cools naturally to room temperature (25℃), slowly add 1g of sodium alginate (SA) to the dissolved polyvinyl alcohol (PVA) solution, mechanically stir for 30min at 400r / min until the sodium alginate (SA) is completely dissolved; add 0.2g of chitosan quaternary ammonium salt (HACC) powder, mechanically stir for 30min at 400r / min, and the 3D printed hydrogel is obtained. The obtained sol-like 3D printing hydrogel was placed in a syringe (BD-5ml), and a printing needle (0.25G) was connected. Under the conditions of ambient temperature of 25℃, humidity of 40%, needle temperature of 37℃, machine temperature of 10℃, printing speed of 6mm / s, extrusion speed of 7mm / s, pullback (SK) of 0.25mm, lift (HP) of 0.2mm, size of 10*10mm, line spacing of 1.2mm, layer height of 0.2mm, and scaffold height of 0.4mm, the printing time lasted for 5 minutes to obtain a composite 3D printing hydrogel scaffold of sodium alginate (SA), polyvinyl alcohol (PVA), and chitosan quaternary ammonium salt (HACC). Sodium alginate (SA) was crosslinked using a 4wt% calcium chloride solution. The scaffold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles of freeze-thaw physical crosslinking of polyvinyl alcohol (PVA), the inner hydrogel scaffold was obtained.

[0021] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a polyvinyl alcohol (PVA) solution. The solution was ultrasonically cleaned for 10min to remove air bubbles. The polyvinyl alcohol (PVA) solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the outer abdominal wall defect repair material was obtained.

[0022] Example 2 The 3D printing method was used to prepare the inner hydrogel scaffold as in Example 1.

[0023] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a PVA solution. The solution was ultrasonically cleaned for 10min in a magnetic ultrasonic cleaner to remove air bubbles. The PVA solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the mold was soaked in 0.1wt% sodium sulfate (Na2SO4) solution for 24h and then soaked in deionized water for 12h to obtain the outer abdominal wall defect repair material.

[0024] Example 3 The 3D printing method was used to prepare the inner hydrogel scaffold as in Example 1.

[0025] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a PVA solution. The solution was ultrasonically cleaned for 10min in a magnetic ultrasonic cleaner to remove air bubbles. The PVA solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the mold was soaked in 0.3wt% sodium sulfate (Na2SO4) solution for 24h and then soaked in deionized water for 12h to obtain the outer abdominal wall defect repair material.

[0026] Example 4 The 3D printing method was used to prepare the inner hydrogel scaffold as in Example 1.

[0027] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a PVA solution. The solution was ultrasonically cleaned for 10min in a magnetic ultrasonic cleaner to remove air bubbles. The PVA solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the mold was soaked in 0.5wt% sodium sulfate (Na2SO4) solution for 24h and then soaked in deionized water for 12h to obtain the outer abdominal wall defect repair material.

[0028] Example 5 The 3D printing method was used to prepare the inner hydrogel scaffold as in Example 1.

[0029] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a PVA solution. The solution was ultrasonically cleaned for 10min in a magnetic ultrasonic cleaner to remove air bubbles. The PVA solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the mold was soaked in 0.7wt% sodium sulfate (Na2SO4) solution for 24h and then soaked in deionized water for 12h to obtain the outer abdominal wall defect repair material.

[0030] Example 6 The 3D printing method was used to prepare the inner hydrogel scaffold as in Example 1.

[0031] Preparation of outer abdominal wall defect repair material: 14.4g of polyvinyl alcohol (PVA) was dissolved in 80ml of deionized water at 90℃ for 2h to obtain a PVA solution. The solution was ultrasonically cleaned for 10min in a magnetic ultrasonic cleaner to remove air bubbles. The PVA solution was then uniformly injected into a custom mold using a 10ml syringe. The mold was frozen at -20℃ for 12h and thawed at -4℃ for 12h. After three cycles, the mold was soaked in 0.9wt% sodium sulfate (Na2SO4) solution for 24h and then soaked in deionized water for 12h to obtain the outer abdominal wall defect repair material.

[0032] The abdominal wall defect repair materials prepared using the above methods do not cause immune rejection or allergic reactions when applied to the human body. Employing a biomimetic approach, combining membrane fabrication and salting-out methods, an outer layer material with excellent mechanical properties and good anti-adhesion performance was prepared. The inner scaffold was fabricated using 3D printing, possessing an extracellular matrix structure that promotes cell growth and migration, and exhibiting good biocompatibility. It shows broad clinical application prospects in the field of soft tissue defect repair.

[0033] Figure 1 The outer membranes prepared by the membrane fabrication method under different concentrations of sodium sulfate solution are shown in Examples 1-6 from left to right. It can be seen that the salting-out method can significantly affect the crystallinity of polyvinyl alcohol molecules. As the concentration of sodium sulfate increases, the opacity of the outer membrane increases and the crystallinity increases.

[0034] Figure 2 The SEM images of Examples 1-6 (a to f are Examples 1-6 respectively) show that as the sodium sulfate concentration increases, the small molecular weight polyvinyl alcohol molecular chains are expelled from the material, resulting in smaller pores on the material surface, but the material surface is generally dense.

[0035] Figure 3The stress-strain diagrams for Examples 1-6 show that the mechanical properties of the material gradually improve with increasing sodium sulfate concentration. The tensile strengths of Examples 1-6 are 0.15 MPa, 1.21 MPa, 2.95 MPa, 5.58 MPa, 5.91 MPa, and 6.57 MPa, respectively, which closely resemble the mechanical parameters of the human abdominal wall.

[0036] Figure 4 The figures show the tensile stress-strain diagrams for sutures in Examples 1-6. As the sodium sulfate concentration increases, the mechanical properties of the material gradually improve. The tensile strengths for sutures are 0.15 MPa, 0.12 MPa, 0.27 MPa, 2.95 MPa, 1.05 MPa, and 1.45 MPa, respectively. Even the material with the lowest tensile strength can withstand a maximum abdominal pressure of 0.02 MPa after being implanted into the human body.

[0037] Figure 5 The cell anti-adhesion diagrams for Examples 1-6 show that, as the sodium sulfate concentration increases, the materials generally exhibit superior anti-adhesion properties.

[0038] Figure 6 The figure shows the antibacterial test results of the inner hydrogel scaffold experimental group (6wt%PVA / 5wt%SA / 1wt%HACC) and the inner hydrogel scaffold blank group (without chitosan quaternary ammonium salt). The experimental group has a good killing effect on Staphylococcus aureus, but has little effect on Escherichia coli.

[0039] Figure 7 The figure shows the blood compatibility test results of the inner hydrogel scaffold experimental group (6wt%PVA / 5wt%SA / 1wt%HACC) and the inner hydrogel scaffold blank group (without chitosan quaternary ammonium salt). It can be seen that the blood compatibility of the blank group and the experimental group is 0.89% and 1.51%, respectively, both of which are less than 5% (ISO 10993-4:2017).

[0040] Figure 8 The CCK-8 plots show the results for the following groups: blank group (material only: culture medium), inner hydrogel scaffold experimental group (6wt% PVA / 5wt% SA / 1wt% HACC), blank group (without chitosan quaternary ammonium salt), outer hydrogel scaffold experimental group (treated with 0.7mol / ml sodium sulfate for salting out), and blank group (without salting out). The results show that the absorbance of the material increases continuously with the extension of culture time, indicating that the material can promote the proliferation of L929 (mouse fibroblasts) and has good biological activity.

Claims

1. A method for preparing a material for repairing abdominal wall defects, characterized in that: A composite material inner scaffold model was constructed. An asymmetric abdominal wall repair material was prepared by combining a 3D-printed sodium alginate / polyvinyl alcohol / chitosan quaternary ammonium salt inner scaffold with a PVA outer scaffold prepared by membrane fabrication and salting-out methods. Specifically, the steps include the following: The first step involves preparing the outer repair material using a combination of membrane fabrication and salting-out methods: (1) Pour 14.4g of polyvinyl alcohol into a three-necked flask, add 80ml of deionized water, set the temperature to 90℃, set the speed to 400r / min, and stir continuously for two hours to obtain a viscous and transparent polyvinyl alcohol liquid. (2) Place the polyvinyl alcohol liquid in a magnetic ultrasonic cleaner and sonicate for 10 minutes to remove the air bubbles and obtain the liquid membrane manufacturing precursor; (3) Place the membrane fabrication precursor in a 10ml syringe and slowly inject it into the mold; (4) The material was subjected to three freeze-thaw cycles: freezing at -20℃ for 12 hours and thawing at -4℃ for 12 hours; (5) Salting out the polyvinyl alcohol film after freezing and thawing the mold by immersing it in sodium sulfate solution for 24 hours and then in deionized water for 12 hours; The second step involves fabricating the hydrogel scaffold using 3D printing. The 3D printing bio-ink was placed in a 5ml syringe, and the printing needle was connected. The printing conditions were: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, retraction 0.25mm, elevation 0.2mm, and size 10. 10 / 15 Under the conditions of 15mm line spacing, 1.2mm line spacing, 0.2mm layer height, and 0.4mm scaffold height, the printing time lasted for 3 minutes to obtain a hydrogel scaffold. After chemical cross-linking with 4wt% calcium chloride solution, the hydrogel scaffold was frozen at -20℃ for 12 hours and thawed at -4℃ for 12 hours, and then subjected to repeated freeze-thaw physical cross-linking three times. The third step involves bonding the outer membrane prepared by the membrane fabrication method combined with the salting-out method to a 3D-printed hydrogel scaffold using 0.5ml~1ml of 3D-printed bio-ink, ultimately obtaining a material for repairing abdominal wall defects.

2. The method for preparing an abdominal wall defect repair material according to claim 1, characterized in that: The sodium sulfate solutions were prepared in concentrations of 0.1, 0.3, 0.5, 0.7, and 0.9 mol / L, in 20 ml portions.

3. The method for preparing an abdominal wall defect repair material according to claim 1, characterized in that: The preparation method of the 3D printing bio-ink is as follows: (1) Weigh out sodium alginate, polyvinyl alcohol and chitosan quaternary ammonium salt powder in a mass ratio of 5:6:1 for later use; (2) Polyvinyl alcohol was continuously stirred in a magnetic stirring pot at 90°C and 600 r / min for 2 hours; (3) Then cool to room temperature, add sodium alginate powder, and mechanically stir at room temperature for 30 minutes at a speed of 400 r / min until the sodium alginate powder is completely dissolved; (4) Slowly add 0.2g of chitosan quaternary ammonium salt powder to the dissolved solution and continue stirring for 30min under the same conditions to obtain 3D printing bio-ink.

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