A method for preparing a collagen-based in-situ gelling bioimplant

Abstract

The application discloses a preparation method of an in-situ gelatinized collagen-based biological patch, which utilizes the in-situ generation of a hydrogel between the surface of a decellularized matrix material and the fiber gaps inside the decellularized matrix material by introducing the multi-chemical reaction activity of dopamine / polydopamine, integrates the hydrogel system and the decellularized matrix material, constructs the in-situ gelatinized biological patch, effectively improves the mechanical support effect, drug loading, cell growth promotion of a defect tissue, and simultaneously has the functions of tissue repair such as healing promotion, adhesion prevention, anti-infection, and the like, and can be widely used for functional modification of the decellularized matrix material.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and in particular to a method for preparing an in-situ gelled collagen-based biopatch. Background Technology

[0002] Abdominal wall injuries, commonly resulting from congenital ruptures, trauma, and resection of primary or secondary tumors, present a significant surgical challenge. Due to the substantial risk of recurrence associated with direct suturing, the use of implantable materials for tension-free healing is currently a common approach for treating abdominal wall defects. Surgeons typically choose permanent synthetic mesh materials, such as polypropylene, to repair the damaged area for maximum support. However, these synthetic mesh materials can significantly accelerate the foreign body reaction and fibrosis process, potentially leading to long-term pain and limited mobility. Furthermore, their non-biodegradability can cause postoperative complications such as intestinal mucosal damage and deformities.

[0003] While natural bioactive patches exhibit good biocompatibility and no toxic side effects, they often demonstrate insufficient mechanical and thermal stability, failing to provide adequate mechanical support for defects and thus failing to meet clinical application requirements. Decellularized matrix materials (dECMs), as natural biomaterials derived from tissues, are primarily composed of collagen and have attracted widespread attention due to their high natural abundance, good biocompatibility, and excellent performance in various biomedical applications. Essentially, dECMs are collagen scaffolds with a three-dimensional network structure, capable of supporting defect sites and have proven to be a good solution for abdominal wall defect repair. However, in practical applications, limitations remain, such as insufficient healing-promoting properties, inadequate anti-inflammatory effects, and insufficient sustained-release drug delivery. Furthermore, the lack of further functionalities (such as anti-inflammatory and antibacterial properties) makes it difficult to address the complex application scenarios of clinical diseases. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing a composite biological patch that can produce a biological repair mesh with good mechanical properties and multiple functional characteristics such as drug delivery, environmental response, drug sustained release, cell carrier, and nutrient supply.

[0005] The purpose of this invention is to provide a method for preparing an in-situ gelled collagen-based biological patch to address the shortcomings of existing technologies. This method is achieved through the following technical measures.

[0006] A method for preparing an in-situ gelled collagen-based biopatch based on dopamine crosslinking includes the following steps:

[0007] (1) Preparation of dopamine-modified decellularized matrix material:

[0008] Weigh a certain amount of decellularized matrix material, add a certain amount of pure water, and under nitrogen protection, add dopamine hydrochloride to treat for a certain period of time. Then adjust the pH of the reaction solution to alkaline, and discard the waste liquid after a certain period of treatment. Add a certain amount of pure water again, wash, discard the waste liquid, and repeat the washing process several times.

[0009] (2) Preparation of in-situ hydrogel precursor solution:

[0010] A certain amount of collagen was dissolved in a buffer solution, and oxidized polysaccharide and phenylboronic acid-modified biomacromolecules were added. (3) Preparation of in-situ gelled collagen-based biopatch based on dopamine crosslinking:

[0011] Dopamine-modified decellularized matrix was placed in hydrogel precursor solution, drug loading was added, stirred and incubated to allow it to fully gel, then PBS buffer was added and washed, and the process was repeated several times.

[0012] In one embodiment, the decellularized matrix material mentioned in step (1) may be a decellularized dermal matrix, a decellularized amniotic matrix, a decellularized submucosal matrix of the small intestine, a decellularized urethral matrix, or a decellularized peritoneal matrix.

[0013] In one embodiment, the reaction temperature in step (1) is controlled at 4 to 40°C.

[0014] In one embodiment, the pH value in step (1) is 7-10.

[0015] In one embodiment, the amount of dopamine hydrochloride used in step (1) is 1%-50% of the decellularized matrix material.

[0016] In one embodiment, the pH adjustment in step (1) can be performed using one of tris(hydroxymethyl)aminomethane hydrochloric acid solution, sodium hydroxide solution, or Tris-HCl solution.

[0017] In one embodiment, the buffer solution mentioned in step (2) refers to one or more of the following: acetate-sodium acetate buffer solution and phosphate buffer solution.

[0018] In one embodiment, the oxidized polysaccharide mentioned in step (2) may be one or more of oxidized chitosan, oxidized hyaluronic acid, oxidized chondroitin sulfate, oxidized sodium alginate, oxidized carboxymethyl chitosan, and oxidized heparin.

[0019] In one embodiment, the phenylboronicized biomacromolecule mentioned in step (2) refers to one or more of the following: gelatin grafted with phenylboronic acid, hyaluronic acid grafted with phenylboronic acid, chondroitin sulfate grafted with phenylboronic acid, sodium alginate grafted with phenylboronic acid, carboxymethyl chitosan grafted with phenylboronic acid, and heparin grafted with phenylboronic acid.

[0020] In one embodiment, the amount of PBS buffer used in step (3) is 5-50 times the weight of the dopamine-modified decellularized matrix.

[0021] In one embodiment, the loaded drug in step (3) may be one or more of water-soluble drugs such as antibiotics, growth factors, and water-soluble natural drugs.

[0022] In one embodiment, the loaded drug in step (3) can be one or more of the poorly soluble drug micelles or microspheres such as apigenin and curcumin.

[0023] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0024] (1) This method takes into account both the cross-linking and in-situ engineering strategies of decellularized matrix materials. First, dopamine is introduced to fully penetrate the decellularized matrix material and then cross-linking is initiated to improve its physicochemical properties, enzyme resistance and mechanical support properties. Then, the multi-reactive hydrogel is constructed in situ using dopamine and the resulting polydopamine, thus achieving the combination of cross-linking and in-situ platform functions.

[0025] (2) In this composite structure, the hydrogel is in situ integrated on the decellularized matrix material, and the two parts are deeply fused together, which can give full play to the advantages of both in terms of structure and performance. Specifically, the decellularized matrix material can provide mechanical strength and structural support at the damaged site, while the hydrogel layer generated in situ on it can realize functions such as drug delivery, environmental response, drug sustained release, cell carrier, and nutrient supply, ultimately forming a composite biological patch that can perform multiple functional properties.

[0026] (3) The in-situ hydrogel is uniform and intact. The porous and irregular structure of the decellularized matrix surface makes the in-situ hydrogel better able to conform to the morphology of the substrate surface than the pre-formed and then bonded hydrogel. The two are organically combined, ensuring that the hydrogel and the substrate will not separate during the entire service of the patch, but can each play their own role, laying a good foundation for subsequent functions and applications.

[0027] (4) In-situ hydrogels create favorable conditions for the multifunctionalization of decellularized matrix materials. Due to their porous structure and excellent biocompatibility, hydrogels have been used to encapsulate various drugs or biomolecules, offering numerous advantages such as high drug encapsulation efficiency, controllable drug release rate, and long delivery time. Constructed hydrogels can serve as carriers for drugs or other bioactive molecules, or achieve multifunctional integration of decellularized matrix materials, including anti-calcification, antibacterial, anti-inflammatory, angiogenesis-promoting, cell proliferation-promoting, microcirculation-promoting, and anticancer properties.

[0028] ( 5It has good controllability. The reaction can be adjusted by regulating the amount of reactants and environmental conditions in the reaction system. The aldehyde groups in the oxidized polysaccharide can also provide chemical cross-linking within the hydrogel. The phenylboronicized biomacromolecules can form borate ester bonds with certain polysaccharides (containing hydroxyl groups), which can be broken in response to the environment (such as pH and glucose), thus achieving the purpose of environmentally responsive drug release, thereby controlling the various performance and functionality of the material.

[0029] (6) Biodegradable. Since the decellularized matrix material itself is biodegradable, and the hydrogel component used is also biodegradable, the biodegradability of the composite biological patch can still be maintained, and its degradation performance can be adjusted by the degree of crosslinking.

[0030] (7) It can be industrialized on a large scale. Decellularized matrix materials are commonly used raw materials in the field of biological patches. The methacrylamide biomolecules required to construct in-situ hydrogels can be prepared from a wide range of biomolecules using mature technologies. Therefore, this method can be applied to large-scale industrial production and is widely used in the field of biomaterials. Attached Figure Description

[0031] Figure 1 This is a cross-sectional view of the OAC-AP / PA scanning electron microscope in Example 1.

[0032] Figure 2 This is an experiment on the anticoagulant properties of OAC-AP / PA in Example 1.

[0033] Figure 3 The image shows the UV absorption curves of the OAC-AP / PA sample and the free radical solution after co-culturing in Example 1.

[0034] Figure 4 This is the expression of oxidative stress in L929 cells of the OAC-AP / PA sample from Example 1.

[0035] Figure 5 The antibacterial properties of OAC-AP / PA in Example 2; (A) Image of contact antibacterial activity; (B) Antibacterial rate.

[0036] Figure 6 This is a cell scratch experiment of L929 fibroblasts co-cultured with OAC-AP / PA in Example 2.

[0037] Figure 7 This is a morphological observation of the OAC-AP / PA patch used in Example 1 to repair abdominal wall defects in rats.

[0038] Figure 8 HE staining observation of OAC-AP / PA patch repairing abdominal wall defects in rats in Example 1. Detailed Implementation

[0039] The present invention will be specifically described below through examples. It should be noted that these examples are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above-described invention.

[0040] Example 1

[0041] (1) At room temperature, under nitrogen protection, add 50g of dopamine hydrochloride and 10kg of water for injection to the drum, adjust the reaction temperature to 4℃, and turn the drum for 10min; weigh 1000g of decellularized porcine dermal matrix, put it into the drum, and turn the drum for 6h; use tris(hydroxymethyl)aminomethane hydrochloric acid solution to adjust the pH of the reaction solution to 8.4, turn the drum, and react at 27℃ for 24h; discard the reaction solution, and at room temperature, use excess water for injection to repeatedly wash the decellularized porcine dermal matrix three times in the drum, which is recorded as PDA-pADM;

[0042] (2) At room temperature, collagen, sodium alginate and oxidized low molecular weight heparin were dissolved in PBS buffer (10 mmol / L phosphate, 100 mmol / L sodium chloride, pH 7.4) at a concentration of 5 mg / mL. Drug-loaded micelles were added to make the concentration 15%, and the mixture was mixed evenly to obtain the hydrogel precursor solution.

[0043] (3) Add 5 kg of the hydrogel precursor solution prepared in the previous step to the drum after step (1), and slowly rotate it at 4°C for 1 hour. Then, take out the material and incubate it at 37°C for 2 hours. After the reaction is complete, wash it three times with excess PBS buffer to obtain the decellularized porcine dermal matrix embedded in the drug-loaded hydrogel. It is denoted as OAC-AP / PA.

[0044] Example 2

[0045] (1) Weigh 10g of decellularized amniotic membrane matrix into an Erlenmeyer flask, immerse it in 50ml of distilled water, add 0.2g of dopamine hydrochloride, and react under nitrogen protection at low temperature on a shaker for 1 hour; remove nitrogen protection, adjust the pH of the reaction system to 8 using 1M sodium hydroxide solution, then raise the temperature to 25℃ and continue to react on a shaker for 10 hours. After the reaction is complete, wash the material thoroughly with distilled water. PDA-pADM;

[0046] (2) Dissolve 2g of collagen, 2g of sodium alginate and 2g of oxidized sodium alginate in 20g of PBS, add an appropriate amount of growth factor, mix thoroughly at low temperature to obtain hydrogel precursor solution;

[0047] (3) The dopamine-crosslinked decellularized amniotic matrix was placed in the solution from the previous step to allow the solution to fully penetrate. The material was then incubated at 37°C for 4 hours. After the reaction was completed, it was washed with excess PBS solution to obtain a growth factor-loaded hydrogel-coated decellularized amniotic matrix, denoted as OAC-AP / PA.

[0048] Data Analysis

[0049] pADM represents decellularized porcine dermal matrix;

[0050] PDA-pADM represents a decellularized porcine dermal matrix that is first permeated by dopamine and then cross-linked.

[0051] OAC-AP / PA indicates that the drug-loaded in-situ hydrogel encapsulates decellularized porcine dermal matrix.

[0052] Morphological analysis

[0053] The cross-sectional morphology of OAC-AP / PA was observed using scanning electron microscopy, such as... Figure 1 As shown, the hydrogel adheres to the surface of the pADM material, exhibiting a good porous structure that provides space for cell migration and growth, enhances the interaction between cells and the material, and promotes cell adhesion, proliferation, and differentiation. Simultaneously, the porous structure of the pADM's longitudinal section is also observed, with its internal collagen fiber bundles stacked and intertwined, providing a suitable growth environment to meet the metabolic needs and migration requirements of cells during growth.

[0054] Evaluation of anticoagulation test

[0055] For implantable materials, good anticoagulant properties can effectively prevent thrombosis, reduce local inflammation, and improve the stability and functional durability of the implant, thereby extending its lifespan. Blood viscosity index (BCI) reflects the material's ability to form a thrombus upon contact with blood. BCI characterizes the in vitro anticoagulant ability of OAC-AP / PA materials; a higher BCI value indicates better anticoagulant performance. Figure 2 As shown, the BCI indices of pADM and OAC-AP / PA were 64.92% and 82.01%, respectively, indicating that OAC-AP / PA had a better anticoagulant effect. This may be because the low molecular weight heparin in the hydrogel can inhibit the production and activity of thrombin, thus exerting an anticoagulant effect to some extent.

[0056] Antioxidant performance evaluation

[0057] Scavenging excess reactive oxygen species (ROS) or reactive nitrogen species (RNS) is an important strategy for removing excess free radicals and mitigating oxidative stress. DPPH and ABTS are typical nitrogen-containing free radicals, while ·OH is a typical oxygen-containing free radical. They are widely used to evaluate the scavenging capacity of materials for reactive nitrogen (RNS) and reactive oxygen species (ROS). Figure 3 Figures (A), (B), and (C) show the UV absorption curves of the OAC-AP / PA sample after co-culturing with DPPH, ABTS, and ·OH radical solutions, respectively. As shown in the figures, the characteristic absorption peaks of the OAC-AP / PA sample solution at 517 nm, 734 nm, and 510 nm are significantly weakened. Figure 3 As shown in (D), OAC-AP / PA shows promise for the treatment of abdominal wall defects. It can quickly respond to the special microenvironment of ROS and RNS at the defect site, thereby intelligently accelerating drug release, reducing inflammatory response, and thus reducing cell damage.

[0058] like Figure 4 As shown in (A), stimulation experiments were conducted using a culture medium containing 5 μg / ml H2O2, and cell viability was measured by MTT assay. The OD values ​​of both the positive control group and the OAC-AP / PA sample group decreased to some extent at 1 and 3 days, indicating that cell viability decreased or even died due to changes in environmental conditions. However, the OD value of the OAC-AP / PA sample group decreased less, indicating that the oxidative stimulation of L929 cells was weakened under its protection, suggesting that OAC-AP / PA has strong antioxidant capacity.

[0059] like Figure 4 As shown in (B), the antioxidant capacity of the OAC-AP / PA sample was comprehensively evaluated by combining the MDA content and GSH / GSSG results in the L929 cell precipitate. The MDA level in the cells stimulated by hydrogen peroxide was significantly upregulated, while the upregulation of MDA content in the OAC-AP / PA sample group was smaller. The GSH / GSSG ratio in the cells stimulated by hydrogen peroxide was significantly decreased, while the decrease in the GSH / GSSG ratio in the OAC-AP / PA sample group was smaller. This indicates that the oxidative stress response of L929 cells was significantly inhibited under the protection of the skin-based hydrogel system, which further proves that OAC-AP / PA has a strong antioxidant capacity.

[0060] Antibacterial performance evaluation

[0061] In the repair of abdominal wall defects, antibacterial properties are crucial for preventing infection, reducing inflammation, prolonging the lifespan of implant materials, and promoting wound healing. Therefore, this study used two representative Gram-negative and Gram-positive bacteria, *Escherichia coli* and *Staphylococcus aureus*, respectively, to evaluate the antibacterial efficacy of OAC-AP / PA. Figure 5As shown in (A), the antibacterial rate of OAC-AP / PA against two bacteria was tested using dilution plate counting. Compared with the control group (E. coli and S. aureus), the colony count in the pADM group showed no significant change, indicating that pADM did not have an antibacterial effect. However, the colony count in the OAC-AP / PA group decreased significantly, indicating that bacterial proliferation was significantly inhibited. This may be because the apigenin encapsulated in the hydrogel of OAC-AP / PA has a certain antibacterial effect. Figure 5 (B) shows that OAC-AP / PA had an inhibition rate of 84.17% against E. coli and 78.63% against S. aureus, demonstrating that OAC-AP / PA has good antibacterial activity.

[0062] Analysis of Cell Scratch Test Results

[0063] Cell scratch assays, also known as wound healing assays or migration assays, simulate the process of cell migration in vivo to some extent. They can be used to study the specific impact of material-cell interactions on cell migration behavior, and are of great significance for evaluating the biocompatibility of materials and optimizing their design for tissue repair, drug delivery, or other biomedical applications. Figure 6 As shown, the shrinkage of the scratch area is the result of the combined effects of cell proliferation and migration. In the 12-hour images, the polygonal cell morphology and tightly packed distribution at the scratch edge indicate that cells are actively proliferating and migrating. The more densely distributed cells at the scratch site in the OAC-AP / PA group suggest a relatively faster migration rate and stronger cell migration activity. Complete repair of the scratch area within 24 hours demonstrates that both pADM and OAC-AP / PA can accelerate cell proliferation and migration and induce wound closure, further proving their excellent biocompatibility.

[0064] Observation on the repair of abdominal wall defects

[0065] Figure 7Morphological observation of pADM, PDA-pADM, and OAC-AP / PA in repairing abdominal wall defects in rats. No animal deaths or hernias were observed. On postoperative day 7, fibrous connective tissue proliferation and angiogenesis were observed, and no significant foreign body reaction was observed in any of the three groups. This is attributed to the excellent biological properties of pADM as a collagen-based scaffold material, exhibiting good healing-promoting capabilities. On postoperative day 14, significant degradation of pADM was clearly observed, while the regenerated tissue thickness in both the pADM and PDA-pADM groups did not reach the normal abdominal wall thickness. The PDA-pADM and OAC-AP / PA materials maintained a certain shape and strength, without shrinkage, and continued to provide tension for the incomplete abdominal wall repair. The original abdominal defect was filled with fibrous connective tissue, and neovascularization was visible within the tissue. This indicates that compared to pADM and PDA-pADM, the OAC-AP / PA patch can provide the greatest support for the regeneration and repair of abdominal wall defects.

[0066] Observation on the repair of abdominal wall defects

[0067] like Figure 8 As shown, histopathological examination of the abdominal wall defect surgical site was performed using hematoxylin and eosin (HE) staining. HE staining results on day fourteen indicated that pADM, PDA-pADM, and OAC-pADM all showed a good trend of neovascularization during the repair of the abdominal wall defect, and also induced varying degrees of inflammatory response. The pADM group showed greater overall inflammatory infiltration, slower muscle layer repair, and invasion by other cells. This may be due to the weak anti-inflammatory ability of the pADM material itself, and the excessively rapid degradation rate of the blank pADM, which significantly weakened the barrier function.

[16] While the PDA-pADM group showed some muscle growth and inflammatory response subsided, the newly formed muscle layer was significantly thinner, falling far short of the required thickness. Furthermore, the OAC-AP / PA group exhibited less inflammatory response, with thicker muscle fibers and a tighter weave, indicating that the OAC-AP / PA patch can rapidly repair and reconstruct abdominal wall defects. This result further confirms its excellent regenerative effect on abdominal wall defects.

Claims

1. A method for preparing an in-situ gelled collagen-based biological patch, characterized in that, Includes the following steps: (1) Preparation of dopamine-modified decellularized matrix materials: Weigh a certain amount of decellularized matrix material, add a certain amount of pure water, and under nitrogen protection, add dopamine hydrochloride to treat for a certain period of time. Then adjust the pH of the reaction solution to weakly alkaline, and discard the waste liquid after treatment for a certain period of time. Add a certain amount of pure water again, wash, discard the waste liquid, and repeat the washing process several times. (2) Preparation of in-situ hydrogel precursor solution: A certain amount of collagen by weight was dissolved in a buffer solution at a concentration of 1–10 mg / mL, and oxidized polysaccharide and phenylboronic acid-modified biomacromolecules were added. (3) Preparation of in-situ gelled collagen-based bio-patterns based on dopamine crosslinking: Dopamine-modified decellularized matrix was placed in hydrogel precursor solution, drug loading was added, and the mixture was stirred and incubated to allow it to fully gel. PBS buffer was added, and the mixture was washed repeatedly.

2. The method for preparing an in-situ gelled collagen-based biological patch as described in claim 1, characterized in that: The decellularized matrix material mentioned in step (1) is one of the following: decellularized dermal matrix, decellularized amniotic matrix, decellularized submucosal matrix of small intestine, decellularized urethral matrix, and decellularized peritoneal matrix.

3. The method for preparing an in-situ gelled collagen-based biological patch as described in claim 1, characterized in that: In step (1), the reaction temperature is controlled at 4 to 40°C.

4. The method for preparing an in-situ gelled collagen-based biological patch as described in claim 1, characterized in that: The pH value in step (1) is 7-10.

5. The method for preparing an in-situ gelled collagen-based biopatch as described in claim 1, characterized in that: The amount of dopamine hydrochloride used in step (1) is 1%-50% of the decellularized matrix material.

6. The method for preparing an in-situ gelled collagen-based biopatch as described in claim 1, characterized in that: The oxidized polysaccharide mentioned in step (2) is one or more of the following: oxidized chitosan, oxidized hyaluronic acid, oxidized chondroitin sulfate, oxidized sodium alginate, oxidized carboxymethyl chitosan, and oxidized heparin.

7. The method for preparing an in-situ gelled collagen-based biological patch as described in claim 1, characterized in that: The phenylboronicized biomacromolecule mentioned in step (2) is one or more of the following: gelatin grafted with phenylboronic acid, hyaluronic acid grafted with phenylboronic acid, chondroitin sulfate grafted with phenylboronic acid, sodium alginate grafted with phenylboronic acid, carboxymethyl chitosan grafted with phenylboronic acid, and heparin grafted with phenylboronic acid.

8. The method for preparing an in-situ gelled collagen-based biopatch as described in claim 1, characterized in that: The amount of PBS buffer used in step (3) is 5-50 times the weight of the dopamine-modified decellularized matrix.

9. The method for preparing an in-situ gelled collagen-based biopatch as described in claim 1, characterized in that: The loaded drug in step (3) is one or more of antibiotics, growth factors, and water-soluble natural drugs.

10. The method for preparing an in-situ gelled collagen-based biological patch as described in claim 1, characterized in that: The loaded drug in step (3) is one or more of the micelle or microsphere morphologies of apigenin, curcumin, etc.

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