Preparation method of in-situ gelation collagen-based biological patch

Abstract

The invention discloses a preparation method of an in-situ gelation collagen-based biological patch, which comprises the following steps: generating hydrogel in situ on the surface of an acellular matrix material and among internal fiber gaps by introducing various chemical reactivity of dopamine / polydopamine, and combining a hydrogel system and the acellular matrix material into one to construct the in-situ gelation biological patch. The material effectively improves the mechanical support effect, drug loading and cell growth promotion of defective tissues, also has the tissue repair functions of healing promotion, adhesion prevention, infection resistance and the like, and can be widely used for functional modification of acellular matrix materials.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a method for preparing an in-situ gelled collagen-based biological patch. Background Art

[0002] Abdominal wall injuries are common in congenital ruptures, trauma, and primary or secondary tumor resections, and are a common surgical challenge. Since direct suture has a significant risk of recurrence, the use of implant materials for tension-free healing is currently a common method for treating abdominal wall defects. Typically, surgeons choose permanent synthetic mesh materials such as polypropylene to repair the damaged area for maximum support. However, the foreign body reaction and fibrosis process caused by such mesh synthetic materials are significantly accelerated, which may lead to long-term pain and limited mobility. At the same time, their non-biodegradability may lead to postoperative complications such as intestinal mucosal damage and deformation.

[0003] Although natural bioactive patches have good biocompatibility and no toxic side effects, they often show insufficient mechanical and thermal stability, cannot provide mechanical support for the defect, and are difficult to meet the requirements of clinical applications. Decellularized matrix material (dECM), as a natural biomaterial derived from tissue, is mainly composed of collagen. It has attracted widespread attention due to its high natural abundance, good biocompatibility, and excellent performance in various biomedical applications. dECM is essentially a collagen scaffold with a three-dimensional network structure that can support the defect site and has been proven to be a good solution for abdominal wall defect repair. However, in actual applications, there are still limitations such as insufficient healing, anti-inflammatory, and sustained-release drugs. In addition, due to the lack of further functionality (such as anti-inflammatory, antibacterial, etc.), it is difficult to cope with the complex application scenarios of clinical diseases. Summary of the invention

[0004] Based on this, it is necessary to provide a method for preparing a composite biological patch that can make the prepared biological repair mesh have good mechanical properties and multiple functional properties such as drug delivery, environmental response, drug sustained release, cell carrier, and nutrient supply.

[0005] The purpose of the present invention is to provide a method for preparing an in-situ gelled collagen-based biological patch in view of the deficiencies of the prior art, which is achieved by the following technical measures.

[0006] A method for preparing an in situ gelling collagen-based biological patch based on dopamine cross-linking comprises the following steps:

[0007] (1) Preparation of dopamine-modified acellular matrix materials:

[0008] Weigh a certain weight of decellularized matrix material, add a certain weight of pure water, add dopamine hydrochloride for a certain time under nitrogen protection, then adjust the pH value of the reaction solution to alkaline, discard the waste liquid after a certain time, then add a certain weight of pure water, wash, discard the waste liquid, and repeat the washing for multiple times.

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

[0010] A certain weight portion of collagen is dissolved in a buffer solution, and oxidized polysaccharide and phenylboronated biomacromolecule are added. (3) Preparation of in situ gelled collagen-based biopatch based on dopamine cross-linking:

[0011] The dopamine-modified decellularized matrix was placed in a hydrogel precursor solution, and the loading drug was added. After stirring, it was allowed to stand and incubate to fully gelate, and then PBS buffer was added and washed, which was repeated several times.

[0012] In one embodiment, the decellularized matrix material in step (1) can be a decellularized dermal matrix, a decellularized amniotic membrane matrix, a decellularized small intestinal submucosa matrix, a decellularized urethral matrix, or a decellularized peritoneal matrix.

[0013] In one embodiment, the reaction temperature in step (1) is controlled at 4-40°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, and Tris-HCl solution.

[0017] In one embodiment, the buffer solution in step (2) is one or more of acetic acid-sodium acetate buffer solution and phosphate buffered saline solution.

[0018] In one embodiment, the oxidized polysaccharide in step (2) can 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 phenylboronic acid-modified biomacromolecule in step (2) refers to one or more of 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) can be one or more 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 drugs such as apigenin, curcumin, etc. in the form of micelles or microspheres.

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

[0024] (1) This method takes into account the implementation of cross-linking and in situ engineering strategies of decellularized matrix materials. Dopamine is first introduced to fully penetrate the decellularized matrix material and then induce cross-linking to improve its physical and chemical properties, resistance to enzyme degradation and mechanical support performance. Subsequently, the multi-reactive properties of dopamine and the formed polydopamine are used to construct a hydrogel in situ, achieving a combination of cross-linking and in situ platform functions.

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

[0026] (3) The in-situ hydrogel is uniform and complete. The porous and irregular structural characteristics of the surface of the decellularized matrix enable the in-situ hydrogel to better conform to the morphology of the substrate surface than the pre-formed and then bonded hydrogel. The organic combination of the two ensures that the hydrogel and the substrate will not separate during the entire patch service process but can play their own roles, laying a good foundation for subsequent functions and applications.

[0027] (4) In situ hydrogelation creates favorable conditions for the multifunctionalization of decellularized matrix materials. Hydrogels have been used to encapsulate a variety of drugs or biomacromolecules due to their porous structure and excellent biocompatibility. Their drug loading has many advantages, such as high drug encapsulation efficiency, controllable drug release rate, and long delivery time. The constructed hydrogels can be used as carriers of drugs or other bioactive molecules, or can achieve multifunctional integration of decellularized matrix materials such as anti-calcification, antibacterial, anti-inflammatory, pro-angiogenic, pro-cell proliferation, pro-microcirculation, and anti-cancer.

[0028] ( 5) Good controllability. The reaction can be adjusted by adjusting the amount of reactants in the reaction system, environmental conditions, etc. Among them, the aldehyde group in the oxidized polysaccharide can also provide chemical cross-linking in the hydrogel, and the phenylborated biomacromolecule can form a borate ester bond with certain polysaccharides (containing hydroxyl groups), which will break in response to the environment (such as pH, glucose), and can achieve the purpose of environmental responsive drug release, thereby regulating the various performance and functionality of the material.

[0029] (6) Biodegradable. Since the acellular matrix material itself is biodegradable and the hydrogel component used is also biodegradable, the biodegradability of the composite biopatch can still be maintained, and its degradation properties can be adjusted by the degree of cross-linking.

[0030] (7) Large-scale industrialization is possible. Acellular matrix materials are commonly used raw materials in the field of biological patches. The methacrylylated biomacromolecules required for constructing in situ hydrogels can be prepared from a wide range of biomacromolecules using mature technologies. Therefore, this method can be applied to large-scale industrial production and is widely used in the field of biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 This is the OAC-AP / PA anticoagulant performance experiment of Example 1.

[0033] Figure 3 This is a graph showing the ultraviolet absorption curve of the OAC-AP / PA sample of Example 1 after co-culture with the free radical solution.

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

[0035] Figure 5 The antibacterial properties of OAC-AP / PA in Example 2; (A) contact antibacterial activity image; (B) inhibition 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 the morphological observation of the OAC-AP / PA patch of Example 1 repairing the abdominal wall defect in rats.

[0038] Figure 8 HE staining observation of the OAC-AP / PA patch of Example 1 repairing the abdominal wall defect in rats. DETAILED DESCRIPTION

[0039] The present invention is described in detail below through examples. It is necessary to point out that this example is only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art in this field can make some non-essential improvements and adjustments based on the content of the above invention.

[0040] Example 1

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

[0042] (2) Dissolve collagen, sodium phenylboronic acid alginate and oxidized low molecular weight heparin at a concentration of 5 mg / mL in PBS buffer (10 mmol / L phosphate, 100 mmol / L sodium chloride, pH 7.4) at room temperature, add drug-loaded micelles to a concentration of 15%, mix well, and obtain a hydrogel precursor solution;

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

[0044] Example 2

[0045] (1) Weigh 10 g of decellularized amniotic membrane matrix in a conical flask, immerse it in 50 ml of distilled water, add 0.2 g of dopamine hydrochloride, and shake on a rocking rack for 1 hour under nitrogen protection and low temperature; remove nitrogen protection, use 1 M sodium hydroxide solution to adjust the pH of the reaction system to 8, then increase the temperature to 25°C, and continue to react on the rocking rack for 10 hours. After the reaction is completed, use distilled water to thoroughly wash the material, PDA-pADM;

[0046] (2) dissolving 2 g collagen, 2 g sodium phenylboronic acid alginate and 2 g oxidized sodium alginate in 20 g PBS, adding appropriate amount of growth factor, and mixing well at low temperature to obtain a hydrogel precursor solution;

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

[0048] Data analysis

[0049] pADM indicates acellular porcine dermal matrix;

[0050] PDA-pADM indicates dopamine-permeated and then cross-linked acellular porcine dermal matrix;

[0051] OAC-AP / PA indicates drug-loaded in situ hydrogel coated acellular porcine dermal matrix;

[0052] Morphology analysis

[0053] The cross-sectional morphology of OAC-AP / PA was observed by scanning electron microscopy. Figure 1 As shown, it can be observed that the hydrogel adheres to the surface of the pADM material, showing a good pore structure, which can provide space for cells to migrate and grow, enhance the interaction between cells and materials, and promote cell adhesion, proliferation and differentiation. At the same time, the longitudinal section porous structure of pADM can also be observed, and the collagen fiber bundles inside it are stacked layer by layer, intertwined, and crisscrossed together, which can meet the material metabolism and cell migration during cell growth and provide cells with a suitable growth environment.

[0054] Anticoagulation test evaluation

[0055] For implantable materials, good anticoagulant properties can effectively prevent thrombosis, reduce local inflammatory reactions, and improve the stability and functional durability of implants, thereby extending the service life of implantable materials. BCI can reflect the ability of materials to form thrombi after contact with blood. BCI is used to characterize the in vitro anticoagulant ability of OAC-AP / PA materials. The higher the BCI value, the better the anticoagulant performance of the material. Figure 2 As shown in the figure, the BCI indexes of pADM and OAC-AP / PA were 64.92% and 82.01%, respectively, indicating that OAC-AP / PA has a better anticoagulant effect. The reason may be that the low molecular weight heparin in the hydrogel can inhibit the generation and activity of thrombin, thus playing an anticoagulant role to a certain extent.

[0056] Evaluation of antioxidant properties

[0057] Scavenging excess reactive oxygen species (ROS) or reactive nitrogen species (RNS) is an important strategy to remove excess free radicals and alleviate oxidative stress. DPPH and ABTS are typical nitrogen-containing free radicals, and ·OH is a typical oxygen-containing free radical. They are widely used to evaluate the scavenging ability of materials for reactive nitrogen species (RNS) and reactive oxygen species (ROS). Figure 3 (A) (B) (C) are the UV absorption curves of OAC-AP / PA samples after co-incubation with DPPH, ABTS and ·OH free radical solutions. As shown in the figure, the characteristic absorption peaks of OAC-AP / PA sample solution at 517nm, 734nm and 510nm are significantly weakened. Figure 3 As shown in (D), OAC-AP / PA is expected to be used for the treatment of abdominal wall defects, quickly responding to the special microenvironment of ROS and RNS at the defect site, thereby intelligently accelerating drug release, slowing down inflammatory responses, and reducing damage to cells.

[0058] like Figure 4 As shown in (A), a stimulation experiment was performed using a culture medium containing 5ug / ml H2O2, and cell viability was determined by MTT. The OD values ​​of the positive control group and the OAC-AP / PA sample group decreased to a certain extent at 1d and 3d, indicating that the cell viability decreased or even died due to changes in environmental conditions, while the OD value of the OAC-AP / PA sample group decreased to a lesser extent, indicating that the oxidative stimulation of L929 cells was weakened under its protection, indicating that OAC-AP / PA has a strong antioxidant capacity.

[0059] like Figure 4 As shown in (B), the antioxidant capacity of OAC-AP / PA samples was comprehensively evaluated by the MDA content and GSH / GSSG results in L929 cell precipitates. The MDA level in cells stimulated by hydrogen peroxide was significantly increased, while the MDA content in the OAC-AP / PA sample group increased less; while the GSH / GSSG ratio in cells stimulated by hydrogen peroxide was significantly decreased, while the GSH / GSSG ratio in the OAC-AP / PA sample group decreased less, indicating that the oxidative stress response of L929 cells was significantly inhibited under the protection of the skin-based hydrogel system, which further proved that OAC-AP / PA has a strong antioxidant capacity.

[0060] Antibacterial performance evaluation

[0061] In the process of abdominal wall defect repair, antibacterial properties are of great significance for preventing infection, reducing inflammatory response, prolonging the life of implant materials, and promoting wound healing. Therefore, this study used Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), two representative Gram-negative and Gram-positive bacteria, to evaluate the antibacterial effect of OAC-AP / PA. Figure 5As shown in (A), the antibacterial rate of OAC-AP / PA against two bacteria was tested by dilution plate counting. Compared with the E. coli colonies and S. aureus colonies in the blank group, the colony count of the pADM group did not change significantly, indicating that pADM had no antibacterial effect. The colony count of the OAC-AP / PA group decreased significantly, indicating that the proliferation of bacteria was significantly inhibited, which may be due to the fact that the apigenin encapsulated in the hydrogel in OAC-AP / PA has a certain antibacterial effect. Figure 5 (B) showed that the inhibition rate of OAC-AP / PA against E. coli was 84.17%, and the inhibition rate against S. aureus was 78.63%, which proved that OAC-AP / PA had good antibacterial activity.

[0062] Analysis of cell scratch assay results

[0063] The cell scratch test is also called the wound healing test or migration test. It simulates the process of cell migration in vivo to a certain extent and can be used to study the specific effects of material-cell interaction on cell migration behavior. It is 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 in the figure, the reduction of the scratch area is the result of the combined effect of cell proliferation and cell migration. In the 12h image, the polygonal cell morphology and tightly packed distribution at the edge of the scratch indicate that the cells are actively undergoing proliferation and migration, while the cells at the scratch in the OAC-AP / PA group are more densely spread, indicating that their migration speed is relatively fast and the cell migration activity is stronger. The scratch area was completely repaired within 24h, indicating that both pADM and OAC-AP / PA can accelerate cell proliferation and migration and induce closure of the injured area, further demonstrating their excellent biocompatibility.

[0064] Observation on the repair of abdominal wall defects

[0065] Figure 7The morphological observation of pADM, PDA-pADM and OAC-AP / PA in repairing abdominal wall defects in rats. No animal death or hernia was observed. On the 7th day after surgery, fibrous connective tissue hyperplasia and neovascularization were observed, and there was no obvious foreign body reaction in the three groups of samples. This is due to the excellent biological properties of pADM as a collagen-based scaffold material, which has good healing-promoting ability. On the 14th day after surgery, it can be clearly observed that pADM has a large degree of degradation, and at this time, the thickness of the regenerated tissue in the pADM and PDA-pADM groups did not reach the normal abdominal wall thickness. The PDA-pADM and OAC-AP / PA materials still maintain a certain shape and strength, without shrinkage, and can continue to provide tension for the repair of the unfinished abdominal wall. The original abdominal defect is filled with fibrous connective tissue, and neovascularization can be seen in the tissue. It shows that compared with pADM and PDA-pADM, the OAC-AP / PA patch can support the regeneration and repair of abdominal wall defects to the greatest extent.

[0066] Observation on the repair of abdominal wall defects

[0067] like Figure 8 As shown, the surgical site of the abdominal wall defect was subjected to histopathological examination using hematoxylin and eosin (HE) staining. The HE staining results on the 14th day showed that pADM, PDA-pADM and OAC-pADM all showed a good trend of new blood vessel growth during the repair of the abdominal wall defect, and also caused different degrees of inflammatory response. The pADM group had more overall inflammatory infiltration, slow muscle layer repair, and invasion by other cells. The reason may be that the anti-inflammatory ability of the pADM material itself is weak, and the degradation rate of blank pADM is too fast, which greatly weakens the barrier effect.

[16] Although the PDA-pADM group grew a certain thickness of muscle layer and the inflammatory response subsided, the new muscle layer was significantly thinner and its thickness was far from the normal thickness requirement. In addition, the OAC-AP / PA group had a smaller inflammatory response, and the muscle layer fibers were thicker and more tightly woven, indicating that the OAC-AP / PA patch can quickly repair and reconstruct the defective abdominal wall. This result further confirms its excellent regenerative effect on the defective abdominal wall.

Claims

1. A method for preparing an in situ gelled collagen-based biological patch, characterized in that: The steps include: (1) Preparation of dopamine-modified acellular matrix materials: Weigh a certain weight of decellularized matrix material, add a certain weight of pure water, add dopamine hydrochloride for a certain time under nitrogen protection, then adjust the pH value of the reaction solution to be weakly alkaline, discard the waste liquid after a certain time, then add a certain weight of pure water, wash, discard the waste liquid, and repeat the washing for multiple times. (2) Preparation of in situ hydrogel precursor solution: A certain weight portion of collagen is dissolved in a buffer solution at a concentration of 1-10 mg / mL, and oxidized polysaccharide and phenylboronated biomacromolecule are added. (3) Preparation of in situ gelled collagen-based biopatch based on dopamine cross-linking: The dopamine-modified decellularized matrix is ​​placed in a hydrogel precursor solution, and loaded drugs are added. After stirring, the solution is allowed to stand and incubate for full gelation. PBS buffer is added, and the solution is washed and repeated for multiple times.

2. The method for preparing an in situ gelling collagen-based biological patch according to claim 1, characterized in that: The decellularized matrix material in step (1) can be one of a decellularized dermal matrix, a decellularized amniotic membrane matrix, a decellularized small intestinal submucosal matrix, a decellularized urethral matrix, and a decellularized peritoneal matrix.

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

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

5. The method for preparing an in situ gelling collagen-based biological patch according to 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 gelling collagen-based biological patch according to claim 1, characterized in that: The oxidized polysaccharide described in step (2) can be one or more of 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 gelling collagen-based biological patch according to claim 1, characterized in that: The phenylboronic acid-modified biomacromolecule described in step (2) can be one or more of 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 gelling collagen-based biological patch according to 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 gelling collagen-based biological patch according to claim 1, characterized in that: The loaded drugs in step (3) can be one or more water-soluble drugs such as antibiotics, growth factors, and water-soluble natural drugs.

10. The method for preparing an in situ gelling collagen-based biological patch according to claim 1, characterized in that: The loaded drug in step (3) can be one or more of the poorly soluble drugs such as apigenin, curcumin, etc. in the form of micelles or microspheres.

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