A PLA-rhCol composite material and its preparation method and application

Abstract

The present invention discloses a PLA-rhCol composite material, its preparation method, and application. The PLA-rhCol composite material of the present invention comprises an NH2 hydrophilic segment molecular chain on a recombinant human-like collagen. Through sodium hydroxide hydrolysis treatment combined with a Schiff base reaction, under the action of EDC and NHS, type I recombinant human-like collagen is successfully grafted onto the surface of a polylactic acid material to form a PLA-rhCol composite material. The synthesis method of the present invention is simple and controllable, and no harmful by-products are generated. The resulting PLA-rhCol composite polymer has enhanced toughness and hydrophilicity, while accelerating the degradation rate of polylactic acid, making it versatile in tissue engineering, biodegradability, and biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of polylactic acid material modification, and specifically relates to a PLA-rhCol (recombinant human-like collagen grafted onto polylactic acid) composite material, its preparation method, and its application. The PLA-rhCol composite material is particularly useful in the fields of medical devices, drug delivery, and tissue engineering. Background Art

[0002] Polylactic acid (PLA) has been certified by the U.S. Food and Drug Administration (FDA) due to its good biocompatibility and biodegradability, as well as the ability of its degradation products to participate in the tricarboxylic acid cycle in the body. It has been widely used in many fields, including surgical sutures, bone fixators, drug delivery systems, tissue engineering materials, and general fully degradable plastics. However, PLA also has some disadvantages, such as its strong hydrophobicity, acid accumulation during degradation that can easily cause swelling and inflammatory reactions, the lack of active groups that further react with bioactive factors, and the lack of cell-specific recognition signals.

[0003] In recent years, to address these shortcomings of PLA, increasing research has focused on modifying it to improve its performance. PLA modification involves introducing different functional groups or structural units to enhance its properties and performance, thereby expanding its use in various applications. These include enhancing its bioactivity, modifying its surface properties to improve cell recognition and adhesion, and controlling its degradation rate to reduce inflammatory responses.

[0004] By selecting appropriate modification strategies, we can effectively address the challenges facing polylactic acid in fields such as medicine and biomaterials, thereby promoting its widespread application and further development in these fields. This diverse modification approach not only improves polylactic acid's mechanical properties, biocompatibility, and degradation behavior, but also expands its functionality, giving it more potential applications. Therefore, modified polylactic acid can better adapt to different application scenarios, bringing more innovation and possibilities to the fields of medicine and biomaterials.

[0005] Traditional collagen mainly comes from animal tissues, such as cowhide, pig skin, fish skin, etc., and collagen from these sources has various limitations. First, animal-derived collagen has the risk of triggering immune rejection, which is a serious consideration, especially in clinical use. Secondly, the extraction process of traditional collagen may involve some risks of biological contamination, which is unacceptable for medical applications that require high purification. In addition, the structure and properties of traditional collagen are largely limited by the source of raw materials, which means that its customizability in clinical and engineering applications is limited.

[0006] Recombinant human-like collagen is synthesized through genetic engineering techniques, allowing for better control of purity, yield, and quality during the production process. The production process primarily involves the following steps: First, collagen mRNA is obtained from human tissue. The mRNA is treated with reverse transcriptase to generate the corresponding cDNA. The cDNA is then digested with enzymes to obtain the desired fragments, which are then ligated. The ligated cDNA fragments are then transferred into expression bacteria, which have high expression capacity. Fermentation techniques are then used to enable the expression bacteria to produce recombinant human-like collagen at high densities. Finally, the fermentation broth is separated and purified to obtain high-purity recombinant human-like collagen (rhCol). This genetically engineered recombinant human-like collagen avoids a number of issues associated with extracting animal-derived collagen, while also allowing for better control of product properties and quality, resulting in significant potential for future applications. Therefore, with the continued development and improvement of genetic engineering techniques, the combination of recombinant human-like collagen with polylactic acid (PLA) materials will have even greater potential for application in pharmaceuticals, food, cosmetics, and other fields.

[0007] Based on this, it is necessary to seek a recombinant human-like collagen grafted polylactic acid composite material and its preparation method and application to solve the above problems. Summary of the Invention

[0008] To address the above-mentioned technical problems, the primary objective of the present invention is to provide a method for preparing PLA-rhCol. This method, through a surface modification grafting process, successfully constructs a PLA composite material grafted with recombinant human-like collagen while retaining the basic structure and properties of the PLA material itself. This method maintains virtually no change in the chemical composition and structure of the material, while significantly improving the surface roughness and hydrophilicity. This modification method not only imparts greater functionality to the material but also enhances its biocompatibility. Furthermore, this material has not been reported in the literature.

[0009] The second object of the present invention is to provide a PLA-rhCol composite material prepared by the above preparation method.

[0010] The third object of the present invention is to provide an application of a PLA-rhCol composite material.

[0011] The primary purpose of the present invention is achieved through the following technical solutions:

[0012] A method for preparing a PLA-rhCol composite material comprises the following steps:

[0013] S1. dissolving polylactic acid particles in an organic reagent to prepare a polylactic acid material (PLA) by solvent evaporation;

[0014] S2. The polylactic acid material obtained in step S1 is treated with a sodium hydroxide solution hydrolysis method to partially hydrolyze the surface of the polylactic acid material, break the main chain, expose the carboxyl group, and prepare a polycarboxyl polylactic acid material (PLA-COOH);

[0015] S3. At room temperature and away from light, the polycarboxylic polylactic acid material obtained in step S2 was added to an aqueous solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and the pH was adjusted to 5 to 6 for the reaction;

[0016] S4. At room temperature and protected from light, add N-hydroxysuccinimide (NHS) aqueous solution and recombinant human collagen (rhCol) aqueous solution to step S3, adjust the pH to 7 to 8, and after the reaction, wash and dry to obtain a PLA-rhCol composite material;

[0017] The polylactic acid particles, organic reagent, sodium hydroxide solution, EDC aqueous solution, NHS aqueous solution, and rhCol aqueous solution are prepared according to the following ratios: 1-100 g: 5-500 mL: 10-500 mmol: 0.05-25 mmol: 0.01-5 mmol: 0.05-1 mg.

[0018] Preferably, the organic reagent in step S1 is at least one of chloroform, dichloromethane, acetone, DMF (N,N-dimethylformamide) and DMSO (dimethyl sulfoxide).

[0019] Preferably, the treatment time of the sodium hydroxide solution in step S2 is 5 to 30 minutes, and the reaction temperature is 25 to 45°C.

[0020] Preferably, the reaction time in step S3 is 3 hours.

[0021] Preferably, the reaction time in step S4 is 12 hours.

[0022] Preferably, the molar ratio of EDC to NHS is (1-5):1.

[0023] Preferably, the concentration of the rhCol aqueous solution in step S4 is 0.05-10 mg / mL.

[0024] Preferably, the molecular weight of the rhCol in step S4 is 25-50 kDa.

[0025] The second object of the present invention is achieved by the following technical solutions:

[0026] A PLA-rhCol composite material is prepared by the above preparation method.

[0027] Preferably, the molecular weight of the PLA-rhCol composite material is 50 to 250 kDa.

[0028] Preferably, the PLA-rhCol composite material is one of silk, film, stent, microsphere or wire.

[0029] The third object of the present invention is achieved through the following technical solutions:

[0030] A PLA-rhCol composite material is used in the fields of medical equipment, drug delivery and tissue engineering.

[0031] Compared with the prior art, the beneficial effects and advantages of the present invention are as follows:

[0032] The present invention uses a surface modification grafting method, first using EDC and then NHS to deactivate the carboxyl groups. While retaining the basic structure and properties of polylactic acid itself, it successfully constructs a polylactic acid composite modified with recombinant human-like collagen. Through this method, the chemical composition and structure of the polylactic acid material remain virtually unchanged, and the roughness and hydrophilicity of the material surface are significantly improved. This modification method not only gives the polylactic acid material more functionality, but also improves its biocompatibility. This innovation is expected to bring new breakthroughs and progress to research and application in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is a Fourier transform attenuated total reflection infrared spectrum of the product of Example 1 of the present invention and the polylactic acid material;

[0035] Figure 2 This is an X-ray photoelectron spectrum of the product of Example 2 of the present invention and a polylactic acid material;

[0036] Figure 3 This is a scanning electron microscope image of the product of Example 4 of the present invention;

[0037] Figure 4 This is a scanning electron microscope image of the product of Example 3 of the present invention;

[0038] Figure 5 This is a scanning electron microscope image of the product of Example 2 of the present invention;

[0039] Figure 6 This is a contact angle measurement diagram of the product of Example 4 of the present invention and the polylactic acid material;

[0040] Figure 7 is the BMSCs cell survival rate of the product of Example 5 of the present invention;

[0041] Figure 8 The results of the BMSCs cell migration experiment of the product of Example 6 of the present invention are as follows;

[0042] Figure 9 is the BMSCs cell migration rate of the product of Example 6 of the present invention;

[0043] Figure 10 This is the result of the chicken embryo allantoic vascularization experiment of the product of Example 4 of the present invention;

[0044] Figure 11 This is a quantitative statistical graph of the blood vessel area of ​​the chicken embryo allantoic vascularization experiment of the product of Example 4 of the present invention;

[0045] Figure 12 This is the application of the product of Example 4 of the present invention in skull defects. DETAILED DESCRIPTION

[0046] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0047] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0048] Example 1

[0049] A PLA-rhCol composite material comprises the following steps:

[0050] S1. 2 g of polylactic acid particles were dissolved in 10 mL of chloroform to prepare polylactic acid material (PLA) by solvent evaporation;

[0051] S2. The polylactic acid material obtained in step S1 was treated with 4 mL of 10 mmol / L sodium hydroxide solution for 30 min to hydrolyze the surface of the polylactic acid material locally, cleaving the main chain and exposing the carboxyl group to obtain a polycarboxyl polylactic acid material (PLA-COOH);

[0052] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 1.5mL 0.5mmol / L EDC aqueous solution, the pH was adjusted to 5.5 and the reaction was continued for 3 hours;

[0053] S4. Add 0.02 mL of 0.1 mmol / L NHS aqueous solution and 1 mL of 0.02 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 7, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 1.

[0054] Figure 1 This is a Fourier transform attenuated total reflection infrared spectrum of the product of Example 1 of the present invention and the polylactic acid material.

[0055] Example 2

[0056] A PLA-rhCol composite material comprises the following steps:

[0057] S1. 5 g of polylactic acid particles were dissolved in 25 mL of chloroform to prepare polylactic acid material by a W / O emulsion method;

[0058] S2. The polylactic acid material obtained in step S1 was treated with 2 mL of 25 mmol / L sodium hydroxide solution for alkaline hydrolysis for 10 min, so that the surface of the polylactic acid material was locally hydrolyzed, the main chain was broken, and the carboxyl groups were exposed to obtain a polycarboxyl polylactic acid material;

[0059] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 15mL 1.25mmol / L EDC aqueous solution, and the pH was adjusted to 6 for 3 hours;

[0060] S4. Add 10 mL of 0.25 mmol / L NHS aqueous solution and 5 mL of 0.05 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 8, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 2.

[0061] Figure 2 This is an X-ray photoelectron spectrum of the product of Example 2 of the present invention and a polylactic acid material; Figure 5 This is a scanning electron microscope image of the product of Example 2 of the present invention.

[0062] Example 3

[0063] A PLA-rhCol composite material comprises the following steps:

[0064] S1. 10 g of polylactic acid particles were dissolved in 100 mL of dichloromethane and polylactic acid material was prepared by 3D printing.

[0065] S2. The polylactic acid material obtained in step S1 was treated with 50 mL of a 50 mmol / L sodium hydroxide solution for 5 min of alkaline hydrolysis to partially hydrolyze the surface of the polylactic acid material, cleaving the main chain and exposing the carboxyl group to obtain a polycarboxyl polylactic acid material;

[0066] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 20mL 2.5mmol / L EDC aqueous solution, the pH was adjusted to 5.5 and the reaction was continued for 3 hours;

[0067] S4. Add 20 mL of 0.5 mmol / L NHS aqueous solution and 20 mL of 0.1 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 8, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 3.

[0068] Figure 4 This is a scanning electron microscope image of the product of Example 3 of the present invention.

[0069] Example 4

[0070] A PLA-rhCol composite material comprises the following steps:

[0071] S1. 2 g of polylactic acid particles were dissolved in 10 mL of dichloromethane to prepare a polylactic acid material by solvent evaporation;

[0072] S2. The polylactic acid material obtained in step S1 was treated with 1 mL of 10 mmol / L sodium hydroxide solution for 30 min of alkaline hydrolysis to partially hydrolyze the surface of the polylactic acid material, break the main chain, and expose the carboxyl group to obtain a polycarboxyl polylactic acid material;

[0073] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 10mL of 0.5mmol / L EDC aqueous solution, and the pH was adjusted to 6 for 3 hours;

[0074] S4. Add 5 mL of 0.1 mmol / L NHS aqueous solution and 2 mL of 0.02 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 8, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 4.

[0075] Figure 3 This is a scanning electron microscope image of the product of Example 4 of the present invention, Figure 6 This is a contact angle measurement diagram of the product of Example 4 of the present invention and the polylactic acid material.

[0076] Example 5

[0077] A PLA-rhCol composite material comprises the following steps:

[0078] S1. 5 g of polylactic acid particles were dissolved in 25 mL of DMF (N, N-dimethylformamide) to prepare a polylactic acid material by solvent evaporation.

[0079] S2. The polylactic acid material obtained in step S1 was treated with 15 mL of a 25 mmol / L sodium hydroxide solution for 15 min by alkaline hydrolysis to partially hydrolyze the surface of the polylactic acid material, cleaving the main chain and exposing the carboxyl group to obtain a polycarboxyl polylactic acid material;

[0080] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 50mL of 1.25mmol / L EDC aqueous solution, and the pH was adjusted to 5 and the reaction was continued for 3 hours;

[0081] S4. Add 30 mL of 0.25 mmol / L NHS aqueous solution and 20 mL of 0.05 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 7.5, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 5.

[0082] Figure 7 This is the BMSCs cell survival rate of the product of Example 5 of the present invention.

[0083] Example 6

[0084] A PLA-rhCol composite material comprises the following steps:

[0085] S1. 15 g of polylactic acid particles were dissolved in 75 mL of DMSO (dimethyl sulfoxide) to prepare a polylactic acid material by solvent evaporation.

[0086] S2. The polylactic acid material obtained in step S1 was treated with 40 mL of 1.5 mol / L sodium hydroxide solution for alkaline hydrolysis for 5 min, so that the surface of the polylactic acid material was locally hydrolyzed, the main chain was broken, and the carboxyl groups were exposed to obtain a polycarboxyl polylactic acid material;

[0087] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 50mL 3.75mmol / L EDC aqueous solution, the pH was adjusted to 6 and the reaction was continued for 3 hours;

[0088] S4. Add 25 mL of 0.75 mmol / L NHS aqueous solution and 10 mL of 0.15 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 8, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 6.

[0089] Figure 8 The results of the BMSCs cell migration experiment of the product of Example 6 of the present invention are as follows: Figure 9 This is the BMSCs cell migration rate of the product of Example 6 of the present invention.

[0090] Example 7

[0091] A PLA-rhCol composite material comprises the following steps:

[0092] S1. 100 g of polylactic acid particles were dissolved in 500 mL of DMSO (dimethyl sulfoxide) to prepare a polylactic acid material by the method;

[0093] S2. The polylactic acid material obtained in step S1 was treated with 100 mL of 500 mmol / L sodium hydroxide solution for 20 min by alkaline hydrolysis, so that the surface of the polylactic acid material was locally hydrolyzed, the main chain was broken, and the carboxyl group was exposed to obtain a polycarboxyl polylactic acid material;

[0094] S3. The polycarboxylic polylactic acid material obtained in step S2 was added to 200mL of a 25mmol / L EDC aqueous solution, and the pH was adjusted to 6 for 3 hours;

[0095] S4. Add 100 mL of 5 mmol / L NHS aqueous solution and 50 mL of 1 mg / L recombinant human collagen aqueous solution to step S3, adjust the pH to 8, and react for 12 hours. After the reaction, wash and dry to obtain the PLA-rhCol composite material of Example 7.

[0096] Example 8: Application of a PLA-rhCol composite material

[0097] Twelve female rats aged 8 to 10 weeks were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg). The skull area was prepared, and the SD rats were fixed on the operating table. The areas around the surgical area on both sides were disinfected and covered with sheets. A 2 cm linear incision was made in the center of the SD rat skull. The muscles and tendons were bluntly separated, and the bone was cut to expose the cortical bone. A 5.0 mm diameter bone drill was used to prepare 5.0 mm and 1 mm deep bone defects on both sides. When preparing the hole, 4°C saline was used to cool it to prevent necrosis of the bone tissue around the defect. Different materials were implanted bilaterally, the wound was rinsed with saline, and the skin was sutured. After the operation was completed, continuous single-unit antibiotic injections were given to prevent infection. After 1 week, 4 weeks, and 8 weeks after the implants were implanted in the animals, the rats were killed and the skull was removed to observe the new bone in the bone defect area. The rats with successful skull modeling were randomly divided into three groups: a, b, and c. Group a was a blank control group, group b was a polylactic acid material group, and group c was a recombinant human-like collagen grafted polylactic acid composite material prepared in Example 4. The skull healing conditions were as follows: Figure 12 As shown. Figure 12It can be seen that the hydrophilic polylactic acid material of the present invention has excellent angiogenic properties and promotes bone regeneration, promotes cell proliferation, and achieves the purpose of bone healing. Its effect is more significant than that of the polylactic acid material group B.

[0098] Detection of modification reaction degree of PLA-rhCol composite material:

[0099] BCA (Bicinchoninic acid method) is a commonly used method for determining protein concentration. Its principle is that under alkaline conditions, protein converts Cu into 2+ Reduction to Cu + , then Cu + It forms a purple complex with the BCA reagent. This complex has a high light absorption value at 562nm, and its color depth is proportional to the protein concentration, so the protein content can be determined by colorimetry. The recombinant human collagen solution before and after the grafting reaction is allowed to react with a quantitative amount of BCA reagent (purchased from Biyuntian). The absorbance of each well is measured at 562nm using an enzyme-linked immunosorbent assay (ELISA). The sample is run in triplicate and the average absorbance value is taken. The degree of modification reaction is calculated using the following formula.

[0100] Modification reaction degree = (OD value of recombinant human-like collagen before grafting - OD value of recombinant human-like collagen after grafting) / OD value of recombinant human-like collagen before grafting × 100%. Specific data are shown in Table 1.

[0101] Table 1 Modification reaction degree of PLA-rhCol composite materials in Examples 1-7

[0102] Test samples Modification reaction degree (%) Blank / Example 1 product 16.48 Example 2 product 14.67 Example 3 product 10.66 Example 4 Product 15.81 Example 5 Product 13.49 Example 6 product 17.43 Example 7 Product 15.42

[0103] The results show that this table primarily compares the degree of modification achieved by different examples of products during the modification reaction of recombinant human-like collagen and polylactic acid composites. Each example represents a different product, and its degree of modification reflects the differences in experimental results. The degree of modification achieved by each example product ranges from 10.66% to 17.43%, representing the degree of modification achieved by each product in the experiment.

[0104] Cytotoxicity assay

[0105] The cytotoxicity experiment used the CCK-8 method (Cell Counting Kit-8) to evaluate the effect of the material on cell activity. First, the cell well plate was removed from the incubator, the old culture medium was removed with a pipette, and PBS was washed once to ensure cleanliness. Subsequently, 400 μL of complete culture medium containing 10% CCK-8 was added to each well. Then, the well plate was placed in a cell culture incubator and incubated for 2 hours in the dark. After incubation, the culture medium was transferred to a 96-well plate, 100 μL was added to each well, and the absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay. Each experimental group included three parallel samples. To reduce errors, CCK-8 was also added to the wells without cells as a blank group, and its absorbance was recorded as OD0. The absorbance of the experimental group was recorded as OD t The absorbance of the control group was recorded as OD1. Finally, the cell survival rate was calculated by the formula:

[0106] Cell viability = (OD t -OD0) / (OD1-OD0)×100%

[0107] The results showed that the cell survival rate of the polylactic acid material group decreased significantly on the 3rd and 5th days, indicating that the polylactic acid material had an adverse effect on cell survival. However, the cell survival rate of the recombinant human-like collagen grafted polylactic acid composite material group was significantly higher than that of the polylactic acid material group at the same time point, but still lower than that of the blank control group. This shows that the addition of recombinant human-like collagen can significantly improve the biocompatibility of polylactic acid. Statistical significance analysis further confirmed the significant differences between the polylactic acid material group and the recombinant human-like collagen grafted polylactic acid composite material group and the blank group at 3 days and 5 days, indicating that these differences were statistically significant. The cell survival rate of the recombinant human-like collagen grafted polylactic acid composite material group was increased by 51.83% compared with the cell survival rate of the polylactic acid material group, indicating that the introduction of rhCol significantly promoted cell proliferation.

[0108] The cell scratch assay procedure is as follows:

[0109] (1) Cell seeding: First, the mouse bone marrow mesenchymal stem cells (BMSCs) in the culture flask were digested and transferred to a 15 mL centrifuge tube. After centrifugation (1600 r, 4 min), the supernatant was removed and the cells were resuspended in cell culture medium. Next, 10 μL of the cell suspension was taken and the cells were counted on a cell counting plate. According to the required total number of cells and cell density, the diluted cell suspension was appropriately diluted and added to a 24-well plate at a cell density of 3 × 10 4 The plate was placed in an incubator for overnight culture.

[0110] (2) Cell scratching: After cells are fully confluent, scratch the bottom of the well plate perpendicular to the ruler using a 200 μL pipette tip. Next, remove the old culture medium and wash the well plate three times with PBS to remove cells that have fallen during the scratching process. Then, add low-serum culture medium containing 2% FBS and place the well plate in the incubator for culture again.

[0111] (3) Cell staining: The culture medium in the well plate was aspirated, and the cells were washed three times with PBS. Then, 500 μL of 4% paraformaldehyde solution was added to each well to fix the cells for 30 min. The paraformaldehyde solution was aspirated, and the cells were washed three times with PBS. Then, the cells were stained with Biyuntian DiO stain (prepared according to the operating instructions in the purchased reagent manual) for 30 min and then washed once with PBS.

[0112] (4) Cell observation: cells were stained at 0, 24, and 48 h after scratching, observed and photographed under an inverted fluorescence microscope, and finally, the images were analyzed using Image J software. The scratch area at 0 h was recorded as (A0), the scratch area at 12 h or 24 h was recorded as (A t ), the cell migration rate was calculated by the formula:

[0113] Cell migration rate (%) = (A0-A t ) / A0×100%

[0114] Chicken embryo allantoic vascularization experiment

[0115] To further evaluate the vascularization-promoting ability of thin films, we used the chick cysto-allantoic membrane (CAM) vascularization assay. The following are the specific experimental steps:

[0116] (1) Egg preparation: Five-day-old SPF-grade eggs (purchased from Guangdong Xinxing Dahua Agricultural and Poultry Egg Co., Ltd.) were thoroughly cleaned with 75% alcohol and air-dried to ensure disinfection. They were then placed in a constant temperature incubator set at 38°C and relative humidity maintained at approximately 65%.

[0117] (2) Opening a window and exposing the CAM: After 7 days of culture, clean the egg shell surface with 75% alcohol to prevent bacterial infection. Use tweezers to open a 2×2 cm window on the egg surface. 2 A small hole was created to expose the allantois. Using an ear bulb, eggshell debris was gently blown away from the air chamber. Next, a 5 mL syringe needle was used to create a small scratch on the air chamber. Normal saline was dripped into the air chamber to moisten it. Then, the air chamber was gently peeled off to expose the CAM. To ensure experimental accuracy, three replicate chick embryos were used in each group.

[0118] (3) Implant material: After sterilization, place the thin sample to be tested on the surface of the allantois, ensuring sufficient contact between the sample and the allantois and maintaining stability. Then, seal the air chamber opening with a fresh-keeping tape and place the treated egg in a constant temperature incubator, maintaining appropriate temperature and humidity conditions during incubation.

[0119] (4) Observation and analysis: The growth of blood vessels in the CAM on days 7 and 8 was observed using a stereomicroscope, and the area of ​​new blood vessels in a fixed field of view was calculated.

[0120] In our experiment, we placed PLA and PLA-rhCol membranes in the CAM, respectively. We observed that by day 7 of incubation, CAM vessels exhibited robust growth. During the following 24 hours of incubation, we noted a significant increase in the number of vessels in both CAM groups by day 8. This is due to the rapid early growth of CAM vessels, resulting in a significant change in vessel number within a short period of time. Compared to the PLA group, the PLA-rhCol group, which also contained rhCol, exhibited faster vessel growth. Arrows mark the same vessels before and after 24 hours of incubation. Statistical analysis of changes in vessel diameter quantified vascular growth rates. Calculation of the ratio of vessel diameters on day 8 to day 7 revealed a value of 1.40 ± 0.05 in the PLA group and 1.74 ± 0.04 in the PLA-rhCol group, indicating that the addition of rhCol significantly promoted vessel diameter growth. We further validated the angiogenic potential of the PLLA-rhCol membrane by measuring vessel area within a fixed field of view. The results showed that the vascular area of ​​the PLA-rhCol group was 2.35±0.09mm 2 , which was significantly higher than that of the PLA group with a vascular area of ​​1.77±0.12mm 2 The CAM test results showed that the prepared PLA-rhCol membrane exhibited better ability to promote the growth and development of blood vessels compared with the PLA membrane.

[0121] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for preparing a PLA-rhCol composite material, characterized in that: The following steps are involved: S1. The polylactic acid particles are dissolved in an organic reagent to prepare a polylactic acid material by solvent evaporation; S2. The polylactic acid material obtained in step S1 is treated with a sodium hydroxide solution hydrolysis treatment method, so that the surface of the polylactic acid material is locally hydrolyzed, the main chain is broken, and the carboxyl group is exposed to prepare a polycarboxyl polylactic acid material; S3 room temperature in the dark, the polycarboxyl polylactic acid material prepared in step S2 was added to the EDC aqueous solution, and the pH was adjusted to 5 to 6 for the reaction; S4 room temperature in the dark, to step S3, add NHS aqueous solution and rhCol aqueous solution, adjust the pH to 7 to 8 reaction, after the reaction is completed, wash and dry to obtain a PLA-rhCol composite material; The polylactic acid particles, organic reagent, sodium hydroxide solution, EDC aqueous solution, NHS aqueous solution, and rhCol aqueous solution are prepared in the following ratios: 1-100 g: 5-500 mL: 10-500 mmol: 0.05-25 mmol: 0.01-5 mmol: 0.05-1 mg.

2. The method for preparing the PLA-rhCol composite material according to claim 1, wherein The organic reagent in step S1 is at least one of chloroform, dichloromethane, acetone, DMF and DMSO.

3. The method for preparing the PLA-rhCol composite material according to claim 1, wherein The treatment time of the sodium hydroxide solution in step S2 is 5 to 30 minutes, and the reaction temperature is 25 to 45°C.

4. The method for preparing the PLA-rhCol composite material according to claim 1, wherein The molar ratio of EDC to NHS is (1-5):

1.

5. The method for preparing the PLA-rhCol composite material according to claim 1, wherein The concentration of the rhCol aqueous solution in step S4 is 0.05-10 mg / mL.

6. The method for preparing the PLA-rhCol composite material according to claim 1, wherein: The molecular weight of rhCol described in step S4 is 25-50 kDa.

7. A PLA-rhCol composite material, characterized in that: Prepared according to any one of claims 1 to 6.

8. The PLA-rhCol composite material according to claim 7, characterized in that The molecular weight of the PLA-rhCol composite material is 50-250 kDa.

9. The method for preparing the PLA-rhCol composite material according to claim 7, wherein: The PLA-rhCol composite material is one of silk, film, stent, microsphere or wire.

10. Use of the PLA-rhCol composite material according to any one of claims 7 to 9 in the fields of medical devices, drug delivery for purposes other than disease diagnosis and treatment, and tissue engineering.

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