Methacrylated gelatin with dual functions

Abstract

A preparation method of the methacrylated gelatin with the dual functions comprises the following steps: firstly, preparing collagen methylated polymethacrylate, then mixing 5% of collagen methylated polymethacrylate, 0.2% of trimethylbenzoyl phosphonate, 10 mg / ml of a herba houttuyniae extract and 20 micrograms / ml of nifedimab, and carrying out uniform mixing, so as to obtain the methacrylated gelatin with the dual functions, the collagen methylated polymethacrylate and the trimethylbenzoyl phosphonate, the herba houttuyniae extract, the nifedimab and the collagen methylated polymethacrylate, the herba houttuyniae extract and the nifedimab. The methacrylated gelatin is prepared under ultraviolet irradiation, and the loose cross-linked network structure of the methacrylated gelatin can realize continuous release of drugs, so that the demand of continuous and stable drug delivery is met; in addition, the gelatin methacrylamide, the herba houttuyniae extract and the nintedanib are combined to relieve hemorrhagic intestinal adhesion and can regulate inflammation and anti-fibrosis functions.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing a methacrylated gelatin with dual functions. BACKGROUND

[0002] Intestinal adhesion is a common clinical condition that affects millions of patients worldwide. Generally, the incidence of intestinal adhesion after injury or surgery involving the abdominal cavity is more than 50%. Patients with intestinal adhesion will experience intermittent abdominal pain, and in severe cases, it can develop into mechanical intestinal obstruction, which requires surgical intervention during the onset of intestinal dysfunction. Therefore, preventing the occurrence of intestinal adhesion is crucial for reducing postoperative adverse symptoms and improving the quality of life of patients. However, strategies to effectively reduce the incidence of intestinal adhesion are still insufficient.

[0003] Tissue engineering and biomaterials provide a promising approach to alleviate intestinal adhesion. According to previous studies, abnormal tissue healing caused by intra-abdominal bleeding and inflammation is the main cause of intestinal adhesion. In addition, the secretion and deposition of fibrinogen are key connecting factors of intestinal adhesion. Therefore, regulating the secretion and deposition of fibrinogen is crucial for preventing the occurrence of intestinal adhesion. At the same time, fibrinogen is mainly synthesized and secreted by fibroblasts and macrophages. Therefore, taking a two-dimensional approach, i.e., reducing the infiltration of inflammatory cells and fibroblasts at the cellular level and reducing the synthesis and secretion of fibrinogen at the molecular level, is a promising and effective strategy to alleviate intestinal adhesion. SUMMARY

[0004] (I) Technical problems solved

[0005] In view of the deficiencies of the prior art, the present application provides a methacrylated gelatin with dual functions.

[0006] (II) Technical solutions

[0007] To achieve the above-mentioned object, the present application provides the following technical solutions: a methacrylated gelatin with dual functions, characterized in that the preparation method is as follows:

[0008] Step 1: To prepare the collagen methylation polymethacrylate, 10 g of gelatin was added to 100 ml of deionized water and stirred in a water bath at 50°C until completely dissolved; then, 6 ml of methacrylic anhydride was slowly added, and the mixture was reacted in a constant temperature shaker at 50°C for 6 hours; after the reaction was completed, the solution was transferred to a dialysis bag with a molecular weight cut-off value of 3.5 kilodaltons and dialyzed in deionized water for 2 days; after dialysis was completed, the solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected, frozen to -80°C, and then freeze-dried for 2 days to obtain the collagen methylation polymethacrylate precursor; the collagen methylation polymethacrylate was prepared by mixing 5% collagen methylation polymethacrylate precursor and 0.2% lithium phenyl, and trimethylbenzoyl phosphonate under 365 nm ultraviolet light irradiation;

[0009] Step 2: The methacrylated gelatin was prepared by mixing 5% collagen methylation polymethacrylate, 0.2% trimethylbenzoyl phosphonate, 10 mg / ml of Houttuynia cordata extract, and 20 μg / ml of nifedipine under ultraviolet light irradiation.

[0010] (III) Beneficial effects

[0011] Compared with the prior art, the Houttuynia cordata extract in the present application is a traditional herbal ingredient with good biocompatibility and anti-inflammatory properties. Nintedanib is an anti-fibrotic drug that can effectively inhibit the production and deposition of fibrinogen. Therefore, the combination of Houttuynia cordata extract and nintedanib can reduce the factors affecting intestinal adhesion at the cellular and molecular levels. In addition, the early stage of acute inflammation and hemorrhagic exudation is a critical period for the accumulation of factors leading to intestinal adhesion, and during this period, it is necessary to continuously regulate local inflammatory factors affecting intestinal adhesion. However, the combination of Houttuynia cordata extract and nintedanib alone may not meet the needs of continuous treatment. Gelatin methacrylamide is a popular hydrogel with good biocompatibility and plasticity, suitable for tissue regeneration and repair. Its loosely cross-linked network structure can achieve sustained release of drugs, thereby meeting the needs of continuous and stable drug delivery. In addition, the combination of gelatin methacrylamide, Houttuynia cordata extract, and nintedanib to relieve hemorrhagic intestinal adhesion can regulate inflammation and anti-fibrotic function. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The structural diagram of the present application is shown in the figure;

[0013] Figure 2This invention relates to the preparation of nhGelMA. (A) Schematic diagram of the nhGelMA synthesis process. (B) 1H NMR spectra of gelatin and GelMA. (C) Macroscopic view of the synthesized nhGelMA. (D) Compressive modulus of 1%, 5%, 10%, and 15% GelMA. (E) CCK8 test results of the blank group, 1% GelMA group, 5% GelMA group, 10% GelMA group, and 15% GelMA group. (F) CCK8 test results of nGelMA at 0, 1, 10, 20, and 50 μg / ml of nitidanib. (G) CCK8 detection results of hGelMA in 0, 1, 10, 100, 1000, 10000, and 100000 μg / ml of Houttuynia cordata.

[0014] Figure 3 The following is a description of the properties of the hydrogels of this invention. (A) Schematic diagram of the preparation of nhGelMA. (B) Macroscopic images of GelMA, nGelMA, and nhGelMA hydrogels before and after UV irradiation. (CF) Colloidal mechanical analysis (C), viscosity (D), swelling ratio (E), and degradation (F) in the GelMA, nGelMA, and nhGelMA groups. (G) Cumulative release of nidonedambar and houttuynia cordata. (H) Compressive modulus of GelMA, nGelMA, and nhGelMA hydrogels;

[0015] Figure 4 The structure and composition of the hydrogels of this invention are shown. (AC) Scanning electron microscope images and elemental distribution images of the GelMA, nGelMA, and nhGelMA groups. (D) Quantitative analysis of carbon, nitrogen, and oxygen. (E, F) XRD and FTIR spectra of GelMA, nGelMA, and nhGelMA;

[0016] Figure 5 This invention relates to the biocompatibility and organ toxicity of the following: (A) Schematic diagram of in vitro assessment of the function of nhGelMA in immunomodulation and antifibrosis. (B, C) Live / dead cell staining of GelMA, nGelMA, and nhGelMA hydrogels carrying fibroblasts and BV2 after 3 days. (DF) Quantitative analysis of live / dead cell staining of fibroblasts and BV2. (H) Organ toxicity of nhGelMA. (IL) Gene expression levels of FN(I), TNFα(J), IL6(K), and IL1(L);

[0017] Figure 6This invention relates to the application of nhGelMA in alleviating hemorrhagic intestinal adhesions. (A) Schematic diagram of the process and mechanism of alleviating hemorrhagic intestinal adhesions. (B) Surgical procedure. (C) Gross appearance of untreated and treated intestinal segments. (DF) Gene expression levels of FN(D), LAMA(E), Col1(F), IL6(H), and IL1(I);

[0018] Figure 7 Histological staining of the intestines of the untreated and treated groups of this invention, including (A) H&E, Masson, FN / LAMA / DAPI, Col1 / DAPI, and CD68 / CD3 / DAPI staining in the Ctrl and nhGelMA groups. (BF) Quantitative analysis of fluorescence intensity of FN (B), LAMA (C), Col1 (D), CD3 (E), and CD68 (F). Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please refer to the attached figure. A methacrylamide gelatin with dual functions is characterized by the following preparation method:

[0021] Step 1: Preparation of collagen methylated polymethyl methacrylate (GelMA) involves adding 10 g of gelatin to 100 mL of deionized water and stirring in a 50°C water bath until completely dissolved. Then, slowly add 6 mL of methacrylic anhydride and react the mixture in a 50°C shaker for 6 hours. After the reaction, transfer the solution to a dialysis bag with a molecular weight cutoff of 3.5 kDaltons and dialyze in deionized water for 2 days. After dialysis, centrifuge the solution at 3000 rpm for 10 minutes, collect the supernatant, freeze it to -80°C, and then freeze-dry it for 2 days to obtain the collagen methylated polymethyl methacrylate precursor. Collagen methylated polymethyl methacrylate (GelMA) is prepared by mixing 5% of the collagen methylated polymethyl methacrylate precursor with 0.2% lithium phenyl and trimethylbenzoylphosphonate (LAP) under 365 nm UV irradiation.

[0022] Step 2 involves preparing methacrylamide gelatin by mixing 5% collagen methylated polymethacrylate, 0.2% trimethylbenzoylphosphonate, 10 mg / mL houttuynia cordata extract, and 20 μg / mL nifedipine, and then irradiating with ultraviolet light.

[0023] Abnormal tissue repair due to injury, bleeding, and inflammatory infection is a major factor leading to intestinal adhesions. Furthermore, fibrinogen secretion and deposition are key connectives in the formation of intestinal adhesions; fibrinogen is primarily synthesized and secreted by fibroblasts and macrophages. Therefore, a two-dimensional approach aimed at reducing the infiltration of inflammatory cells and fibroblasts, and reducing fibrinogen synthesis and secretion, holds great potential for developing effective repair strategies to alleviate intestinal adhesions. The development of hydrogels and pharmaceuticals provides the necessary technical support to meet these requirements for reducing intestinal adhesions.

[0024] Gelatin is a natural material widely used in daily life and clinical applications due to its excellent biocompatibility and biosafety. The exposed amino (-NH2) and hydroxyl (-OH) groups on the amino acid chains of gelatin possess excellent plasticity, providing a basis for the chemical modifications required to prepare methacrylated gelatin (GelMA). The carbon-carbon double bonds of GelMA can be activated by the combined excitation of a photoinitiator (LAP) and ultraviolet light, forming new connections and thus creating a network structure, macroscopically manifested as methacrylated gelatin (GelMA). Figure 1 A). The ease of gelation and the post-gel cross-linked network of GelMA hydrogels provide an operational and structural basis for drug loading and release, making them an excellent drug carrier. In this study, GelMA was synthesized, and the gelatin and GelMA fractions were analyzed by 1H NMR spectroscopy to confirm the chemical grafting. The results showed that the GelMA fraction exhibited a bimodal distribution in the 5–6 ppm range, corresponding to a successful grafting reaction (…). Figure 2 B). In addition, Figure 2 C presents a macroscopic view of the preparation process of GelMA and the hydrogel formation process to confirm the successful preparation of the hydrogel. The results show that polymethyl methacrylate (GelMA) was successfully synthesized and a hydrogel was formed. The concentration of the hydrogel significantly affects the density of the cross-linked network within the scaffold, thereby influencing its mechanical strength and nutrient permeability. When the hydrogel concentration is 1%, the internal cross-linked network cannot form sufficient mechanical strength to maintain its overall macroscopic morphology, resulting in a fluid state unsuitable for subsequent drug loading. With increasing concentration, the mechanical strength of the hydrogel also increases. Figure 2 D). However, in order to enhance strength to accommodate cells, an increase in mechanical strength leads to a decrease in cell viability with increasing concentration. Figure 2E). Therefore, considering the feasibility of the operation and the impact on cell viability, a 5% GelMA concentration was used in subsequent experiments. In this study, Houttuynia cordata (a traditional natural herbal ingredient) exhibited excellent biocompatibility and anti-inflammatory functions for modulating inflammatory cells after intestinal injury. However, the optimal dosage still needs to be confirmed. Therefore, we used different concentrations of Houttuynia cordata (hGelMA) to prepare GelMA and evaluated its toxicity to fibroblasts. The results showed that Houttuynia cordata is safe at concentrations below 10 mg / mL. Figure 2 G). Therefore, we selected 10 mg / mL of Houttuynia cordata for subsequent experiments. Similarly, in this study, we used the proven anti-fibrotic drug nintedanib to reduce fibronectin (FN) production. Cytotoxicity tests at different concentrations showed that nintedanib was safe at concentrations below 20 μg / mL. Figure 2 F). Therefore, we determined 20 μg / mL nintedanib for subsequent experiments. Finally, nhGelMA hydrogel was successfully prepared by mixing 20 μg / mL nintedanib and 10 mg / mL houttuynia cordata into 5% GelMA. Figure 3 A).

[0025] Description of the properties of hydrogels:

[0026] To assess the safety and feasibility of these substances, it is necessary to further verify the effects of Houttuynia cordata and nintedanib on the hydrogel properties. By observing the transformation process of GelMA, hGelMA, and nhGelMA from sol to gel, and through rheological and viscosity assessments, the results show that the addition of Houttuynia cordata and nintedanib does not affect their rheological properties. Figure 3 (BD). The results of the expansion test showed that the expansion rate of these hydrogels was less than 150%, which is considered a safe range for intraperitoneal expansion. Figure 3 E). Furthermore, degradation experiments showed that GelMA, hGelMA, and nhGelMA exhibited similar degradation rates, exceeding 50% degradation within 2 days, thus confirming their biodegradability and safety. Figure 3 F). The sustained release of Houttuynia cordata and nintedanib in nhGelMA is an important characteristic of current repair strategies. In vitro sustained-release experiments confirmed that Houttuynia cordata and nintedanib can release more than 60% of the drug within 3 days. Figure 3 (G), indicating that nhGelMA has the potential to achieve time-matched release, thereby modulating inflammatory cells and reducing fibrin production after intestinal injury. Furthermore, the compressive moduli of GelMA, hGelMA, and nhGelMA suggest that the addition of houttuynia cordata and nintedanib does not affect its mechanical properties. Figure 3H). Furthermore, scanning electron microscopy analysis revealed that these hydrogels possess similar porous network structures (H). Figure 4 Elemental analysis showed that the addition of houttuynia cordata and nintedanib increased the ratio of carbon (C) to oxygen (O). Figure 4 Finally, XRD and FTIR results showed that GelMA, hGelMA, and nhGelMA hydrogels have similar crystal structures and chemical bonds. Figure 4 E and F).

[0027] Biocompatibility of hydrogels:

[0028] The biocompatibility of nhGelMA is crucial for its potential in vivo applications, a point that requires further clarification. In this study, fibroblasts (progenitor cells responsible for producing fibronectin) and macrophages (cells primarily secreting inflammatory factors) were selected as validation seed cells to assess its biocompatibility. Figure 5 A). Live / dead staining results showed that fibroblasts and macrophages survived well in GelMA, hGelMA, and nhGelMA, with only a small number of cells dying. Figure 5 BG). Furthermore, assessing whether the hydrogel's application in vivo would lead to organ toxicity was also important. H&E staining results showed that, compared to the untreated control group, the application of nhGelMA in vivo did not cause significant changes in the heart, liver, spleen, lungs, or kidneys, thus confirming the absence of organ toxicity. Figure 5 Finally, gene expression analyses related to fibronectin and inflammatory factors were performed in the GelMA and nhGelMA groups to further investigate the in vitro functions of Houttuynia cordata and nintedanib in nhGelMA. The results showed that nhGelMA could reduce the expression level of FN genes in fibroblasts, and also reduce the expression levels of inflammatory factors TNFα, IL6, and IL1 in macrophages. Figure 5 Therefore, nhGelMA possesses pre-programmed anti-fibrotic and anti-inflammatory modulatory properties, making it suitable for further in vivo experiments to alleviate intestinal adhesions.

[0029] Application of nhGelMA in relieving hemorrhagic intestinal adhesions:

[0030] The ability of nhGelMA to effectively relieve intestinal adhesions was the most important aspect of this study. The untreated intestinal injury group served as the control group, while the experimental group received nhGelMA treatment. Figure 6 B). Gross observation 3 days post-surgery showed severe intestinal adhesions in the untreated group, while no adhesions were observed in the nhGelMA group. Figure 6C). Further histological staining for H&E, Masson, FN, LAMA, and Col1 in both the untreated and nhGelMA groups revealed significant accumulation of adhesion-related proteins in the adherent intestinal wall of the untreated group. In contrast, the levels of these proteins were very low in the treated group, suggesting that nhGelMA hydrogel plays a crucial role in reducing adhesions. Figure 7 AD). Furthermore, inflammation-related staining of CD68 and CD3 showed that inflammatory infiltration was significantly higher in the untreated group than in the control group (AD). Figure 7 A, E, and F). Furthermore, PCR analysis was performed in the control group and the nhGelMA group to further elucidate the mechanism by which nhGelMA alleviates adhesions. The results showed that nhGelMA not only reduced adhesion-related genes (i.e., FN...) Figure 6 D), LAMA Figure 6 E) and Col( Figure 6 The expression level of F) was also downregulated, as were genes related to inflammation (i.e., TNFα). Figure 6 H), IL6 ( Figure 6 I) and IL1 Figure 6 The expression level of J). Therefore, nhGelMA can alleviate intestinal adhesions by reducing adhesions and inflammation. Figure 6 A).

[0031] in conclusion

[0032] Intestinal adhesions are one of the most common clinical conditions, affecting millions of patients worldwide. However, effective strategies to reduce the incidence of intestinal adhesions remain insufficient. In this study, a dual-functional methacrylamide gelatin (nhGelMA) was developed by integrating GelMA, Houttuynia cordata extract, and nifedipine. This material can modulate inflammation and anti-fibrosis functions. First, the optimal concentration of GelMA hydrogel was prepared and screened. Then, appropriate concentrations of Houttuynia cordata extract and nifedipine were selected to prepare the final nhGelMA. Subsequently, nhGelMA was characterized, and its biocompatibility and biological functions were analyzed. The results showed that nhGelMA has good biocompatibility, no organ toxicity, and can modulate the expression levels of inflammation and fibronectin (FN). Finally, the nhGelMA hydrogel was applied to the prevention and treatment of hemorrhagic intestinal adhesions. Results showed that nhGelMA effectively reduced the deposition of fibronectin (FN), laminin (LAMA), and collagen 1 (Col1), and decreased the infiltration of macrophages and neutrophils, thereby preventing intestinal adhesions. In summary, we have developed a novel, dual-functional nhGelMA hydrogel that alleviates hemorrhagic intestinal adhesions through immunomodulation and anti-fibrotic effects, providing a promising strategy for clinical translation.

[0033] To confirm the successful preparation of gel methyl methacrylate (GelMA), 1H NMR spectra of gelatin and GelMA were analyzed. 1 Detection was performed using 10 mg of gelatin and GelMA by 1.5 mL of deionized dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, 151831) containing 0.03% tetramethylsilane (TMS) as an internal standard. Subsequently, 0.4 mL of each solution was transferred to 5 mm NMR tubes and analyzed using a Bruker 600 MHz NMR spectrometer (Germany). 1 ¹H NMR measurements were performed. After data collection, the spectra were processed using MestReNova software. Several characteristic peaks were identified: GelMA showed an additional peak at approximately 5.3–5.8 ppm, corresponding to the vinyl protons of the methacrylamide group, thus confirming the methacrylation process.

[0034] Mechanical properties

[0035] These hydrogel samples were 1 cm in diameter and 3 mm thick. Compression tests were performed using a universal testing machine (INSTRON 5982) equipped with a 500 N loading device. In short, the samples were placed on a compression plate and compressed at a rate of 1 mm / min until 80% of the original thickness was reached. The compressive modulus was obtained from the stress-strain curve, where the modulus was defined as the slope of this linear segment

[38] .

[0036] CCK-8 test

[0037] To assess the cytotoxicity of the hydrogel, a CCK-8 assay was performed using fibroblasts. First, 20 μL of hydrogel was added to the wells of a 96-well plate and cured under 365 nm UV irradiation. Subsequently, 5 × 10⁶ cells were cultured per well. 3 Fibroblasts were seeded at a density of [number] cells per well and 100 μL of culture medium was added. The culture plate was incubated at 37°C for 24 hours. Then, 10 μL of CCK-8 reagent (DOJINDO, CK04) was added to each well and mixed with 90 μL of culture medium, and incubation was continued under the same conditions for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The relative cell viability was calculated based on the control group.

[0038] Rheological analysis and viscosity measurement

[0039] Rheological tests were performed at 25 °C using a HAAKE MARS rotational rheometer equipped with a parallel plate geometry (P20 TiL, 20 mm diameter). Rheological experiments involved exposing the samples to light (365 nm, 20 mW / cm²). A 120-second time-domain oscillation test was conducted under 10% stress, 1 Hz frequency, and a 0.5 mm gap, during which the storage modulus (G') and loss modulus (G”) were recorded. All tests were performed triplicate, and the data were analyzed using the instrument software to generate correlation curves and viscoelastic parameters.

[0040] Swelling test of hydrogel

[0041] The swelling of the hydrogel was tested using a gravimetric method. The initial wet weight of the hydrogel sample was recorded as W0. The hydrogel was then immersed in PBS at 37°C for 2 hours, 4 hours, 8 hours, and 12 hours. After these periods, the hydrogel was carefully removed, and its wet weight was measured and recorded as Wt. The swelling rate at each time point was calculated using the formula: Swelling rate = (Wt / Wt) t -W0) / W0×100%.

[0042] Degradation test of hydrogel

[0043] The degradation of the hydrogel was tested by gravimetric method. The initial dry weight of the lyophilized hydrogel sample was recorded as W0. The hydrogel was then immersed in PBS containing 10 μg / ml collagenase (Aladdin, C754929) at 37°C. At specified time intervals (12 h, 24 h, 48 h, and 72 h), the hydrogel was carefully removed, lyophilized, and its weight was measured and recorded as W0. t The degradation rate at each time point is calculated using the formula: Degradation rate = (W0 - W t ) / W0×100%.

[0044] Continuous release test

[0045] The sustained release of Houttuynia cordata extract from hydrogels was tested using spectrophotometry. nhGelMA hydrogels containing 10 mg / mL Houttuynia cordata extract were immersed in PBS at 37°C, and supernatants were collected at 12, 24, 48, and 72 hours. Standard curves were established by measuring PBS solutions containing 0 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL, 1000 μg / mL, 10 mg / mL, and 100 mg / mL Houttuynia cordata extract. The standard curves were then plotted with concentration on the x-axis and OD value on the y-axis. The release rate of Houttuynia cordata extract from the supernatant was calculated based on the standard curves. Similarly, the sustained release of nintedanib from nhGelMA hydrogels was tested using liquid chromatography. nhGelMA hydrogels loaded with nintedanib were immersed in PBS at 37°C, and supernatants were collected at 12, 24, 48, and 72 hours. Standard curves were obtained by measuring concentrations of 0 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL in the release medium solution. The standard curves were then plotted with concentration on the x-axis and peak area on the y-axis. The release rate of nintedanib in the supernatant was calculated based on the standard curves.

[0046] Standard deviation (SEM) and elemental analysis

[0047] Scanning electron microscopy (SEM) observation and elemental analysis were performed using a Zeiss GeminiSEM 300 instrument (ZEISS, Germany). Lyophilized samples were sputtered with gold to improve conductivity. SEM images were acquired at an accelerating voltage of 5 kV. Elemental distribution was analyzed using energy-dispersive spectroscopy (EDS), which aided in the detection and visualization of the elemental composition of the sample surface.

[0048] XRD and FTIR

[0049] XRD analysis was performed using a Rigaku SmartLab SE instrument (Japan), with a scanning angle range of 10-80° and a scanning speed of 2° / min. FTIR measurements were performed using a Nicolet iS20 instrument (USA) from Thermaffer Scientific (USA), with a wavenumber range of 400-4000 cm⁻¹. -1 To characterize the functional groups of the sample.

[0050] Isolation and culture of fibroblasts

[0051] Rabbit skin tissue was collected and rinsed with PBS solution containing antibiotics. The tissue was then cut into small pieces using sterile scissors. These samples were then immersed in PBS solution containing 0.2% Dispase II (Aladdin, D743370) and incubated at 37°C for 8 hours to promote the separation of the epidermis from the dermis. 0.1% collagenase solution (Merck, C0773) was added, and the mixture was incubated at 37°C for 2 hours to digest the dermal tissue. The cell suspension was filtered through a cell filter to remove undigested tissue fragments, and the filtrate was then centrifuged at 1000 rpm for 5 minutes to separate the cells. These cells were then cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics.

[0052] live / dead staining

[0053] Cells were seeded onto a hydrogel and cultured for 72 hours. After removing the culture medium, the samples were gently rinsed twice with PBS to remove any residual medium. The hydrogel was then covered with a live / dead staining solution (DOJINDO) and incubated at 37°C for 10 minutes. After incubation, the staining solution was aspirated, and the samples were rinsed with PBS to remove any excess dye. The stained cells were then immediately observed under a fluorescence microscope (Leica). Under this microscope, live cells exhibited green fluorescence, while dead cells emitted red fluorescence.

[0054] PCR

[0055] Total RNA was extracted from the samples using TRIzol reagents according to the manufacturer's instructions. cDNA was then synthesized from the total RNA using a reverse transcription kit based on the kit protocol. PCR amplification was performed in a 20 μL reaction system comprising 10 μL of 2×PCR major mixture, 1 μL of forward primer (10 μmol), 1 μL of reverse primer (10 μmol), 2 μL of cDNA template, and 6 μL of nuclease-free water. The relative expression level of the target gene was calculated using the 2^(-ΔΔCt) method. Primers were designed and synthesized by Shanghai SangGene Biotechnology Co., Ltd.

[0056] Surgery and Ethics

[0057] Two-month-old New Zealand white rabbits weighing 2 kg were purchased from Shanghai Jiajian Biotechnology Co., Ltd. The rabbits were anesthetized with isoflurane, then their fur was removed and the abdominal area was disinfected. An incision was made in the center of the abdomen to open the abdominal cavity, and the appendix was carefully identified and exposed. The appendix was scraped with the handle of a scalpel to create a bleeding model. In the experimental group, nhGelMA was applied to the injured area for treatment, while the control group received no treatment. After the surgery, the abdominal cavity was closed layer by layer by suture. All animal experiments were approved by the Animal Care and Experimentation Committee of Nantong University (S20250818-001).

[0058] Immunofluorescence staining

[0059] Three days later, the rabbits were euthanized, and their intestines were removed. The samples were fixed in 4% paraformaldehyde for 24 hours, then dehydrated and cut into 4-5 micrometer thick sections. The sections were dewaxed with tap water, antigen retrieval was performed, and then blocked with 5% BSA for 30 minutes at room temperature. Antibodies against FN (Abcam, ab286324), LAMA (Abcam, ab151715), Col1 (Abcam, ab270994), CD3 (Abcam, ab16669), and CD68 (HUABIO, HA601292) were added to the sections, and incubated overnight (12 hours) at 4°C. The sections were washed with phosphate-buffered saline (PBST) containing Tween 20, then fluorescently labeled secondary antibodies were added, and incubated in the dark at 37°C for 1 hour. Finally, the sections were counterstained with DAPI for 5 minutes and observed under a fluorescence microscope. Images were taken for subsequent analysis.

[0060] All data are presented as mean ± standard deviation (SD). The t-test was used for comparisons between two groups, while one-way ANOVA was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A dual-functional methacrylamide gelatin, characterized in that, The preparation method is as follows: Step 1: Preparation of collagen methylated polymethyl methacrylate. Add 10 g of gelatin to 100 mL of deionized water and stir in a 50°C water bath until completely dissolved. Then, slowly add 6 mL of methacrylic anhydride and react the mixture in a 50°C shaker for 6 hours. After the reaction, transfer the solution to a dialysis bag with a molecular weight cutoff of 3.5 kDaltons and dialyze in deionized water for 2 days. After dialysis, centrifuge the solution at 3000 rpm for 10 minutes, collect the supernatant, freeze it to -80°C, and then freeze-dry for 2 days to obtain the collagen methylated polymethyl methacrylate precursor. Collagen methylated polymethyl methacrylate is prepared by mixing 5% of the collagen methylated polymethyl methacrylate precursor with 0.2% lithium phenyl and trimethylbenzoylphosphonate under 365 nm UV irradiation. Step 2 involves preparing methacrylamide gelatin by mixing 5% collagen methylated polymethacrylate, 0.2% trimethylbenzoylphosphonate, 10 mg / mL houttuynia cordata extract, and 20 μg / mL nifedipine, and then irradiating with ultraviolet light.

Images