Multifunctional biological scaffold as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional biological scaffold as well as a preparation method and application thereof. The method comprises the following steps: performing proteinase K treatment on a natural egg membrane to obtain a pure substrate; performing gas foaming in the wet substrate by hydrolyzing sodium borohydride to release hydrogen so as to construct a three-dimensional porous scaffold; then, a polydopamine coating is formed on the surface of the stent through dopamine oxidation auto-polymerization, and nano-silver particles are loaded through in-situ reduction; and finally, through simulated body fluid mineralization, a bone-like apatite layer is deposited on the surface of the coating. A uniformly interconnected three-dimensional porous structure is created through gas foaming, and a physical platform is provided for subsequent functionalization. The obtained final material integrates a three-dimensional porous structure, rapid physical-chemical synergistic hemostasis, lasting broad-spectrum antibacterial property and strong osteogenic activity promotion, and is especially suitable for repairing infectious bone defects or refractory soft tissue wounds. The preparation technology is simple, raw materials are easy to obtain, and high-value utilization of waste resources is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials technology, specifically to a multifunctional biological scaffold, its preparation method, and its application. Background Technology

[0002] Egg membrane, as a natural biomaterial, is mainly composed of protein fibers such as keratin and possesses unique semi-permeability, biocompatibility, and certain bioactivity. Traditionally, due to its good water absorption and barrier function, egg membrane has been attempted for use in dressings for small-area burns and scalds, or as a topical material in folk remedies. However, its applications are mostly limited to a two-dimensional membrane structure. This dense fibrous arrangement of egg membrane presents significant limitations: First, the dense structure results in poor water solubility and degradation performance, making it difficult to be rapidly absorbed and metabolized in vivo, thus limiting its potential as a biodegradable implant material; second, the two-dimensional membrane structure is not conducive to cell ingrowth, nutrient penetration, and vascularization, making it difficult to meet the three-dimensional spatial requirements of complex tissue regeneration; furthermore, the function of egg membrane is relatively singular, lacking active biological functions for specific regenerative scenarios (such as osteogenic activity required for bone repair, and antibacterial and hemostatic functions required for wound healing). Therefore, a large amount of egg membrane is considered waste in the food and processing industries, failing to realize its high-value biomedical utilization.

[0003] To expand the application of egg membranes in tissue engineering, researchers have attempted to transform them from two-dimensional membrane structures into three-dimensional porous scaffolds. Three-dimensional scaffolds can mimic the structure of the extracellular matrix, providing a three-dimensional space for cell attachment, proliferation, and differentiation, facilitating the transport of nutrients and metabolites, and promoting the ingrowth of new tissue. However, the keratin fibers in egg membranes are tightly entangled physically and chemically cross-linked, forming a dense network that current technologies struggle to effectively expand and form a stable three-dimensional porous structure.

[0004] On the other hand, to endow biomaterials with additional functions, it is often necessary to modify their surface or incorporate functional components. Silver ions ( It has attracted widespread attention due to its broad-spectrum and efficient antibacterial properties, but its direct loading onto the surface of materials often results in problems such as burst release and poor stability.

[0005] Therefore, developing a multifunctional tissue engineering scaffold that overcomes the structural limitations of egg membranes and transforms them into structures with three-dimensional porous structures, good biocompatibility, antibacterial activity, and bone-promoting functions is of great significance for the efficient utilization of egg membrane resources and expanding their applications in regenerative medicine fields such as wound repair and bone defect filling. Currently, there is a lack of a systematic preparation method that can effectively dissociate the cross-linking of egg membrane fibers, achieve stable three-dimensionalization, and integrate antibacterial and mineralization functions. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a multifunctional biological scaffold, its preparation method, and its applications. This invention first selectively weakens the cross-linking of egg membrane fibers using proteases, then constructs a three-dimensional porous framework using controlled chemical foaming technology. Next, it utilizes polydopamine for silver ion loading and functional coating construction, and finally employs an integrated preparation strategy to impart bioactivity through in vitro mineralization. To achieve the above objectives, this invention is implemented through the following technical solutions:

[0007] According to a first aspect of the present invention, a method for preparing a multifunctional biological scaffold is provided, comprising the following steps: S1, treating an egg membrane to weaken the cross-linking structure between its protein fibers; S2, subjecting the egg membrane treated in S1 to gas foaming treatment to form a porous scaffold with three-dimensional channels; S3, constructing a polymer coating on the surface of the porous scaffold obtained in S2, the polymer coating having both adhesive and reducing properties, and using the coating to load antibacterial metal components.

[0008] Preferably, the method further includes step S4: placing the scaffold loaded with antibacterial metal components obtained in S3 in a mineralization solution to deposit a calcium phosphate biomineralization layer on its surface.

[0009] Preferably, in step S1, the egg membrane is treated with proteinase K at a concentration of 0.01-0.1 mg / mL, and the reaction is carried out at 55-65°C for 12-24 hours in a Tris-HCl buffer system containing NaCl at pH 8.0.

[0010] Preferably, in step S2, the gas foaming treatment is carried out using a sodium borohydride solution with a concentration of 0.5-5 wt%, and the foaming treatment is carried out at 25-30°C for 5-15 minutes.

[0011] Preferably, after step S2, the scaffold forms a three-dimensional porous structure with an average thickness of 150-350 μm.

[0012] Preferably, step S3 includes: S3.1, immersing the three-dimensional porous scaffold in a weakly alkaline buffer solution containing 0.1-1 wt% dopamine hydrochloride and 0.05-0.5 wt% sodium periodate, and reacting at 25-30°C for 6-12 hours to form a polydopamine coating on the scaffold surface; S3.2, immersing the scaffold coated with polydopamine in a 0.001-0.05 mmol / L silver nitrate solution, and reacting at 25-30°C for 6-12 hours to reduce and load silver nanoparticles in situ.

[0013] Preferably, in step S4, the mineralization solution is a simulated body fluid with a concentration of 1 to 5 times, and the mineralization treatment is carried out at 25-37°C for 24-48 hours; the main component of the calcium phosphate biomineralization layer is bone-like apatite.

[0014] According to a second aspect of the present invention, a multifunctional biological scaffold is provided, the substrate of which is an egg membrane pretreated with proteinase K and gas-foamed, having a three-dimensional porous network structure; the surface of the three-dimensional porous network is sequentially coated with a polydopamine-silver nanoparticle antibacterial coating and a calcium phosphate biomineralization layer.

[0015] Preferably, the average thickness of the support is 150-350 μm.

[0016] According to a third aspect of the present invention, the application of the above-described multifunctional biological scaffold in the preparation of medical devices for wound repair or bone defect repair is provided.

[0017] This invention transforms waste egg membranes into a series of high-performance, application-oriented biomaterials, exhibiting significant multi-level beneficial effects. Utilizing controlled gas foaming technology, this invention reconstructs dense proteinase K-treated egg membranes (KEM, approximately 40 μm thick) into foamed scaffolds (FEM, approximately 250 μm thick) with a highly interconnected three-dimensional porous structure. This creates an ideal microenvironment conducive to cell migration, nutrient delivery, and tissue growth, and endows the material with excellent physical adsorption and procoagulant capabilities, laying a crucial physical platform for subsequent functional integration.

[0018] Building upon this foundation, a FEM@DAg scaffold was successfully prepared by constructing a polydopamine (PDA) coating on a porous surface and in-situ reducing loaded silver nanoparticles. This material combines the healing-promoting capabilities of a three-dimensional porous structure with the adhesiveness of PDA and the potent antibacterial properties of silver nanoparticles. Animal experiments showed that FEM@DAg exhibited the best healing rate in an infected wound model. Furthermore, a bone-like apatite active layer was introduced by biomimetic mineralization on the surface of FEM@DAg, resulting in the FEM@DAgM composite material. The superior performance of this invention stems from the inherent synergy of its specific technical pathways, rather than the simple superposition of steps. Comparative experiments showed that the two-dimensional membrane material (2D-EM@Ag-M) with the same surface chemical modification but without foaming treatment had a mineralization layer limited to the surface and weak hemostatic and osteogenic effects; while the control material (FEM-FD@DAgM) with a three-dimensional structure constructed by freeze-drying had unstable functional coating distribution and limited bioactivity due to uneven pore size. In summary, the materials of this invention exhibit comprehensive performance advantages in complex tissue repair scenarios, and have significant clinical translational potential and application value. Attached Figure Description

[0019] Figure 1These are the macroscopic and microscopic structural features of different scaffolds provided according to embodiments of the present invention. Figure 1 a is a photograph of the scaffold samples prepared according to different embodiments of the present invention, which intuitively shows the differences in macroscopic morphology, color and thickness of the egg membrane scaffold treated only with proteinase K (KEM), the foamed three-dimensional egg membrane scaffold (FEM), the polydopamine silver-coated three-dimensional egg membrane scaffold (FEM@DAg), and the mineralized multifunctional three-dimensional egg membrane scaffold (FEM@DAgM). Figure 1 b represents scanning electron microscope (SEM) images prepared according to different embodiments provided in this invention, clearly showing the cross-sectional microstructure of KEM, FEM, FEM@DAg and FEM@DAgM scaffolds, including their thickness, fiber structure and porous features, especially the three-dimensional porous structure of the FEM series scaffolds.

[0020] Figure 2 Porosity (a) and density (b) of scaffolds prepared according to different embodiments of the present invention are analyzed. KEM has low porosity and high density, while FEM, FEM@DAg, and FEM@DAgM scaffolds have high porosity and low density after foaming treatment, which is beneficial to the infiltration behavior of tissue cells.

[0021] Figure 3 To evaluate the in vitro cell (HUVECs and BMSCs) compatibility of the scaffolds of different embodiments provided in this invention, cell live / dead staining (a) and CCK-8 metabolism (bc) tests were conducted, demonstrating that the FEM, FEM@DAg, and FEM@DAgM scaffolds treated by the method of this invention all have good biocompatibility and can support cell growth.

[0022] Figure 4 (a) Antimicrobial performance testing and quantitative analysis results of scaffolds of different embodiments provided according to the present invention. By comparing colony growth and bacterial survival rate, it is shown that the FEM@DAg and FEM@DAgM scaffolds loaded with polydopamine silver coating have significant antimicrobial capabilities, while the KEM and FEM scaffolds do not have this function.

[0023] Figure 5 This study investigates the in vivo biocompatibility and degradation of scaffolds according to different embodiments of the present invention in animal models. Histological staining and other analyses at 3 and 6 weeks demonstrated the inflammatory response, tissue integration, and degradation rate of each scaffold in vivo, verifying their safety as implantable materials.

[0024] Figure 6The above figures show a comparison of the repair effects of the three scaffolds (KEM, FEM, and FEM@DAg) provided in the embodiments of the present invention in an animal skin wound model. The ability of different scaffolds to promote wound regeneration and repair was evaluated by indicators such as wound healing area and tissue regeneration.

[0025] Figure 7 The results of in vitro coagulation performance evaluation of stents according to different embodiments of the present invention are presented. The hemostatic capabilities of KEM, FEM, FEM@DAg, and FEM@DAgM stents are visually compared using hemolysis (a) and coagulation index (b), highlighting the superior procoagulant properties of the FEM@DAgM stent.

[0026] Figure 8 The results are the in vitro and in vivo osteogenic performance evaluation results of the scaffold provided according to the embodiments of the present invention. Figure 8 a) To evaluate the in vitro osteogenic performance of scaffolds provided in different embodiments according to the present invention, the ability of each scaffold to promote osteoblast differentiation and matrix mineralization was qualitatively and semi-quantitatively demonstrated by alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) calcium nodule staining. Figure 8 b is an evaluation of the in vivo cranial regeneration effect of the scaffold provided in different embodiments according to the present invention. The cranial regeneration and repair experiment shows that the FEM@DAgM scaffold has a significant effect on promoting cranial tissue regeneration in vivo and has significant advantages in promoting bone formation in vivo. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The experimental methods of the present invention are as follows:

[0028] Porosity and density testing: The scaffold was prepared in a size of 20 mm × 20 mm and its mass was recorded as m0. Then, the sample was transferred to anhydrous ethanol for thorough immersion, removed, and placed on filter paper to remove any remaining ethanol. The sample mass was weighed again and recorded as m1. The porosity P of the scaffold was calculated using the following formula:

[0029]

[0030] m0 and m1 are the weights of the stent before and after ethanol immersion, respectively; ρ is the density of anhydrous ethanol at room temperature; and V is the volume of the stent. The density of the stent is obtained from the dry mass m0 and the calculated stent volume V using the following formula:

[0031] .

[0032] BCI Test: In the dynamic whole blood coagulation assay, samples were prepared into 10 mm diameter discs (control group: double-layer gauze). 100 µL of fresh anticoagulated rabbit whole blood solution was then added to the surface of each sample, and the sample was incubated in a shaker (37℃, 100 rpm) for 5 min. Finally, 10 mL of deionized water was slowly added, and after unbound RBCs were lysed, 100 µL of the supernatant was taken and the OD value was measured at 545 nm. 100 µL of fresh anticoagulated rabbit blood was added to 10 mL of DI water and physiological saline, and after thorough mixing, the OD value at 545 nm was measured as the positive control (PC) and negative control group (NC). The in vitro coagulation index (BCI) of each group of samples was calculated using the following formula:

[0033]

[0034] Among them, I T I P and I N The absorbance values ​​are for the sample group, positive control group, and negative control group.

[0035] In vitro biocompatibility analysis: 10 mm diameter discs were prepared using a circular punch and sterilized by UV irradiation. HUVECs and BMSCs were activated and passaged in DMEM medium (containing 10% FBS and 1% penicillin antibiotics) before use. Cell metabolic activity was detected using a CCK-8 assay kit, and cell viability was assessed by AM / PI staining.

[0036] Antimicrobial activity assessment: E. coil and S. aures bacteria were activated to the logarithmic growth phase in LB broth for later use. The logarithmic growth phase bacterial solution was diluted and co-cultured with the material for 12 h. The co-cultured bacterial solution was diluted 1000X and spread onto LB agar plates. After incubation at 37 ℃ for 12 h, colony observation and bacterial viability analysis were performed.

[0037] In vivo biocompatibility: The in vivo biocompatibility of the scaffold was evaluated using a subcutaneous implantation model in 8-week-old SD rats (250-300 g). Sodium pentobarbital (… SD rats were anesthetized by intramuscular injection, and a subcutaneous pocket model was created by removing hair from their backs. The sample was implanted subcutaneously and the wound was sutured. Animals were sacrificed at 3 and 6 weeks post-implantation, and the subcutaneous implanted sample and surrounding tissue were collected. The sample was then fixed, dehydrated, embedded, sectioned, and stained with H&E and Masson staining.

[0038] Wound regeneration assessment: The potential of scaffolds for wound healing was evaluated using a full-thickness skin defect model in SD rats. Twenty-four male SD rats (450-500 g) were randomly divided into four groups and treated with sodium pentobarbital (…). Intramuscular anesthesia was administered. After shaving the hair on the back, full-thickness skin defects with a diameter of 10 mm were created on symmetrical areas on both sides of the back, and the initial state of the wounds was recorded. Subsequently, appropriate scaffolds were applied to each group of wounds and sutured in place. Wound healing was recorded by photograph on days 3, 7, 10, and 14.

[0039] ALP and ARS staining analysis: BMSCs were seeded in 48-well plates and cultured for 14 days in osteogenic induction medium containing extracts from each example. Cells were fixed by adding paraformaldehyde, and the osteogenic properties of BMSCs were analyzed using an alkaline phosphatase assay kit (ALP) and an alizarin red (ARS) staining kit.

[0040] Cranial repair model: Six-week-old SD rats were selected and anesthetized by intraperitoneal injection of sodium pentobarbital. A circular cranial tectum defect with a diameter of 5 mm was then created. The defects were subsequently treated with scaffolds from Examples 1, 2, and 4. Rats in the group without material treatment served as a blank control group.

[0041] Example 1: Preparation and performance of proteinase K-treated egg membrane scaffolds (KEM)

[0042] This embodiment provides a method for preparing a basic, pure egg membrane protein scaffold, and evaluates its basic physicochemical properties and biological performance as a reference for subsequent functional modification.

[0043] Preparation method

[0044] (1) Raw material acquisition and pretreatment: Collect fresh eggshells and wash them with deionized water to remove surface impurities. Immerse the washed eggshells in a 5% (v / v) formic acid aqueous solution and let them stand at room temperature for 24 hours to partially dissolve the eggshells (the main component of which is calcium carbonate) and loosen and peel off the inner egg membrane.

[0045] (2) Separation and cleaning: The eggshell membrane and residual eggshell are separated manually, and the eggshell membrane is repeatedly rinsed with sufficient deionized water until the rinsing water is neutral, thus obtaining a clean natural eggshell membrane.

[0046] (3) Enzyme treatment and purification: The above-mentioned natural egg membrane was placed in proteinase K buffer (200 mM NaCl solution and 50 mM Tris-HCl buffer (pH 8.0) mixed at a volume ratio of 1:1), and the final concentration of proteinase K was 0.05 mg / mL. Enzymatic hydrolysis was carried out in a water bath at 60℃ for 12 hours.

[0047] (4) Inactivation and post-treatment: After enzymatic hydrolysis, the system was kept at 70°C for 10 minutes to inactivate proteinase K. Then, it was rinsed three times with deionized water to remove residual enzymes, salts and other soluble impurities, and finally the proteinase K-treated egg membrane scaffold was obtained, denoted as KEM.

[0048] Performance and Characterization Results

[0049] The obtained KEM scaffold is uniformly light yellow and translucent. Figure 1 (KEM group). Scanning electron microscopy (SEM) showed that its cross-sectional structure was dense with no obvious pores, and the average thickness was about 40 μm. Figure 2 (KEM group). Direct cell contact assays showed that the KEM scaffold extract had no significant toxicity to L929 fibroblasts or MC3T3-E1 osteoblast-like cells, with high cell viability, demonstrating good biocompatibility. Figure 3 KEM group). Inhibition zone method or bacterial co-culture method tests showed that the KEM scaffold had no significant inhibitory effect on Staphylococcus aureus or Escherichia coli, indicating that it lacks inherent antibacterial function. Figure 4 (KEM group).

[0050] Subcutaneous implantation experiments in mice showed that the KEM scaffold degraded slowly in vivo, and retained most of its structural integrity after 4 weeks. Figure 5 (KEM group). In a rat full-thickness skin defect model, the wound healing speed of the implanted KEM scaffold was not significantly better than that of the untreated group or the commercial gauze group, and the healing performance was generally poor. Figure 6 (KEM group). In the whole blood clotting time test, the KEM group test solution turned distinctly red after standing, similar in color depth to the negative control (gauze) group, indicating that a large number of red blood cells ruptured without aggregation, resulting in a higher clotting index (BCI) and poorer clotting ability. Figure 7 (KEM group).

[0051] After co-culturing with osteoblasts, alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed. The results showed that the staining was relatively light, indicating that the KEM scaffold had a weak ability to induce osteoblast differentiation and extracellular matrix mineralization, resulting in poor in vivo cranial repair. Figure 8 (KEM group).

[0052] Example 2: Preparation and performance of foamed three-dimensional porous egg membrane scaffold (FEM)

[0053] Based on Example 1 (KEM), a three-dimensional porous structure was constructed by gas foaming to improve the physical morphology of the material and enhance its ability to absorb liquid, coagulate blood, and grow cells.

[0054] Preparation method

[0055] (1) Prepare the KEM scaffold according to steps (1)–(4) of Example 1.

[0056] (2) Gas foaming treatment: The wet egg membrane scaffold (wet KEM) obtained in the above steps was completely immersed in 1 wt% sodium borohydride ( In an aqueous solution, let it stand at 25°C for 10 minutes to react. The reaction with water continuously produces hydrogen gas, which forms bubbles inside and on the surface of the egg membrane, thus achieving foaming.

[0057] (3) Termination of reaction and cleaning: Immediately after foaming, remove the support and rinse it repeatedly with plenty of deionized water 5 times to completely terminate the reaction and remove residue. Borates and metal ions were used to obtain a foamed egg membrane scaffold with a three-dimensional porous structure, denoted as FEM.

[0058] Performance and Characterization Results

[0059] The FEM scaffold is light yellow, significantly enlarged in volume, and increased in thickness. Figure 1 (FEM group). SEM showed that its average cross-sectional thickness increased to approximately 250 μm, with an internal interconnected micron-sized pore structure. Figure 1 (FEM group). SEM images were analyzed using the alcohol immersion method and image analysis software. The porosity of the foamed scaffold was greater than 70%, and the average pore size distribution was in the range of 20-80 μm. Figure 2 Similar to KEM, the FEM scaffold extract was non-toxic to the test cells, supported cell growth during a 5-day evaluation, and exhibited good biocompatibility. Figure 3 FEM group). FEM stents still do not have antibacterial capabilities; bacterial survival rate exceeds 100%. Figure 4 (FEM group).

[0060] Subcutaneous implantation experiments showed that the FEM scaffold degraded significantly faster than the KEM scaffold. Figure 5 (FEM group). In the rat skin defect model, its effect on promoting wound healing was still only moderate. Figure 6 In the whole blood coagulation test, the test solution in the FEM group was lighter in color than that in the KEM group and the gauze group, and the coagulation index (BCI) was lower, demonstrating a physical coagulation-promoting effect. Figure 7 (FEM group). ALP and ARS staining results were still relatively light, indicating that it still lacked the biological activity to promote bone differentiation, and the in vivo skull repair effect was poor. Figure 8 (FEM group).

[0061] Example 3: Preparation and performance of silver-loaded polydopamine-coated foamed egg membrane scaffold (FEM@DAg)

[0062] Based on the three-dimensional porous structure obtained in Example 2 (FEM), the material was endowed with surface activity, tissue adhesion and antibacterial function by constructing a polydopamine (PDA) coating and in-situ reducing loaded silver nanoparticles.

[0063] Preparation method

[0064] (1) Prepare the FEM scaffold according to steps (1)-(3) of Example 2.

[0065] (2) Construction of polydopamine (PDA) coating: The FEM scaffold was immersed in Tris-HCl buffer (10 mM, pH 8.5) containing 0.1 wt% sodium periodate (oxidant) and 0.5 wt% dopamine hydrochloride. The reaction was carried out at 30°C in the dark with gentle shaking for 6 hours to allow dopamine to oxidize and self-polymerize, forming a firm PDA coating on the scaffold surface and pore walls. After the reaction, the scaffold was thoroughly rinsed with deionized water to obtain the PDA-coated foamed scaffold, denoted as FEM@DA.

[0066] (3) In-situ deposition of nano-silver: The FEM@DA scaffold was immersed in a 0.01 mmol / L silver nitrate aqueous solution and left to stand at 30°C in the dark for 6 hours. The catechol / quinone groups in the PDA coating reduced Ag⁺ to Ag. 0 Nanoparticles were fixed in the coating. Unreacted ions were removed by rinsing three times with deionized water to obtain a silver-loaded polydopamine-coated foamed egg membrane scaffold, denoted as FEM@DAg.

[0067] Performance and Characterization Results

[0068] The scaffold is yellowish-brown (a typical color of PDA and nano-silver) and its thickness is comparable to that of FEM. Figure 1 (FEM@DAg group). SEM showed that the scaffold maintained a three-dimensional porous structure, and a PDA film and uniformly distributed silver nanoparticles were visible on the surface. Figure 2 (FEM@DAg group). At effective antibacterial concentrations, the FEM@DAg scaffold extract showed manageable cytotoxicity and acceptable biocompatibility. Figure 3 (FEM@DAg group).

[0069] FEM@DAg scaffolds exhibited significant inhibition zones and bactericidal effects against both Staphylococcus aureus and Escherichia coli, demonstrating excellent broad-spectrum antibacterial function. Figure 4 (FEM@DAg group). Subcutaneous implantation experiments showed that its degradation rate was further accelerated compared to FEM. Masson trichrome staining indicated richer collagen fiber regeneration at the implantation site, suggesting that this structure promotes tissue integration and repair. Figure 5 (FEM@DAg group).

[0070] In rat models of infected and non-infectious wounds, the FEM@DAg scaffold, with its dual antibacterial and porous healing-promoting effects, demonstrated superior wound healing rates and histological repair scores compared to KEM and FEM. Figure 6 (FEM@DAg group). In vitro coagulation ability was comparable to FEM ( Figure 7(FEM@DAg group). ALP and ARS staining were still relatively light, indicating that although the material can promote soft tissue repair, its contribution to bone activity is limited. Figure 8 (FEM@DAg group).

[0071] Example 4: Preparation and performance of mineralized silver-loaded polydopamine foamed egg membrane scaffold (FEM@DAgM)

[0072] Based on the three-dimensional porous, antibacterial, and soft tissue healing-promoting functions of Example 3 (FEM@DAg), we will further develop a composite material suitable for bone defect repair.

[0073] Preparation method

[0074] (1) Prepare the FEM@DAg scaffold according to steps (1)–(3) of Example 3.

[0075] (2) Biomimetic mineralization treatment: The FEM@DAg scaffold was immersed in a 1X concentration of simulated body fluid (SBF, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and mineralized at 25°C for 48 hours. Calcium ions and phosphate ions in SBF underwent heterogeneous nucleation and growth on the surface of the PDA / Ag-activated material, forming a bone-like apatite mineralization layer. After mineralization, the material was gently rinsed with deionized water to obtain a mineralized silver-loaded polydopamine foamed egg membrane scaffold, denoted as FEM@DAgM.

[0076] Performance and Characterization Results

[0077] The support is yellowish-brown overall, with visible white mineralization deposits on the surface, giving it a whitish appearance. Figure 1 FEM@DAgM group). SEM showed that it maintained a porous structure, with the surface and pore walls densely covered by plate-like or spherical calcium phosphate crystals. Figure 1 (FEM@DAgM group). Porosity remained above 60%, and the average pore size ranged from 10 to 60 μm. Figure 2 At osteogenic concentrations, the material extract was non-toxic to osteogenic precursor cells (such as MC3T3-E1) and promoted cell proliferation, exhibiting good biocompatibility. Figure 3 (FEM@DAgM group). It exhibits sustained antibacterial activity against common orthopedic infectious pathogens. Figure 4 (FEM@DAgM group).

[0078] The in vitro coagulation performance was significantly enhanced, with coagulation tests showing a coagulation index (BCI) of 61%, and the test solution was clear. Figure 7The FEM@DAgM group showed extremely deep staining (ALP staining, an early marker of osteogenic development, and ARS staining, a marker of calcium nodules and late mineralization) after co-culturing with osteoblasts, significantly stronger than the previous three examples. Quantitative analysis showed a significant increase in ALP activity and calcium nodule content, demonstrating that FEM@DAgM can strongly promote osteoblast differentiation and matrix mineralization, and has a significant effect on in vivo skull regeneration. Figure 8 (FEM@DAgM group).

[0079] FEM@DAgM integrates four key properties: three-dimensional porous structure (facilitating cell ingrowth and material exchange), antibacterial function (preventing infection), active hemostasis (controlling intraoperative bleeding), and strong osteogenic activity (guiding new bone formation). It also exhibits an appropriate degradation rate in vivo, making it a highly promising multifunctional bone repair material for the repair of infected or difficult-to-heal bone defects.

[0080] Example 5: Mineralized two-dimensional silver-loaded polydopamine egg membrane (2D-EM@Ag-M) and its performance evaluation

[0081] 1. Preparation of two-dimensional silver-loaded polydopamine egg membrane (2D-EM@Ag)

[0082] This comparative example is prepared according to the steps of prior art embodiments:

[0083] (1) Raw material acquisition and pretreatment: Take fresh eggs, separate the eggshells and carefully peel off and collect the inner natural egg membrane. Wash thoroughly with deionized water to remove residual egg white and other impurities. Then cut the egg membrane into pieces of appropriate size (e.g., 3 cm × 3 cm).

[0084] (2) Acid solution treatment: The cleaned egg membrane was immersed in a 100 mM, pH 3 hydrochloric acid aqueous solution and left to stand at room temperature (about 25°C) for 4 days to carry out preliminary cleaning and surface modification.

[0085] (3) Cleaning: After acid treatment, remove the egg membrane and wash it with deionized water by shaking 5 times for 3 minutes each time to thoroughly remove residual acid and dissolved substances.

[0086] (4) Polydopamine coating deposition: Prepare a 4 mg / mL dopamine hydrochloride solution (solvent is 100 mM Tris-HCl buffer, pH adjusted to 9.5). Immerse the acid-treated egg membrane in this solution and let it stand at room temperature in the dark for 20 hours to allow dopamine to oxidize and self-polymerize to form a PDA coating.

[0087] (5) Clean again: After polymerization, remove the membrane and clean it with deionized water 5 times for 3 minutes each time to remove unreacted monomers and loosely attached polymers.

[0088] (6) In-situ loading of nano-silver: The egg membrane coated with PDA was immersed in 50 nM silver nitrate aqueous solution and left to stand at room temperature in the dark for 20 hours. The reducing and adhesive properties of PDA were used to reduce silver ions in situ to nano-silver particles and fix them on the coating surface.

[0089] (7) Final treatment: After silver loading treatment, wash with deionized water 5 times for 3 minutes each time to remove unreacted silver ions, and air dry at room temperature to obtain two-dimensional silver-loaded polydopamine egg membrane, denoted as 2D-EM@Ag.

[0090] (8) Biomimetic mineralization treatment: 2D-EM@Ag was immersed in 1X simulated body fluid (SBF) and mineralized at 25°C for 48 hours to deposit calcium phosphate on the membrane surface.

[0091] (9) Post-treatment: After mineralization, the surface mineralized two-dimensional silver-loaded polydopamine egg membrane was obtained by gently rinsing it three times with deionized water to remove loose attached crystals. It was denoted as 2D-EM@Ag-M.

[0092] 2. Performance comparison with FEM@DAgM

[0093] Parallel comparative tests were conducted between 2D-EM@Ag-M and FEM@DAgM prepared in Example 4 of this invention. The results are as follows: After drying, 2D-EM@Ag-M is brittle and easily curled. SEM showed that its surface is covered with a layer of discontinuous plate-like or spherical calcium phosphate crystals; cross-section showed that the upper layer was a mineralized layer, the middle layer was a dense egg membrane protein / PDA / Ag composite layer, and the lower layer was unmineralized or very shallowly mineralized, and the overall structure was still a dense two-dimensional structure without internal pores. Compared with FEM@DAgM, the mineralization of 2D-EM@Ag-M only occurs on the two-dimensional surface, forming an outer shell, while the interior remains a dense, non-porous core; while the mineralization of Example 4 occurs on the surface in three-dimensional space, and the pores remain open, resulting in a difference in the interaction mode between the two and biological tissues. In terms of hemostasis, 2D-EM@Ag-M performed poorly, the test solution remained red, the physical hemostasis ability was weak, and the overall hemostasis speed was slow. This indicates that although both contain the same chemical coagulant components, the three-dimensional porous structure is an indispensable synergistic factor, and the efficiency of simple surface chemical action is limited. Therefore, the excellent hemostatic, antibacterial, and osteogenic properties obtained by this invention are not simply the sum of surface chemical modification (PDA / Ag coating) and mineralization treatment. The high porosity and highly interconnected three-dimensional network structure created by the gas foaming step is an essential prerequisite and key physical platform. This structure not only significantly improves the material's liquid absorption and cell-carrying capacity but also greatly increases the specific surface area, allowing subsequent polydopamine coating, nano-silver loading, and biomimetic mineralization to proceed efficiently and uniformly within a three-dimensional space. This results in a synergistic enhancement effect between the physical structure (three-dimensional porosity) and chemical functions (antibacterial and mineralization), making the overall performance far exceed that of two-dimensional membrane materials or three-dimensional materials prepared by other methods (such as freeze-drying).

[0094] Example 6: Mineralized freeze-dried egg membrane scaffold and its performance evaluation

[0095] This embodiment aims to investigate the effects of different three-dimensional processing methods on the performance of the final mineralized egg membrane scaffold. The preparation of a three-dimensional porous scaffold by homogenizing, suspending, and freeze-drying the egg membrane is a mature and common method in the prior art.

[0096] Preparation method

[0097] (1) Egg membrane acquisition and enzyme treatment: The steps (1) and (2) of Example 4 are exactly the same, and the egg membrane scaffold (KEM) after proteinase K treatment is obtained.

[0098] (2) Three-dimensional processing of egg membrane (using existing freeze-drying technology): The wet KEM scaffold was blotted with filter paper to remove excess surface moisture, and then rapidly frozen in liquid nitrogen to instantly solidify the internal moisture into ice crystals. The sample was then quickly transferred to a freeze dryer and dried at -50℃ and 0.1 mBar vacuum for 48 hours. The sublimation of the ice crystals left pores, forming a three-dimensional structure. This scaffold was named freeze-dried three-dimensional egg membrane scaffold (FEM-FD).

[0099] (3) Egg membrane coating treatment: exactly the same as step (4) in Example 4, the FEM-FD scaffold was sequentially subjected to PDA coating deposition and nano silver loading to obtain FEM-FD@DAg.

[0100] (4) Egg membrane mineralization treatment: exactly the same as step (5) in Example 4, the FEM-FD@DAg scaffold was immersed in 1X SBF and mineralized at 25°C for 48 hours to obtain mineralized freeze-dried egg membrane scaffold (FEM-FD@DAgM).

[0101] Parallel comparative tests were conducted between FEM-FD@DAgM and FEM@DAgM prepared in Example 4 of this invention: Regarding pore structure, the lyophilized FEM-FD@DAgM scaffold had larger and unevenly distributed pores. This irregular macroporous system made it difficult for cells to attach evenly and stably, physically limiting cell activity and tissue ingrowth. In contrast, the FEM@DAgM scaffold prepared in Example 4 had a uniform pore structure and high internal interconnection, significantly improving cell attachment, spreading, and proliferation efficiency. Regarding antibacterial properties, both were endowed with antibacterial function through a silver coating. However, due to the uneven pore and surface structure, the silver coating distribution of FEM-FD@DAgM was also uneven, resulting in local fluctuations in antibacterial efficacy and relatively weaker overall stability. FEM@DAgM, with its uniform pore and surface structure, was able to support and maintain a more uniformly distributed and stable silver ion antibacterial coating. Regarding mineralization capacity and bone regeneration potential, the mineralization layer formed on the surface of the FEM-FD@DAgM scaffold is thin and unevenly deposited, resulting in insufficient ability to provide osteogenic induction signals and serve as a template for bone deposition. In vitro ALP and ARS staining results are also shallow, limiting its ability to promote bone differentiation and mineralization. In contrast, the FEM@DAgM surface can achieve uniform and deep mineralization deposition.

[0102] 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 variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of protection of the claims.

Claims

1. A method for preparing a multifunctional biological scaffold, characterized in that, Includes the following steps: S1. Treat the egg membrane to weaken the cross-linking structure between its protein fibers; S2. The egg membrane treated by S1 is subjected to gas foaming to form a porous support with three-dimensional channels. S3. Construct a polymer coating on the surface of the porous scaffold obtained in S2. The polymer coating has both adhesive and reductive properties, and is used to load antibacterial metal components.

2. The preparation method according to claim 1, characterized in that, It also includes step S4: placing the scaffold loaded with antibacterial metal components obtained in S3 in a mineralization solution, and depositing a calcium phosphate biomineralization layer on its surface.

3. The preparation method according to claim 1, characterized in that, In step S1, the egg membrane is treated with proteinase K at a concentration of 0.01-0.1 mg / mL at 55-65°C for 12-24 hours in a Tris-HCl buffer system containing NaCl at pH 8.

0.

4. The preparation method according to claim 1, characterized in that, In step S2, the gas foaming treatment is carried out using a sodium borohydride solution with a concentration of 0.5-5 wt%, and the foaming treatment is carried out at 25-30°C for 5-15 minutes.

5. The preparation method according to claim 4, characterized in that, After step S2, the scaffold forms a three-dimensional porous structure with an average thickness of 150-350 μm.

6. The preparation method according to claim 1, characterized in that, Step S3 includes: S3.1 The three-dimensional porous scaffold is immersed in a weakly alkaline buffer solution containing 0.1-1 wt% dopamine hydrochloride and 0.05-0.5 wt% sodium periodate, and reacted at 25-30°C for 6-12 hours to form a polydopamine coating on the scaffold surface. S3.2 Immerse the scaffold coated with polydopamine in a 0.001-0.05 mmol / L silver nitrate solution and react at 25-30℃ for 6-12 hours to reduce and load silver nanoparticles in situ.

7. The preparation method according to claim 2, characterized in that, In step S4, the mineralization solution is a simulated body fluid with a concentration of 1 to 5 times, and the mineralization treatment is carried out at 25-37°C for 24-48 hours; the main component of the calcium phosphate biomineralization layer is bone-like apatite.

8. A multifunctional biological scaffold, characterized in that, Its substrate is an egg membrane pretreated with proteinase K and foamed with gas, which has a three-dimensional porous network structure; the surface of the three-dimensional porous network is sequentially coated with a polydopamine-silver nanoparticle antibacterial coating and a calcium phosphate biomineralization layer.

9. The multifunctional biological scaffold according to claim 8, characterized in that, The average thickness of the scaffold is 150-350 μm.

10. The use of the multifunctional biological scaffold of claim 8 or 9 in the preparation of medical devices for wound repair or bone defect repair.

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