A collagen-based porous microsphere implant and a preparation method and application thereof

Abstract

This invention provides a collagen-based porous microsphere implant, its preparation method, and its application, relating to the field of tissue engineering materials technology. The implant comprises collagen-based porous microspheres; the raw material components of the collagen-based porous microspheres include collagen, silk fibroin, and mucopolysaccharides; the collagen-based porous microspheres have a interconnected hierarchical porous structure; the hierarchical porous structure includes micron-sized pores and mesopores. In the implant provided by this invention, the stable framework constructed by the cross-linking of silk fibroin and collagen provides long-term chemical support; the interconnected hierarchical porous structure, synergistically with internally and externally grafted RGD peptides, actively recruits tissue cells to migrate and grow into the deeper layers of the microspheres, providing an ideal three-dimensional microenvironment for cell adhesion, proliferation, and collagen secretion. Simultaneously, the stable framework constructed by the cross-linking of silk fibroin and collagen provides long-term chemical support, and it is expected that after implantation in vivo, it will help achieve in-situ isovolumetric regeneration of tissues.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering materials technology, and more specifically, to a collagen-based porous microsphere implant, its preparation method, and its application. Background Technology

[0002] Tissue-engineered scaffold materials and implantable materials are widely used in the field of medical repair, often for filling and regenerating soft tissue defects. Ideal implantable materials should possess good biocompatibility, provide stable physical support at the implantation site, and effectively guide the in-situ regeneration of the body's own tissues.

[0003] Currently, traditional biodegradable microsphere implant materials mainly include polylactic acid, polycaprolactone, and hydroxyapatite. Their mechanism of action typically involves providing immediate physical filling through the microsphere carrier, and then continuously stimulating the body's immune response as a foreign body, guiding macrophages to aggregate and secrete new collagen. In addition, injectable microsphere materials based on natural macromolecules (such as single collagen) have also emerged in existing technologies, aiming to utilize the properties of natural materials for tissue filling.

[0004] However, existing implant materials have significant limitations in practical applications. On the one hand, the mechanism of action, which relies on foreign body inflammatory stimulation, is time-consuming, and its regeneration degree is greatly affected by individual factors such as physical condition, making it difficult to predict and control precisely. Furthermore, excessive and continuous inflammatory stimulation can easily lead to safety issues such as delayed-onset nodules and granulomas. On the other hand, existing collagen gels or microspheres often have a dense internal structure and lack a permeable pore network, preventing surrounding tissue cells from migrating and growing into them, essentially limiting them to physical filling. At the same time, the mechanical strength of single collagen raw materials is poor, and their degradation rate in vivo is too fast, failing to provide long-term scaffold template support during the long tissue regeneration cycle, resulting in limited regenerated tissue.

[0005] In summary, existing microsphere implantation materials generally suffer from several technical defects, including reliance on excessive inflammatory stimulation leading to high safety risks, unfavorable microstructure for deep cell migration and ingrowth, insufficient mechanical properties, mismatch between degradation rate and tissue regeneration rate, dense internal structure lacking physical channels, and insufficient cell affinity. These defects make it difficult to actively guide the large-scale ingrowth and deep colonization of tissue cells, and the mechanical properties of the materials themselves are insufficient to provide long-term scaffold support.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The present invention aims to provide a collagen-based porous microsphere implant, its preparation method, and its applications. This collagen-based porous microsphere implant utilizes a stable framework of silk fibroin, a hierarchical porous structure, and the synergistic effect of RGD peptides to actively recruit cells for deep ingrowth. Unlike traditional physically filled microsphere products with dense internal structures, this invention aims to solve the problems of existing microsphere implantation materials, such as difficulty in guiding cells to grow deeply and poor mechanical support. It provides a structurally stable collagen-based porous microsphere implant that can actively recruit cell adhesion and migration. Utilizing its unique hierarchical porous structure and modified RGD peptides to exert a synergistic chemotactic effect, it guides cells into the interior of the microsphere scaffold, possessing the potential to serve as an ideal in-situ isochoric regeneration scaffold.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a collagen-based porous microsphere implant, wherein the collagen-based porous microsphere implant comprises collagen-based porous microspheres; The raw material components of the collagen-based porous microspheres include: collagen, silk fibroin, and mucopolysaccharides; wherein, the silk fibroin is cross-linked with the collagen to form a supporting framework; The collagen-based porous microspheres have a through-hole hierarchical pore structure; the hierarchical pore structure includes micron-sized pores and mesopores distributed on the pore walls of the micron-sized pores; RGD peptides are grafted onto at least a portion of the outer surface of the collagen-based porous microspheres and at least a portion of the pore walls of the hierarchical pore structure.

[0009] In an optional embodiment, the pore size of the micron-sized pore is 10 μm to 50 μm; and / or, The pore size of the mesopore is 2nm~50nm; and / or, The collagen-based porous microspheres have a particle size of 50 μm to 250 μm; and / or, The porosity of the collagen-based porous microspheres is greater than 90%; and / or, The collagen includes bovine type I collagen; and / or, The RGD peptide comprises arginyl-glycyl-aspartic acid; and / or, The mucopolysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, heparin, and their salts; and / or, The silk fibroin is degummed silk fibroin, and its degumming rate, determined by the weight loss method before and after degumming, is between 20% and 30%.

[0010] In an optional embodiment, the mass ratio of collagen to silk fibroin is (2~10):(1~7).

[0011] In an optional embodiment, the collagen-based porous microspheres are subjected to chemical cross-linking and irradiation cross-linking treatments; Preferably, the crosslinking agent used in the chemical crosslinking includes one of glutaraldehyde, genipin, and EDC / NHS; Preferably, the irradiation crosslinking is performed using high-energy electron beam irradiation crosslinking; more preferably, the irradiation crosslinking dose is 5 kGy to 25 kGy.

[0012] In an optional embodiment, the storage modulus of the collagen-based porous microsphere implant is 4000 Pa to 7000 Pa at a frequency of 1 Hz; and / or, the loss modulus is 700 Pa to 1500 Pa.

[0013] Secondly, the present invention provides a method for preparing a collagen-based porous microsphere implant as described in any of the foregoing embodiments, comprising: A suspension made of collagen and mucopolysaccharide is mixed with a solution made of silk fibroin, and a cross-linking agent solution is added to react and obtain a composite solution. The composite solution is injected into the oil phase via a high-pressure gas jet spray method to simultaneously form microspheres and undergo emulsification and solidification reactions; wherein, the high-pressure gas jet spray method is used to maintain the initial spherical shape of the droplets, and the oil phase is used to simultaneously emulsify and solidify the droplets to obtain microspheres with regular morphology. The composite solution was added to the oil phase via a high-pressure gas spray method for emulsification, and then collected and washed to obtain porous microspheres. The cleaned porous microspheres were immersed in an acidic crosslinking agent solution, and the RGD peptide was added to carry out a grafting reaction. After cleaning, the modified microspheres were obtained. The modified microspheres were subjected to gradient temperature-controlled freezing and freeze-drying, followed by high-energy electron beam irradiation for cross-linking and sterilization to obtain the collagen-based porous microsphere implant.

[0014] In some embodiments, the gradient temperature-controlled freezing process sequentially includes: a first freezing stage, a thawing growth stage, and a second freezing stage; Wherein, the temperature of the first freezing stage is lower than the temperature of the second freezing stage; the temperature of the second freezing stage is lower than the temperature of the warm-up growth stage, and the temperature of the warm-up growth stage is lower than 0°C; Preferably, the temperature of the first freezing stage is -90℃ to -70℃; and / or, the temperature of the warm-up growth stage is -30℃ to -10℃; and / or, the temperature of the second freezing stage is -70℃ to -50℃; more preferably, the temperature of the first freezing stage is -80℃; and / or, the temperature of the warm-up growth stage is -20℃; and the temperature of the second freezing stage is -60℃. Preferably, the processing time of the first freezing stage is 1h to 2h; and / or, the processing time of the warm-up growth stage is 1h to 2h; and / or, the processing time of the second freezing stage is not less than 12 hours.

[0015] In some embodiments, the volume ratio of the suspension made from the collagen and the mucopolysaccharide to the solution made from the silk fibroin is (10~40):(1~10); and / or, The spray pressure of the high-pressure airflow spray method is 0.1 bar to 10 bar; preferably 2 bar to 6 bar; and / or, The oil phase comprises liquid paraffin; and / or, The emulsification reaction also includes an emulsifier, which comprises sorbitan oleate and polysorbate 80; and / or, The cleaning agents used include ethanol and deionized water.

[0016] In some embodiments, the acidic crosslinking agent solution is prepared by mixing a PBS buffer at pH 5.0 with 20 mM to 60 mM EDC and 5 mM to 30 mM NHS.

[0017] Thirdly, the present invention provides the application of the collagen-based porous microsphere implant as described in any of the foregoing embodiments in the preparation of tissue engineering materials, medical devices or cosmetic injection products.

[0018] Compared with existing technologies, the implant provided by this invention uses collagen, silk fibroin, and mucopolysaccharides as raw material components, wherein silk fibroin and collagen are cross-linked to form a stable supporting framework. This structure effectively overcomes the natural defects of insufficient mechanical strength and excessively rapid degradation in vivo caused by single collagen components, endowing the microspheres with excellent mechanical support properties, enabling them to act as a long-lasting and stable scaffold template in vivo, ensuring that the degradation rate of the material matches the regeneration rate of human tissue, and avoiding structural collapse due to insufficient support in the early stages of implantation.

[0019] The microspheres possess a hierarchical porous structure consisting of interconnected micron-sized pores and mesopores distributed on the pore walls. Combined with RGD peptides grafted onto the outer surface and at least part of the pore walls inside the microspheres, this structure achieves deep synergy between physical spatial channels and biochemical signals. The hierarchical porous structure completely breaks down the traditional dense and closed physical barrier inside microspheres, providing an ample three-dimensional physical network for cell ingrowth, migration, and nutrient exchange. At the same time, the RGD peptides distributed on the outer surface and deep pore walls exert a high-affinity biological traction effect, actively and efficiently recruiting surrounding tissue cells to adhere and migrate deeper into the microspheres along the pores.

[0020] These microspheres, constructed through a high-strength material framework, three-dimensional hierarchical channels, and surfactant peptides, significantly enhance the material's chemotaxis and recruitment capabilities for cells, effectively overcoming the physical barrier that prevents cell ingrowth in traditional solid microspheres. After implantation, cells can rapidly and actively ingrow into the microspheres along the hierarchical channels for proliferation. Furthermore, this highly biomimetic cellular microenvironment and long-lasting scaffold support are expected to effectively reduce the risk of severe inflammatory rejection by tissues from simple physical filling of foreign materials, facilitating the gradual replacement of degraded microspheres by newly formed tissue. This provides an optimal material science solution for achieving in-situ isochoric regeneration of tissues.

[0021] The core technological contribution of this application lies in the construction of a three-in-one synergistic system of 'physical channel-biochemotaxis-mechanical support'. Its synergistic mechanism is as follows: the interconnected hierarchical porous structure provides a 'physical highway' for cell ingrowth, but without guidance, the intrinsic motivation for cells to actively enter the pores is insufficient; while the specific distribution of RGD peptides forms 'bionavigation signals' inside and outside the pores, actively attracting cells inward. If the microspheres collapse due to lack of silk fibroin support, the physical channel becomes blocked, and the bionavigation signals also fail. Therefore, all three are indispensable, jointly achieving a leap from 'passive filling' to 'active recruitment and regeneration ingrowth,' which is also the key technological threshold that distinguishes this application from simple feature superposition. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a scanning electron microscope (SEM) image (200 μm) of the overall morphology of the collagen-based porous microspheres prepared in Example 1 of the present invention. Figure 2 This is a magnified SEM image (10 μm) of the pore wall of the collagen-based porous microspheres prepared in Example 1 of the present invention. Figure 3 This is a high-magnification SEM image (1 μm) of the pore walls of the collagen-based porous microspheres prepared in Example 1 of the present invention, showing its nanoscale wrinkles and mesoporous morphology. Figure 4 This is a graph showing the detection results (CCK-8 method) of the effect of collagen-based porous microspheres on cell proliferation described in the experimental examples of this invention. Figure 5A crystal violet-stained photograph of the Transwell cell migration assay of the microspheres (without RGD peptide) obtained in Comparative Example 1 of this invention; Figure 6 This is a crystal violet-stained image of the Transwell cell migration assay of the microspheres (grafted with RGD peptide) obtained in Example 2 of this invention. Figure 7 The graph shows the degradation rate of the microspheres prepared in Comparative Example 2 and Example 2 of this invention during in vitro enzymatic hydrolysis. Figure 8 The image shows the BJH pore size distribution curve of the collagen-based porous microspheres prepared in Example 1 of this invention (obtained by nitrogen adsorption-desorption test). Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] The present invention provides a collagen-based porous microsphere implant, comprising collagen-based porous microspheres as the core entity.

[0026] Furthermore, the raw material components of the collagen-based porous microspheres include: collagen, silk fibroin, and mucopolysaccharides; wherein the silk fibroin is cross-linked with the collagen to form a supporting framework.

[0027] Specifically, due to the rapid degradation rate and inherent mechanical limitations of single collagen materials in vivo, this application introduces silk fibroin, which possesses excellent mechanical strength, and initiates a cross-linking reaction between the two to construct a three-dimensional scaffold network with high compressive modulus and a suitable degradation cycle. This supporting scaffold can effectively resist physical compression from surrounding tissues at the implantation site, preventing premature collapse of microspheres during cell ingrowth and self-degradation, thereby providing a long-lasting and stable physical scaffold template during the lengthy process of in-situ isochoric regeneration of tissue.

[0028] It is important to note that the introduction of silk fibroin in this application is not only to enhance the macroscopic mechanical strength of the microspheres, but also a necessary prerequisite for preparing a interconnected hierarchical porous structure. Experimental verification shows that if pure collagen is used in the preparation process described in this application, it is difficult to form a high-porosity interconnected hierarchical porous structure due to the limitations of collagen's film-forming properties and structural strength (porosity is significantly reduced and the pores are not interconnected). This application achieves the desired interconnected physical channels by combining silk fibroin and collagen, utilizing the specific nucleation effect of silk fibroin in the ice crystal template method.

[0029] The collagen-based porous microspheres have a through-hole hierarchical pore structure; the hierarchical pore structure includes micron-sized pores and mesopores distributed on the pore walls of the micron-sized pores.

[0030] It should be specifically noted that the 'micron-sized pores' mentioned in the embodiments of this application specifically refer to physical pores with pore sizes on the micron scale (especially in the range of 10μm to 50μm). This term is defined based on the spatial scale that provides a three-dimensional physical channel for tissue cell ingrowth, and it differs from the 'micropores' defined by the IUPAC standard in traditional materials science, which have pore sizes smaller than 2nm. The specific 10μm to 50μm micron-sized pores of this application match the volume of targeted tissue repair cells such as fibroblasts, thereby allowing and guiding cells to smoothly grow into the deep layers of the microspheres in physical space.

[0031] It should be noted that the 'mesoporosis' described in the embodiments of this application is not limited to closed or semi-closed standard cylindrical pores with regular geometric shapes. In the specific protein backbone crosslinking and freeze-drying system of this embodiment, the 'mesoporosis' also encompasses nanoscale dense wrinkles, microcracks, and three-dimensional rough textures with extremely high specific surface areas formed on the micron-scale pore walls due to ice crystal sublimation and polymer chain contraction. These nanoscale micromorphologies are comparable to traditional mesopores in spatial scale (2nm~50nm) and functionally play the same role in greatly increasing specific surface area, providing high-density grafting sites for RGD peptides, and promoting capillary penetration of nutrients.

[0032] To overcome the structural defects of traditional microsphere materials, such as dense internal structures that hinder deep cell migration, the collagen-based porous microspheres are configured with a continuous hierarchical porous structure. Specifically, the hierarchical porous structure includes large-sized micrometer-scale pores and smaller mesopores distributed on the walls of the micrometer-scale pores. In this specific hierarchical topology, the continuous micrometer-scale pores form a three-dimensional main channel for the migration and colonization of surrounding tissue cells into the deeper layers of the microsphere; while the densely distributed mesopores on the walls of the micrometer-scale pores significantly increase the specific surface area of ​​the microsphere and form a microscopic fluid permeation network, providing ample channels for nutrient delivery and metabolic waste removal for cells penetrating deep into the microsphere, ensuring high cell survival rates and extracellular matrix secretory activity.

[0033] RGD peptides are grafted onto at least a portion of the outer surface of the collagen-based porous microspheres and at least a portion of the pore walls of the hierarchical pore structure.

[0034] Furthermore, RGD peptides (arginyl-glycyl-aspartic acid) are grafted onto at least a portion of the outer surface of the collagen-based porous microspheres and at least a portion of the pore walls of the hierarchical pore structure. As bioactive molecules that can be specifically recognized by cell membrane surface receptors, RGD peptides can significantly promote cell adhesion and proliferation. In the structural system of this application, the RGD peptides and the interconnected hierarchical pore structure produce a significant synergistic effect: the RGD peptides grafted onto the outer surface of the microspheres can rapidly capture and anchor free surrounding tissue cells, while the RGD peptides extended onto the inner pore walls constitute guiding signals, actively attracting and recruiting attached cells to grow deeper into the microspheres along the hierarchical channels. Through this synergistic cooperation of physical spatial channels and biochemical chemotactic signals, this application completely changes the passive mechanism of traditional materials that rely on foreign body inflammatory stimulation for tissue proliferation, effectively reducing the risk of delayed nodules and granulomas caused by excessive inflammatory responses, and achieving safe, efficient, and controllable in-situ isovolumetric tissue regeneration.

[0035] In summary, the implant provided in this embodiment utilizes a stable framework constructed by cross-linking silk fibroin and collagen, overcoming the shortcomings of poor mechanical properties and rapid degradation associated with single materials, and providing a long-lasting and stable scaffold template. The multi-level porous structure formed by interconnected micron-sized pores and mesopores breaks down the dense physical barrier of traditional materials. Combined with the biochemical traction effect exerted by RGD peptides grafted onto the inner and outer surfaces, the two work synergistically to actively and efficiently recruit surrounding tissue cells to migrate and grow deeper into the microspheres. This design completely changes the passive mechanism of traditional microspheres that rely on foreign body inflammatory stimulation for efficacy, significantly reducing the risk of inflammatory nodules and granulomas, and achieving safe and controllable in-situ isovolumetric tissue regeneration.

[0036] In some embodiments of this application, the physical size, microstructure parameters and specific chemical composition of the collagen-based porous microspheres have been further optimized and preferred to construct a highly biomimetic extracellular microenvironment.

[0037] In some embodiments, the pore size of the micron-sized pore is 10μm to 50μm. For example, it can be 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 48μm, 50μm, etc.

[0038] In some embodiments, the pore size of the mesopore is 2nm to 50nm. For example, it can be 2nm, 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 38nm, 45nm, 50nm, etc.

[0039] In some embodiments, the particle size of the collagen-based porous microspheres is 50 μm to 250 μm. For example, it can be 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 240 μm, 250 μm, etc.

[0040] In some embodiments, the porosity of the collagen-based porous microspheres is greater than 90%.

[0041] Specifically, in the hierarchical porous structure, the pore size of the micron-sized pores is 10μm~50μm, and the pore size of the mesopores is 2nm~50nm. This pore size range is designed with specific biological considerations in mind: the 10μm~50μm micron-sized pores match the volume of targeted tissue repair cells such as fibroblasts, thus allowing and guiding cells to smoothly grow into the deeper layers of the microspheres in physical space; while the 2nm~50nm mesopores are distributed on the pore walls of the micron-sized pores, forming a dense material exchange network, ensuring the uptake of nutrients and the removal of metabolic waste by deep cells, maintaining a high cell survival rate. Combined with an ultra-high porosity of over 90%, the microspheres provide ample three-dimensional space for the ingrowth of new tissue and microvessels. Simultaneously, the overall particle size of the collagen-based porous microspheres is controlled within the range of 50μm~250μm. This particle size range avoids microspheres that are too small to be rapidly engulfed and cleared by macrophages, while also ensuring excellent rheological injection properties, allowing them to pass smoothly through extremely fine clinical injection needles without agglomeration or blockage.

[0042] In some embodiments, the collagen includes bovine type I collagen.

[0043] In some embodiments, the RGD peptide comprises arginyl-glycyl-aspartic acid.

[0044] In some embodiments, the mucopolysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, heparin, and their salts.

[0045] In some embodiments, the silk fibroin is degummed silk fibroin, and its degumming rate, determined by the weight loss method before and after degumming, is between 20% and 30%. For example, it can be 20%, 22%, 24%, 26%, 28%, 30%, etc.

[0046] In this application, the degumming rate of the silk fibroin refers to the percentage of the mass of silk lost during the degumming process relative to the original silk mass, which mainly reflects the degree of removal of sericin. The specific testing method is as follows: Weigh the original mulberry silk before degumming (W1), perform boiling degumming and drying according to the process described in this application, weigh the resulting silk fibroin (W2), and calculate (W1-W2) / W1×100%. In a preferred embodiment of this application, the degumming rate is between 20% and 30%. This range ensures that the sericin component is sufficiently removed, effectively reducing the immunogenicity of the material.

[0047] Regarding the selection of chemical and biological raw materials, the collagen preferably includes bovine type I collagen, which highly retains the natural triple helix structure and provides excellent biocompatibility. The mucopolysaccharide preferably includes at least one of hyaluronic acid, chondroitin sulfate, heparin, and their salts. These substances, as important components of the natural extracellular matrix, significantly enhance the tissue hydration capacity of the material due to their abundant hydrophilic groups. For the silk fibroin providing the supporting framework, this embodiment strictly limits its degumming rate to greater than 95%. This maximizes the removal of sericin components that easily trigger immune rejection and inflammatory responses, ensuring high purity and high mechanical strength of the framework. Furthermore, the RGD peptide grafted onto the surface and pore walls of the microspheres is specifically defined as an arginyl-glycyl-aspartic acid tripeptide sequence. This specific sequence can be precisely recognized by integrin receptors on the cell surface, thereby exerting a powerful cell-targeting anchoring and active chemotactic recruitment effect.

[0048] In a specific embodiment of this application, the ratio of the two core macromolecular materials constituting the porous microsphere framework is precisely optimized and defined. The mass ratio of collagen to silk fibroin is (2~10):(1~7). For example, it can be 2:1, 2:7, 3:2, 4:1, 5:3, 6:5, 8:3, 8:7, 10:1, 10:7, etc.

[0049] This specific mass ratio forms the material basis for the microspheres to achieve ideal mechanical support and isochoric regeneration. Since a single collagen raw material degrades too quickly in vivo and has insufficient compressive strength, while a single silk fibroin degrades too slowly and has relatively limited biocompatibility, this embodiment controls the ratio of the two within the aforementioned range, constructing a cross-linked network framework that combines rigidity and flexibility at the microscopic level. Within this mass ratio range, silk fibroin rich in β-sheet structures provides excellent mechanical strength to the microspheres, ensuring that the framework does not collapse due to physical compression from surrounding tissues after implantation, thus maintaining the physical unobstructed flow of the internal hierarchical porous structure. Simultaneously, sufficient collagen not only ensures high cell affinity on the framework surface but also precisely regulates the overall material degradation rate. This synergistic effect of the ratio allows for a perfect dynamic match between the material's degradation cycle and the regeneration cycle of the body's own cells ingrowing and secreting new extracellular matrix, avoiding support failure caused by excessively rapid implant degradation or long-term inflammation caused by excessively slow degradation, ultimately achieving high-quality in-situ isochoric tissue regeneration.

[0050] In some embodiments of this application, the collagen and silk fibroin backbone system constituting the porous microspheres undergoes a specific pattern of dual cross-linking treatment to ensure that the implant material possesses both excellent mechanical support properties and biocompatibility.

[0051] Specifically, the collagen-based porous microspheres undergo sequential chemical cross-linking and irradiation cross-linking treatments. A single cross-linking process is insufficient to achieve the high degree of cross-linking required for degradation resistance while maintaining low cytotoxicity. This embodiment first uses a chemical cross-linking agent to perform initial cross-linking of the mixed matrix, resulting in stable covalent bonds between collagen and silk fibroin molecular chains, thereby constructing a primary three-dimensional framework that maintains the hierarchical porous structure from collapsing.

[0052] Preferably, the crosslinking agent used in the chemical crosslinking includes one of glutaraldehyde, genipin, and EDC / NHS; Preferably, the irradiation crosslinking is performed using high-energy electron beam irradiation crosslinking; more preferably, the irradiation crosslinking dose is 5 kGy to 25 kGy. For example, it can be 5 kGy, 8 kGy, 10 kGy, 12 kGy, 14 kGy, 16 kGy, 18 kGy, 20 kGy, 22 kGy, 25 kGy, etc.

[0053] The chemical crosslinking agent preferably includes one of glutaraldehyde, genipin, or EDC / NHS (1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide). Using highly biocompatible crosslinking agents such as genipin or EDC / NHS can effectively prevent the release of cytotoxic free small molecules during in vivo degradation while improving structural strength.

[0054] After chemical cross-linking and curing the structure, the microspheres were further cross-linked by irradiation using a high-energy electron beam. High-energy electron beam irradiation can excite the polymer chains to generate free radicals and couple, constructing a more compact secondary network structure on the basis of the primary skeleton, which significantly improves the energy storage modulus and enzyme resistance of the microspheres.

[0055] More importantly, this embodiment strictly controls the irradiation crosslinking dose between 5 kGy and 25 kGy. Within this specific dose range, the high-energy electron beam not only effectively achieves terminal sterilization to meet the sterility requirements of implantable medical devices, but also promotes appropriate and effective crosslinking of macromolecular chains. Simultaneously, the upper dose limit (25 kGy) effectively avoids the destruction of collagen helical structures and the breakage and degradation of macromolecular backbones caused by excessive radiation energy. This dual synergy of "chemical + irradiation" and precise dose control endows the porous microspheres with excellent long-lasting chemical properties and a precisely controllable in vivo degradation cycle.

[0056] In some embodiments of this application, the macroscopic mechanical and viscoelastic parameters of the collagen-based porous microsphere implant have been optimized and limited to ensure the structural stability and biological function of the implant under complex mechanical environments in vivo.

[0057] Specifically, at a dynamic testing frequency of 1 Hz, the storage modulus of the collagen-based porous microsphere implant is limited to 4000 Pa to 7000 Pa (e.g., 4000 Pa, 4300 Pa, 4500 Pa, 4800 Pa, 5000 Pa, 5500 Pa, 6000 Pa, 6200 Pa, 6600 Pa, 7000 Pa, etc.), and the loss modulus is limited to 700 Pa to 1500 Pa (e.g., 700 Pa, 800 Pa, 900 Pa, 1000 Pa, 1100 Pa, 1200 Pa, 1300 Pa, 1350 Pa, 1450 Pa, 1500 Pa, etc.).

[0058] In rheology, storage modulus characterizes a material's solid-state elastic support capability, while loss modulus characterizes its fluid viscous buffering capability. This application constructs a robust interpenetrating network framework by crosslinking silk fibroin and collagen in a specific ratio, thereby macroscopically endowing the implant with a significantly enhanced storage modulus.

[0059] The energy storage modulus of 4000Pa~7000Pa ensures that the microspheres can fully resist the physical compression and deformation of surrounding muscles and soft tissues after being implanted under the skin or in areas of tissue defects. This excellent compressive support performance is the core physical prerequisite for maintaining the internal interconnected hierarchical porous structure and preventing premature closure and collapse, thereby ensuring the physical space for free cells to migrate smoothly into the deeper layers of the microspheres.

[0060] The loss modulus of 700Pa to 1500Pa gives the material both good flexibility and certain fluid lubrication properties. The suitable viscoelasticity not only makes the implant highly simulate the feel of natural human soft tissue in macroscopic mechanics, but also ensures that it can pass smoothly through ultra-fine injection needles with appropriate deformation in clinical applications, achieving smooth minimally invasive injection. After reaching the target site, it quickly rebounds to restore its porous scaffold morphology, ultimately providing a long-lasting and stable mechanical microenvironment for cell adhesion growth and in-situ isochoric regeneration of tissues.

[0061] This application also provides a method for preparing a collagen-based porous microsphere implant. This method organically combines the chemical cross-linking, physical morphology shaping, and bioactive modification of macromolecular materials, and specifically includes the following steps: S1, a suspension made of collagen and mucopolysaccharide is mixed with a solution made of silk fibroin, and a cross-linking agent solution is added to react and obtain a composite solution; S2, the composite solution is injected into the oil phase via a high-pressure gas spray method to carry out simultaneous spheroidization and emulsification solidification reactions; wherein, the high-pressure gas spray method is used to maintain the initial spherical shape of the droplets, and the oil phase is used to simultaneously emulsify and solidify the droplets to obtain microspheres with regular morphology.

[0062] A suspension prepared by mixing the collagen and the mucopolysaccharide is uniformly mixed with a silk fibroin solution, and a cross-linking agent solution is added to initiate a preliminary cross-linking reaction to obtain a composite solution. In this step, collagen, silk fibroin, and mucopolysaccharide with different physicochemical properties achieve uniform dispersion at the molecular level in the liquid system, and a macromolecular interpenetrating network framework is initially constructed under the action of the cross-linking agent, giving the fluid suitable viscosity and structural stability, laying the material basis for subsequent atomization molding to prevent phase separation of components.

[0063] S3, the composite solution is added to the oil phase by high-pressure gas spraying to carry out emulsification reaction, and then collected and washed to obtain porous microspheres.

[0064] The composite solution obtained in the above steps is added to the oil phase of the continuous phase via high-pressure gas spraying for emulsification. The generated microparticles are then collected and thoroughly washed to obtain porous microsphere embryos. This step utilizes the aerodynamic shear force of the high-pressure gas flow to atomize the composite solution into micron-sized droplets. Upon entering the oil phase, these droplets spontaneously shrink into regular spherical shapes under surface tension. Using high-pressure gas spraying instead of traditional mechanical stirring yields microspheres with more uniform particle size distribution and spherical morphology, and facilitates large-scale continuous production. The subsequent washing step aims to extract and remove residual oil phase and emulsifiers from the microsphere surface, ensuring the biocompatibility of the material.

[0065] S4, the cleaned porous microspheres are immersed in an acidic crosslinking agent solution, the RGD peptide is added to carry out a grafting reaction, and after cleaning, the modified microspheres are obtained.

[0066] The cleaned porous microspheres were immersed in a prepared acidic cross-linking agent solution, followed by the addition of the RGD peptide for a grafting reaction. After the reaction, the microspheres were washed again to obtain the modified microspheres. This step constitutes the core chemical basis for the active recruitment of cells. In a specific acidic buffer environment, the cross-linking agent can efficiently activate the exposed carboxyl groups on the porous microsphere backbone (especially the collagen and silk fibroin macromolecular chains), causing them to form strong covalent amide bonds with the amino groups of the RGD peptide molecules. Compared with physical adsorption, this chemical grafting method greatly improves the binding stability of the RGD peptide on the outer surface and internal pore walls of the microspheres, ensuring that it can continue to exert its biological functions of cell anchoring and chemotactic recruitment even under the flushing of body fluids in vivo.

[0067] S5, the modified microspheres are subjected to gradient temperature-controlled freezing and freeze-drying in sequence, followed by high-energy electron beam irradiation crosslinking sterilization to obtain the collagen-based porous microsphere implant.

[0068] The modified microspheres were subjected to gradient temperature-controlled freezing and freeze-drying (lyophilization) treatments, followed by high-energy electron beam irradiation crosslinking sterilization, to finally obtain the collagen-based porous microsphere implant.

[0069] In this step, gradient temperature-controlled freezing is the key physical mechanism for forming the material's interconnected hierarchical porous structure (i.e., the ice crystal template method). By setting different freezing temperatures at different stages, the nucleation rate of water molecules and the size of ice crystals inside the microspheres are precisely controlled. Subsequently, under vacuum conditions, these ice crystals with specific morphologies are directly sublimated and dehydrated, transforming the three-dimensional space originally occupied by the ice crystals into physically interconnected micron-sized pores and mesoporous structures. Finally, high-energy electron beam irradiation not only penetrates the microspheres to kill all potential microorganisms, achieving strict terminal sterilization standards, but its high-energy radiation also further excites the protein macromolecular chains to generate free radicals and undergo secondary cross-linking, significantly improving the macroscopic mechanical storage modulus and in vivo enzymatic resistance of the microsphere implant.

[0070] In a specific embodiment of this application, the shaping of the hierarchical porous structure inside the microspheres does not rely on traditional porogen elution, but rather employs a gradient temperature-controlled freezing procedure based on the "ice crystal template mechanism." Through precise human intervention in the thermodynamic evolution of the freezing process, a high degree of control over pore size and permeability is achieved.

[0071] Specifically, the gradient temperature-controlled freezing process sequentially includes: a first freezing stage, a warm-up growth stage, and a second freezing stage. In this process, the temperature of each stage is strictly controlled as follows: the temperature of the first freezing stage is lower than the temperature of the second freezing stage; the temperature of the second freezing stage is lower than the temperature of the warm-up growth stage, and the temperature of the warm-up growth stage is below 0°C. Preferably, the temperature of the first freezing stage is -90℃ to -70℃; for example, it can be -90℃, -88℃, -85℃, -82℃, -80℃, -78℃, -75℃, -72℃, -70℃, etc. And / or, the temperature of the warm-up growth stage is -30℃ to -10℃; for example, it can be -30℃, -28℃, -25℃, -22℃, -20℃, -18℃, -15℃, -12℃, -10℃, etc. And / or, the temperature of the second freezing stage is -70℃ to -50℃; for example, it can be -70℃, -68℃, -65℃, -62℃, -60℃, -58℃, -55℃, -52℃, -50℃, etc. More preferably, the temperature of the first freezing stage is -80°C; and / or, the temperature of the warm-up growth stage is -20°C; and the temperature of the second freezing stage is -60°C; Preferably, the processing time of the first freezing stage is 1 hour to 2 hours; for example, it can be 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours, etc. And / or, the processing time of the warm-up growth stage is 1 hour to 2 hours; for example, it can be 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours, etc. And / or, the processing time of the second freezing stage is not less than 12 hours.

[0072] This stepped temperature-changing logic has a specific microscopic phase transition synergistic mechanism.

[0073] In the first freezing stage (the temperature is preferably -90℃ to -70℃, more preferably -80℃, and the processing time is 1h to 2h), the extremely low temperature provides the system with a huge degree of supercooling, which causes the water inside the microspheres to generate an extremely high nucleation rate, instantly forming a large number of dense and extremely small initial crystal nuclei.

[0074] The system then enters a warming growth stage (preferably -30℃ to -10℃, more preferably -20℃, for 1 to 2 hours). The moderate temperature rise triggers a ripening effect at the microscale. Under this thermodynamic environment, the extremely small ice crystals with poor thermal stability undergo phase transformation and melt, attaching to larger ice crystals to further grow and fuse. The growing ice crystals squeeze and merge with each other, thus forming a larger and interconnected ice crystal network. This is the key process for the formation of the micron-sized pores and mesoporous multi-level structure in porous microspheres.

[0075] Finally, the system enters the second freezing stage (preferably at -70℃ to -50℃, more preferably -60℃, for a processing time of no less than 12 hours), where another rapid cooling aims to instantly terminate the continued disordered growth of ice crystals and completely solidify the constructed interconnected network. This prolonged cryogenic treatment further ensures the rigid fixation of the protein polymer chains' physical conformation around the ice crystal template, thereby guaranteeing that the microspheres maintain their intact hierarchical porous structure without pore wall collapse during the subsequent sublimation freeze-drying process.

[0076] It should be noted that during the alternation between the warming growth stage (-20℃) and the second freezing stage (-60℃), in addition to the formation of macroscopic interconnected micron-sized pores, the local polymer network of cross-linked silk fibroin and collagen will inevitably undergo microscale stress contraction on the surface of the pore wall matrix under the compression of the ice crystal boundary and the subsequent vacuum sublimation, thereby generating in situ the aforementioned nanoscale wrinkles and mesoporous morphologies with characteristic sizes of 2nm~50nm.

[0077] In some embodiments of this application, the specific parameters of the preparation process and the auxiliary media used have been further optimized and defined.

[0078] In some embodiments, the volume ratio of the suspension made of the collagen and the mucopolysaccharide to the solution made of the silk fibroin is (10~40):(1~10). For example, it can be 10:1, 15:2, 20:3, 25:5, 30:7, 35:8, 40:1, 40:5, 40:10, etc.

[0079] In some embodiments, the spray pressure of the high-pressure airflow spray method is 0.1 bar to 10 bar; preferably 2 bar to 6 bar. For example, it can be 0.1 bar, 0.5 bar, 1 bar, 2 bar, 3 bar, 4 bar, 5 bar, 6 bar, 8 bar, 10 bar, etc.

[0080] Specifically, in preparing the composite solution, the volume ratio of collagen to the suspension made of the mucopolysaccharide and the solution made of silk fibroin is controlled at (10~40):(1~10). This precise control of the ratio achieves optimal compatibility between the bioactive matrix and the mechanically reinforcing components, resulting in a composite slurry with suitable rheological parameters, which is beneficial for subsequent atomization molding. In the molding step, a high-pressure airflow spray method is used to inject the composite solution into the oil phase. During this process, the spray pressure is controlled at 0.1 bar to 10 bar, preferably 2 bar to 6 bar. By adjusting the aerodynamic shear force generated by the spray pressure, the degree of droplet fragmentation can be precisely controlled, thereby obtaining well-defined microsphere embryos with a particle size distribution between 50 μm and 250 μm.

[0081] In some embodiments, the oil phase comprises liquid paraffin.

[0082] In some embodiments, an emulsifier is also added to the emulsification reaction, the emulsifier including sorbitan oleate and polysorbate 80.

[0083] In some embodiments, the cleaning agents used include ethanol and deionized water.

[0084] To ensure the morphological stability of the microspheres during the emulsification process, the oil phase is preferably a chemically inert and biocompatible liquid paraffin. Simultaneously, a composite emulsifier composed of sorbitan oleate (Span-80) and polysorbate 80 (Tween-80) is introduced into the emulsion system. This emulsion system significantly reduces the tension at the water / oil interface, constructing a stable physical barrier on the surface of the microsphere droplets and effectively preventing microsphere aggregation during collection. Furthermore, to thoroughly remove residual reagents from the microsphere surface, this embodiment uses ethanol and deionized water as cleaning agents. The introduction of ethanol effectively extracts and removes adsorbed liquid paraffin and oily components from the surface, while subsequent deionized water rinsing ensures the cleanliness of the microsphere environment, thereby guaranteeing that the final collagen-based porous microsphere implant meets stringent biomedical application requirements.

[0085] In some specific embodiments of this application, the specific chemical reaction environment and reagent concentrations on which the RGD peptide surface modification of porous microspheres depends have been precisely optimized and limited.

[0086] In some embodiments, the acidic crosslinking agent solution is prepared by mixing a PBS buffer at pH 5.0 with 20 mM to 60 mM of EDC (e.g., 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 52 mM, 55 mM, 60 mM, etc.) and 5 mM to 30 mM of NHS (e.g., 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 25 mM, 28 mM, 30 mM, etc.).

[0087] Specifically, the acidic cross-linking agent solution used for RGD peptide grafting is prepared by mixing PBS buffer (pH 5.0), 20mM–60mM EDC, and 5mM–30mM NHS. In the process of surface chemical modification of macromolecular biological scaffolds, precisely controlling the microenvironment of the reaction system at a weakly acidic state of pH 5.0 is of paramount thermodynamic importance. This specific pH value not only maintains the three-dimensional structural stability of the collagen-silk fibroin composite framework and prevents acid-induced protein denaturation, but also provides the most suitable proton environment for the cross-linking agent activation reaction.

[0088] In the aforementioned specific weakly acidic buffer system, EDC and NHS constitute a highly efficient covalently coupled synergistic system. EDC at concentrations of 20 mM to 60 mM initially acts as an activator, activating the free carboxyl groups on the surface of the porous microsphere framework into highly reactive intermediates. Simultaneously, NHS at concentrations of 5 mM to 30 mM rapidly intervenes, transforming these highly unstable intermediates into NHS-ester reactive intermediates with longer half-lives and higher stability in the aqueous phase. This synergistic transformation mechanism significantly reduces the probability of hydrolytic failure of the activated carboxyl groups. When RGD peptides are introduced, their terminal primary amino groups can efficiently undergo nucleophilic substitution reactions with stable NHS-esters, thereby forming exceptionally strong amide covalent bonds on the outer surface of the microspheres and the pore walls of the hierarchical porous structure. Controlling the concentrations of EDC and NHS within the aforementioned specific range ensures that the microsphere surface receives a sufficiently high density of RGD peptide grafts to exert a powerful chemotactic recruitment function, while effectively avoiding excessive self-crosslinking side reactions between protein macromolecules caused by excessively high crosslinking agent concentrations. Furthermore, since EDC and NHS, as zero-length crosslinking agents, do not participate in the formation of the final covalent bond, their reaction byproducts are all highly water-soluble and can be completely removed in subsequent cleaning steps, ensuring the extremely high biosafety of the prepared implant.

[0089] In some embodiments of this application, the specific applications and implementation paths of the above-mentioned collagen-based porous microsphere implants in different biomedical fields are disclosed in detail. Specifically, the applications of collagen-based porous microsphere implants in the preparation of tissue engineering materials, medical devices, or cosmetic injection products are described.

[0090] In the application of tissue engineering materials, the collagen-based porous microsphere implant can be used as a three-dimensional cell culture scaffold. During implementation, seed cells (such as fibroblasts and chondrocytes) can be co-cultured with the porous microspheres in vitro, using RGD peptides on the microsphere surface to guide cell adhesion and growth within the hierarchical pores. Due to the microspheres' porosity exceeding 90% and their interconnected micron-sized pores, efficient nutrient permeation within the scaffold is ensured, resulting in a highly viable biological scaffold for the regenerative repair of skin defects or cartilage damage.

[0091] In the application of medical devices, the implant can be aseptically packaged and prepared into implantable medical devices. Due to its robust supporting framework constructed from silk fibroin and its preferred energy storage modulus range of 4000 Pa to 7000 Pa, the microspheres can serve as a durable structural support material. For example, in the preparation of pressure-support implants, this material can resist tissue compression and maintain specific anatomical space. Furthermore, its dual cross-linking process ensures a controllable degradation cycle in vivo after implantation, achieving stable support for 3 to 18 months according to the needs of different medical scenarios.

[0092] In the application of cosmetic injectable products, during the preparation of these products, collagen-based porous microspheres can be mixed with pharmaceutically acceptable carriers (such as physiological saline, cross-linked sodium hyaluronate gel, etc.) to create an injectable suspension. In clinical applications, this product is injected into the deep dermis or subcutaneous tissue using fine needles such as 27G or 30G.

[0093] After implantation, the microspheres initially provide immediate physical filling to improve local depressions. Subsequently, RGD peptides grafted onto the hierarchical pore walls of the microspheres exert chemotactic activity, actively recruiting endogenous cells from surrounding tissues to migrate into the microspheres. As the ingrown cells secrete new autologous extracellular matrix in situ within the microspheres, combined with the controllable degradation cycle of the silk fibroin-collagen scaffold, this implant is expected to guide the gradual replacement of the degraded scaffold material by newly generated autologous tissue. This design approach, which replaces passive stimulation with active recruitment, provides a new way to reduce the safety risks caused by traditional foreign body reactions and has significant application value in achieving natural and safe in-situ isovolumetric tissue regeneration.

[0094] In clinical practice, the rigid-flexible cross-linked network framework constructed in this application has an actual complete absorption period of approximately 12 months at the implantation site. This long-lasting support characteristic allows the interconnected hierarchical porous structure to maintain physical patency during the dynamic process of microsphere degradation, providing a sufficiently ample time window for the continuous inward migration of endogenous cells, capillary ingrowth, and stable secretion of autologous collagen. Ultimately, this results in a dynamic balance between the amount of newly generated autologous tissue and the amount of microsphere degradation, achieving truly safe, long-lasting filling and in-situ volumetric regeneration.

[0095] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0096] Example 1 This embodiment provides a collagen-based porous microsphere for actively recruiting cell ingrowth, used to investigate the microsphere forming effect and basic characteristics under specific processes and formulations. The preparation process is as follows: S1, Preparation of collagen solution: Take bovine Achilles tendon type I collagen aqueous solution, add physical cross-linking agent mucopolysaccharide solution, stir at 2~30℃ for 2h, centrifuge at 9000rpm, and collect collagen-mucopolysaccharide precipitate. Emulsify the collagen-mucopolysaccharide precipitate using a disperser to obtain a collagen-mucopolysaccharide suspension with a mass fraction of 2%.

[0097] S2, Preparation of silk fibroin solution: Silkworm cocoons were added to sodium carbonate solution and boiled three times to remove the glue. The solution was dissolved using lithium bromide, dialyzed through a dialysis membrane, centrifuged at 10,000 rpm to remove impurities, and then concentrated in a 60℃ oven to obtain a 4% (w / w) silk fibroin solution.

[0098] S3, Preparation of the composite solution: The obtained collagen-mucopolysaccharide suspension and silk fibroin solution were mixed at a ratio of 14:3 and stirred at 400 rpm for 3 hours. Then, the glutaraldehyde crosslinking agent solution was added to make the content of glutaraldehyde in the system 0.005%, and stirring was continued for 3 hours.

[0099] S4, Oil phase preparation: Liquid paraffin was used as the oil phase, and 10% of Tween-80 and Span-80 were added in a ratio of 1:3. Then, glutaraldehyde crosslinking agent was added to make its content in the system 0.005%. After mixing evenly, the oil phase was obtained.

[0100] S5, Collagen-based microspheres were prepared by high-pressure airflow spray emulsification: The S3 composite solution was added to the oil phase by high-pressure airflow spray (pressure adjusted to 2 bar) and stirred continuously at 400 rpm for 2 h.

[0101] S6, Collection of collagen-based microspheres: Filter the solution obtained in S5 and collect the collagen-based microspheres.

[0102] S7, Washing of collagen-based microspheres: The obtained microspheres were washed three times with 95% ethanol, 30 min each time. Then they were washed three times with purified water, 30 min each time.

[0103] S8, RGD modification: The washed microspheres were immersed in pH 5.0 PBS buffer containing 40 mM EDC and 10 mM NHS, and stirred at 400 rpm for 10 min. Then, RGD peptide (concentration 0.15 mg / mL) was added, and stirring was continued for 10 h. Finally, pH 8.0 Tris-HCl buffer was added, and stirring was carried out at 400 rpm for 2 h. The collagen-based microspheres were collected by filtration and washed three times with purified water for 30 min each time.

[0104] In this process, an acidic buffer system at pH 5.0 is used to initiate an EDC / NHS-mediated covalent bonding reaction, enabling efficient amidation of RGD peptides with the microsphere backbone. After the reaction, a pH 8.0 Tris-HCl buffer is added to rapidly quench unreacted active intermediates, terminating the chemical reaction. Subsequent high-intensity rinsing and extraction with purified water completely removes any free RGD peptide molecules and reaction byproducts that are weakly physically adsorbed to the microsphere surface via non-covalent means such as hydrogen bonding or electrostatic attraction. Therefore, the RGD peptides retained and immobilized on the porous microspheres after this rigorous cleaning process are stably grafted onto the hierarchical pore walls and outer surface via covalent bonds, effectively ensuring the stability and persistence of the product's subsequent biological activity expression.

[0105] S9, freeze-drying: The cleaned microspheres were frozen at -80℃ for 1-2 hours, then at -20℃ for 1-2 hours, and then at -60℃ for 12 hours. The microspheres were then freeze-dried to obtain collagen-based microspheres.

[0106] S10, sieving: using a sieve to sieve out particles with a size of 50~250μm.

[0107] S11, Irradiation sterilization: The obtained collagen-based particles are dispensed into vials and subjected to high-energy electron beam irradiation cross-linking sterilization.

[0108] Example 2 This embodiment provides a collagen-based porous microsphere for actively recruiting cell ingrowth. Process parameters such as the concentration ratio of macromolecular raw materials, the concentration of cross-linking agent, and the spray pressure were adjusted to further optimize the preparation method. The preparation process is as follows: S1, Preparation of collagen solution: Take bovine Achilles tendon type I collagen aqueous solution, add physical cross-linking agent mucopolysaccharide solution, stir at 2~30℃ for 2h, centrifuge at 9000rpm, and collect collagen-mucopolysaccharide precipitate. Emulsify the collagen-mucopolysaccharide precipitate using a disperser to obtain a collagen-mucopolysaccharide suspension with a mass fraction of 3.5%.

[0109] S2, Preparation of silk fibroin solution: Silkworm cocoons were added to sodium carbonate solution and boiled three times to remove the glue. The solution was dissolved using lithium bromide, dialyzed through a dialysis membrane, centrifuged at 10,000 rpm to remove impurities, and then concentrated in a 60℃ oven to obtain a 6% (w / w) silk fibroin solution.

[0110] S3, Preparation of the composite solution: The obtained collagen-mucopolysaccharide suspension and silk fibroin solution were mixed at a ratio of 12:7 and stirred at 400 rpm for 2-4 hours. Then, the glutaraldehyde crosslinking agent solution was added to make the content of glutaraldehyde in the system 0.001%, and stirring was continued for 2 hours.

[0111] S4, Oil phase preparation: Liquid paraffin was used as the oil phase, and 10% of Tween-80 and Span-80 were added in a ratio of 1:3. Then, glutaraldehyde crosslinking agent was added to make its content in the system 0.1%. After mixing evenly, the oil phase was obtained.

[0112] S5, Collagen-based microspheres were prepared by high-pressure airflow spray emulsification: The S3 composite solution was added to the oil phase by high-pressure airflow spray (pressure adjusted to 4 bar) and stirred continuously at 400 rpm for 4 h.

[0113] S6, Collection of collagen-based microspheres: Filter the solution obtained in S5 and collect the collagen-based microspheres.

[0114] S7, Washing of collagen-based microspheres: The obtained microspheres were washed three times with 95% ethanol, 30 min each time. Then they were washed three times with purified water, 30 min each time.

[0115] S8, RGD modification: The washed microspheres were immersed in pH 5.0 PBS buffer containing 40 mM EDC and 10 mM NHS, and stirred at 400 rpm for 10 min. Then, RGD peptide (concentration 0.2 mg / mL) was added, and stirring was continued for 16 h. Finally, pH 8.0 Tris-HCl buffer was added, and stirring was carried out at 400 rpm for 2 h. The collagen-based microspheres were collected by filtration and washed three times with purified water for 30 min each time.

[0116] In this process, an acidic buffer system at pH 5.0 is used to initiate an EDC / NHS-mediated covalent bonding reaction, enabling efficient amidation of RGD peptides with the microsphere backbone. After the reaction, a pH 8.0 Tris-HCl buffer is added to rapidly quench unreacted active intermediates, terminating the chemical reaction. Subsequent high-intensity rinsing and extraction with purified water completely removes any free RGD peptide molecules and reaction byproducts that are weakly physically adsorbed to the microsphere surface via non-covalent means such as hydrogen bonding or electrostatic attraction. Therefore, the RGD peptides retained and immobilized on the porous microspheres after this rigorous cleaning process are stably grafted onto the hierarchical pore walls and outer surface via covalent bonds, effectively ensuring the stability and persistence of the product's subsequent biological activity expression.

[0117] S9, freeze-drying: The cleaned microspheres were frozen at -80℃ for 1-2 hours, then at -20℃ for 1-2 hours, and then at -60℃ for 12 hours. The microspheres were then freeze-dried to obtain collagen-based microspheres.

[0118] S10, sieving: using a sieve to sieve out particles with a size of 50~100μm.

[0119] S11, Irradiation sterilization: The obtained collagen-based particles are dispensed into vials and subjected to high-energy electron beam irradiation cross-linking sterilization.

[0120] Comparative Example 1 This comparative example provides collagen-based porous microspheres without surface biochemical modification to investigate the effect of RGD peptide surface modification steps on the cell affinity of the microspheres.

[0121] The preparation process includes steps S1 to S7, and each step and its parameters are the same as in Example 2. After completing the cleaning step S7, the RGD modification step S8 is not performed; instead, subsequent operations are carried out directly. The specific subsequent operations are as follows: S8, Freeze-drying: The cleaned microspheres were frozen at -80℃ for 1-2 hours, then at -20℃ for 1-2 hours, and then at -60℃ for 12 hours. The microspheres were then freeze-dried to obtain collagen-based microspheres.

[0122] S9, Sieving: Use a sieve to sieve out particles with a size of 50~100μm.

[0123] S10, Irradiation sterilization: The obtained collagen-based particles are dispensed into vials and subjected to high-energy electron beam irradiation cross-linking sterilization.

[0124] Comparative Example 2 This comparative example provides a pure collagen microsphere (without a silk fibroin backbone) to investigate the contribution of the silk fibroin component to the mechanical properties and anti-degradation properties of the microsphere.

[0125] Its preparation process is as follows: S1, Preparation of collagen solution: Take bovine Achilles tendon type I collagen aqueous solution, add physical cross-linking agent mucopolysaccharide solution, stir at 2~30℃ for 2h, centrifuge at 9000rpm, and collect collagen-mucopolysaccharide precipitate. Emulsify the collagen-mucopolysaccharide precipitate using a disperser to obtain a collagen-mucopolysaccharide suspension with a mass fraction of 3.5%.

[0126] S2, Preparation of the composite solution: Glutaraldehyde cross-linking agent solution was added to the obtained collagen-mucopolysaccharide suspension to make the content of glutaraldehyde in the system 0.001%, and stirring was continued for 2 hours. (Note: Silk fibroin solution was not added in this step.) S3, Oil phase preparation: Liquid paraffin was used as the oil phase, and 10% of Tween-80 and Span-80 were added in a ratio of 1:3. Then, glutaraldehyde crosslinking agent was added to make its content in the system 0.1%. After mixing evenly, the oil phase was obtained.

[0127] S4, Collagen microspheres were prepared by high-pressure airflow spray emulsification: The S3 composite solution was added to the oil phase by high-pressure airflow spray (pressure adjusted to 4 bar) and stirred continuously at 400 rpm for 4 h.

[0128] S5, Collagen Microsphere Collection: Filter the solution obtained in S5 and collect the collagen microspheres.

[0129] S6, Collagen Microsphere Washing: Wash the obtained microspheres three times with 95% ethanol, 30 min each time. Then wash three times with purified water, 30 min each time.

[0130] S7, RGD Modification: The washed microspheres were immersed in pH 5.0 PBS buffer containing 40 mM EDC and 10 mM NHS, and stirred at 400 rpm for 10 min. Then, RGD peptide (concentration 0.2 mg / mL) was added, and stirring was continued for 16 h. Finally, pH 8.0 Tris-HCl buffer was added, and stirring was carried out at 400 rpm for 2 h. The collagen microspheres were collected by filtration and washed three times with purified water for 30 min each time.

[0131] S8, Freeze-drying: The cleaned microspheres were frozen at -80℃ for 1-2 hours, then at -20℃ for 1-2 hours, and then at -60℃ for 12 hours. The microspheres were then freeze-dried to obtain collagen-based microspheres.

[0132] S9, Sieving: Use a sieve to sieve out particles with a size of 50~100μm.

[0133] S10, Irradiation sterilization: The obtained collagen particles are dispensed into vials and subjected to high-energy electron beam irradiation cross-linking sterilization.

[0134] Experiment Example 1: Investigation of Microsphere Morphology and Porosity This experiment comprehensively examines the macroscopic particle size, interconnected porous structure, and microscopic surface morphology of the prepared composite porous microspheres to verify whether they possess multi-level physical channel space suitable for cell ingrowth and a high specific surface area for biomolecular modification.

[0135] 1. Experimental methods and results: The microspheres obtained in Example 1 were subjected to morphological observation and porosity testing.

[0136] First, refer to Figure 1 (SEM image of the microspheres) Under low magnification electron microscopy, the composite microspheres exhibit a regular three-dimensional morphology with particle sizes ranging from 50 to 250 μm. The surface and interior of the microspheres have a rich interconnected porous structure with micron-sized pores of approximately 20 to 30 μm in diameter. The overall porosity of the microspheres can reach 90%.

[0137] Further, refer to Figure 2 (10μm scale bar) and Figure 3 (1μm scale bar) is a magnified SEM image of the pore wall. The supporting pore wall of the above micron-sized pores is observed at a high magnification. It can be seen that the surface of the pore wall is not smooth, but exhibits extremely dense nanoscale wrinkles and micro-porous morphology (i.e., mesoporous structure).

[0138] 2. Experimental conclusions: The composite porous microspheres prepared in this application successfully constructed an excellent hierarchical porous structure system. Macroscopically (e.g., Figure 1 The high porosity and micron-sized interconnected micron-sized pores (as shown in the image) provide unobstructed physical channels for the ingrowth, migration, and three-dimensional proliferation of tissue cells; simultaneously, the dense nanoscale folds and mesoporous morphology (as shown in the magnified image) on the walls of the micron-sized pores... Figure 2 and Figure 3 As shown in the figure, this endows the material with an extremely high specific surface area. This multi-level microtexture not only constitutes a nutrient exchange network required for cell metabolism, but also provides an extremely rich number of active sites for the subsequent high-density chemical grafting of bioactive molecules (RGD peptides). The two work synergistically in structure and function to jointly constitute an ideal microenvironment for in-situ tissue regeneration.

[0139] Experiment Example 2: Cell Proliferation and Migration Detection Experiment This experiment investigated the promoting effect of RGD peptide modification on cell adhesion, proliferation and migration activities on the surface of microspheres through in vitro cell co-culture.

[0140] 1. Experimental methods and results: Cell proliferation was assessed using microspheres obtained from Example 2 (with RGD modification) and Comparative Example 1 (without RGD modification). L929 cells were seeded into 96-well plates at a seeding density of 1 × 10⁻⁶. 4 Incubate at 37℃ in a 5% CO2 incubator for 24 hours. Take 30 mg of each of the corresponding microspheres, add 3 mL of high-glucose DMEM, soak at 37℃ for 72 hours, then dilute 4-fold and add to 96-well plates. Co-culture with cells at 37℃ for 1, 3, and 5 days. At each time point, add 10 μL of CCK-8 solution and continue culturing at 37℃ for 2 hours. Samples are taken for cell proliferation rate detection.

[0141] Furthermore, the microspheres obtained in Example 2 and Comparative Example 1 were used for Transwell cell migration assays. Specifically, the microspheres were prepared into a suspension with a concentration of 0.025 mg / mL using complete culture medium containing 10% FBS and 1% penicillin-dextrose antibody. 200 μL of a 2.5 × 10⁻⁶ mg / mL solution was added to the upper chamber of the Transwell chamber. 5 L929 cell suspension at cell / mL was added to the lower chamber with 600 μL of complete culture medium containing microspheres and incubated at 37°C with 5% CO2 for 24 h. The chamber was then removed, the culture medium discarded, and both chambers were washed twice with PBS. The chamber was transferred to an empty well, and 600 μL of 4% paraformaldehyde was added to the lower chamber (to immerse the membrane) for fixation at room temperature for 15–20 min. The fixative was discarded, and the cells were washed with PBS and air-dried. Then, 600 μL of 0.1% crystal violet staining solution was added to the lower chamber, and staining was performed in the dark for 20–30 min. The cells were washed several times with PBS until the eluent was colorless and then air-dried again. Finally, the unmigrated cells on the upper surface of the Transwell chamber membrane were wiped clean with a cotton swab (only the migrated cells on the lower surface were preserved), and the chamber was observed and photographed under a light microscope (results are shown in Figure 1). Figure 5 , Figure 6 (As shown).

[0142] 2. Experimental conclusions: refer to Figure 4 (Quantitative data: results of cell proliferation experiments), and Figure 5 (Comparative Example 1) Figure 6 (Example 2). Wherein, Figure 5 (Comparative Example 1 - Without RGD) Due to the lack of RGD peptide's biochemical chemotaxis, fewer purple cells migrated to the lower layers, and their distribution was sparse. Meanwhile... Figure 6 (Example 2 - Containing RGD) Because the surface of the microspheres and the pore walls are grafted with a high density of RGD peptides, a strong "active recruitment" signal is generated, attracting a large number of cells to migrate to the lower layer. Therefore, the purple stained areas in the photo are significantly denser and more numerous.

[0143] The test results showed that RGD-modified microspheres were more conducive to cell adhesion and proliferation. Through the above... Figure 5 , Figure 6 The comparison directly proves that after RGD modification and hierarchical pore structure work together, it does have the ability to "actively recruit" cells, and is not just due to the random growth of physical pores.

[0144] The microspheres of this invention grafted with RGD peptides showed significantly higher cell migration rates than the comparative example. This significant chemotactic effect indirectly confirms that the numerous mesopores (and nanoscale wrinkled morphology) present on the walls of the micron-sized pores play a crucial structural support role. It is precisely because this specific microstructure provides an extremely high specific surface area that the RGD peptides can be grafted efficiently at a high density, thereby releasing a powerful 'active recruitment' signal sufficient to overcome physical spatial barriers.

[0145] Experiment Example 3: Microsphere Mechanical Property Testing Experiment This experiment uses rheological testing to investigate the effect of introducing silk fibroin into the ternary composite system on improving the macroscopic mechanical support performance of the microsphere skeleton.

[0146] 1. Experimental methods and results: Microspheres prepared in Example 2 (collagen-silk fibroin composite microspheres) and Comparative Example 2 (pure collagen microspheres) were used to test their mechanical properties using a rheometer. The tests were conducted at an angular frequency of 1 Hz. The rheological parameter test results are as follows: Comparative Example 2 (pure collagen): Elastic modulus (storage modulus) G' is 4728.3 Pa, loss modulus G'' is 800.62 Pa, and loss factor is 0.169.

[0147] Example 2 (composite microspheres): The elastic modulus (storage modulus) G' was increased to 6660.3 Pa, the loss modulus G'' was 1089.1 Pa, and the loss factor was 0.164.

[0148] 2. Experimental conclusions: Table 1: Mechanical properties of microspheres

[0149] Referring to Table 1, the rheological data fully demonstrate that the silk fibroin introduced in this application can effectively enhance the mechanical properties of microspheres, such as elastic modulus, making the porous microsphere skeleton more conducive to providing solid support and shaping within tissues.

[0150] In the observation of Comparative Example 2 (pure collagen microspheres), it was found that under the same gradient freezing process, the pore structure distribution inside the microspheres of the pure collagen group was extremely uneven, the number of pores was significantly less than that of the Example group, and most of them were closed pores, which did not have good permeability.

[0151] Experiment Example 4: Determination of the In Vitro Degradation Performance of Microspheres This experiment investigated the control effect of composite framework materials on the degradation rate and morphological stability of microspheres through in vitro enzymatic hydrolysis, and verified its potential to achieve long-term support for "isochoric regeneration".

[0152] 1. Experimental methods and results: The degradation performance of microspheres prepared in Example 2 (collagen-silk fibroin composite microspheres) and Comparative Example 2 (pure collagen microspheres) was determined by weighing. The concentration of collagenase degradation solution was 500 U / mL. Residual samples were collected at 1h, 6h, 9h, 12h, and 24h, respectively. After washing and freeze-drying, the residual weight was measured, and the degradation rate was calculated.

[0153] 2. Experimental conclusions: Table 2: Appearance changes of collagen microspheres during degradation.

[0154] Refer to Table 2 and Figure 7 The results of macroscopic appearance changes showed that, after 12 hours of degradation, the pure collagen microspheres (Comparative Example 2) had been mostly degraded, while the composite microspheres (Example 2) still had a large number of fragments remaining.

[0155] Degradation rate data also demonstrate that the composite microspheres with added silk fibroin have significantly better in vitro enzymatic hydrolysis performance, which can greatly extend the support time to match the tissue regeneration cycle.

[0156] Furthermore, based on the aforementioned in vitro accelerated degradation experimental data and relevant literature model calculations, the actual biodegradation period (complete absorption time) of the collagen-silk fibroin composite porous microspheres prepared in this invention after implantation under the human skin is estimated to be approximately 12 months. This degradation period is highly consistent with the physiological cycle of human soft tissue regeneration and repair, ensuring that the microspheres can provide long-term and stable mechanical support during the process of guiding autologous tissue ingrowth as a scaffold template, thereby truly achieving in-situ isochoric regeneration of tissue.

[0157] Furthermore, based on the aforementioned in vitro accelerated degradation experimental data and relevant literature model calculations, the actual biodegradation period (complete absorption time) of the collagen-silk fibroin composite porous microspheres prepared in this invention after implantation under the human skin is estimated to be approximately 12 months. This degradation period is highly consistent with the physiological cycle of human soft tissue regeneration and repair, ensuring that the microspheres can provide long-term and stable mechanical support during the process of guiding autologous tissue ingrowth as a scaffold template, thereby truly achieving in-situ isochoric regeneration of tissue.

[0158] Experimental Example 5: Microsphere Pore Size Distribution and Chemical Structure Characterization 1. Experimental methods and results: The porous microspheres prepared in Example 1 were analyzed for their micropore structure using a nitrogen adsorption-desorption method. Figure 8 The BJH desorption pore size distribution curve shows that the pore size distribution of the microspheres exhibits a highly concentrated multi-peak pattern in the range of 2nm to 50nm. Specifically, extremely significant pore volume characteristic peaks appear near 2nm to 3nm, 4nm to 5nm, and 20nm, indicating that micropores in this size range are densely distributed inside the material.

[0159] 2. Experimental conclusions: The quantitative pore size distribution detection data mentioned above are corroborated by the nanoscale micro-wrinkle morphology observed under high-magnification SEM. Figure 8 The experimental results provide direct and objective quantitative evidence from the perspective of material physics and structure, confirming that the collagen-based porous microspheres described in this application do not merely have surface roughness, but actually possess a large number of typical mesoporous (2nm~50nm) structures. Since the matrix framework (collagen and silk fibroin) of the porous microspheres in this application are themselves large molecular proteins rich in a large number of high-density peptide bonds (amide bonds), the characteristic absorption peaks of the amide I and amide II bands in their infrared spectra are extremely strong and highly superimposed. However, the amide bond signals of the trace amounts of RGD tripeptides grafted on the surface are often masked by the background signal of the bulk macromolecules in the infrared spectrum, making it difficult to distinguish them explicitly using conventional Fourier transform infrared spectroscopy (FTIR).

[0160] Therefore, this application confirms the hierarchical pore structure of the RGD peptide through the aforementioned BJH desorption pore size distribution curve, and further confirms, in conjunction with the directed migration and ingrowth behavior of cells in Experimental Example 2, that the RGD peptide achieves efficient loading and biological function expression on the mesoporous pore walls and outer surface. This interconnected hierarchical physical network not only provides the RGD peptide with an extremely rich internal surface area and chemically active sites, but also lays a decisive microstructural foundation for the subsequent capillary penetration of nutrients and the active deep ingrowth of cells. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A collagen-based porous microsphere implant, characterized by, The collagen-based porous microsphere implant includes collagen-based porous microspheres; The raw material components of the collagen-based porous microspheres include: collagen, silk fibroin, and mucopolysaccharides; wherein, the silk fibroin is cross-linked with the collagen to form a supporting framework; The collagen-based porous microspheres have a through-hole hierarchical pore structure; the hierarchical pore structure includes micron-sized pores and mesopores distributed on the pore walls of the micron-sized pores; RGD peptides are grafted onto at least a portion of the outer surface of the collagen-based porous microspheres and at least a portion of the pore walls of the hierarchical pore structure.

2. The collagen-based porous microsphere implant as described in claim 1, characterized in that, The pore size of the micron-sized pores is 10 μm to 50 μm; and / or, The pore size of the mesopore is 2nm~50nm; and / or, The collagen-based porous microspheres have a particle size of 50 μm to 250 μm; and / or, The porosity of the collagen-based porous microspheres is greater than 90%; and / or, The collagen includes bovine type I collagen; and / or, The RGD peptide comprises arginyl-glycyl-aspartic acid; and / or, The mucopolysaccharide includes at least one of hyaluronic acid, chondroitin sulfate, heparin, and their salts; and / or, The silk fibroin is degummed silk fibroin, and its degumming rate, determined by the weight loss method before and after degumming, is between 20% and 30%.

3. The collagen-based porous microsphere implant as described in claim 1, characterized in that, The mass ratio of collagen to silk fibroin is (2~10):(1~7).

4. The collagen-based porous microsphere implant as described in claim 1, characterized in that, The collagen-based porous microspheres are subjected to chemical cross-linking and irradiation cross-linking treatments. Preferably, the crosslinking agent used in the chemical crosslinking includes one of glutaraldehyde, genipin, and EDC / NHS; Preferably, the irradiation crosslinking is performed using high-energy electron beam irradiation crosslinking; more preferably, the irradiation crosslinking dose is 5 kGy to 25 kGy.

5. The collagen-based porous microsphere implant as described in claim 1, characterized in that, At a frequency of 1 Hz, the storage modulus of the collagen-based porous microsphere implant is 4000 Pa to 7000 Pa; and / or, the loss modulus is 700 Pa to 1500 Pa.

6. A method of preparing the collagen-based porous microsphere implant of any one of claims 1 to 5, wherein, include: A suspension made of collagen and mucopolysaccharide is mixed with a solution made of silk fibroin, and a cross-linking agent solution is added to react and obtain a composite solution. The composite solution is injected into the oil phase via a high-pressure gas jet spray method to simultaneously form microspheres and undergo emulsification and solidification reactions; wherein, the high-pressure gas jet spray method is used to maintain the initial spherical shape of the droplets, and the oil phase is used to simultaneously emulsify and solidify the droplets to obtain microspheres with regular morphology. The composite solution was added to the oil phase via a high-pressure gas spray method for emulsification, and then collected and washed to obtain porous microspheres. The cleaned porous microspheres were immersed in an acidic crosslinking agent solution, and the RGD peptide was added to carry out a grafting reaction. After cleaning, the modified microspheres were obtained. The modified microspheres were subjected to gradient temperature-controlled freezing and freeze-drying, followed by high-energy electron beam irradiation for cross-linking and sterilization to obtain the collagen-based porous microsphere implant.

7. The method for preparing the collagen-based porous microsphere implant as described in claim 6, characterized in that, The gradient temperature-controlled freezing procedure includes, in sequence, a first freezing stage, a warm-up growth stage, and a second freezing stage; Wherein, the temperature of the first freezing stage is lower than the temperature of the second freezing stage; the temperature of the second freezing stage is lower than the temperature of the warm-up growth stage, and the temperature of the warm-up growth stage is lower than 0°C; Preferably, the temperature of the first freezing stage is -90℃ to -70℃; and / or, the temperature of the warm-up growth stage is -30℃ to -10℃; and / or, the temperature of the second freezing stage is -70℃ to -50℃; more preferably, the temperature of the first freezing stage is -80℃; and / or, the temperature of the warm-up growth stage is -20℃; and the temperature of the second freezing stage is -60℃. Preferably, the processing time of the first freezing stage is 1h to 2h; and / or, the processing time of the warm-up growth stage is 1h to 2h; and / or, the processing time of the second freezing stage is not less than 12 hours.

8. The method for preparing the collagen-based porous microsphere implant as described in claim 6, characterized in that, The volume ratio of the suspension made from the collagen and the mucopolysaccharide to the solution made from the silk fibroin is (10~40):(1~10); and / or, The spray pressure of the high-pressure airflow spray method is 0.1 bar to 10 bar; preferably 2 bar to 6 bar; and / or, The oil phase comprises liquid paraffin; and / or, The emulsification reaction also includes an emulsifier, which comprises sorbitan oleate and polysorbate 80; and / or, The cleaning agents used include ethanol and deionized water.

9. The method for preparing the collagen-based porous microsphere implant as described in claim 6, characterized in that, The acidic crosslinking agent solution is prepared by mixing PBS buffer at pH 5.0 with 20 mM to 60 mM EDC and 5 mM to 30 mM NHS.

10. The use of the collagen-based porous microsphere implant according to any one of claims 1-5 in the preparation of tissue engineering materials, medical devices or cosmetic injection products.

Images