Recombinant collagen and bioglass composite and method of making

Abstract

The application discloses a recombinant collagen and bio-glass composite material and a preparation method, relates to the technical field of artificial tissue materials, and comprises recombinant humanized collagen and bioactive glass powder, wherein the particle size of the bioactive glass powder is 50-500 nm; the recombinant humanized collagen is crosslinked to form a three-dimensional organic network structure, the bioactive glass powder is uniformly dispersed in the three-dimensional organic network structure, and a stable organic composite skeleton is formed. The application has good biocompatibility, bioactivity, mechanical properties and tissue induction capacity, and can be widely applied to the fields of bone tissue defect repair, diabetic foot wound repair and tissue engineering regeneration.

Description

Technical Field

[0001] This invention relates to the field of artificial tissue materials technology, and in particular to a recombinant collagen and bioglass composite material and its preparation method. Background Technology

[0002] With the development of tissue engineering and regenerative medicine, biomedical materials that combine good biocompatibility, bioactivity, and biodegradability have become an important research direction in fields such as bone tissue repair and chronic wound repair. Especially in applications such as bone defect repair, diabetic foot wound repair, and synergistic regeneration of soft and hard tissues, materials not only need to have good mechanical support capabilities, but also need to provide a suitable microenvironment for cell adhesion, growth, and tissue regeneration, thereby promoting rapid repair and functional reconstruction of damaged tissues.

[0003] Bone defects are a common clinical problem in orthopedics, dental implantology, and trauma repair. Currently, clinical treatment primarily utilizes autologous bone grafting, allogeneic bone grafting, and artificial bone substitutes. While autologous bone grafting exhibits good osteogenic properties, it faces challenges such as limited donor availability, donor site damage, and the need for secondary surgery. Allogeneic bone grafting carries risks of immune rejection and disease transmission. Artificial bone substitutes have garnered significant attention due to their stable availability and ease of preparation; however, existing materials generally suffer from insufficient bioactivity, suboptimal mechanical properties, and degradation rates that cannot keep pace with new bone formation, making them unsuitable for the repair of complex bone tissue.

[0004] Bioactive glass, as an inorganic biomaterial with excellent osteoinductive properties, can release bioactive ions such as calcium, silicon, and phosphorus in the body fluid environment and form a hydroxyapatite layer on the material surface, thereby promoting osteoblast adhesion, proliferation, and differentiation, and improving bone tissue regeneration capacity. However, bioactive glass itself has defects such as high brittleness, insufficient toughness, and poor processing and forming properties. It is prone to breakage during the repair of complex-shaped tissue defects, which limits its further application.

[0005] On the other hand, diabetic foot, a common and serious chronic complication of diabetes, suffers from persistent microcirculatory disturbances, nerve damage, and inflammation due to long-term hyperglycemia, making the wound difficult to heal and even leading to amputation in severe cases. Currently, clinical treatment mainly employs a comprehensive approach including debridement, anti-infection therapy, negative pressure wound therapy, and wound dressings. However, existing repair materials typically only provide a covering and protective function, failing to simultaneously promote cell migration, induce angiogenesis, improve the wound microenvironment, and facilitate tissue regeneration. Therefore, the treatment effectiveness still needs improvement.

[0006] Collagen is widely used in tissue engineering and regenerative medicine due to its excellent biocompatibility, biodegradability, and cell adhesion properties. Traditional natural collagen is mainly derived from animal tissues, which presents challenges such as unstable sources, significant batch-to-batch variability, and potential immunogenicity and pathogen transmission risks. In recent years, recombinant humanized collagen has gradually become an important development direction for tissue repair materials due to its advantages such as designable structure, high purity, good safety, low immunogenicity, and stable production. However, when used alone, recombinant collagen has relatively insufficient mechanical properties and structural stability, making it difficult to meet the needs of bone tissue repair and complex tissue engineering applications.

[0007] Currently, some technologies have attempted to combine collagen with bioactive glass to leverage the performance advantages of both. However, these technologies generally suffer from problems such as insufficient interfacial bonding strength between collagen and bioactive glass, uneven dispersion of bioactive glass particles, poor stability of the composite structure, difficulty in controlling the pore structure, and challenges in synergistically optimizing the material's mechanical properties and bioactivity. Furthermore, most existing composite materials are designed for single bone repair applications, lacking applicability to fields such as chronic wound repair of diabetic foot and tissue engineering regeneration, thus failing to meet the application needs of diverse tissue repair scenarios.

[0008] Therefore, there is an urgent need to provide a recombinant collagen and bioglass composite material and its preparation method that has excellent biocompatibility, bioactivity, mechanical properties and controllable degradation properties, so as to realize multiple applications such as bone tissue defect repair, diabetic foot wound repair and tissue engineering regeneration, and provide safer and more efficient biomedical materials for clinical tissue repair. Summary of the Invention

[0009] In order to at least solve one of the above-mentioned technical problems, the present invention aims to provide a recombinant collagen and bioglass composite material and its preparation method, which has good biocompatibility, bioactivity, mechanical properties and tissue induction ability, and can be widely used in the fields of bone tissue defect repair, diabetic foot wound repair and tissue engineering regeneration.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A recombinant collagen and bioglass composite material includes recombinant humanized collagen, bioactive glass powder, and a crosslinking agent, wherein the particle size of the bioactive glass powder is 50-500 nm; the recombinant humanized collagen is crosslinked to form a three-dimensional organic network structure, and the bioactive glass powder is uniformly dispersed in the three-dimensional organic network structure to form a stable organic composite framework.

[0012] Preferably, the recombinant humanized collagen has a mass fraction of 1% to 20%.

[0013] Preferably, the bioactive glass powder has a mass fraction of 10% to 80%.

[0014] Preferably, the crosslinking agent is one or more of the EDC / NHS crosslinking system, genipin, or other biocompatible crosslinking agents.

[0015] Preferably, the recombinant collagen and bioglass composite material has a three-dimensional interconnected pore structure.

[0016] A method for preparing a composite material of recombinant collagen and bioglass includes the following steps:

[0017] S1. Add recombinant humanized collagen to phosphate buffer and stir to dissolve at low temperature to obtain a homogeneous collagen solution.

[0018] S2. Add the bioactive glass powder to the collagen solution, and disperse the bioactive glass powder evenly in the collagen solution by ultrasonic dispersion and mechanical stirring.

[0019] S3. Add a crosslinking agent to the mixture and carry out a crosslinking reaction under preset temperature and preset time conditions to form a stable three-dimensional organic network structure of recombinant humanized collagen and fix the bioactive glass powder in the three-dimensional organic network structure.

[0020] S4. Place the cross-linked composite system in a mold and shape it using freeze molding, gel molding or 3D printing.

[0021] S5. The molded material is pre-frozen and then vacuum freeze-dryed to form a composite material with a three-dimensional interconnected pore structure.

[0022] S6. The obtained composite material is sterilized by any one of ethylene oxide sterilization, gamma-ray sterilization or electron beam sterilization to obtain sterile recombinant collagen and bioglass composite material.

[0023] Preferably, in step S3, one or more of the following are used as crosslinking agents: EDC / NHS crosslinking system and genipin.

[0024] Preferably, in step S4, the composite system is prepared into a scaffold, gel, or dressing by any one of cryogenic molding, gel molding, or 3D printing.

[0025] Preferably, in step S6, the sterilization method is one or more of ethylene oxide sterilization, gamma ray sterilization, or electron beam sterilization.

[0026] The present invention has the following beneficial effects:

[0027] I. Improving the Comprehensive Mechanical Properties of Composite Materials: This invention uses recombinant humanized collagen as the organic matrix, forming a stable three-dimensional organic network structure through cross-linking. Nanoscale bioactive glass powder is uniformly dispersed within the network, constructing a stable organic-inorganic composite framework. Compared to using collagen or bioactive glass alone, this invention fully leverages the synergistic effect of the two materials, effectively improving the brittleness and insufficient toughness of bioactive glass, while simultaneously enhancing the structural stability and load-bearing capacity of the collagen system. This results in a composite material with superior mechanical properties, meeting the support requirements of bone tissue repair and tissue engineering scaffolds.

[0028] II. Enhancing the bioactivity and tissue repair capabilities of the material; This invention utilizes bioactive glass to continuously release bioactive ions such as calcium and silicon ions, which can induce the formation of a hydroxyapatite layer on the material surface, promoting osteoblast adhesion, proliferation, differentiation, and mineralization, thereby enhancing bone tissue regeneration. Simultaneously, recombinant humanized collagen possesses excellent cell affinity and cell adhesion capabilities, promoting cell migration, growth, and tissue reconstruction. The synergistic effect of both significantly improves the tissue-inducing properties of the material, making the composite material suitable for various applications such as bone tissue repair, diabetic foot wound repair, and tissue engineering regeneration.

[0029] Third, improve the structural stability of composite materials; This invention uses a biocompatible crosslinking agent to crosslink recombinant humanized collagen, so that the collagen forms a stable three-dimensional network structure and firmly fixes the bioactive glass particles inside the network, enhancing the interfacial bonding ability between collagen and bioactive glass, avoiding the shedding or local aggregation of inorganic particles, improving the overall structural stability of composite materials, thereby ensuring the long-term performance and tissue repair effect of the material after implantation.

[0030] IV. Improving the dispersion uniformity of bioactive glass: In the preparation process, this invention adopts a combination of ultrasonic dispersion and mechanical stirring to fully disperse the bioactive glass powder in the collagen solution and maintain a uniform distribution during the subsequent cross-linking process. This effectively avoids the problems of easy agglomeration of inorganic particles and excessively high local concentration in traditional composite materials, making the overall composition of the material more uniform and conducive to the continuous release of bioactive ions and the stable performance of tissue repair effects.

[0031] Fifth, it is conducive to cell ingrowth and tissue regeneration; the present invention constructs a composite material with a three-dimensional interconnected pore structure by combining pre-freezing treatment with vacuum freeze-drying process. This structure is not only conducive to cell adhesion, migration, proliferation and blood vessel ingrowth, but also promotes the exchange of oxygen, nutrients and metabolites, providing a good growth space for new tissue, thereby improving the efficiency of bone tissue repair and chronic wound healing.

[0032] VI. Improved Material Safety and Biocompatibility: This invention uses recombinant humanized collagen to replace traditional animal-derived collagen, effectively reducing immunogenicity and pathogen transmission risks, and improving material source stability and batch consistency. Simultaneously, the use of a cross-linking system with good biocompatibility and standardized sterilization processes ensures the composite material has good biocompatibility, meeting the application requirements of implantable materials and wound repair materials.

[0033] VII. Improving the matching between material degradation and tissue regeneration: This invention, through the synergistic regulation of the degree of cross-linking, the content of bioactive glass, and the three-dimensional network structure, enables the composite material to have good degradability. After implantation, it can gradually degrade with the formation of new tissue, so as to coordinate the material degradation rate with the bone tissue formation and wound repair process, avoid long-term material residue or excessively rapid degradation causing tissue repair failure, and improve the quality of tissue regeneration.

[0034] 8. Improve the adaptability of the preparation process and its industrial application value; This invention adopts processes such as low-temperature dissolution, ultrasonic dispersion, cross-linking reaction, freeze drying and sterilization. Each process step is simple, stable and easy to control. Different product forms such as scaffolds, gels or dressings can be prepared by cryo-molding, gel molding or three-dimensional printing according to different clinical needs. It is suitable for bone tissue defect repair, diabetic foot wound repair and other tissue engineering regeneration fields, and has good industrialization and promotion value. Attached Figure Description

[0035] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts. Wherein:

[0036] Figure 1 This is a process flow diagram of an embodiment of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the implementation cases of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This invention addresses the problems of high brittleness and insufficient flexibility in existing bioactive glass materials, as well as the problems of insufficient mechanical strength and poor structural stability in recombinant collagen materials. It proposes an organic-inorganic composite material based on recombinant humanized collagen and bioactive glass.

[0039] The basic principle of this invention is as follows: using recombinant humanized collagen as an organic matrix, a cross-linking agent is used to promote the formation of stable connections between collagen molecular chains, thereby forming a continuous three-dimensional network structure; at the same time, bioactive glass powder is uniformly embedded in the above-mentioned collagen network, so that inorganic particles are coated and fixed by the collagen network, avoiding the agglomeration of traditional bioactive glass particles and improving the overall stability of the material.

[0040] Among them, recombinant humanized collagen can provide a microenvironment similar to the natural extracellular matrix. Its molecular structure contains a large number of hydrophilic groups, which are beneficial to cell adhesion, growth, and tissue formation. After cross-linking treatment, the stability of the collagen network structure is enhanced, which can reduce the rapid swelling and degradation of the material in the body fluid environment.

[0041] As an inorganic active component, bioactive glass powder gradually releases active ions such as calcium and silicon after implantation or contact with the wound environment. Calcium ions participate in bone mineralization and promote hydroxyapatite deposition, while silicon ions promote osteoblast-related cell activity and improve the local tissue regeneration microenvironment. Therefore, incorporating bioactive glass into the collagen network structure can enhance the material's osteoinductive and tissue repair capabilities while maintaining collagen biocompatibility.

[0042] The recombinant humanized collagen used in this invention is preferably a recombinant collagen with high purity and low immunogenicity. During preparation, the recombinant humanized collagen is added to a buffer solution to fully hydrate it and form a homogeneous solution.

[0043] Specifically, recombinant humanized collagen powder is slowly added to phosphate buffer and stirred at a low temperature of 2°C to 8°C. The stirring speed is controlled at 100 r / min to 500 r / min and stirred continuously for 1 h to 6 h to fully dissolve the collagen.

[0044] The reason for using a low-temperature dissolution method is that collagen is a natural high-molecular-weight protein material, and high-temperature environments can easily cause changes in its protein structure, reducing its biological activity. Low-temperature conditions can maintain the integrity of the collagen molecular structure, allowing it to form a stable network structure during subsequent cross-linking.

[0045] The bioactive glass powder used in this invention is preferably a nanoscale bioactive glass material with a particle size range of 50 nm to 500 nm.

[0046] Before adding the collagen solution, the bioactive glass powder can be pretreated. This pretreatment includes:

[0047] Bioactive glass powder is added to deionized water and then ultrasonically treated to fully disperse the particles for 20 to 60 minutes to reduce particle aggregation.

[0048] Subsequently, the dispersed bioactive glass suspension was slowly added to the collagen solution and further mixed using mechanical stirring to ensure that the bioactive glass particles were evenly distributed within the collagen system.

[0049] The above dispersion method allows the bioactive glass particles to be uniformly fixed within the collagen network during subsequent cross-linking, avoiding internal defects in the material caused by local particle aggregation.

[0050] The crosslinking agent used in this invention is preferably the EDC / NHS crosslinking system or genipin. EDC / NHS is a zero-length crosslinking system, which can promote the formation of stable amide bonds between collagen molecules and will not introduce significant harmful residues into the material. Genipin is a natural crosslinking agent with good biocompatibility, which can reduce the risk of cytotoxicity.

[0051] By controlling the amount of crosslinking agent added and the reaction time, the collagen network density can be adjusted, thereby achieving regulation of the material's mechanical properties, water absorption properties, and degradation properties.

[0052] After completing the above raw material preparation, composite materials of different forms can be further prepared by cryogenic molding, gel molding or 3D printing, depending on different application requirements. These include porous scaffolds, gel materials and wound dressing materials.

[0053] like Figure 1 As shown, the specific implementation is as follows:

[0054] Example 1

[0055] This embodiment provides a low-content recombinant collagen and bioglass composite material to verify that the composite system of the present invention can still form a stable structure and exert a biological repair function under conditions of low component content.

[0056] The specific preparation method is as follows:

[0057] First, recombinant humanized collagen was added to phosphate buffer and stirred at 4°C to fully dissolve the collagen, thus preparing a 1% recombinant humanized collagen solution.

[0058] The low-temperature dissolution process can reduce the thermal degradation of collagen molecular chains, allowing collagen to maintain a better natural spatial structure and providing a basis for subsequent cross-linking to form a stable network.

[0059] Subsequently, bioactive glass powder with a particle size of 50 nm was added to the collagen solution to achieve a bioactive glass powder mass fraction of 10%. During the addition process, the bioactive glass powder was first dispersed in deionized water and then ultrasonically treated for 30 minutes to fully wet the particles and reduce agglomeration.

[0060] The dispersed bioactive glass suspension was slowly added to the collagen solution and stirred continuously at 300 r / min for 2 hours to ensure that the bioactive glass particles were evenly distributed inside the collagen solution.

[0061] Subsequently, an EDC / NHS cross-linking system was added to the mixture, in which EDC activated the carboxyl active sites of collagen and promoted the formation of stable cross-linked structures between collagen molecules. The reaction temperature was controlled at 25℃ and the reaction time was 6 hours, allowing collagen to gradually form a continuous three-dimensional network structure while simultaneously fixing bioactive glass particles within the network.

[0062] After crosslinking, the resulting composite system is placed in a mold for freeze-forming. Specifically, the material is pre-frozen at -20°C for 12 hours, then transferred to a vacuum freeze-drying equipment and dried under vacuum conditions for 48 hours to sublimate the internal moisture of the material, forming a porous composite material with a three-dimensional interconnected pore structure.

[0063] Finally, the obtained material was sterilized with ethylene oxide to obtain the recombinant collagen and bioglass composite material described in Example 1.

[0064] In this embodiment, the composite material retains a high proportion of collagen characteristics due to its low content of bioactive glass, resulting in good flexibility and cell affinity. Simultaneously, the 50nm bioactive glass particles have a large specific surface area, which improves the ion release efficiency after the material comes into contact with bodily fluids.

[0065] The principle behind its technical effect lies in:

[0066] The three-dimensional network structure formed by recombinant humanized collagen provides cells with an attachment environment similar to the natural extracellular matrix, which is conducive to cell migration and proliferation. The nanoscale bioactive glass dispersed within it can release active ions such as calcium and silicon, improve the local microenvironment, and promote tissue repair. Therefore, even at a low proportion of bioactive glass, the material still possesses certain bioactivity and tissue-inducing capabilities.

[0067] This embodiment is applicable to soft tissue repair scenarios that require high flexibility, such as wound covering materials in the repair of diabetic foot wounds.

[0068] Example 2

[0069] This embodiment provides a composite material of medium proportion of recombinant collagen and bioglass to verify the comprehensive performance of material structure, properties and bioactivity under preferred component ratio conditions.

[0070] The specific preparation method is as follows:

[0071] Recombinant humanized collagen was added to phosphate buffer and stirred at 4°C to dissolve, thus preparing a collagen solution with a mass fraction of 10%.

[0072] Bioactive glass powder with a particle size of 250 nm was added to the above collagen solution to make the mass fraction of bioactive glass powder reach 45%.

[0073] Before addition, the bioactive glass powder was first added to deionized water and ultrasonically dispersed for 40 minutes to ensure full dispersion of the particles. Then, the dispersion was slowly added to the collagen solution and mixed using mechanical stirring at a speed of 400 rpm for 3 hours.

[0074] After thorough mixing, genipin is added as a cross-linking agent to induce a cross-linking reaction between collagen molecules. The cross-linking temperature is controlled at 30℃, and the reaction time is 12 hours, allowing the collagen to gradually form a stable three-dimensional network structure.

[0075] After cross-linking, the composite system was prepared into a tissue repair gel using a gel molding method. Subsequently, the obtained gel was pre-frozen and then a composite material with a three-dimensional porous structure was prepared by vacuum freeze-drying. Finally, it was sterilized by gamma rays.

[0076] In this embodiment, the content of recombinant humanized collagen and the content of bioactive glass are in a relatively optimal balance.

[0077] The collagen protein content is 10%, which ensures the formation of a continuous and stable organic support network; the bioactive glass content is 45%, which provides sufficient inorganic active components while avoiding increased material brittleness due to excessive content.

[0078] Bioactive glass particles with a diameter of 250 nm achieve a good balance between uniform dispersion and active ion release capability. On the one hand, their particle size will not cause serious agglomeration due to being too small; on the other hand, their large specific surface area ensures that the material has a continuous ion release capability.

[0079] The composite material obtained in this embodiment has good comprehensive performance in terms of mechanical properties, bioactivity and structural stability.

[0080] The principle behind its technical effect lies in:

[0081] The cross-linked collagen network structure can restrict the movement of bioactive glass particles, so that inorganic particles are uniformly fixed inside the material, thereby improving the overall stability of the material. At the same time, the calcium and silicon ions released by the bioactive glass can promote the activity of osteoblast-related cells and improve the bone tissue formation capacity.

[0082] Therefore, the material obtained in this embodiment has both the good cell compatibility of collagen and the excellent osteoinductive properties of bioactive glass, and can be used for bone tissue defect repair and tissue engineering scaffold preparation.

[0083] Example 3

[0084] This embodiment provides a composite material with high content of recombinant collagen and bioglass to verify the material's structural retention ability and bone repair performance under conditions of highly active components.

[0085] The specific preparation method is as follows:

[0086] Recombinant humanized collagen was added to a buffer solution and stirred at 4°C to dissolve, thus preparing a collagen solution with a mass fraction of 20%.

[0087] Bioactive glass powder with a particle size of 500 nm was added to the above collagen solution to make the mass fraction of bioactive glass powder reach 80%.

[0088] Before addition, the bioactive glass powder was ultrasonically dispersed for 60 minutes to ensure thorough dispersion of the particles. It was then added to the collagen system and mechanically stirred at 500 rpm for 4 hours to ensure uniform distribution of the bioactive glass.

[0089] Add the EDC / NHS cross-linking system to the mixture and react at 25°C for 8 hours to form a high-density cross-linked network of collagen.

[0090] Subsequently, the resulting composite system was shaped using 3D printing, and a bone tissue engineering scaffold with a regular pore structure was prepared according to the preset structure.

[0091] The shaped material is pre-frozen and then freeze-dried under vacuum to form a stable porous structure. Finally, it is sterilized by electron beam sterilization.

[0092] In this embodiment, the higher content of bioactive glass increases the proportion of inorganic active components in the material, giving it a stronger mineralization-inducing ability. When the material comes into contact with body fluids, the bioactive glass can rapidly release active ions, promoting hydroxyapatite deposition and enhancing bone tissue formation.

[0093] Meanwhile, since the collagen content reaches 20%, it can form a relatively dense network structure after cross-linking, which can coat and fix a large number of bioactive glass particles, avoiding the loose material structure caused by high content of inorganic particles.

[0094] The principle behind the technical effect achieved in this embodiment is as follows:

[0095] High-content bioactive glass provides a continuous source of inorganic ions, enhancing the material's bone-inducing ability; a high-crosslinking-density collagen network improves the overall stability of the material, enabling it to maintain high bioactivity while retaining good structural integrity.

[0096] Therefore, the material obtained in this embodiment is suitable for tissue repair scenarios with large bone defects that require high support performance and bone induction capacity.

[0097] Example 4

[0098] This embodiment provides a recombinant collagen and bioglass composite material suitable for the repair of diabetic foot wounds.

[0099] Since diabetic foot wounds often suffer from long-term inflammatory response, insufficient local blood supply, decreased cell migration ability, and reduced tissue regeneration ability, this embodiment focuses on optimizing the flexibility, cell affinity, and wound microenvironment regulation capabilities of the material.

[0100] The specific preparation method is as follows:

[0101] First, recombinant humanized collagen was added to phosphate buffer and stirred at 4°C to dissolve it, thus preparing a collagen solution with a mass fraction of 5%.

[0102] Subsequently, bioactive glass powder with a particle size of 100 nm was added to the collagen solution to make the mass fraction of bioactive glass powder reach 30%.

[0103] During the addition process, the bioactive glass powder is first pre-dispersed in deionized water and then ultrasonically treated for 40 minutes to ensure thorough dispersion of the bioactive glass particles. Then, it is slowly added to the collagen solution and mechanically stirred at 300 rpm for 2 hours to ensure the bioactive glass particles are evenly distributed within the collagen system.

[0104] Subsequently, genipin was added as a cross-linking agent to carry out the cross-linking reaction. The reaction temperature was controlled at 25°C, and the reaction time was 18 hours, so that a stable cross-linked structure could be formed between collagen molecules.

[0105] After cross-linking, the composite system is prepared into a gel-like wound dressing. Further, the resulting gel material is pre-frozen at low temperature and then freeze-dried under vacuum to form a flexible composite dressing with a certain porous structure.

[0106] Finally, the material was sterilized using ethylene oxide to obtain a sterile material for repairing diabetic foot wounds.

[0107] The material obtained in this embodiment has good flexibility and liquid absorption properties, which can conform to the shape of irregular wounds and absorb wound exudate, providing a moist repair environment for the wound.

[0108] The principle behind its technical effect is as follows:

[0109] Recombinant humanized collagen, as the main organic component, can mimic the structure of the natural extracellular matrix, providing adhesion sites for fibroblasts, keratinocytes, etc., and promoting cell migration and tissue remodeling.

[0110] Meanwhile, the calcium and silicon ions released by the bioactive glass can improve the local microenvironment of the wound, promote cell activity, and promote the formation of new tissue.

[0111] In addition, the composite network structure formed by collagen and bioactive glass can improve the stability of the material, avoid the problem of traditional collagen dressings degrading too quickly in a moist environment, and allow the material to remain in place for an appropriate period of time during wound repair.

[0112] Therefore, the composite material obtained in this embodiment is particularly suitable for the repair of chronic diabetic foot wounds, skin tissue defects, and other difficult-to-heal wounds.

[0113] Example 5

[0114] This embodiment provides a three-dimensional printed recombinant collagen and bioglass composite scaffold for bone tissue defect repair.

[0115] This embodiment adjusts the material composition and molding method to form a tissue engineering scaffold with a regular pore structure from composite materials, so as to meet the needs of bone defect areas for mechanical support and tissue ingrowth.

[0116] The specific preparation method is as follows:

[0117] Recombinant humanized collagen was added to phosphate buffer and stirred at 4°C to dissolve, thus preparing a collagen solution with a mass fraction of 15%.

[0118] Bioactive glass powder with a particle size of 400 nm was added to the above collagen solution to make the mass fraction of bioactive glass powder reach 60%.

[0119] Before addition, the bioactive glass powder was ultrasonically pretreated for 45 minutes to reduce particle aggregation. It was then added to the collagen solution and mechanically stirred to ensure uniform dispersion of the bioactive glass.

[0120] Add the EDC / NHS cross-linking system to the mixture and react at 30°C for 10 hours to allow collagen to form a stable network structure.

[0121] The cross-linked composite slurry is loaded into a 3D printing device, and the printing path is designed according to the preset bone defect shape to prepare a bone repair scaffold with a continuous pore structure.

[0122] After printing, the bracket is pre-frozen and then further stabilized by vacuum freeze drying.

[0123] Finally, gamma ray sterilization was used to obtain a sterile bone tissue repair scaffold.

[0124] The support obtained in this embodiment has good spatial structure retention capability and channel connectivity.

[0125] The principle behind its technical effect is as follows:

[0126] 3D printing can design scaffold structures based on the morphology of bone defect areas, giving the materials a higher structural matching ability and improving post-implantation stability.

[0127] The three-dimensional interconnected pore structure formed inside the scaffold can promote cell migration into the material and facilitate blood vessel ingrowth, providing a spatial basis for the formation of new bone tissue.

[0128] Among them, bioactive glass can continuously release active ions such as calcium and silicon, promote the proliferation and differentiation of osteoblast-related cells, and improve bone formation capacity; while recombinant collagen network provides flexible support and cell adhesion environment.

[0129] Therefore, the composite scaffold obtained in this embodiment can simultaneously meet the needs of mechanical support, biological induction, and tissue fusion during the bone defect repair process.

[0130] Comparative Example 1

[0131] This comparative example provides a material containing only recombinant humanized collagen to verify the effect of bioactive glass components on improving the performance of composite materials.

[0132] The specific preparation method is as follows:

[0133] Recombinant humanized collagen was added to phosphate buffer to prepare a collagen solution with a mass fraction of 10%.

[0134] Adding an EDC / NHS cross-linking system to it causes a cross-linking reaction between collagen molecules, forming a three-dimensional collagen network structure.

[0135] Subsequently, the obtained adhesive raw material was freeze-dried to form a porous adhesive raw material.

[0136] Compared with Example 2, this comparative example does not contain any bioactive glass powder.

[0137] Tests and analyses revealed that the material has a certain degree of cell compatibility, but its overall mechanical properties and osteoinductive ability are significantly lower than those of the composite material obtained in Example 2.

[0138] The reason is:

[0139] While recombinant collagen alone can provide a cell adhesion environment, it lacks the support of inorganic active components, resulting in insufficient mineralization induction capacity of the material. Furthermore, the collagen network is prone to rapid degradation in body fluids, leading to a decline in the material's structural integrity.

[0140] In contrast, this invention introduces bioactive glass and uses a collagen network to fix it, enabling the material to possess both organic support properties and inorganic bioactivity.

[0141] Comparative Example 2

[0142] This comparative example provides a material containing only bioactive glass powder to verify the effect of recombinant collagen components on improving the structural properties of the material.

[0143] The specific preparation method is as follows:

[0144] Bioactive glass powder with a particle size of 250 nm was selected, and block materials were prepared by compression molding and then sterilized.

[0145] This comparative example did not contain recombinant humanized collagen, nor did it form an organic network structure.

[0146] Analysis revealed that the material has high inorganic activity, which can promote the deposition of hydroxyapatite. However, the material is generally brittle and lacks compressive and impact resistance, making it prone to structural damage in complex tissue repair environments.

[0147] The reason is:

[0148] Although bioactive glass possesses excellent osteoinductive properties, it is a brittle inorganic material lacking a flexible supporting structure. When subjected to external loads, cracks easily propagate within the material, affecting its long-term stability.

[0149] This invention overcomes the shortcomings of single bioactive glass materials by embedding bioactive glass into a three-dimensional network structure of recombinant collagen, allowing collagen to play a buffering and connecting role, thereby improving the overall toughness of the material.

[0150] Material performance testing and technical effect verification

[0151] To further verify the structural properties, bioactivity, and tissue repair effects of the recombinant collagen and bioglass composite materials provided by this invention, performance tests were conducted on the composite materials obtained in Examples 1 to 5, as well as the materials obtained in Comparative Examples 1 and 2.

[0152] It should be noted that the following test methods and results are only used to illustrate that the technical solution of the present invention can achieve the expected technical effect, and do not constitute a limitation on the scope of protection of the present invention.

[0153] I. Biocompatibility Testing

[0154] Cell culture experiments were used to evaluate the effects of different materials on cell growth.

[0155] The specific method is as follows:

[0156] Materials prepared in Examples 1 to 5, as well as Comparative Examples 1 and 2, were sterilized and then extracted to obtain material extracts.

[0157] Bone marrow mesenchymal stem cells were selected as test cells. The cells were seeded in a culture system containing extracts of different materials and cultured at 37°C and 5% carbon dioxide.

[0158] Cell proliferation was detected using cell viability assays after 1, 3, and 7 days of culture.

[0159] The test results show that:

[0160] The composite materials obtained in Examples 1 to 5 all exhibited good cell compatibility, and cells were able to adhere to and continuously proliferate on the material surface.

[0161] The material obtained in Example 2 showed better cell proliferation effect because the ratio of recombinant humanized collagen to bioactive glass in this example was in a better balance, which could provide sufficient cell adhesion sites and release active ions through bioactive glass to promote cell metabolism.

[0162] The diabetic foot repair material obtained in Example 4 also showed good cell compatibility, indicating that the material can meet the needs of cell migration and tissue reconstruction during the repair of chronic wounds.

[0163] In contrast, Comparative Example 2, which contains only bioactive glass and lacks the cell adhesion environment provided by collagen, has a weaker initial cell adhesion ability.

[0164] This demonstrates that the use of recombinant humanized collagen as an organic substrate in this invention helps to improve the biocompatibility of the material.

[0165] II. Testing of the Mineralization Induction Performance of Bioactive Glass

[0166] To verify the osteoinductive effect of bioactive glass in the composite system, in vitro mineralization experiments were conducted on each group of materials.

[0167] The specific method is as follows:

[0168] The materials from Examples 1 to 5 and the comparative examples were immersed in simulated body fluid environments and cultured at 37°C for 14 days.

[0169] After cultivation, the surface of the material is observed and analyzed to detect whether a hydroxyapatite-like deposition layer has formed on the surface.

[0170] The test results show that:

[0171] Different degrees of mineralization deposition could be observed on the surface of the materials obtained in Examples 1 to 5.

[0172] Among them, Examples 3 and 5 have a higher content of bioactive glass, resulting in a more obvious degree of mineralization deposition.

[0173] The reason is:

[0174] When bioactive glass enters the body fluid environment, it can undergo ion exchange reactions and continuously release active ions such as calcium ions and silicon ions.

[0175] Among them, calcium ions can promote calcium phosphate deposition, forming a structure similar to hydroxyapatite in natural bone tissue; silicon ions can promote osteoblast-related cell activity and improve bone formation capacity.

[0176] In contrast, Comparative Example 1, lacking bioactive glass, relied solely on the collagen's own structure, resulting in significantly insufficient mineralization induction ability.

[0177] This demonstrates that the present invention, by introducing bioactive glass, enables the composite material to achieve excellent osteoinductive properties.

[0178] III. Mechanical Property Testing

[0179] To evaluate the structural stability of composite materials, compression performance tests were conducted on different materials.

[0180] The specific method is as follows:

[0181] The materials from Examples 1 to 5 and the comparative examples were prepared into porous material samples of the same size and subjected to compression tests at room temperature.

[0182] Record the maximum load-bearing capacity of the material when significant structural damage occurs during the test.

[0183] The test results show that:

[0184] The materials obtained in Examples 1 to 5 all have certain compressive strength.

[0185] The material obtained in Example 2 exhibits better overall mechanical properties, which is due to the following reasons:

[0186] When the collagen protein mass fraction is 10% and the bioactive glass mass fraction is 45%, the collagen network structure can fully encapsulate and fix inorganic particles, while the bioactive glass can enhance the overall rigidity of the material, thus achieving a good balance between toughness and strength.

[0187] The three-dimensional printed scaffold obtained in Example 5 has a strong ability to maintain its overall structure due to its regular channel structure design, making it suitable for bone defect repair environments.

[0188] Although Comparative Example 1 has a certain degree of flexibility, its overall load-bearing capacity is low due to the lack of inorganic reinforcing phase.

[0189] Although Comparative Example 2 has a high content of inorganic components, it is brittle due to the lack of a flexible collagen network, and is prone to breakage when subjected to external forces.

[0190] This demonstrates that the present invention effectively improves the performance defects of single materials by constructing a composite structure of collagen and bioactive glass.

[0191] IV. Degradation Performance Test

[0192] To evaluate the compatibility between the material degradation process and the tissue formation process, in vitro degradation experiments were conducted on each group of materials.

[0193] The specific method is as follows:

[0194] Different materials were placed in a simulated body fluid environment and cultured at 37°C.

[0195] Periodically remove the material, clean and dry it, weigh the remaining mass, and calculate the material mass retention rate.

[0196] The test results show that:

[0197] The materials obtained in Examples 1 to 5 all exhibited controllable degradation characteristics.

[0198] Among them, Example 1 has a higher proportion of collagen and a lower content of bioactive glass, so the degradation rate is relatively fast, making it more suitable for wound repair scenarios that require rapid tissue coverage.

[0199] Because of the high proportion of bioactive glass and the high degree of collagen cross-linking in Examples 3 and 5, the material structure is maintained for a longer time, making it more suitable for bone tissue repair.

[0200] The reason is:

[0201] The cross-linked collagen network can reduce the rapid swelling and degradation of materials in aqueous environments, while the bioactive glass inorganic components can improve the stability of materials.

[0202] By adjusting the collagen content, bioactive glass content, and degree of cross-linking, the degradation rate of the material can be controlled to match the process of new tissue formation.

[0203] V. Verification of the Repair Effect of Diabetic Foot Wounds

[0204] To verify the application effect of the material of the present invention in the field of chronic wound repair, a diabetic animal wound model was used for evaluation.

[0205] The specific method is as follows:

[0206] A diabetic animal foot skin defect model was established, and the composite gel dressing obtained in Example 4 was applied to the wound area.

[0207] Meanwhile, a standard collagen dressing was used as a control.

[0208] During the experiment, we observed the wound closure, granulation tissue formation, and neovascularization.

[0209] The experimental results show that:

[0210] The wound treated with the material in Example 4 showed a significantly improved closure speed and more complete granulation tissue formation in the wound area.

[0211] The main reason is:

[0212] Recombinant humanized collagen can mimic the natural extracellular matrix, providing a favorable environment for fibroblast migration; at the same time, the active ions released by bioactive glass can improve the local microenvironment, promote cell activity, and promote tissue repair.

[0213] In addition, the porous structure formed by composite materials can improve the transport capacity of oxygen and nutrients, which is beneficial to the recovery of chronic wounds.

[0214] Therefore, the material of the present invention is not only suitable for repairing bone tissue defects, but also meets the needs of repairing diabetic foot wounds.

[0215] VI. Comprehensive Technical Effect Analysis

[0216] The above embodiments and performance tests demonstrate that the recombinant collagen and bioglass composite material provided by this invention achieves a comprehensive improvement in multiple properties through the synergistic effect between organic and inorganic components.

[0217] Among them, recombinant humanized collagen mainly plays the roles of cell adhesion, tissue support and flexibility enhancement; bioactive glass mainly plays the roles of active ion release, mineralization induction and bone formation promotion; the cross-linked three-dimensional network structure improves the binding stability between the two.

[0218] Compared with single collagen materials, the material of the present invention has higher mechanical stability and bioactivity; compared with single bioactive glass materials, the material of the present invention has better flexibility, structural integrity and cell compatibility.

[0219] Furthermore, by adjusting the material composition ratio and molding method, porous scaffold materials suitable for bone tissue defect repair and gel or dressing materials suitable for diabetic foot wound repair can be prepared, realizing the multifunctional application of a single material system in the fields of hard tissue repair and soft tissue repair. Therefore, this invention has good clinical application value and promising prospects for industrialization.

[0220] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A composite material of recombinant collagen and bioglass, characterized in that, The product comprises recombinant humanized collagen, bioactive glass powder, and a crosslinking agent, wherein the particle size of the bioactive glass powder is 50-500 nm; the recombinant humanized collagen is crosslinked to form a three-dimensional organic network structure, and the bioactive glass powder is uniformly dispersed in the three-dimensional organic network structure to form a stable organic composite framework.

2. The recombinant collagen and bioglass composite material according to claim 1, characterized in that, The recombinant humanized collagen has a mass fraction of 1% to 20%.

3. The recombinant collagen and bioglass composite material according to claim 1, characterized in that, The bioactive glass powder has a mass fraction of 10% to 80%.

4. The recombinant collagen and bioglass composite material according to claim 1, characterized in that, The crosslinking agent is one or more of the EDC / NHS crosslinking system, genipin, or other biocompatible crosslinking agents.

5. The recombinant collagen and bioglass composite material according to claim 1, characterized in that, The recombinant collagen and bioglass composite material has a three-dimensional interconnected pore structure.

6. A method for preparing a recombinant collagen and bioglass composite material according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Add recombinant humanized collagen to phosphate buffer and stir to dissolve at low temperature to obtain a homogeneous collagen solution. S2. Add the bioactive glass powder to the collagen solution, and disperse the bioactive glass powder evenly in the collagen solution by ultrasonic dispersion and mechanical stirring. S3. Add a crosslinking agent to the mixture and carry out a crosslinking reaction under preset temperature and preset time conditions to form a stable three-dimensional organic network structure of recombinant humanized collagen and fix the bioactive glass powder in the three-dimensional organic network structure. S4. Place the cross-linked composite system in a mold and shape it using freeze molding, gel molding or 3D printing. S5. The molded material is pre-frozen and then vacuum freeze-dryed to form a composite material with a three-dimensional interconnected pore structure. S6. The obtained composite material is sterilized by any one of ethylene oxide sterilization, gamma-ray sterilization or electron beam sterilization to obtain sterile recombinant collagen and bioglass composite material.

7. The preparation method according to claim 6, characterized in that, In S3, one or more of the following are used as crosslinking agents: EDC / NHS crosslinking system and genipin.

8. The preparation method according to claim 6, characterized in that, In step S4, the composite system is prepared into a scaffold, gel, or dressing by any of the following methods: cryogenic molding, gel molding, or 3D printing.

9. The preparation method according to claim 6, characterized in that, In step S6, the sterilization method is one or more of ethylene oxide sterilization, gamma ray sterilization, or electron beam sterilization.

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