Collagen nerve repair material based on fusion protein controlled release and preparation method and application thereof

Abstract

The application relates to a collagen nerve repair material based on fusion protein controlled release and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The collagen nerve repair material comprises a type I collagen protein matrix and a CBD-TIMP-FGF2 fusion protein; the CBD-TIMP-FGF2 fusion protein comprises a collagen binding domain, an MMP-cleavable TIMP peptide segment and an alkaline fibroblast growth factor; wherein the CBD-TIMP-FGF2 fusion protein is combined with the type I collagen protein matrix to form an MMP-responsive controlled release system, and the alkaline fibroblast growth factor is released by cleavage of the TIMP peptide segment. The collagen nerve repair material can realize on-demand release of bFGF, realize spatiotemporal synergistic regulation of vascularization and nerve regeneration at the same time, promote vascular reconstruction and nerve repair at a damage site, and has excellent biocompatibility and clinical application potential.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to collagen nerve repair materials based on controlled release of fusion proteins, their preparation methods and applications. Background Technology

[0002] Neurological trauma repair (especially large-segment peripheral nerve defects and spinal cord injuries) has always been a major clinical challenge. While autologous nerve transplantation is currently the gold standard, it is limited by donor shortages, donor site dysfunction, and nerve type mismatch, making it difficult to meet clinical needs. Tissue engineering strategies, such as artificial nerve conduits, offer alternatives, but existing material systems still have significant shortcomings, such as: 1. Insufficient bioactivity: Natural materials like collagen and chitosan, and synthetic materials like polylactic acid, only provide physical scaffolds and lack the ability to actively regulate cell behavior, failing to effectively promote axonal regeneration; 2. Degradation-regeneration timeline mismatch: The degradation rate of materials is difficult to synchronize with the nerve regeneration cycle, either prematurely degrading and losing support function, or remaining in the body and causing chronic inflammation; 3. Imbalance in the mechanical microenvironment: The mechanical strength (such as elastic modulus) of materials deviates significantly from that of natural nerve tissue; excessive strength compresses regenerated tissue, while insufficient strength fails to maintain structural integrity; 4. Lack of vascularization mechanisms: The high metabolic demands of nerve tissue contradict the interruption of blood supply after injury, and traditional materials lack vascularization-promoting functions, leading to cell necrosis in the graft's central region.

[0003] To improve vascularization, researchers have attempted to add angiogenic factors (such as VEGF and bFGF) to materials. However, existing technologies using physical encapsulation or heparin adsorption result in the burst release of growth factors (>70% release within 24 hours), making it difficult to maintain effective therapeutic concentrations. Furthermore, there are risks of activity loss during preparation and systemic exposure due to non-targeted release. While smart responsive materials (such as pH / temperature-sensitive hydrogels) have emerged, their synthetic polymer substrates have poor biocompatibility, and the physical signal response is not directly related to the pathological process of nerve injury. In recent years, enzyme-responsive systems based on matrix metalloproteinases (MMPs) have shown promise, but existing MMP-responsive materials mostly rely on synthetic carriers (such as peptide-modified polyethylene glycol), lacking biomimetic extracellular matrix structures and failing to provide the biological signals required by nerve cells. Crucially, controlled release technology for angiogenic factors remains at the physical mixing stage, failing to address the core issue of precise spatiotemporal release. This leads to a disconnect between nerve regeneration and vascularization processes, becoming a key bottleneck restricting clinical translation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the objectives of this application include providing a collagen nerve repair material based on controlled release of fusion protein, its preparation method, and its application, so as to achieve on-demand release of bFGF and promote vascular reconstruction and nerve repair at the site of injury.

[0005] In a first aspect, embodiments of this application provide a collagen-based neurorepair material based on controlled release of a fusion protein, comprising a type I collagen matrix and a CBD-TIMP-FGF2 fusion protein; the CBD-TIMP-FGF2 fusion protein comprises a collagen-binding domain, a cleavable TIMP peptide of MMP, and basic fibroblast growth factor; wherein, the CBD-TIMP-FGF2 fusion protein binds to the type I collagen matrix to form an MMP-responsive controlled release system, releasing basic fibroblast growth factor through TIMP peptide cleavage.

[0006] This application's innovative CBD-TIMP-FGF2 fusion protein design anchors to collagen fibers with high affinity via the collagen-binding domain (CBD), avoiding the loss of growth factors caused by physical adsorption. Simultaneously, the TIMP peptide acts as a specific "molecular switch" for MMP-2 and MMP-9, cleaving only when MMP activity is upregulated at the nerve injury site. This enables precise, on-demand release of basic fibroblast growth factor (bFGF), fundamentally overcoming the shortcomings of traditional materials in terms of growth factor burst release and lack of targeting. Furthermore, the released bFGF promotes vascular endothelial cell proliferation and Schwann cell activation by activating FGFR receptors, forming a synergistic effect of vascular-neural regeneration. Therefore, the collagen-based nerve repair material provided in this application, through its molecular-level intelligent response design, achieves for the first time the spatiotemporal synergistic regulation of vascularization and axonal regeneration during nerve repair.

[0007] In some embodiments of this application, the TIMP peptide includes the common recognition sequence PLGLAG or GPQGIAGQ of MMP-2 and MMP-9.

[0008] When nerve injury occurs, the release of inflammatory factors promotes the upregulation of MMP-2 and MMP-9 expression, with local MMP concentrations reaching 5-10 times the normal level. High concentrations of MMP compete with TIMP for binding, leading to the cleavage of the TIMP peptide at the PLGLAG or GPQGIAGQ sequence, releasing free bFGF. bFGF, as an effector molecule, promotes angiogenesis and nerve repair.

[0009] In some embodiments of this application, the molecular weight of the CBD-TIMP-FGF2 fusion protein is 35-40 kDa, and the loading amount of the CBD-TIMP-FGF2 fusion protein in the type I collagen matrix is ​​10-50 μg / mL.

[0010] In the above technical solution, the precisely controlled molecular weight range (35-40kDa) enables the fusion protein to diffuse efficiently and distribute evenly in the collagen matrix, while avoiding steric hindrance caused by excessive molecular weight; the loading window of 10-50μg / mL perfectly balances the therapeutic effect and material stability, with the lower limit of 10μg / mL ensuring the minimum effective bFGF release concentration (>1ng / mL) and the upper limit of 50μg / mL preventing excessive loading from damaging the collagen network structure.

[0011] In some embodiments of this application, the type I collagen matrix is ​​derived from the tendon, skin, or bone matrix of animals under 6 months of age.

[0012] In the above technical solutions, the collagen from these young animals (under 6 months old) has a significantly lower degree of cross-linking than that of adult individuals. This allows the Gly-XY repeat sequence to be fully preserved, thereby exposing more integrin binding sites (such as GFOGER sequences) and promoting cell adhesion. The low cross-linking makes the collagen easier to be hydrolyzed by collagenases, forming channels suitable for the migration of vascular endothelial cells and Schwann cells. Moreover, the collagen fibers in young tissues are more loosely arranged, which can preserve the natural conformation to the maximum extent, thereby preserving the heparin binding domain (promoting bFGF stability) and MMP cleavage sites (regulating degradation).

[0013] In some embodiments of this application, the collagen nerve repair material has a three-dimensional network structure with a porosity of 80-90% and a pore size of 10-100 μm; and / or, the tensile strength of the collagen nerve repair material is 0.1-1.0 MPa and the elastic modulus is 10-100 kPa; and / or, the degradation period of the collagen nerve repair material is 8-12 weeks, and the degradation products are amino acids and small peptides.

[0014] In the aforementioned technical solution, the high porosity (80-90%) provides ample space for cell migration and nutrient diffusion, significantly promoting the infiltration of vascular endothelial cells and Schwann cells. The pore size range of 10-100 μm accommodates capillary bud growth and provides a directional guiding topology for axonal extension, resulting in a significant increase in the average extension length of neurites. Furthermore, the porous structure of this nerve repair material enhances tissue fluid exchange through capillary action, accelerates the removal of metabolic waste, and maintains the material's mechanical stability. This achieves a synergistic balance among vascularization, nerve regeneration, and mechanical support, providing a suitable microenvironment for nerve cell growth and vascular invasion.

[0015] In some embodiments of this application, the dosage form of the collagen nerve repair material includes a nerve conduit, a nerve bridging scaffold, and an injectable hydrogel; wherein, the parameters of the nerve conduit include: an inner diameter of 1-15 mm, a conduit wall thickness of 0.5-2 mm, and a length of 10-80 mm; and / or, the inner surface of the nerve conduit is provided with a longitudinal guide groove or filled with a fiber scaffold to guide the directional growth of axons; and / or, the concentration of the injectable hydrogel is 1-3 mg / mL, the gelation time is 5-15 min, and the elastic modulus is 10-100 kPa.

[0016] The multi-form adaptable design of collagen nerve repair materials in this application (nerve conduit, bridging scaffold, and injectable hydrogel) significantly improves clinical applicability through precise structure-function matching. Specifically, the longitudinal guide groove structure of the nerve conduit (inner diameter 1-15mm, wall thickness 0.5-2mm) provides a directional growth channel for large peripheral nerve defects (10-80mm), and its biomimetic mechanical strength effectively resists suture tension. The three-dimensional porous structure of the bridging scaffold supports cell infiltration and vascularization, suitable for anastomosing irregular nerve ends. The injectable hydrogel (concentration 1-3mg / mL, gelation time 5-15min) precisely fills the spinal cord injury cavity through minimally invasive injection; its shear-thinning properties ensure that the injection process does not damage fragile nerve tissue, and the in-situ formed three-dimensional network perfectly conforms to the central nervous system microenvironment. All three forms retain the core function of MMP responsive controlled release, achieving full-scale repair coverage from millimeter-level peripheral nerves to micrometer-level central nervous systems, demonstrating high clinical adaptability.

[0017] Secondly, embodiments of this application provide a method for preparing the above-mentioned collagen nerve repair material based on controlled release of fusion protein, comprising: dissolving type I collagen powder in acetic acid solution to obtain collagen solution; subsequently, adding CBD-TIMP-FGF2 fusion protein and adjusting the pH to 7.0-7.4 to obtain collagen nerve repair material.

[0018] This preparation method achieves efficient synthesis of type I collagen and CBD-TIMP-FGF2 fusion protein, forming an MMP-responsive intelligent release system to achieve on-demand release of bFGF, promoting vascular remodeling and nerve repair at the injury site. The entire preparation process is simple to operate, has good reproducibility, and fully preserves the bioactivity and functional properties of the material.

[0019] In some embodiments of this application, the preparation of the CBD-TIMP-FGF2 fusion protein includes: constructing a recombinant expression vector containing a collagen-binding domain, a TIMP peptide, and a basic fibroblast growth factor coding sequence; transforming it into Escherichia coli BL21 or transfecting it into mammalian cells for protein expression; and purifying it to obtain the CBD-TIMP-FGF2 fusion protein.

[0020] In the above technical solution, the CBD-TIMP-FGF2 fusion protein is expressed by genetic engineering technology, and high-purity (≥90%) mass production is achieved using E. coli or mammalian systems. The directional modification of its collagen-binding domain (CBD) ensures stable binding with collagen. The mild compounding process with pH regulation (pH 7.0-7.4) enables collagen and fusion protein to self-assemble through electrostatic interaction, which maintains the MMP-responsive controlled release function and preserves the three-dimensional porous structure of the material.

[0021] In some embodiments of this application, the preparation method includes: adding CBD-TIMP-FGF2 fusion protein to a final concentration of 20-100 μg / mL; and / or, the concentration of collagen solution is 10-20 mg / mL.

[0022] A lower concentration of 20 μg / mL allows for the effective release of bFGF to reach the threshold concentration for promoting angiogenesis, while an upper concentration of 100 μg / mL prevents excessive fusion protein from occupying cross-linking sites between collagen fibers and compromising the material's mechanical properties, while also avoiding the risk of abnormal proliferation caused by excessive release of growth factors. In other words, within this concentration range, the fusion protein achieves uniform distribution through the specific binding of its CBD domain to collagen, optimizing the temporal resolution of the MMP-responsive controlled-release system and ultimately promoting synergistic regeneration of blood vessels and nerves. The 10-20 mg / mL collagen solution window perfectly balances material formability and bioactivity; the lower limit of 10 mg / mL ensures injectability, while the upper limit of 20 mg / mL provides sufficient foundation for fiber self-assembly, enabling the formation of a three-dimensional network with suitable mechanical strength and ideal pore structure after gelation, while also providing a uniform loading space for the fusion protein.

[0023] Thirdly, this application provides an application of the above-mentioned collagen nerve repair material based on controlled release of fusion protein in the preparation of drugs or medical devices for treating nerve injury diseases, including peripheral nerve defects, spinal cord injuries, traumatic brain injury, or neuroinflammatory diseases.

[0024] The collagen-based nerve repair material provided in this application demonstrates excellent clinical suitability in drugs or medical devices for nerve injury diseases. For example, in peripheral nerve defects, its nerve conduit form guides precise axon regeneration through directional pore structures; for spinal cord injuries, the injectable hydrogel fills irregular injury cavities while promoting vascularization and axon regeneration through MMP-responsive controlled release of bFGF; in traumatic brain injury and neuroinflammatory diseases, the material's low immunogenicity and spatiotemporally specific drug release characteristics can target and inhibit the inflammatory microenvironment (downregulating TNF-α / IL-6 expression) while avoiding systemic side effects. Through an integrated mechanism of "damage microenvironment sensing - on-demand drug release - synergistic vascular and nerve regeneration," this material overcomes the technical bottlenecks of traditional repair devices with limited functionality and indications, providing a comprehensive solution for complex neurotrauma. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The electrophoretic analysis results are for type I collagen in Experimental Example 1 of this application; Figure 2 The results of thermal denaturation analysis for Test Example 1 of this application; Figure 3 The hydroxyproline retention rate of Experimental Example 1 in this application; Figure 4 The electrophoretic analysis results are for the CBD-TIMP-FGF2 fusion protein of Experimental Example 2 of this application; Figure 5 This is a plasmid map of the CBD-TIMP-FGF2 fusion protein from Experimental Example 2 of this application; Figure 6 This is an assessment of angiogenesis function in Experiment Example 2 of this application; Figure 7 This is the second assessment of angiogenesis function in Experiment Example 2 of this application; Figure 8 This is the sciatic nerve function recovery status of Experimental Example 3 in this application; Figure 9 The results of nerve conduction velocity 12 weeks post-operation in Case 3 of this application; Figure 10 The results of the complex muscle action potentials at 12 weeks post-operation in Case 3 of this application are shown. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] The following describes a collagen nerve repair material based on controlled release of fusion protein, its preparation method, and its application, according to embodiments of this application.

[0029] This application provides a collagen-based neurorepair material with controlled release of a fusion protein, comprising a type I collagen matrix and a CBD-TIMP-FGF2 fusion protein. The CBD-TIMP-FGF2 fusion protein includes a collagen-binding domain, a cleavable TIMP peptide, and basic fibroblast growth factor. The CBD-TIMP-FGF2 fusion protein binds to the type I collagen matrix to form an MMP-responsive controlled release system, releasing basic fibroblast growth factor through TIMP peptide cleavage.

[0030] The collagen-based nerve repair material provided in this application achieves precise spatiotemporal release of bFGF through a CBD-TIMP-FGF2 fusion protein sequence. When nerve injury leads to upregulation of local MMP activity, the TIMP peptide is cleaved at specific sites, releasing free bFGF. The released bFGF activates FGFR1 and FGFR2 receptors on the surface of vascular endothelial cells, initiating angiogenesis cascade reactions. Activated endothelial cells secrete vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and angiopoietin (Ang-1), forming an angiogenesis factor network. This, in turn, promotes endothelial cell proliferation, migration, and the formation of capillary buds, guiding blood vessels to grow towards the nerve injury site via a chemokine gradient. New blood vessels provide essential oxygen, nutrients, and neurotrophic factors for nerve regeneration, improving the local microenvironment. Angiogenesis and nerve regeneration have a synergistic effect; neurotrophic factors secreted by vascular endothelial cells, such as BDNF, NGF, and NT-3, promote axonal growth and myelin formation.

[0031] The TIMP peptide includes the common recognition sequence PLGLAG or GPQGIAGQ for both MMP-2 and MMP-9.

[0032] The MMP-responsive controlled-release mechanism of the neural repair material provided in this application is based on the specific interaction between TIMP and MMP. The TIMP peptide contains the common recognition sequence PLGLAG or GPQGIAGQ for MMP-2 and MMP-9. Under normal physiological conditions, TIMP and MMP form a stable 1:1 stoichiometric complex, inhibiting the proteolytic activity of MMP. When nerve injury occurs, the release of inflammatory factors such as TNF-α, IL-1β, and IL-6 promotes the upregulation of MMP-2 and MMP-9 expression, and the local MMP concentration can reach 5-10 times the normal level. The high concentration of MMP competes with TIMP for binding, causing the TIMP peptide to be cleaved at a specific site, namely the aforementioned sequence PLGLAG or GPQGIAGQ, releasing free bFGF.

[0033] The bFGF described in this application is a recombinant human basic fibroblast growth factor with a molecular weight of approximately 18 kDa and containing 146 amino acid residues. It is produced using a *Pyrzybum pylori* or *Escherichia coli* expression system and purified by affinity chromatography and ion exchange chromatography to a purity ≥95%. Its bioactivity was verified by in vitro cell proliferation experiments, EC50... 50 The concentration ranges from 0.1 to 1.0 ng / mL. The bFGF molecular structure comprises 12 β-sheets and linked ring structures, possessing a highly conserved heparin-binding domain. bFGF loading in this collagen-based neurorepair material utilizes three strategies: physical adsorption, electrostatic interaction, and fusion protein expression. Covalent linkage via the CBD-TIMP-FGF2 fusion protein ensures the stability and controlled release of bFGF in the matrix. The biological functions of bFGF include promoting vascular endothelial cell proliferation, migration, and tubular formation, and activating the PI3K / Akt and MAPK signaling pathways.

[0034] In this application, the CBD-TIMP-FGF2 fusion protein has a molecular weight of 35-40 kDa, and its loading amount in the type I collagen matrix is ​​10-50 μg / mL. As an example, the loading amounts of the CBD-TIMP-FGF2 fusion protein in the type I collagen matrix include, but are not limited to, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, and 50 μg / mL. This loading amount indicates that the mass of CBD-TIMP-FGF2 fusion protein contained in each milliliter of the prepared collagen nerve repair material is 10-50 μg (i.e., 0.01-0.05 mg / mL).

[0035] In this application, the type I collagen matrix is ​​derived from the tendon, skin, or bone matrix of animals under 6 months of age. Examples of animals include, but are not limited to, cattle, pigs, and rats. The type I collagen contains ≥8% hydroxyproline to ensure the thermal stability of its triple-helix structure; has a denaturation temperature ≥37°C to maintain a stable triple-helix conformation at physiological temperatures; and has a molecular weight of 280-300 kDa, with the α1 / α2 chain ratio maintained at a natural 2:1 ratio.

[0036] Type I collagen matrix is ​​derived from the tendons, skin, or bone matrix of young animals under 6 months of age (e.g., cattle, pigs, or rats). It retains the advantageous properties of young ECM, namely, maintaining the natural triple helix structure and bioactivity of collagen, with a protein retention rate of ≥85%. The molecular weight of this Type I collagen matrix is ​​concentrated in the range of 280-320 kDa, and the α1 / α2 chain ratio remains at the natural 2:1 ratio, which can improve the structural stability of collagen molecules.

[0037] In this application, the collagen nerve repair material has a three-dimensional network structure with a porosity of 80-90% and a pore size of 10-100 μm; the tensile strength of the collagen nerve repair material is 0.1-1.0 MPa, and the elastic modulus is 10-100 kPa; the degradation period of the collagen nerve repair material is 8-12 weeks, and the degradation products are amino acids and small peptides. As an example, the porosity includes, but is not limited to, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and 90%. As an example, the pore size range includes, but is not limited to, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, and 100 μm. This collagen-based nerve repair material possesses excellent physicochemical and biological properties. Its mechanical strength meets the physiological needs of nerve tissue, with a tensile strength of 0.1-1.0 MPa and an elastic modulus of 10-100 kPa, closely resembling the mechanical properties of natural nerve tissue. Simultaneously, this collagen-based nerve repair material exhibits good biocompatibility, and its degradation performance matches the nerve regeneration timeline. In vivo, it is gradually degraded by collagenases and other matrix metalloproteinases, producing amino acids and small peptides that are non-toxic and completely metabolized by the body, with complete degradation occurring within 8-12 weeks. The degradation rate can be adjusted by the degree of cross-linking, controlled through methods such as glutaraldehyde, EDC / NHS, or recombinant protein cross-linking.

[0038] In this application, the collagen nerve repair material takes the form of a nerve conduit, a nerve bridging scaffold, or an injectable hydrogel. The parameters of the nerve conduit include: an inner diameter of 1-15 mm, a wall thickness of 0.5-2 mm, and a length of 10-80 mm. The inner surface of the nerve conduit has longitudinal guide grooves or is filled with a fibrous scaffold to guide the directional growth of axons. For spinal cord injuries, this collagen nerve repair material is applied in the form of an injectable hydrogel, precisely delivered to the injury site using a microinjector. As an example, the concentration of the injectable hydrogel is 1-3 mg / mL, and the gelation time is 5-15 min, ensuring rapid formation of a stable three-dimensional scaffold after injection, with an elastic modulus of 10-100 kPa.

[0039] The type I collagen matrix of this application has a negatively charged surface, which is conducive to forming electrostatic interactions with positively charged growth factors; the fiber network formed after gelation has a nanofiber structure similar to the natural extracellular matrix, with a fiber diameter of 50-200 nm.

[0040] The neural repair material provided in this application can be combined with neural stem cells, Schwann cells or mesenchymal stem cells to provide a carrier platform for cell therapy.

[0041] The preparation method of the collagen nerve repair material based on controlled release of fusion protein provided in this application is described below.

[0042] A method for preparing the above-mentioned collagen nerve repair material based on controlled release of fusion protein includes the following steps: (1) Extraction and purification of type I collagen matrix Collagen was extracted from tissues of mammals under 6 months of age using dilute acid extraction or enzymatic extraction. After salting out, dialysis, and freeze-drying, type I collagen powder was obtained.

[0043] The optimal temperature for extracting collagen using dilute acid or enzymatic methods is 4-15℃. Extracting collagen at this temperature minimizes protein activity loss. A specific concentration of weakly acid (0.08-1.2 mol / L) effectively dissociates collagen fibers while preventing irreversible damage to the triple-helix structure from strong acids (such as concentrated hydrochloric acid), thus preserving the integrity of collagen's natural conformation.

[0044] As one implementation method, tendon or skin tissue from healthy animals under 6 months of age (e.g., rats) is selected, and after pretreatment to remove fat and impurities, collagen is extracted using dilute acid extraction or enzymatic extraction. The extraction temperature is controlled at 4-15°C to avoid collagen denaturation. The collagen is then purified through steps such as salting out, dialysis, and freeze-drying to obtain type I collagen powder with a purity ≥95%.

[0045] (2) Construction and expression of CBD-TIMP-FGF2 fusion protein S2. A recombinant expression vector containing the coding sequences for collagen-binding domain, TIMP, and basic fibroblast growth factor was constructed, and the vector was transformed into E. coli or transfected into mammalian cells for protein expression. After purification, the CBD-TIMP-FGF2 fusion protein was obtained.

[0046] The purification process included affinity chromatography, ion exchange chromatography, and gel filtration chromatography. The purified CBD-TIMP-FGF2 fusion protein had a purity ≥90% and retained ≥85% of its biological activity.

[0047] Among the usable mammalian cells are 293T cells.

[0048] In the CBD-TIMP-FGF2 fusion protein, the amino acid sequence of CBD is shown in SEQ ID NO.1, the amino acid sequence of the TIMP peptide is shown in SEQ ID NO.2, the amino acid sequence of FGF2 is shown in SEQ ID NO.3, and the complete amino acid sequence of CBD-TIMP-FGF2 is shown in SEQ ID NO.4.

[0049] (3) Preparation of collagen nerve repair materials (construction of MMP responsive controlled release mechanism) The type I collagen powder obtained in step (1) was dissolved in acetic acid solution to obtain collagen solution; then, the CBD-TIMP-FGF2 fusion protein obtained in step (2) was added, and the pH was adjusted to 7.0-7.4 to obtain collagen nerve repair material.

[0050] The concentration of the acetic acid solution was 0.08-1.2 mol / L, the concentration of the collagen solution was 10-20 mg / mL, and the final concentration of the CBD-TIMP-FGF2 fusion protein was 20-100 μg / mL. The pH was adjusted to 7.0-7.4, and the ionic strength was controlled at 150-200 mM. The collagen molecule retained the complete Gly-XY repeat sequence, with the amino acid composition at the X and Y positions close to the natural ratio.

[0051] For example, the concentration of acetic acid includes, but is not limited to, 0.08 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, 1.1 mol / L, and 1.2 mol / L.

[0052] As an example, the concentration of the collagen solution includes, but is not limited to, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, and 20 mg / mL.

[0053] As an example, the final concentration of the CBD-TIMP-FGF2 fusion protein includes, but is not limited to, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, and 100 μg / mL.

[0054] In this application, collagen nerve repair materials of different shapes can be prepared by molding or injection molding.

[0055] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0056] Example 1 This example describes the extraction and purification of type I collagen, including the following steps: Tail tendons from healthy SD rats aged 3-6 months were selected as the collagen source. The tendon tissue was minced into 1-2 mm pieces and thoroughly washed with physiological saline to remove blood and impurities. The tissue pieces were placed in 0.5M acetic acid solution and extracted with stirring at 4°C for 48 hours. Undissolved tissue residue was removed by filtration through a 200-mesh sieve to obtain a collagen solution. Subsequently, collagen was precipitated by salting out with 3.5M sodium chloride solution, and the precipitate was collected by centrifugation. The precipitate was redissolved in 0.1M acetic acid solution and dialyzed against distilled water for 72 hours, with the dialysate changed every 12 hours. The dialyzed collagen solution was freeze-dried to obtain type I collagen powder.

[0057] Example 2 This example describes the expression and purification of the CBD-TIMP-FGF2 fusion protein, including the following steps: A recombinant expression vector pET-28a-CBD-TIMP-FGF2 containing the coding sequences for CBD, TIMP, and FGF2 was constructed. The CBD sequence is derived from the collagen-binding domain of Clostridium collagenase and contains 110 amino acids; the TIMP sequence contains the common recognition site PLGLAG for MMP-2 and MMP-9; and the FGF2 sequence is mature human basic fibroblast growth factor, containing 146 amino acids.

[0058] The recombinant vector was transformed into Escherichia coli BL21(DE3) competent cells, and positive clones were obtained by kanamycin selection. The bacteria were cultured in LB medium until OD200. 600=0.6, add 1mM IPTG to induce protein expression, and incubate at 37°C with shaking for 4h. Collect bacteria by centrifugation, sonicate to disrupt, and centrifuge again to collect the supernatant.

[0059] His-tagged fusion protein was purified using a Ni-NTA affinity chromatography column. The column was equilibrated with binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), and protein solution was loaded. The protein was washed with washing buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, pH 8.0) and eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, pH 8.0).

[0060] Example 3 This embodiment describes the construction of an MMP-responsive controlled-release system, including the following steps: The type I collagen powder prepared in Example 1 was dissolved in 0.1M acetic acid solution to prepare a collagen solution of 15 mg / mL. The CBD-TIMP-FGF2 fusion protein prepared in Example 2 was added, bringing the final concentration to 50 μg / mL. The mixture was stirred at 4°C for 2 hours to ensure complete binding of the fusion protein with the collagen.

[0061] The pH of the mixed solution was adjusted to 7.2 using 1M NaOH solution, and then immediately transferred to a 37°C incubator and allowed to stand for 30 minutes to form a collagen hydrogel. The prepared hydrogel was transparent and had a certain degree of elasticity.

[0062] The distribution of the fusion protein in the collagen matrix was observed using confocal microscopy, and the results showed that the protein was uniformly distributed without obvious aggregation. Rheological analysis of the hydrogel revealed a storage modulus G' of 156 Pa and a loss modulus G" of 23 Pa, which are close to the mechanical properties of natural nerve tissue.

[0063] In vitro release experiments were conducted by treating the hydrogel with different concentrations of MMP-2, and the released bFGF content was detected by ELISA. The results showed that in the absence of MMP, the cumulative release of bFGF within 24 hours was only 3.2%; when the MMP-2 concentration was 100 ng / mL, the cumulative release reached 47.8% within 24 hours; and when the MMP-2 concentration was 500 ng / mL, the cumulative release reached 78.4%. This indicates that the material exhibits good MMP-responsive properties.

[0064] Experimental Example 1 This experimental example is for characterizing the type I collagen powder prepared in Example 1, including: 1. The molecular weight distribution of collagen was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the molecular weights of α1 and α2 chains were 130 kDa and 115 kDa, respectively, with a ratio of approximately 2:1 (e.g., ...). Figure 1 As shown in the figure, it conforms to the characteristics of type I collagen. Figure 1 In the image, from left to right, the following are the molecular weight standard (MW), control group (Ctrl, no collagen), collagen extraction 1 (Ext1), collagen extraction 2 (Ext2), purified collagen (Pure), degraded collagen (Deg), heat-treated (Heat), and enzymatically digested sample (Enz). In the Ext1, Ext2, and Pure columns, two clear bands of 130 kDa (α1) and 115 kDa (α2) are clearly visible, with α1 intensity approximately twice that of α2, consistent with the theoretical α1:α2 ratio of 2:1 for type I collagen. Low molecular weight fragments appear in the Deg, Heat, and Enz columns, corresponding to collagen degradation, thermal denaturation, and enzymatic digestion products, respectively.

[0065] 2. Differential scanning fluorescence (DSF) was used to determine the thermal denaturation temperature, a highly efficient and sensitive classic technique for assessing protein thermal stability. This experiment was performed on a real-time quantitative PCR instrument. Its core principle is based on a fluorescent dye (such as SYPRO Orange) that is highly sensitive to hydrophobic environments. At low temperatures, the protein structure is compact, and the dye cannot bind to the internal hydrophobic regions, resulting in a weak fluorescence signal. As the temperature increases at a programmed, uniform rate (e.g., from 25°C to 95°C), the protein gradually unfolds and denatures, exposing the previously encapsulated hydrophobic amino acid residues, which then bind to the dye, leading to a significant increase in fluorescence intensity. The instrument acquires fluorescence signals at each temperature, ultimately obtaining a temperature-dependent fluorescence change curve. The temperature corresponding to the inflection point is defined as the thermal denaturation temperature Tm (referred to as Td in this study). Figure 2 The results of the thermal denaturation analysis show that the thermal denaturation temperature Td is 36.4°C, indicating that the collagen structure is intact.

[0066] 3. The determination of hydroxyproline content employed an acid hydrolysis-spectrophotometric method, the gold standard for the specific quantification of collagen and its derivatives, as hydroxyproline is a highly characteristic amino acid in the collagen family. The detection procedure begins with rigorous acid hydrolysis of the sample: the protein sample is hydrolyzed for 16-24 hours under concentrated hydrochloric acid (e.g., 6M HCl), high temperature (110°C), and anaerobic conditions, thereby completely breaking down the protein into free amino acid components, including hydroxyproline. After hydrolysis, the hydrolysate is neutralized and diluted, then subjected to specific colorimetric reactions sequentially with an oxidizing agent (e.g., chloramine-T) and a chromogenic agent (e.g., p-dimethylaminobenzaldehyde). Hydroxyproline is quantitatively converted into a rose-red product with strong absorption at a specific wavelength (typically around 560 nm). Finally, the absorbance of the solution is measured using a UV-Vis spectrophotometer, and the absolute content of hydroxyproline in the sample can be accurately calculated by comparing it with a standard curve of known concentrations of hydroxyproline. By comparing this content with the theoretical value or the content of the initial raw materials, its retention rate during the preparation process can be calculated. This indicator directly reflects the purity of the collagen product and the degree to which its unique amino acid structure is retained during the processing. Figure 3 As can be seen from the above, the type I collagen prepared in Example 1 of this application has a protein retention rate of 87.3%, indicating that the structure of the type I collagen molecule is highly stable and meets the design requirements.

[0067] Experimental Example 2 This experimental example characterizes the CBD-TIMP-FGF2 fusion protein constructed in Example 2, including: 1. SDS-PAGE analysis showed that the fusion protein had a molecular weight of approximately 38 kDa and a purity ≥90%; Western blot analysis confirmed correct protein expression (e.g., ...). Figure 4 (As shown). Figure 4 In the image, from left to right, are the molecular weight standard (MW), control group (Ctrl, no collagen), collagen extraction 1 (Ext1), collagen extraction 2 (Ext2), purified collagen (Pure), degraded collagen (Deg), heat-treated (Heat), and enzymatically digested sample (Enz). In the Ext1, Ext2, and Pure columns, two clear bands of 130 kDa (α1) and 115 kDa (α2) are clearly visible, with α1 intensity approximately twice that of α2, consistent with the theoretical α1:α2 ratio of 2:1 for type I collagen. Low molecular weight fragments appear in the Deg, Heat, and Enz columns, corresponding to collagen degradation, thermal denaturation, and enzymatic digestion products, respectively.

[0068] 2. The plasmid map of the CBD-TIMP-FGF2 fusion protein is shown below. Figure 5As shown, the vector is approximately 4576 bp in length. The origin (ori) is the sequence necessary for the plasmid to self-replicate in the host cell. AmpR (ampicillin resistance gene) is an ampicillin resistance gene that allows bacteria containing this plasmid to grow in a selection medium containing ampicillin for screening successful transformants. This gene is driven by the AmpR promoter. The CMV enhancer and CMV promoter are the cytomegalovirus enhancer and promoter, respectively, used to drive transcription of downstream genes at high expression levels, typically used in gene expression in mammalian cells. The IL-2 signal sequence, following the CMV promoter, guides the secretion of the fusion protein extracellularly. The core expression framework of the CBD-TIMP-FGF2 fusion protein consists of three functional modules: 1. A collagen-binding domain (CBD), which endows the protein with specific binding ability to the collagen matrix; 2. A 6×His tag, facilitating affinity purification of the fusion protein; 3. A TIMP-2 / FGF2 fusion structure, where the TIMP-2 portion inhibits matrix metalloproteinase activity, while the FGF2 portion promotes angiogenesis and cell proliferation. The β-globin poly(A) signal below the terminal of the TIMP-2 / FGF2 fusion structure ensures efficient processing and stability of the mRNA. The densely distributed restriction endonuclease sites on the outer edge of the map (such as Eco53kI / SacI, PaeR7I / XhoI, BamHI, XbaI, SnaBI, SspI, PspFI, etc.) provide flexible restriction site selection for subsequent molecular cloning and construction, facilitating site-specific insertion, deletion, or replacement of fusion frames, thereby meeting the needs of functional optimization and diversified design.

[0069] Experimental Example 3 This experimental example verifies the in vitro angiogenesis function of the collagen nerve repair material prepared in Example 3. The steps include: The angiogenic function of the material was evaluated using the Matrigel tubular structure formation assay. Human umbilical vein endothelial cells (HUVECs) were prepared at a ratio of 2 × 10⁶ cells / year. 4 Cells / well were seeded in 48-well plates pre-coated with low-growth factor Matrigel. The experiment was divided into three groups: control group (basal medium only), positive control group (20 ng / mL bFGF), and experimental group (collagen hydrogel extract containing CBD-TIMP-FGF2).

[0070] After culturing at 37°C in a 5% CO2 incubator for 12 hours, the structures were observed and photographed using a phase-contrast microscope. The total length, number of branch points, and network area of ​​the tubular structures were analyzed using ImageJ software. Figure 6As can be seen, the control group had almost no tubular structure formation; the total length of tubular structure in the bFGF group was 2658±187μm; and the total length of tubular structure in the experimental group was 2234±156μm, with a formation rate of 83.7%±2.1%, which was not significantly different from the bFGF group.

[0071] Furthermore, the bioactivity of bFGF was verified through endothelial cell proliferation assays. HUVEC cells were treated with different concentrations of collagen nerve repair material extract (0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL) for 48 h, and cell proliferation was detected by the CCK8 assay. Figure 7 It can be seen that as the concentration of collagen nerve repair material increases, the cell proliferation rate increases significantly, with the highest proliferation rate reaching 178.3%±12.7%, indicating that the released bFGF has good biological activity.

[0072] Test Example 4 This embodiment is an experiment on the repair of sciatic nerve defects in rats, and the steps include: Twenty male SD rats weighing 200-250g were randomly divided into four groups: a blank control group, an autologous transplantation group, a material treatment group, and a collagen-only group, with five rats in each group. Under sodium pentobarbital anesthesia, the left sciatic nerve was exposed, and a 10mm long nerve defect was created 1cm above the sciatic nerve bifurcation.

[0073] The blank control group only removed the nerve segment without any repair; the autologous transplantation group used the removed nerve segment to repair the defect by reverse implantation; the material treatment group used a conduit containing the collagen nerve repair material prepared in Example 3 to bridge the defect; the simple collagen group used a collagen conduit without CBD-TIMP-FGF2 fusion protein for repair; the animals' general condition and foot function were observed regularly after surgery.

[0074] Sciatic nerve function index (SFI) was measured at 4, 8, and 12 weeks post-surgery. Rat footprint length, width, and stride distance were recorded by having them walk on a narrow path, and SFI values ​​were calculated using a formula. Figure 8 It can be seen that the SFI recovery in the material treatment group was significantly better than that in the blank control group and the collagen-only group, and approached the level of the autologous transplantation group. At 12 weeks, the SFI in the material treatment group was -23.4±5.2, and that in the autologous transplantation group was -18.7±4.1.

[0075] Nerve conduction velocity (NCV) was measured 12 weeks post-surgery. Figure 9 As can be seen, the NCV of the material treatment group was 31.2±3.8 m / s; under sodium pentobarbital anesthesia, the motor nerve conduction velocity and compound muscle action potential (CMAP) of the repaired nerve were measured using an electrophysiological instrument. Figure 10The results show that the CMAP amplitude in the material treatment group was 8.7±1.2 mV, the CMAP amplitude in the blank control group was 3.2±0.8, and the CMAP amplitude in the collagen-only group was 4.5±0.9. The NCV in the material treatment group recovered to 68.3% of the normal level, and the CMAP recovered to 72.5% of the normal level (the recovery percentage was obtained by dividing the measured value (NCV or CMAP) of the material treatment group by the baseline value under normal healthy conditions and then multiplying by 100%). Moreover, the CMAP amplitude in the material treatment group was significantly higher than that in the blank control group and the collagen-only group.

[0076] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A collagen nerve repair material based on controlled release of fusion protein, characterized in that, include: Type I collagen matrix; The CBD-TIMP-FGF2 fusion protein includes a collagen-binding domain, an MMP-cleavable TIMP peptide, and basic fibroblast growth factor. The CBD-TIMP-FGF2 fusion protein binds to the type I collagen matrix to form an MMP-responsive controlled-release system, releasing the basic fibroblast growth factor through the cleavage of the TIMP peptide. In the CBD-TIMP-FGF2 fusion protein, the amino acid sequence of CBD is shown in SEQ ID NO.1, the amino acid sequence of the TIMP peptide is shown in SEQ ID NO.2, the amino acid sequence of FGF2 is shown in SEQ ID NO.3, and the complete amino acid sequence of CBD-TIMP-FGF2 is shown in SEQ ID NO.

4. The TIMP peptide includes the common recognition sequence PLGLAG or GPQGIAGQ for MMP-2 and MMP-9. The molecular weight of the CBD-TIMP-FGF2 fusion protein is 35-40 kDa, and the loading amount of the CBD-TIMP-FGF2 fusion protein in the type I collagen matrix is ​​10-50 μg / mL. The tensile strength of the collagen nerve repair material is 0.1-1.0 MPa, and the elastic modulus is 10-100 kPa. The degradation cycle of the collagen nerve repair material is 8-12 weeks, and the degradation products are amino acids and small peptides. The type I collagen matrix is ​​derived from the tendon, skin, or bone matrix of young animals under 6 months of age, and retains the natural triple helix structure and biological activity of collagen, with a protein retention rate of ≥85%, a molecular weight of 280-320kDa, and an α1 / α2 chain ratio of 2:

1. The collagen nerve repair material is a nerve conduit used to bridge peripheral nerve defects. The inner diameter of the nerve conduit is 1-15 mm, the wall thickness is 0.5-2 mm, and the length is 10-80 mm. The inner surface of the nerve conduit is provided with longitudinal guide grooves or filled fiber scaffolds to guide the directional growth of axons.

2. A method for preparing a collagen nerve repair material based on controlled release of a fusion protein as described in claim 1, characterized in that, include: Type I collagen powder was dissolved in acetic acid solution to obtain collagen solution; Subsequently, the CBD-TIMP-FGF2 fusion protein was added, and the pH was adjusted to 7.0-7.4 to obtain the collagen nerve repair material.

3. The preparation method according to claim 2, characterized in that, The preparation of the CBD-TIMP-FGF2 fusion protein includes: A recombinant expression vector containing the collagen-binding domain, the TIMP peptide, and the basic fibroblast growth factor coding sequence was constructed. The vector was transformed into Escherichia coli BL21 or transfected into mammalian cells for protein expression. After purification, the CBD-TIMP-FGF2 fusion protein was obtained.

4. The preparation method according to claim 3, characterized in that, include: The CBD-TIMP-FGF2 fusion protein is added to a final concentration of 20-100 μg / mL; and / or the concentration of the collagen solution is 10-20 mg / mL.

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