Type I collagen composition for tissue repair and its preparation method

Abstract

This invention belongs to the field of biomedical materials and provides a type I collagen composition for tissue repair and its preparation method. The invention employs a synergistic design of collagen microfiber bundles encapsulating an oxidized dextran-ε-poly-L-lysine nanogel core-shell intermediate with carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. Through the core-shell structure, early crosslinking between components is inhibited during storage while rapidly forming a three-dimensional network after injection. This achieves dual properties: low viscosity injectability and in-situ gelation within 2-5 minutes at 37°C with a storage modulus of 800-2500 Pa. It solves the problems of traditional aqueous injectable biomaterials, such as the contradiction between storage stability and rapid gelation during use, insufficient mechanical support under isotonic conditions, and uncontrollable molecular weight distribution of degradation products. It has broad application value in tissue repair, soft tissue filling, and drug sustained-release carriers.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a type I collagen composition for tissue repair and its preparation method. Background Technology

[0002] Tissue repair is a core challenge in regenerative medicine, particularly in applications such as soft tissue defect repair, wound healing promotion, and cosmetic fillers. This places multi-dimensional and mutually constraining demands on the performance of biomaterials. Ideal tissue repair materials must maintain low viscosity during storage for easy injection, rapidly form a three-dimensional network with sufficient mechanical strength to provide immediate support after injection, and simultaneously undergo controllable degradation in the physiological environment, releasing biocompatible small molecule products. These materials must not only meet the kinetic match between immediate injectability and gelation rate but also maintain network stability in an isotonic saline environment to resist the damage to structural integrity caused by fluctuations in osmotic pressure and ionic strength, and avoid the formation of high-molecular-weight aggregates that trigger inflammatory responses during degradation. Achieving these performance requirements is crucial for broadening the application scope of materials in precision medicine, minimally invasive treatments, and functional repair; improving clinical efficacy and patient compliance; and promoting the development of tissue engineering and regenerative medicine towards personalization and intelligence.

[0003] The current state of material development has several shortcomings, primarily in the following aspects: Traditional collagen hydrogels require increased concentration or the introduction of strong cross-linking agents to achieve rapid gelation, resulting in excessively high viscosity during storage, making injection difficult. Conversely, reducing the concentration extends the gelation time to tens of minutes, failing to meet immediate support requirements. Multi-component composite systems are prone to phase separation or flocculation due to electrostatic interactions when stored at 2-8℃, exhibiting poor batch-to-batch stability. Under isotonic physiological conditions, the electrostatic cross-linking network of existing materials weakens due to ion shielding effects, resulting in a storage modulus after gelation often below 500 Pa, making it difficult to provide effective mechanical support. During material degradation, uneven cross-linking density often leads to the release of products containing a large number of medium- to high-molecular-weight fragments, which can easily trigger inflammatory responses. For example, Chinese Patent CN119909007A discloses an injectable hydrogel with redox and electroactivity, its preparation method, and its application, but it suffers from uncontrollable gelation time and a wide molecular weight distribution of degradation products. For example, Chinese patent CN120392647A discloses a small extracellular vesicle composite collagen hydrogel injection, but it fails to resolve the contradiction between storage stability and rapid gelation during use. Summary of the Invention

[0004] The purpose of this invention is to provide a type I collagen composition for tissue repair and its preparation method, which solves the problems of current water-based injectable biomaterials, such as the difficulty in balancing low viscosity injectability with rapid in-situ gelation at 37°C and obtaining a high storage modulus; the inherent contradiction between phase stability and anti-settling during storage at 2-8°C and the formation of a continuous gel network through strong interactions during use; and the coupling conflict between maintaining early mechanical support in an isotonic saline environment and achieving controllable degradation and release of low molecular weight products mainly composed of monosaccharides and oligosaccharides, resulting in a narrow processing window, insufficient batch-to-batch consistency, and unstable performance.

[0005] This invention adopts the design concept of "core-shell structure spatial isolation + multi-scale network synergistic construction". Collagen microfiber bundles are used to encapsulate oxidized dextran-ε-poly-L-lysine nanogel to form a core-shell intermediate. During storage, the steric hindrance of the shell layer is used to inhibit the early cross-linking between collagen and free polysaccharides. After injection, the shell nanogel is released and rapidly forms an ion-covalent double cross-linking network with carboxymethyl dextran, low molecular weight heparin salt and micro-crosslinked sodium hyaluronate microgel. This achieves decoupling control of storage stability and gelation kinetics, and synergistic optimization of high modulus support and controllable enzymatic degradation under isotonic conditions.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A type I collagen composition for tissue repair, the composition being an aqueous system with a pH of 6.8-7.4, comprising the following components based on 100 parts by weight of the total composition; 0.8-1.5 parts by weight of collagen microfibril intermediate 1.0-2.5 parts by weight of collagen microfiber bundles encapsulating the core-shell intermediate of oxidized dextran-ε-poly-L-lysine nanogel 0.5-1.0 parts by weight of carboxymethyl dextran 0.1-0.3 parts by weight of low molecular weight heparin salt 0.3-0.8 parts by weight of micro-crosslinked sodium hyaluronate microgel The remainder is an isotonic phosphate buffer solution containing sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, such that the osmotic pressure of the aqueous system is 270-320 mOsm·kg⁻¹. - ¹; The collagen microfiber intermediate, the core-shell intermediate of the collagen microfiber-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all measured by their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of the collagen microfiber-encapsulated oxidized dextran-ε-poly-L-lysine nanogel is 160-460 nm, the shell thickness is 20-80 nm, and the weight percentage of oxidized dextran in the shell is 20-50% based on the total dry matter mass of the shell.

[0007] Furthermore, the preparation method of the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel includes the following steps: A1. The collagen microfibril intermediate is dispersed in purified water at 4-10℃ to obtain a collagen microfibril suspension with a solid content of 0.5-2.0 wt% based on the total mass of the suspension. A2. The oxidized dextran-ε-poly-L-lysine nanogel intermediate is added to the collagen microfibril suspension, and the mass ratio is 0.5-3:1, based on the ratio of the mass of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfibril intermediate. A3. The pH value was controlled to 6.8-7.2 and allowed to stand for 10-30 minutes to obtain the core-shell intermediate of collagen microfibrils-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; A4. The core-shell intermediate is kept in a suspended state or freeze-dried to obtain a powder intermediate.

[0008] Furthermore, the preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate includes the following steps: B1. Dissolve oxidized dextran in an isotonic phosphate buffer solution, wherein the isotonic phosphate buffer solution is a phosphate buffer solution with a pH of 7.0-7.4, to prepare an oxidized dextran solution with a mass concentration of 20-40 mg / mL; B2. Dissolve ε-poly-L-lysine in the isotonic phosphate buffer solution described in step B1 to prepare an ε-poly-L-lysine solution with a mass concentration of 5-15 mg / mL; B3. Under conditions of 4-10℃ and pH value of 7.0-7.4, the ε-poly-L-lysine solution is added dropwise to the oxidized dextran solution over 10-30 min, so that the molar ratio of aldehyde group to primary amine group is 0.6-1.0∶1; B4. Stir the reaction for 20-40 minutes to form a nanogel; B5. Remove free small molecules and uncrosslinked ε-poly-L-lysine by dialysis or ultrafiltration; B6. The solid content of the nanogel suspension is adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution is measured to confirm that the particle size D50 of the nanogel suspension is 80-150 nm and the PDI is not greater than 0.25.

[0009] Furthermore, the preparation method of oxidized dextran includes the following steps: C1. Dissolve dextran in purified water to obtain a dextran solution with a mass concentration of 50-150 g / L; C2. Under conditions of 0-10℃ and protection from light, add 20-100 g / L sodium periodate aqueous solution dropwise to the dextran solution, so that the molar ratio of sodium periodate to repeating glucose units in dextran is 0.1-0.5:1, adjust the pH value to 3.0-4.5, and stir the reaction for 1-3 hours. C3. After the reaction is complete, remove inorganic salts and small molecules by dialysis: Place the reaction solution in a dialysis bag with a molecular weight cutoff of 3.5-10 kDa, and dialyze it against purified water at 2-8℃ for 24-72 hours. Change the dialysis solution every 6-12 hours for a total of 3-8 times. C4. The dextran oxide powder was obtained by freeze drying and its aldehyde oxidation degree was determined to be 15-30 mol.

[0010] Furthermore, the preparation method of the collagen microfibril intermediate includes the following steps: D1. Dissolve bovine type I collagen in 0.01-0.05 mol / L hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 3-6 mg / mL; D2. Add 0.1-0.5 mol / L sodium hydroxide solution dropwise to the acidic collagen solution to adjust the pH value to 7.2-7.4, and incubate at 37±1℃ for 1-3 h to induce collagen fiber self-assembly; D3. Apply the resulting fiber suspension for 500-800 seconds. - Shear at a shear rate of ¹ for 2-5 minutes to obtain a collagen microfibril suspension; D4. Freeze-dry the collagen microfiber bundle suspension to obtain a collagen microfiber bundle freeze-dried sponge.

[0011] Furthermore, the collagen microfibril intermediate is made from bovine type I collagen, and the diameter D50 of a single fiber is 50-300 nm, the fiber length is 5-30 μm, and the collagen triple helix structure retention rate is not less than 80%.

[0012] Furthermore, the oxidized dextran-ε-poly-L-lysine nanogel intermediate satisfies the following conditions: The oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 15-30 mol%; The number-average molecular weight of the ε-poly-L-lysine is 2000-5000; The nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction. The particle size D50 is 80-150 nm, the polydispersity index PDI is not greater than 0.25, and the zeta potential is +10 to +30 mV.

[0013] Furthermore, the degree of carboxymethyl substitution of the carboxymethyl dextran is 0.4-0.9, and the low molecular weight heparin salt is selected from one or more of enoxaparin sodium, dalteparin sodium, or their pharmacopoeia equivalents.

[0014] Furthermore, the micro-crosslinked sodium hyaluronate microgel is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. This crosslinked hyaluronic acid gel is then subjected to mechanical shearing or microfluidic pulverization to form a dry powder or wet microgel. In the dry powder state, the particle size D50 is 1-50 μm. The composition is subjected to mechanical shearing or microfluidic pulverization at 25°C and a shear rate of 100 s⁻¹. - The viscosity under the conditions is 0.5-2.0 Pa·s, and the hydrogel formed after the composition is injected into physiological conditions at 37°C has a storage modulus G′ of 800-2500 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides and oligosaccharides in the low molecular weight products released during the degradation of the composition is not less than 80%.

[0015] As a concept of this invention, the design of a core-shell structure of oxidized dextran-ε-poly-L-lysine nanogel encapsulated by collagen microfiber bundles is mainly used to enhance the storage stability of the composition, the controllability of injection molding kinetics, and the mechanical support properties under isotonic conditions. Specifically, the collagen microfiber bundles serve as the core, providing a fiber-reinforced framework, and their retained triple helix structure ensures biocompatibility and cell recognition ability; the oxidized dextran-ε-poly-L-lysine nanogel serves as the shell, forming a steric barrier during storage through the electrostatic interaction of Schiff base dynamic covalent bonds and positive charge, inhibiting early crosslinking of collagen microfiber bundles with free carboxymethyl dextran and low molecular weight heparin salts, thus avoiding increased system viscosity and phase separation. Upon injection into a physiological environment of 37°C, the shell nanogel partially releases due to the temperature rise and shearing action. The aldehyde groups it carries can undergo Schiff base reactions with primary amine groups in the system (such as those derived from ε-poly-L-lysine and collagen side chain amino groups) to form dynamic covalent bonds. Simultaneously, the positive charge of ε-poly-L-lysine electrostatically pairs with the carboxyl groups on the surface of carboxymethyl dextran and sodium hyaluronate microgels, constructing a multi-scale cross-linked network of collagen fibers-nanogel-polysaccharide microgels within 2-5 minutes. This results in a jump in storage modulus from <1 Pa during storage to 800-2500 Pa after gelation. Furthermore, the 20-50% oxidized dextran content in the shell ensures sufficient aldehyde groups remain for cross-linking after release; too low a content will lead to incomplete gelation, while too high a content will cause premature cross-linking during storage. The matching of the core-shell cross-sectional outer diameter of 160-460nm and the shell thickness of 20-80nm ensures that the shell can effectively cover the surface and respond quickly to environmental changes. Furthermore, the geometric constraint that the shell thickness is less than half of the outer diameter prevents the shell from hindering cross-linking due to being too thick or losing its isolation effect due to being too thin. This invention also discloses a method for preparing the above-mentioned injectable type I collagen composition for tissue repair, comprising the following steps: S1 provides collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution. S2 was used to prepare the first and second components at 2-8℃. S3 fills the first component and the second component into a double-barrel syringe or a two-way needle device with a Y-type mixer, so that the contents of the two barrels are mixed at the tip of the needle during injection to form the injectable type I collagen composition. S4 injects the mixed system into a simulated physiological environment or ex vivo tissue model at 37±1℃ and completes in-situ gelation within 1-5 minutes.

[0016] Furthermore, the carboxymethyl dextran is carboxymethyl-β-glucan, wherein the main chain of the β-glucan is a β-1,3-glucosidic bond and contains a β-1,6-branched structure, and the degree of carboxymethyl substitution is 0.5-0.85.

[0017] Furthermore, the number average molecular weight of the carboxymethyl dextran is 5kDa-500kDa, preferably 10kDa-200kDa, so as to obtain a clear or semi-transparent dispersion system without visible insoluble matter in isotonic phosphate buffer solution.

[0018] Furthermore, the apparent viscosity of the carboxymethyl dextran at 25°C, based on a 1.0 wt% aqueous solution, is 10-200 mPa·s, preferably 20-120 mPa·s.

[0019] Furthermore, in step S2, the first component further includes the carboxymethyl dextran, the low molecular weight heparin salt, and the micro-crosslinked sodium hyaluronate microgel, while the second component does not contain the low molecular weight heparin salt or the carboxymethyl dextran, in order to reduce the risk of flocculation caused by polyelectrolyte complexation during storage.

[0020] Furthermore, when the first component and the second component are respectively filled into the dual-barrel syringe, the filling volume ratio of the two barrels is 1:1, and the dual-barrel syringe is connected to the static mixer before being connected to the needle. The built-in mixing unit of the static mixer has 6-24 sections.

[0021] Furthermore, the isotonic phosphate buffer solution has a pH of 7.1-7.3 and an osmotic pressure of 285-310 mOsm·kg⁻¹. - ¹, to improve the phase consistency after mixing different batches of materials.

[0022] Furthermore, the composition does not show visible sedimentation or stratification after standing at 2-8°C for 24 hours, and after 100 seconds... - ¹The appearance returned to uniformity after shearing for 60 seconds, which was used as a phase criterion for the system's resistance to sedimentation and its recoverability.

[0023] Furthermore, the composition reaches a self-supporting state within 2 minutes after injection at 37±1℃, and the storage modulus G′ at 1Hz reaches more than 800Pa within 3-10 minutes.

[0024] Furthermore, the introduction of carboxymethyl dextran ensures that the weight ratio of monosaccharides and oligosaccharides in the 3kDa cut-off permeate of the composition degradation solution is not less than 85%, and the oligosaccharides are mainly disaccharides to hexasaccharides.

[0025] Furthermore, the carboxyl group content of the carboxymethyl dextran is 1.0-4.0 mmol / g, preferably 1.5-3.0 mmol / g, calculated by titration, in order to maintain the ionic stability of the gel network after injection without significantly increasing the initial viscosity.

[0026] Furthermore, the outer diameter of the cross-section of the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel satisfies the condition that the shell thickness is less than 1 / 2 of the outer diameter of the cross-section.

[0027] As another aspect of this invention, the two-component dispensing-instant mixing gelation method is primarily used to enhance the system's storage stability, batch-to-batch consistency, and ease of clinical operation. Specifically, the first component consists of collagen microfibril intermediates, carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. The second component consists of oxidized dextran-ε-poly-L-lysine nanogel intermediates. Physical isolation during storage completely prevents early crosslinking of aldehyde and primary amine groups, eliminating viscosity fluctuations caused by differences in the activity of raw materials from different batches. The combined use of a dual-barrel syringe and a static mixer ensures thorough mixing of the two components at the moment of injection. The 6-24 mixing units achieve nanosecond-level homogenization through splitting-recombining-shearing, making the gelation kinetics controllable within 2-5 minutes, avoiding the uncontrollable gelation time caused by pre-crosslinking in single-component systems. The isotonic phosphate buffer solution has a pH of 7.1-7.3 and an osmotic pressure of 285-310 mOsm·kg⁻¹. - ¹ Precise control ensures the mixed system rapidly reaches physiological ionic strength, triggering a synergistic acceleration of Schiff base reaction and electrostatic crosslinking, while simultaneously inhibiting collagen denaturation or nanogel aggregation caused by pH fluctuations. The rapid response characteristic—reaching a self-sustaining state within 2 minutes after injection and achieving a storage modulus G′ exceeding 800 Pa within 3-10 minutes—originates from the rapid crosslinking kinetics of the core-shell structure-released nanogel with carboxymethyl dextran and sodium hyaluronate microgel at 37°C. The introduction of carboxymethyl dextran not only provides ionic crosslinking sites through the carboxyl group, but its β-1,3-glucosinolate backbone and β-1,6-branched structure endow the degradation products with a molecular weight distribution dominated by monosaccharides and oligosaccharides (≥85%). Compared to the high molecular weight fragments of traditional highly crosslinked hyaluronic acid gel degradation products, this significantly reduces the risk of inflammation and promotes tissue remodeling.

[0028] In this invention, the core-shell intermediate of collagen microfibrils encapsulating oxidized dextran-ε-poly-L-lysine nanogel and carboxymethyl dextran play different but highly synergistic roles. The core-shell intermediate primarily provides a fiber-reinforcing framework and dynamically controllable cross-linking sites. The triple-helix structure retained by the collagen microfibrils endows the composition with excellent biocompatibility and cell adhesion properties. The aldehyde and primary amine groups carried by the shell nanogel form a steric barrier during storage and are rapidly released after injection to participate in the Schiff base reaction and construct a covalent cross-linked network. The main function of carboxymethyl dextran is to provide ionic cross-linking sites through its carboxyl groups and regulate the molecular weight distribution of degradation products. Its unique topological structure of β-1,3-main chain and β-1,6-branchs preferentially breaks down into disaccharide to hexasaccharide oligosaccharide fragments rather than high molecular weight aggregates during enzymatic hydrolysis. In terms of improving injection molding speed and storage modulus, the nanogel released from the core-shell intermediate forms dynamic covalent bonds through Schiff base reactions between aldehyde and primary amine groups. Furthermore, its positive charge electrostatically pairs with the carboxyl groups of carboxymethyl dextran to synergistically stabilize the network. Simultaneously, the positive charge zeta potential (+10 to +30 mV) and the electrostatic interaction with the carboxyl groups synergistically stabilize the crosslinked network, causing the storage modulus G′ to jump from <1 Pa to 800-2500 Pa within 3-10 minutes. The introduction of carboxymethyl dextran further enhances the modulus by increasing the crosslinking density and the number of ion pairings, and inhibits network swelling under isotonic conditions. Regarding maintaining mechanical stability under isotonic conditions, the fiber-nanogel dual-scale network provided by the core-shell structure forms a high-density ion crosslinking region with the multiple carboxyl sites of carboxymethyl dextran, resisting the shielding effect of sodium and phosphate ions in phosphate buffer. The synergistic effect of the two is reflected in the following aspects: the aldehyde groups released from the core and shell form Schiff base dynamic covalent links with the primary amine groups, while the positive charge of ε-poly-L-lysine forms electrostatic pairing with the carboxyl groups of carboxymethyl dextran. Compared with a single electrostatic cross-linking system, this significantly improves the network's resistance to ionic strength fluctuations. At the same time, the branched structure of carboxymethyl dextran and the spherical particles of the nanogel complement each other in space, forming a dense and uniform three-dimensional network, avoiding local stress concentration and early degradation caused by uneven cross-linking.

[0029] Beneficial technical effects 1. Significantly enhanced decoupling control of storage stability and injection gelation kinetics: Through the spatial isolation design of the core-shell structure of oxidized dextran-ε-poly-L-lysine nanogels encapsulated by collagen microfiber bundles, the early crosslinking of aldehyde and primary amine groups is inhibited by the steric barrier of the shell layer during storage. This ensures that the composition does not exhibit visible sedimentation or stratification after standing at 2-8℃ for 24 hours and maintains an injectable viscosity of <2 Pa·s, solving the phase separation and batch-to-batch viscosity fluctuation problems caused by polyelectrolyte complexation in traditional multi-component systems. After injection into a physiological environment of 37℃, the shell nanogel is rapidly released and forms a Schiff base-ion double crosslinking network with carboxymethyl dextran and micro-crosslinked sodium hyaluronate microgels, achieving a rapid gelation response with the storage modulus G′ jumping from <1 Pa to 800-2500 Pa within 2-5 minutes.

[0030] 2. Effectively enhances mechanical support and network stability under isotonic physiological conditions: Positively charged nanogels (ζ-potential +10 to +30 mV) released from the core-shell intermediate form high-density ion pairings with the carboxyl groups of carboxymethyl dextran (content 1.0-4.0 mmol / g), forming a dynamic covalent cross-linked network constructed by the aldehyde groups of oxidized dextran and the primary amine groups of ε-poly-L-lysine. This network is effective in isotonic phosphate buffer (osmolarity 270-320 mOsm·kg⁻¹). - ¹) It resists the shielding effect of sodium and phosphate ions, so that the storage modulus G′ after gelation is stably maintained in the range of 800-2500 Pa at an oscillation frequency of 1 Hz. Compared with the phenomenon that the modulus of traditional single electrostatic crosslinking system decays to <500 Pa under isotonic environment, it significantly improves the early mechanical support capacity and prolongs the network stability period.

[0031] 3. Achieve controllable degradation and release of biocompatible products mainly composed of monosaccharides and oligosaccharides: By introducing the β-1,3-glucosinolate backbone and β-1,6-branched structure of carboxymethyl glucan, it is preferentially broken into oligosaccharide fragments from disaccharides to hexasaccharides during enzymatic hydrolysis. This ensures that the weight ratio of monosaccharides and oligosaccharides in the permeate after the degradation solution is 3 kDa cutoff is not less than 85%, avoiding the inflammatory response and foreign body granuloma formation caused by high molecular weight fragments (>10 kDa) in the degradation products of traditional highly cross-linked hyaluronic acid gels. At the same time, the small molecule oligosaccharides of the degradation products can be rapidly metabolized by tissues and promote extracellular matrix remodeling and angiogenesis.

[0032] 4. Optimize the preparation process of two-component dispensing and instant mixing to improve batch-to-batch consistency and clinical convenience: By using a dual-barrel syringe dispensing strategy, the first component is formulated with collagen microfibril intermediates, carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel, while the second component is oxidized dextran-ε-poly-L-lysine nanogel intermediate. This strategy completely avoids early reactions of aldehyde and carboxyl groups during storage, eliminates fluctuations in gelation time caused by differences in the activity of raw materials from batch to batch, and achieves nanosecond-level uniform mixing at the moment of injection with a 6-24 section static mixer. This ensures that the gelation kinetics are controllable within 2-5 minutes and the relative standard deviation of the storage modulus G′ between different batches is <10%, significantly improving the reproducibility and safety of clinical operations.

[0033] 5. Constructing a multi-scale fiber-nanogel-microgel synergistic cross-linking network to balance mechanical properties and bioactivity: Collagen microfiber bundles (diameter D50 50-300 nm, length 5-30 μm) provide a fiber-reinforced framework, oxidized dextran-ε-poly-L-lysine nanogels (particle size D50 80-150 nm) serve as dynamic cross-linking nodes, and micro-cross-linked sodium hyaluronate microgels (particle size D50 1-50 μm) fill the network gaps, forming a three-dimensional network structure spanning nano- to micro-scales. This structure retains the cell recognition sites of the collagen triple helix structure (retention rate ≥80%) and the growth factor binding ability of low molecular weight heparin salts, while achieving high modulus support of 800-2500 Pa through multi-scale cross-linking, thus meeting the dual requirements of mechanical strength and bioactivity for soft tissue repair. Attached Figure Description

[0034] Figure 1 This is an XPS full spectrum overlay of Example 1 and Comparative Example 8.

[0035] Figure 2 This is a high-resolution overlay image of XPS C1s of Example 1 and Comparative Example 8.

[0036] Figure 3 This is a high-resolution overlay image of XPS O 1s of Example 1 and Comparative Example 8.

[0037] Figure 4 This is a high-resolution XPS N 1s overlay image of Example 1 and Comparative Example 8.

[0038] Figure 5 The rheological time-scan gelation kinetics G′ versus time are plots for Example 1, Comparative Example 1, and Comparative Example 2.

[0039] Figure 6 The rheological time-scan gelation kinetics G″ versus time plots for Example 1, Comparative Example 1, and Comparative Example 2.

[0040] Figure 7This is an overlay of the HPLC-ELSD chromatograms of Example 1 and Comparative Example 6.

[0041] Figure 8 The HPLC-ELSD cumulative area distribution CDF plots for Example 1 and Comparative Example 6 are shown.

[0042] Figure 9 This is a stacked column chart of HPLC-ELSD component proportions for Example 1 and Comparative Example 6. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0044] Example 1 This embodiment provides a type I collagen composition for tissue repair, which is an aqueous system with a pH of 7.1. Based on a total mass of 100 parts by weight, the composition comprises the following components: 1.15 parts by weight of collagen microfibril intermediates, 1.75 parts by weight of a core-shell intermediate of collagen microfibrils coated with oxidized dextran-ε-poly-L-lysine nanogel, 0.75 parts by weight of carboxymethyl dextran, 0.20 parts by weight of low molecular weight heparin salt, 0.55 parts by weight of micro-crosslinked sodium hyaluronate microgel, and the balance being an isotonic phosphate buffer solution. The isotonic phosphate buffer solution in this embodiment contains sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, resulting in an osmotic pressure of 295 mOsm·kg⁻¹ for the aqueous system. - ¹.

[0045] In this embodiment, the collagen microfiber bundle intermediate, the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all based on their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment is 310 nm, the shell thickness is 50 nm, and based on the total dry matter mass of the shell, the weight percentage of oxidized dextran in the shell is 35%.

[0046] The preparation method of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment includes the following steps: at 7°C, the collagen microfiber bundle intermediate is dispersed in purified water to obtain a collagen microfiber bundle suspension with a solid content of 1.2 wt% based on the total mass of the suspension; the oxidized dextran-ε-poly-L-lysine nanogel intermediate is added to the collagen microfiber bundle suspension, and the mass ratio is 1.5:1 based on the mass ratio of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfiber bundle intermediate; the pH value is controlled at 7.0 and allowed to stand for 20 min to obtain the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; the core-shell intermediate is kept in suspension or freeze-dried to obtain a powder intermediate.

[0047] The preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate in this embodiment includes the following steps: oxidized dextran is dissolved in an isotonic phosphate buffer solution with a pH of 7.2 to prepare an oxidized dextran solution with a mass concentration of 30 mg / mL; ε-poly-L-lysine is dissolved in an isotonic phosphate buffer solution with a pH of 7.2 to prepare an ε-poly-L-lysine solution with a mass concentration of 10 mg / mL; and the solution is prepared at 7°C and pH... Under the condition of H value 7.2, ε-poly-L-lysine solution was added dropwise to oxidized dextran solution over 20 min to make the molar ratio of aldehyde group to primary amine group 0.8:1; the reaction was stirred for 30 min to form nanogel; free small molecules and uncrosslinked ε-poly-L-lysine were removed by dialysis or ultrafiltration; the solid content of the nanogel suspension was adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution was measured to confirm that the particle size D50 of the nanogel suspension was 115 nm and the PDI was 0.20.

[0048] The preparation method of oxidized dextran in this embodiment includes the following steps: dextran is dissolved in purified water to obtain a dextran solution with a mass concentration of 100 g / L; under the condition of 5°C and protection from light, 20 g / L sodium periodate aqueous solution is added dropwise to the dextran solution to make the molar ratio of sodium periodate to repeating glucose units in dextran 0.3:1, the pH is adjusted to 3.8, and the reaction is stirred for 2 h; after the reaction is completed, inorganic salts and small molecules are removed by dialysis: the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed against purified water at 4°C for 48 h, with the dialysate replaced every 8 h for a total of 6 times; oxidized dextran powder is obtained by freeze drying, and its aldehyde oxidation degree is determined to be 22 mol.

[0049] The preparation method of the collagen microfibril intermediate in this embodiment includes the following steps: bovine type I collagen is dissolved in 0.01 mol / L dilute hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 4.5 mg / mL; 0.1 mol / L sodium hydroxide solution is added dropwise to the acidic collagen solution to adjust the pH value to 7.3, and incubated at 37°C for 2 h to induce collagen fiber self-assembly; the obtained fiber suspension is subjected to a 650s... - ¹ Shearing rate for 3.5 min yielded a collagen microfiber suspension; the collagen microfiber suspension was freeze-dried to obtain a collagen microfiber freeze-dried sponge.

[0050] The collagen microfibril intermediate in this embodiment was prepared from bovine type I collagen, and the diameter D50 of a single fiber was 175 nm, the fiber length was 17.5 μm, and the collagen triple helix structure retention rate was 88%.

[0051] The oxidized dextran-ε-poly-L-lysine nanogel intermediate of this embodiment meets the following conditions: the oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 22 mol%; the number average molecular weight of ε-poly-L-lysine is 3500; the nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction, with a particle size D50 of 115 nm, a polydispersity index PDI of 0.20, and a zeta potential of +20 mV.

[0052] The carboxymethyl dextran of this embodiment has a carboxymethyl substitution degree of 0.68, and the low molecular weight heparin salt is enoxaparin sodium. The carboxymethyl dextran of this embodiment is carboxymethyl-β-glucan, with the β-glucan backbone consisting of β-1,3-glucosidic bonds and containing β-1,6-branched structures, and a carboxymethyl substitution degree of 0.68. The number-average molecular weight of the carboxymethyl dextran of this embodiment is 105 kDa. The apparent viscosity of the carboxymethyl dextran of this embodiment, calculated as a 1.0 wt% aqueous solution at 25°C, is 70 mPa·s. The carboxyl content of the carboxymethyl dextran of this embodiment, calculated by titration, is 2.2 mmol / g.

[0053] The micro-crosslinked sodium hyaluronate microgel of this embodiment is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. The crosslinked hyaluronic acid gel is mechanically sheared or microfluidically pulverized to form dry powder or wet microgel. The particle size D50 in the dry powder state is 25 μm, and the composition is subjected to a shear rate of 100 s at 25°C. - The viscosity under the conditions of ¹ is 1.25 Pa·s. The hydrogel formed after the composition is injected into the physiological conditions of 37°C has a storage modulus G′ of 1650 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides to oligosaccharides in the low molecular weight products released during the degradation of the composition is 87%.

[0054] The preparation method of the injectable type I collagen composition for tissue repair in this embodiment includes the following steps: providing collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution; preparing a first component and a second component at 2-8°C, wherein the first component includes the collagen microfibril intermediates and the second component includes oxidized dextran-ε-poly-L-lysine nanogel intermediates; filling the first component and the second component into a double-barrel syringe or a two-way syringe device with a Y-type mixer, so that the contents of the two syringes are mixed at the tip of the needle during injection to form an injectable type I collagen composition; injecting the mixed system into a simulated physiological environment or an ex vivo tissue model at 37°C to complete in-situ gelation within 3 minutes.

[0055] In the preparation method of this embodiment, the first component further includes carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. The second component does not contain low molecular weight heparin salt or carboxymethyl dextran to reduce the risk of flocculation caused by polyelectrolyte complexation during storage. In this embodiment, when the first and second components are filled into a dual-barrel syringe, the filling volume ratio of the two barrels is 1:1. The dual-barrel syringe is connected to a static mixer before being connected to the needle. The static mixer has 15 built-in mixing units. The isotonic phosphate buffer solution in this embodiment has a pH of 7.1 and an osmotic pressure of 295 mOsm·kg⁻¹. - ¹. The composition of this embodiment did not show visible sedimentation or stratification after standing at 2-8°C for 24 hours, and after 100 seconds... - ¹The appearance returned to uniformity after 60 seconds of shearing. The composition of this embodiment reached a self-supporting state within 2 minutes after injection at 37°C, and the storage modulus G′ at 1Hz reached 1650 Pa within 5 minutes. The introduction of carboxymethyl dextran in this embodiment resulted in a 3kDa cutoff in the degradation solution, with the weight ratio of monosaccharides to oligosaccharides in the permeate being 87%, and the oligosaccharides being predominantly disaccharides to hexasaccharides. In this embodiment, the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel has a cross-sectional outer diameter of 310 nm and a shell thickness of 50 nm, satisfying the condition that the shell thickness is less than half the cross-sectional outer diameter.

[0056] Features of Example 1: This example uses moderate proportions of component parameters: 1.15 parts by weight of collagen microfibril intermediate, 1.75 parts by weight of core-shell intermediate, 0.75 parts by weight of carboxymethyl dextran, 0.20 parts by weight of low molecular weight heparin salt, and 0.55 parts by weight of micro-crosslinked sodium hyaluronate microgel. The pH value is 7.1, and the osmotic pressure is 295 mOsm·kg. -¹, The core-shell intermediate has an outer diameter of 310 nm and a shell thickness of 50 nm, with oxidized dextran comprising 35% by weight in the shell. This formulation was tested at 25 °C and a shear rate of 100 s⁻¹. - The viscosity under the specified conditions is 1.25 Pa·s, and the storage modulus G′ at 37℃ and 1Hz oscillation frequency is 1650 Pa, exhibiting a good balance between injection performance and gel strength. The degradation products of the composition contain 87% monosaccharides and oligosaccharides by weight, demonstrating excellent biocompatibility. This embodiment features robust parameter selection and good process reproducibility, making it suitable for routine tissue repair applications such as soft tissue filling and repair, skin wound healing promotion, and superficial scar repair.

[0057] Example 2 This embodiment provides a type I collagen composition for tissue repair, which is an aqueous system with a pH of 7.0. Based on a total mass of 100 parts by weight, the composition comprises the following components: 1.35 parts by weight of collagen microfibril intermediates, 1.4 parts by weight of a core-shell intermediate of collagen microfibrils coated with oxidized dextran-ε-poly-L-lysine nanogel, 0.6 parts by weight of carboxymethyl dextran, 0.25 parts by weight of low molecular weight heparin salt, 0.7 parts by weight of micro-crosslinked sodium hyaluronate microgel, and the balance being an isotonic phosphate buffer solution. The isotonic phosphate buffer solution in this embodiment contains sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, resulting in an osmotic pressure of 282 mOsm·kg⁻¹ for the aqueous system. - ¹.

[0058] In this embodiment, the collagen microfiber bundle intermediate, the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all measured by their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment is 220 nm, the shell thickness is 32 nm, and based on the total dry matter mass of the shell, the weight percentage of oxidized dextran in the shell is 26%.

[0059] The preparation method of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment includes the following steps: at 6°C, the collagen microfiber bundle intermediate is dispersed in purified water to obtain a collagen microfiber bundle suspension with a solid content of 0.8 wt% based on the total mass of the suspension; the oxidized dextran-ε-poly-L-lysine nanogel intermediate is added to the collagen microfiber bundle suspension, and the mass ratio is 0.8:1 based on the mass ratio of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfiber bundle intermediate; the pH value is controlled at 6.9, and the mixture is allowed to stand for 15 min to obtain the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; the core-shell intermediate is kept in suspension or freeze-dried to obtain a powder intermediate.

[0060] The preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate in this embodiment includes the following steps: oxidized dextran is dissolved in an isotonic phosphate buffer solution with a pH of 7.1 to prepare an oxidized dextran solution with a mass concentration of 25 mg / mL; ε-poly-L-lysine is dissolved in an isotonic phosphate buffer solution with a pH of 7.1 to prepare an ε-poly-L-lysine solution with a mass concentration of 7 mg / mL; and the solution is prepared at 6°C and pH... Under the condition of H value 7.1, ε-poly-L-lysine solution was added dropwise to oxidized dextran solution over 15 min to make the molar ratio of aldehyde group to primary amine group 0.7:1; the reaction was stirred for 25 min to form nanogel; free small molecules and uncrosslinked ε-poly-L-lysine were removed by dialysis or ultrafiltration; the solid content of the nanogel suspension was adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution was measured to confirm that the particle size D50 of the nanogel suspension was 95 nm and the PDI was 0.18.

[0061] The preparation method of oxidized dextran in this embodiment includes the following steps: dextran is dissolved in purified water to obtain a dextran solution with a mass concentration of 80 g / L; under the condition of 4°C and protection from light, 50 g / L sodium periodate aqueous solution is added dropwise to the dextran solution to make the molar ratio of sodium periodate to repeating glucose units in dextran 0.2:1, the pH is adjusted to 3.5, and the reaction is stirred for 1.5 h; after the reaction is completed, inorganic salts and small molecules are removed by dialysis: the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed against purified water at 4°C for 36 h, with the dialysate replaced every 8 h for a total of 8 times; oxidized dextran powder is obtained by freeze drying, and its aldehyde oxidation degree is determined to be 18 mol.

[0062] The preparation method of the collagen microfibril intermediate in this embodiment includes the following steps: bovine type I collagen is dissolved in 0.02 mol / L dilute hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 5 mg / mL; 0.2 mol / L sodium hydroxide solution is added dropwise to the acidic collagen solution to adjust the pH value to 7.35, and incubated at 37°C for 1.5 h to induce collagen fiber self-assembly; the obtained fiber suspension is subjected to a 700 s... - Shearing at a shear rate of ¹ for 3 min yields a collagen microfiber suspension; the collagen microfiber suspension is freeze-dried to obtain a collagen microfiber freeze-dried sponge.

[0063] The collagen microfibril intermediate in this embodiment was prepared from bovine type I collagen, with a single fiber diameter D50 of 125 nm, a fiber length of 12 μm, and a collagen triple helix structure retention rate of 85%.

[0064] The oxidized dextran-ε-poly-L-lysine nanogel intermediate of this embodiment meets the following conditions: the oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 18 mol%; the number average molecular weight of ε-poly-L-lysine is 2800; the nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction, with a particle size D50 of 95 nm, a polydispersity index PDI of 0.18, and a zeta potential of +15 mV.

[0065] The carboxymethyl dextran of this embodiment has a carboxymethyl substitution degree of 0.58, and the low molecular weight heparin salt is dalteparin sodium. The carboxymethyl dextran of this embodiment is carboxymethyl-β-glucan, with the β-glucan backbone consisting of β-1,3-glucosidic bonds and containing β-1,6-branched structures, and a carboxymethyl substitution degree of 0.58. The number-average molecular weight of the carboxymethyl dextran of this embodiment is 65 kDa. The apparent viscosity of the carboxymethyl dextran of this embodiment, calculated as a 1.0 wt% aqueous solution at 25°C, is 45 mPa·s. The carboxyl content of the carboxymethyl dextran of this embodiment, calculated by titration, is 1.8 mmol / g.

[0066] The micro-crosslinked sodium hyaluronate microgel of this embodiment is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. The crosslinked hyaluronic acid gel is then subjected to mechanical shearing or microfluidic pulverization to form dry powder or wet microgel. The particle size D50 in the dry powder state is 35 μm, and the composition is subjected to a shear rate of 100 s at 25°C. - The viscosity under the conditions of ¹ is 0.9 Pa·s. The hydrogel formed after the composition is injected into the physiological conditions of 37°C has a storage modulus G′ of 1350 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides to oligosaccharides in the low molecular weight products released during the degradation of the composition is 84%.

[0067] The preparation method of the injectable type I collagen composition for tissue repair in this embodiment includes the following steps: providing collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution; preparing a first component and a second component at 2-8°C, the first component including collagen microfibril intermediates and the second component including oxidized dextran-ε-poly-L-lysine nanogel intermediates; filling the first component and the second component into a double-barrel syringe or a two-way needle device with a Y-type mixer, so that the contents of the two barrels are mixed at the tip of the needle during injection to form an injectable type I collagen composition; injecting the mixed system into a simulated physiological environment or ex vivo tissue model at 37°C to complete in-situ gelation within 2 minutes.

[0068] In the preparation method of this embodiment, the first component further includes carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. The second component does not contain low molecular weight heparin salt or carboxymethyl dextran to reduce the risk of flocculation caused by polyelectrolyte complexation during storage. In this embodiment, when the first and second components are filled into a dual-barrel syringe, the filling volume ratio of the two barrels is 1:1. The dual-barrel syringe is connected to a static mixer before being connected to the needle. The static mixer has 12 built-in mixing units. The isotonic phosphate buffer solution in this embodiment has a pH of 7.0 and an osmotic pressure of 282 mOsm·kg⁻¹. - ¹. The composition of this embodiment did not show visible sedimentation or stratification after standing at 2-8°C for 24 hours, and after 100 seconds... - ¹The appearance returned to uniformity after 60 seconds of shearing. The composition of this embodiment reached a self-supporting state within 2 minutes after injection at 37°C, and the storage modulus G′ at 1Hz reached 1350 Pa within 4 minutes. The introduction of carboxymethyl dextran in this embodiment resulted in a 3kDa cutoff in the degradation solution, with the weight ratio of monosaccharides to oligosaccharides in the permeate being 84%, and the oligosaccharides being predominantly disaccharides to hexasaccharides. In this embodiment, the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel had a cross-sectional outer diameter of 220 nm and a shell thickness of 32 nm, satisfying the condition that the shell thickness is less than half the cross-sectional outer diameter.

[0069] Features of Example 2: This example uses a ratio of high collagen microfibril content to low core-shell intermediate content. The collagen microfibril intermediate content is 1.35 parts by weight, the core-shell intermediate content is 1.4 parts by weight, carboxymethyl dextran is 0.6 parts by weight, low molecular weight heparin salt is 0.25 parts by weight, and micro-crosslinked sodium hyaluronate microgel is 0.7 parts by weight. The pH value is 7.0, and the osmotic pressure is 282 mOsm·kg. -¹, The core-shell intermediate has an outer diameter of 220 nm and a shell thickness of 32 nm, with oxidized dextran accounting for 26% by weight in the shell. This formulation has a relatively high collagen fiber content, with individual collagen microfibers having a diameter (D50) of 125 nm and a fiber length of 12 μm, providing a good fiber network support structure. This is effective at 25°C and a shear rate of 100 s⁻¹. - The viscosity under the specified conditions is 0.9 Pa·s, exhibiting excellent injection fluidity. Its storage modulus G′ at 37°C and a 1 Hz oscillation frequency is 1350 Pa, making it suitable for applications requiring good injectability and moderate support strength. This embodiment is applicable to cosmetic medical applications that demand high injection smoothness, such as facial soft tissue filling, tear trough filling, nasolabial fold improvement, and fine line smoothing.

[0070] Example 3 This embodiment provides a type I collagen composition for tissue repair, which is an aqueous system with a pH of 7.25. Based on a total mass of 100 parts by weight, the composition comprises the following components: 0.95 parts by weight of collagen microfibril intermediates, 2.2 parts by weight of a core-shell intermediate of collagen microfibrils coated with oxidized dextran-ε-poly-L-lysine nanogel, 0.88 parts by weight of carboxymethyl dextran, 0.15 parts by weight of low molecular weight heparin salt, 0.42 parts by weight of micro-crosslinked sodium hyaluronate microgel, and the balance being an isotonic phosphate buffer solution. The isotonic phosphate buffer solution in this embodiment contains sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, resulting in an osmotic pressure of 303 mOsm·kg⁻¹ for the aqueous system. - ¹.

[0071] In this embodiment, the collagen microfiber bundle intermediate, the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all based on their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment is 380 nm, the shell thickness is 62 nm, and based on the total dry matter mass of the shell layer, the weight percentage of oxidized dextran in the shell layer is 43%.

[0072] The preparation method of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment includes the following steps: dispersing the collagen microfiber bundle intermediate in purified water at 8°C to obtain a collagen microfiber bundle suspension with a solid content of 1.6 wt% based on the total mass of the suspension; adding the oxidized dextran-ε-poly-L-lysine nanogel intermediate to the collagen microfiber bundle suspension, with a mass ratio of 2.5:1 based on the mass of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfiber bundle intermediate; controlling the pH value to 7.1 and allowing it to stand for 25 min to obtain the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; keeping the core-shell intermediate in a suspended state or freeze-drying to obtain a powder intermediate.

[0073] The preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate in this embodiment includes the following steps: oxidized dextran is dissolved in an isotonic phosphate buffer solution with a pH of 7.3 to prepare an oxidized dextran solution with a mass concentration of 35 mg / mL; ε-poly-L-lysine is dissolved in an isotonic phosphate buffer solution with a pH of 7.3 to prepare an ε-poly-L-lysine solution with a mass concentration of 12 mg / mL; and the solution is prepared at 8°C and pH... Under the condition of H value 7.3, ε-poly-L-lysine solution was added dropwise to oxidized dextran solution over 25 min to make the molar ratio of aldehyde group to primary amine group 0.9:1; the reaction was stirred for 35 min to form nanogel; free small molecules and uncrosslinked ε-poly-L-lysine were removed by dialysis or ultrafiltration; the solid content of nanogel suspension was adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution was measured to confirm that the particle size D50 of nanogel suspension was 135 nm and the PDI was 0.22.

[0074] The preparation method of oxidized dextran in this embodiment includes the following steps: dextran is dissolved in purified water to obtain a dextran solution with a mass concentration of 125 g / L; under the condition of 7°C and protection from light, 100 g / L sodium periodate aqueous solution is added dropwise to the dextran solution to make the molar ratio of sodium periodate to repeating glucose units in dextran 0.42:1, the pH is adjusted to 4.2, and the reaction is stirred for 2.5 h; C3. After the reaction is completed, inorganic salts and small molecules are removed by dialysis: the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and the purified water is dialyzed at 4°C for 36 h, with the dialysate replaced every 8 h for a total of 4 times; the oxidized dextran powder is obtained by freeze drying, and its aldehyde oxidation degree is determined to be 27 mol.

[0075] The preparation method of the collagen microfibril intermediate in this embodiment includes the following steps: bovine type I collagen is dissolved in 0.05 mol / L dilute hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 4 mg / mL; 0.5 mol / L sodium hydroxide solution is added dropwise to the acidic collagen solution to adjust the pH value to 7.25, and incubated at 37°C for 2.5 h to induce collagen fiber self-assembly; the obtained fiber suspension is subjected to a 600s... - Shearing at a shear rate of ¹ for 4 min yields a collagen microfiber suspension; the collagen microfiber suspension is freeze-dried to obtain a collagen microfiber freeze-dried sponge.

[0076] The collagen microfibril intermediate in this embodiment was prepared from bovine type I collagen, with a single fiber diameter D50 of 230 nm, a fiber length of 23 μm, and a collagen triple helix structure retention rate of 83%.

[0077] The oxidized dextran-ε-poly-L-lysine nanogel intermediate of this embodiment meets the following conditions: the oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 27 mol%; the number average molecular weight of ε-poly-L-lysine is 4200; the nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction, with a particle size D50 of 135 nm, a polydispersity index PDI of 0.22, and a zeta potential of +26 mV.

[0078] The carboxymethyl dextran of this embodiment has a carboxymethyl substitution degree of 0.78, and the low molecular weight heparin salt is enoxaparin sodium. The carboxymethyl dextran of this embodiment is carboxymethyl-β-glucan, with the β-glucan backbone consisting of β-1,3-glucosidic bonds and containing β-1,6-branched structures, and a carboxymethyl substitution degree of 0.78. The number-average molecular weight of the carboxymethyl dextran of this embodiment is 155 kDa. The apparent viscosity of the carboxymethyl dextran of this embodiment, calculated as a 1.0 wt% aqueous solution at 25°C, is 95 mPa·s. The carboxyl content of the carboxymethyl dextran of this embodiment, calculated by titration, is 2.7 mmol / g.

[0079] The micro-crosslinked sodium hyaluronate microgel of this embodiment is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. The crosslinked hyaluronic acid gel is mechanically sheared or microfluidically pulverized to form dry powder or wet microgel. The particle size D50 in the dry powder state is 18 μm, and the composition is subjected to a shear rate of 100 s at 25°C. - The viscosity under the conditions of ¹ is 1.65 Pa·s. The hydrogel formed after the composition is injected into the physiological conditions of 37°C has a storage modulus G′ of 2100 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides to oligosaccharides in the low molecular weight products released during the degradation of the composition is 89%.

[0080] The preparation method of the injectable type I collagen composition for tissue repair in this embodiment includes the following steps: providing collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution; preparing a first component and a second component at 2-8°C, wherein the first component includes the collagen microfibril intermediates and the second component includes oxidized dextran-ε-poly-L-lysine nanogel intermediates; filling the first component and the second component into a double-barrel syringe or a two-way syringe device with a Y-type mixer, so that the contents of the two syringes are mixed at the tip of the needle during injection to form an injectable type I collagen composition; injecting the mixed system into a simulated physiological environment or ex vivo tissue model at 37°C to complete in-situ gelation within 4 minutes.

[0081] In the preparation method of this embodiment, the first component further includes carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. The second component does not contain low molecular weight heparin salt or carboxymethyl dextran to reduce the risk of flocculation caused by polyelectrolyte complexation during storage. In this embodiment, when the first and second components are filled into a dual-barrel syringe, the filling volume ratio of the two barrels is 1:1. The dual-barrel syringe is connected to a static mixer before being connected to the needle. The static mixer has 18 built-in mixing units. The isotonic phosphate buffer solution in this embodiment has a pH of 7.25 and an osmotic pressure of 303 mOsm·kg⁻¹. - ¹. The composition of this embodiment did not show visible sedimentation or stratification after standing at 2-8°C for 24 hours, and after 100 seconds... - ¹The appearance returned to uniformity after 60 seconds of shearing. The composition of this embodiment reached a self-supporting state within 2 minutes after injection at 37°C, and the storage modulus G′ at 1Hz reached 2100 Pa within 6 minutes. The introduction of carboxymethyl dextran in this embodiment resulted in a 3kDa cutoff in the degradation solution, with the weight ratio of monosaccharides to oligosaccharides in the permeate being 89%, and the oligosaccharides being predominantly disaccharides to hexasaccharides. In this embodiment, the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel has a cross-sectional outer diameter of 380 nm and a shell thickness of 62 nm, satisfying the condition that the shell thickness is less than half the cross-sectional outer diameter.

[0082] Features of Example 3: This example uses a ratio of lower collagen microfibril content to higher core-shell intermediate content. The collagen microfibril intermediate content is 0.95 parts by weight, the core-shell intermediate content is 2.2 parts by weight, carboxymethyl dextran is 0.88 parts by weight, low molecular weight heparin salt is 0.15 parts by weight, and micro-crosslinked sodium hyaluronate microgel is 0.42 parts by weight. The pH value is 7.25, and the osmotic pressure is 303 mOsm·kg. -¹, The core-shell intermediate has an outer diameter of 380 nm and a shell thickness of 62 nm, with oxidized dextran accounting for 43% of the shell by weight. This formulation has a high content of core-shell intermediates, a nanogel particle size D50 of 135 nm, and a zeta potential of +26 mV, providing strong electrostatic stability and cell adhesion promotion. It is effective at 25 °C and a shear rate of 100 s⁻¹. - The viscosity under the specified conditions is 1.65 Pa·s, and the storage modulus G′ at 37℃ and 1 Hz oscillation frequency is 2100 Pa, exhibiting high gel strength and mechanical support properties. The degradation products of the composition contain 89% monosaccharides and oligosaccharides by weight, and the degradation products are highly metabolizable. This embodiment is suitable for applications requiring strong mechanical support and long-lasting filling effects, including deep soft tissue collapse repair, nasolabial fold filling, chin enhancement, and cheekbone shaping, among other deep tissue filling and shaping applications.

[0083] Example 4 This embodiment provides a type I collagen composition for tissue repair, which is an aqueous system with a pH of 6.88. Based on a total mass of 100 parts by weight, the composition comprises the following components: 1.42 parts by weight of collagen microfibril intermediates, 2.35 parts by weight of a core-shell intermediate of collagen microfibrils coated with oxidized dextran-ε-poly-L-lysine nanogel, 0.53 parts by weight of carboxymethyl dextran, 0.28 parts by weight of low molecular weight heparin salt, 0.74 parts by weight of micro-crosslinked sodium hyaluronate microgel, and the balance being an isotonic phosphate buffer solution. The isotonic phosphate buffer solution in this embodiment contains sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, resulting in an osmotic pressure of 313 mOsm·kg⁻¹ for the aqueous system. - ¹.

[0084] In this embodiment, the collagen microfiber bundle intermediate, the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all based on their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment is 195 nm, the shell thickness is 73 nm, and based on the total dry matter mass of the shell, the weight percentage of oxidized dextran in the shell is 22%.

[0085] The preparation method of the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel in this embodiment includes the following steps: at 5°C, the collagen microfiber bundle intermediate is dispersed in purified water to obtain a collagen microfiber bundle suspension with a solid content of 1.85 wt% based on the total mass of the suspension; the oxidized dextran-ε-poly-L-lysine nanogel intermediate is added to the collagen microfiber bundle suspension, and the mass ratio is 0.6:1 based on the mass ratio of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfiber bundle intermediate; the pH value is controlled at 6.85 and allowed to stand for 28 min to obtain the core-shell intermediate of collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; the core-shell intermediate is kept in suspension or freeze-dried to obtain a powder intermediate.

[0086] The preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate in this embodiment includes the following steps: oxidized dextran is dissolved in an isotonic phosphate buffer solution with a pH of 7.05 to prepare an oxidized dextran solution with a mass concentration of 22 mg / mL; ε-poly-L-lysine is dissolved in an isotonic phosphate buffer solution with a pH of 7.05 to prepare an ε-poly-L-lysine solution with a mass concentration of 13.5 mg / mL; and the solution is prepared at 5°C. Under pH 7.05 conditions, ε-poly-L-lysine solution was added dropwise to oxidized dextran solution over 28 min to achieve a molar ratio of aldehyde to primary amine groups of 0.65:1. The mixture was stirred for 38 min to form a nanogel. Free small molecules and uncrosslinked ε-poly-L-lysine were removed by dialysis or ultrafiltration. The solid content of the nanogel suspension was adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution was measured to confirm that the particle size D50 of the nanogel suspension was 87 nm and the PDI was 0.24.

[0087] The preparation method of oxidized dextran in this embodiment includes the following steps: dextran is dissolved in purified water to obtain a dextran solution with a mass concentration of 140 g / L; under the condition of 2°C and protection from light, 80 g / L sodium periodate aqueous solution is added dropwise to the dextran solution to make the molar ratio of sodium periodate to repeating glucose units in dextran 0.15:1, the pH is adjusted to 3.2, and the reaction is stirred for 2.8 h; after the reaction is completed, inorganic salts and small molecules are removed by dialysis: the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 10 kDa, and dialyzed against purified water at 4°C for 72 h, with the dialysate replaced every 12 h for a total of 6 times; oxidized dextran powder is obtained by freeze drying, and its aldehyde oxidation degree is determined to be 16 mol.

[0088] The preparation method of the collagen microfibril intermediate in this embodiment includes the following steps: bovine type I collagen is dissolved in 0.04 mol / L dilute hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 5.5 mg / mL; 0.4 mol / L sodium hydroxide solution is added dropwise to the acidic collagen solution to adjust the pH value to 7.38, and incubated at 37°C for 1.2 h to induce collagen fiber self-assembly; the obtained fiber suspension is subjected to a 750 s... - ¹ Shearing rate for 2.2 min yielded a collagen microfiber suspension; the collagen microfiber suspension was freeze-dried to obtain a collagen microfiber freeze-dried sponge.

[0089] The collagen microfibril intermediate in this embodiment was prepared from bovine type I collagen, with a single fiber diameter D50 of 85 nm, a fiber length of 8 μm, and a collagen triple helix structure retention rate of 81%.

[0090] The oxidized dextran-ε-poly-L-lysine nanogel intermediate of this embodiment meets the following conditions: the oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 16 mol%; the number-average molecular weight of ε-poly-L-lysine is 4600; the nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction, with a particle size D50 of 87 nm, a polydispersity index PDI of 0.24, and a zeta potential of +12 mV.

[0091] The carboxymethyl dextran of this embodiment has a carboxymethyl substitution degree of 0.52, and the low molecular weight heparin salt is a mixture of enoxaparin sodium and dalteparin sodium in a mass ratio of 1:1. The carboxymethyl dextran of this embodiment is carboxymethyl-β-glucan, with a β-1,3-glucosidic bond in the main chain and containing β-1,6-branched structures, and a carboxymethyl substitution degree of 0.52. The number-average molecular weight of the carboxymethyl dextran of this embodiment is 38 kDa. The apparent viscosity of the carboxymethyl dextran of this embodiment, calculated as a 1.0 wt% aqueous solution at 25°C, is 28 mPa·s. The carboxyl content of the carboxymethyl dextran of this embodiment, calculated by titration, is 1.6 mmol / g.

[0092] The micro-crosslinked sodium hyaluronate microgel of this embodiment is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. The crosslinked hyaluronic acid gel is then subjected to mechanical shearing or microfluidic pulverization to form dry powder or wet microgel. The particle size D50 in the dry powder state is 45 μm, and the composition is subjected to mechanical shearing at 25°C and a shear rate of 100 s. -The viscosity under the conditions is 0.75 Pa·s. The hydrogel formed after the composition is injected into the physiological conditions at 37°C has a storage modulus G′ of 1180 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides to oligosaccharides in the low molecular weight products released during the degradation of the composition is 82%.

[0093] The preparation method of the injectable type I collagen composition for tissue repair in this embodiment includes the following steps: providing collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution; preparing a first component and a second component at 2-8°C, wherein the first component includes the collagen microfibril intermediates and the second component includes oxidized dextran-ε-poly-L-lysine nanogel intermediates; filling the first component and the second component into a double-barrel syringe or a two-way syringe device with a Y-type mixer, so that the contents of the two syringes are mixed at the tip of the needle during injection to form an injectable type I collagen composition; injecting the mixed system into a simulated physiological environment or an ex vivo tissue model at 37°C to complete in-situ gelation within 3 minutes.

[0094] In the preparation method of this embodiment, the first component further includes carboxymethyl dextran, low molecular weight heparin salt, and micro-crosslinked sodium hyaluronate microgel. The second component does not contain low molecular weight heparin salt or carboxymethyl dextran to reduce the risk of flocculation caused by polyelectrolyte complexation during storage. In this embodiment, when the first and second components are filled into a dual-barrel syringe, the filling volume ratio of the two barrels is 1:1. The dual-barrel syringe is connected to a static mixer before being connected to the needle. The static mixer has 20 built-in mixing units. The isotonic phosphate buffer solution in this embodiment has a pH of 6.88 and an osmotic pressure of 313 mOsm·kg⁻¹. - ¹. The composition of this embodiment did not show visible sedimentation or stratification after standing at 2-8°C for 24 hours, and after 100 seconds... - ¹The appearance returned to uniformity after 60 seconds of shearing. The composition of this embodiment reached a self-supporting state within 2 minutes after injection at 37°C, and the storage modulus G′ at 1Hz reached 1180 Pa within 7 minutes. The introduction of carboxymethyl dextran in this embodiment resulted in a 3kDa cutoff in the degradation solution, with the weight ratio of monosaccharides to oligosaccharides in the permeate being 82%, and the oligosaccharides being predominantly disaccharides to hexasaccharides. In this embodiment, the core-shell intermediate of the collagen microfiber bundle-encapsulated oxidized dextran-ε-poly-L-lysine nanogel had a cross-sectional outer diameter of 195 nm and a shell thickness of 73 nm, satisfying the condition that the shell thickness is less than half the cross-sectional outer diameter.

[0095] Features of Example 4: This example uses a combination of multiple components in proportions close to their endpoints. The collagen microfibril intermediate is 1.42 parts by weight, the core-shell intermediate is 2.35 parts by weight, carboxymethyl dextran is 0.53 parts by weight, low molecular weight heparin salt is 0.28 parts by weight (using a 1:1 mixture of enoxaparin sodium and dalteparin sodium), and micro-crosslinked sodium hyaluronate microgel is 0.74 parts by weight. The pH value is 6.88, and the osmotic pressure is 313 mOsm·kg⁻¹. - ¹, The core-shell intermediate has an outer diameter of 195 nm and a shell thickness of 73 nm, with oxidized dextran accounting for 22% of the shell by weight. The formulation contains collagen microfiber bundles with a single fiber diameter (D50) of 85 nm and a fiber length of 8 μm. The nanogel particles have a D50 of 87 nm, forming a composite network structure of fine fibers and nanoparticles. This structure is effective at 25°C and a shear rate of 100 s⁻¹. - The viscosity under the specified conditions is 0.75 Pa·s, exhibiting excellent flowability and injection smoothness. The storage modulus G′ at 37°C and a 1Hz oscillation frequency is 1180 Pa, providing adequate mechanical support. In this embodiment, the carboxymethyl dextran has a number-average molecular weight of 38 kDa and an apparent viscosity of 28 mPa·s. The degradation products of the composition contain 82% monosaccharides and oligosaccharides by weight, and the degradation products can be fully metabolized and eliminated. This embodiment is suitable for applications requiring extremely high injection precision, including fine-tuning procedures such as filling fine lines around the eyes, precise lip shaping, minimally invasive rhinoplasty, and earlobe repair—all requiring precise injection and a delicate texture in fine cosmetic medicine and tissue repair applications.

[0096] Comparative Example 1: It is basically the same as Example 1, except that the amount of collagen microfibril intermediate is 0.65 parts by weight, while the amount of other components and preparation conditions remain unchanged.

[0097] Comparative Example 2: It is basically the same as Example 1, except that the amount of the core-shell intermediate of the collagen microfiber bundles encapsulating the oxidized dextran-ε-poly-L-lysine nanogel is 0.75 parts by weight, while the amount of other components and preparation conditions remain unchanged.

[0098] Comparative Example 3: It is basically the same as Example 1, except that the amount of carboxymethyl dextran is 0.35 parts by weight, while the amounts of other components and preparation conditions remain unchanged.

[0099] Comparative Example 4: It is basically the same as Example 1, except that the cross-sectional outer diameter of the core-shell intermediate is 135 nm and the shell thickness is 15 nm, while the amount of other components and preparation conditions remain unchanged.

[0100] Comparative Example 5: Basically the same as Example 1, except that the pH value of the composition is 6.5, and the amount of other components and preparation conditions remain unchanged.

[0101] Comparative Example 6: It is basically the same as Example 1, except that carboxymethyl dextran was not added, the amount of other components was adjusted proportionally to maintain the total solid content basically the same, and other preparation conditions remained unchanged.

[0102] Comparative Example 7: Basically the same as Example 1, except that the degree of aldehyde oxidation of dextran was 10 mol%, and the amounts of other components and preparation conditions remained unchanged.

[0103] Comparative Example 8: It is basically the same as Example 1, except that the weight percentage of oxidized dextran in the shell is 15%, while the amount of other components and preparation conditions remain unchanged.

[0104] Performance testing: Experiment 1 Evaluation of Injection Flow Properties This experiment aimed to evaluate the injection flow properties of an injectable type I collagen composition at 25°C. The apparent viscosity of the composition at a constant shear rate was measured using a cone-plate rheometer (cone angle 2°, gap 50 μm). Based on the rheological behavior of Newtonian or non-Newtonian fluids under shear, the flow resistance during injection was characterized. The composition was loaded onto the test platform at 25 ± 0.5°C and injected at 100 s⁻¹. - ¹Shear rate was continuously sheared for 60 s, and steady-state apparent viscosity was recorded. Each sample was tested three times, and the mean ± standard deviation was taken. Key parameters were temperature 25 ± 0.5℃ and shear rate 100 s. - ¹ The test duration is 60 seconds. During data processing, the average apparent viscosity (Pa·s) and standard deviation are calculated. A steady-state viscosity fluctuation of <5% is considered as flow stability to evaluate the injection smoothness and clinical ease of use of the composition.

[0105] Experiment 2 In-situ gel strength evaluation This experiment aimed to evaluate the in-situ gelation strength of an injectable type I collagen composition under physiological conditions at 37°C. The storage modulus G′ of the gel was determined using a plate rheometer via oscillatory rheological testing, reflecting the elastic strength and cross-linking density of the gel network based on linear viscoelasticity theory. The composition was injected into parallel plates (1 mm gap) preheated to 37±0.5°C, and an oscillatory load of 1 Hz frequency and 1% strain was immediately applied. The time evolution curve of the storage modulus G′ was continuously recorded over 10 min. Each sample was tested three times, and the mean ± standard deviation was used. Key parameters were: temperature 37±0.5°C, oscillation frequency 1 Hz, strain amplitude 1%, and test duration 10 min. During data processing, the gelation time (time to reach 800 Pa) and the final G′ value (Pa) at 10 min were recorded to evaluate the composition's rapid gelation ability and the mechanical support strength of the gel network.

[0106] Experiment 3 Evaluation of storage stability and anti-settlement performance This experiment aimed to evaluate the phase stability and anti-sedimentation properties of injectable type I collagen compositions stored at 2-8°C. The phase separation, sedimentation, and stratification phenomena of the compositions during low-temperature storage were observed visually and detected using an optical microscope. The interaction equilibrium between components was assessed based on colloidal stability theory. The compositions were placed in transparent glass vials and stored at 2-8°C for 24 hours to observe for visible sedimentation and stratification. Subsequently, a 100-second pressure was applied to the samples. - ¹Shearing for 60 s, then observing the recovery of appearance. Microstructural uniformity was observed using an optical microscope (100× magnification). Each sample was tested three times. Key parameters were: storage temperature 2-8℃, resting time 24 h, and shear rate 100 s. - ¹, Shearing time: 60 s. During data processing, record whether visible sedimentation occurs (yes / no) and the degree of appearance recovery after shearing (homogeneous / non-homogeneous) to evaluate the storage stability and reusability of the composition.

[0107] Experiment 4 Evaluation of the molecular weight distribution of degradation products This experiment aimed to evaluate the composition of low-molecular-weight products released during the degradation of an injectable type I collagen composition, verifying that the proportion of monosaccharides and oligosaccharides was not less than 80%. The content of monosaccharides and oligosaccharides with a molecular weight <3 kDa in the degradation solution was quantitatively analyzed by ultrafiltration and high-performance liquid chromatography (HPLC), and the biometabolizability of the degradation products was assessed based on the principles of polysaccharide degradation kinetics. The composition was degraded in phosphate buffer at 37°C and pH 7.4 for 14 days. Samples were taken periodically, filtered through a 3 kDa ultrafiltration membrane, and the permeate was collected. The content of monosaccharides (glucose, mannose, etc.) and oligosaccharides (disaccharides to hexasaccharides) in the permeate was analyzed by HPLC-ELSD, and quantification was performed using external standard method. Each sample was tested three times, and the mean ± standard deviation was used. Key parameters were degradation temperature 37°C, pH 7.4, degradation time 14 days, and ultrafiltration molecular weight cutoff 3 kDa. The weight percentage (%) of monosaccharides and oligosaccharides was calculated during data processing, requiring ≥80% to verify the regulatory role of carboxymethyl dextran in the controlled release of degradation products.

[0108] Experiment 5 Osmotic Pressure Evaluation This experiment aimed to evaluate the osmotic pressure of injectable type I collagen compositions to ensure isotonicity with the physiological environment and avoid osmotic damage to local tissues after injection. The osmotic pressure was determined using the freezing point depression method, and calculations were performed based on the linear relationship between freezing point depression and osmotic pressure (ΔTf = Kf·m, where Kf is the freezing point depression constant and m is the molality). Using an osmoremeter, a 0.2 mL sample of the composition was cooled below its freezing point in the sample cell of the instrument, and the freezing point temperature was recorded. The instrument automatically calculated the osmotic pressure value (mOsm·kg⁻¹). -¹). Each sample was tested three times, and the mean ± standard deviation was used. Key parameters were: sample volume 0.2 mL, test temperature range 0 to -7℃, and instrument accuracy ±2 mOsm·kg. - ¹. Calculate the average osmotic pressure (mOsm·kg⁻¹) during data processing. - ¹), requiring a concentration of 270-320 mOsm·kg - ¹Within a range that ensures the composition is isotonic with plasma, reducing post-injection irritation and adverse reactions.

[0109] Figure 1 The images show the XPS full spectrum overlays of Example 1 and Comparative Example 8. The XPS testing method, energy range, data processing, and plotting aperture were kept constant. The only change was the sample number, which was switched from Example 1 to Comparative Example 8, resulting in differences in surface elemental composition and signal intensity distribution. The full spectrum profiles of both examples show distinguishable differences in intensity distribution and characteristic segments, reflecting that Example 1 and Comparative Example 8 are not identical in surface chemical composition. This demonstrates that Example 1 indeed achieved the surface chemical characteristics related to the target shell, validating the effectiveness of the scheme from the perspective of the overall surface spectrum.

[0110] Figure 2 The images shown are XPS C 1s high-resolution overlay plots of Example 1 and Comparative Example 8. The XPS high-resolution testing conditions, binding energy range, and peak shape display processing were kept constant. The only change was the sample number, which was switched from Example 1 to Comparative Example 8. The peak shapes and relative intensity distributions in the C 1s region of Example 1 and Comparative Example 8 differ, indicating differences in the carbon-containing functional group environment and the proportion of surface organic components. This demonstrates that the surface chemical environment constructed in Example 1 is different from that in Comparative Example 8, supporting the correctness of the shell chemical composition regulation from the perspective of carbon chemical states.

[0111] Figure 3 The images shown are XPS O 1s high-resolution overlay images of Example 1 and Comparative Example 8. The parameters were kept constant: XPS high-resolution test conditions, binding energy range, and data processing flow were consistent. The only change was that the sample number was switched from Example 1 to Comparative Example 8. The differences in the O 1s peak shape and its relative distribution indicate that there are changes in oxygen-containing functional groups and surface oxygen-related components. Example 1 exhibits characteristics more consistent with the oxygen-containing environment that should appear after the introduction of the shell, further demonstrating from the perspective of oxidation state that this method can effectively change and stabilize the particle surface composition.

[0112] Figure 4The image shows the XPS N 1s high-resolution overlay images of Example 1 and Comparative Example 8. The parameters were kept constant: XPS high-resolution testing conditions, binding energy range, and data processing flow were consistent. The only change was the sample number, which was switched from Example 1 to Comparative Example 8. The differences in peak shape and relative intensity in the N 1s interval indicate that Example 1 and Comparative Example 8 differ in nitrogen-containing species or nitrogen-related chemical environments. This suggests that Example 1 introduced or retained different nitrogen chemical states on its shell surface, consistent with the change in the target shell composition, thus supporting the correctness of the surface chemical construction of this scheme.

[0113] Figure 5 The rheological time-scan gelation kinetics G′ versus time plots for Example 1, Comparative Example 1, and Comparative Example 2 are shown. The parameters were fixed as follows: test temperature 37°C, frequency 1Hz, strain 1%, and consistent rheological testing procedure. The parameter variation was that switching the sample number from Example 1 to Comparative Example 1 or Comparative Example 2 resulted in different network formation kinetics and final storage modulus. Example 1 showed a faster increase in G′ over time and a higher final plateau, indicating more complete gelation and a stronger elastic network. In contrast, Comparative Example 1 and Comparative Example 2 showed lower G′ growth and plateau values, indicating insufficient network construction or weaker structural stability. Macroscopic mechanical response validates that this scheme is beneficial for forming a stronger gel network.

[0114] Figure 6 The rheological time-scan gelation kinetics G″ versus time plots for Example 1, Comparative Example 1, and Comparative Example 2 are shown. The parameters were fixed as follows: test temperature 37°C, frequency 1Hz, strain 1%, and consistent rheological testing procedure. The parameters varied by switching the sample number from Example 1 to Comparative Example 1 or Comparative Example 2, resulting in differences in viscous dissipation and structural evolution. The change of G″ over time in Example 1 matches its G′ evolution and tends to stabilize, reflecting a more defined transition from a fluid state to a stable viscoelastic network during gelation. The G″ level and evolution characteristics of the comparative sample differ, suggesting that its structure formation degree and dissipation behavior are not as consistent as in Example 1. This strengthens the evidence chain of gelation kinetics from the perspective of viscous response, proving that this method is more likely to obtain a stable network.

[0115] Figure 7 The image shows a superimposed HPLC-ELSD chromatogram of Example 1 and Comparative Example 6. The parameters were kept constant: the HPLC-ELSD characterization method, retention time range, data processing, and plotting rules were consistent. The only change was the sample number, which was switched from Example 1 to Comparative Example 6, resulting in different peak shapes and relative response distributions of the degradation products. The response distribution of Example 1 in different retention time ranges differed significantly from that of Comparative Example 6, indicating that the composition and distribution of the degradation products were different. Example 1 exhibited a product distribution pattern more consistent with expectations. From a separation characterization perspective, this demonstrates that the proposed method can effectively control the degradation product spectrum and improve system controllability.

[0116] Figure 8The HPLC-ELSD cumulative area distribution (CDF) plots for Example 1 and Comparative Example 6 are shown. The method for obtaining the cumulative area distribution by integrating the same chromatographic mean curve was kept constant, consistent with the data processing workflow. The only change was switching the sample number from Example 1 to Comparative Example 6. The rising rhythm of the cumulative curve in Example 1 during the key retention period differs from that in Comparative Example 6, reflecting a difference in the contribution range and accumulation rate of the major product. This indicates that the distribution of degradation products in Example 1 over time better matches the target composition and structure. The cumulative integration evidence further verifies that the proposed scheme is effective and reproducible in regulating the product spectrum.

[0117] Figure 9 The HPLC-ELSD component proportion stacked column chromatograms of Example 1 and Comparative Example 6 are shown. The parameters were kept constant: the component classification criteria, proportion calculation method, and plotting rules were consistent. The variable parameter was the sample number, which was changed from Example 1 to Comparative Example 6, resulting in different proportions of monosaccharides, oligosaccharides, and macromolecular polysaccharides. Example 1 and Comparative Example 6 exhibit distinguishable proportions of the three types of components. The component composition of Example 1 is closer to the target distribution characteristics, while Comparative Example 6 leans towards a different compositional pattern. This indicates that Example 1 has a more controllable structural result in the macroscopic composition of the degradation products, consistent with the chromatographic overlay and CDF chromatograms, thus proving the correctness and feasibility of this scheme.

[0118] As can be seen from the performance of the examples and comparative examples in Table 1, the compositions of the present invention exhibit significant advantages in injection performance, gelation performance, and degradation product quality. The apparent viscosity of Examples 1-4 was controlled within the range of 0.75-1.65 Pa·s, achieving good injection flowability; the storage modulus reached 1180-2100 Pa, providing excellent mechanical support strength; and the gelation time was controlled within 2.5-3.5 min, achieving rapid in-situ gelation. Comparative Examples 1 and 2, due to insufficient collagen microfibrils or core-shell intermediate content, had storage moduli of only 680 Pa and 820 Pa, respectively; the gelation time was prolonged to 5.2-6.8 min; and slight sedimentation was observed, indicating that the gel network strength and stability significantly decreased after the composition ratio deviated from the optimized range. Comparative Example 6, without the addition of carboxymethyl dextran, although exhibiting acceptable gelation performance, saw a decrease in the proportion of monosaccharides and oligosaccharides in the degradation product to 62%, and significant sedimentation occurred, indicating that carboxymethyl dextran plays a crucial role in improving the quality and storage stability of the degradation product. Comparative Examples 4, 7, and 8, due to deviations of core-shell structure parameters, oxidation degree, and shell composition from the optimized range, had storage modulus reduced to 950-1220 Pa and gelation time extended to 4.3-4.8 min, respectively. This verifies the importance of fine structure regulation of core-shell intermediates for the gelation kinetics and gel strength of the composition.

[0119] Table 1 Performance Comparison Summary Table Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A type I collagen composition for tissue repair, characterized in that, The composition is an aqueous system with a pH of 6.8-7.4, and based on 100 parts by weight of the total composition, the composition comprises the following components; 0.8-1.5 parts by weight of collagen microfibril intermediates; 1.0-2.5 parts by weight of collagen microfibrils encapsulating the core-shell intermediate of oxidized dextran-ε-poly-L-lysine nanogel; 0.5-1.0 parts by weight of carboxymethyl dextran; 0.1-0.3 parts by weight of low molecular weight heparin salt; 0.3-0.8 parts by weight of micro-crosslinked sodium hyaluronate microgel; The remainder is an isotonic phosphate buffer solution containing sodium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate, such that the osmotic pressure of the aqueous system is 270-320 mOsm·kg⁻¹. -1 ; The collagen microfiber intermediate, the core-shell intermediate of the collagen microfiber-encapsulated oxidized dextran-ε-poly-L-lysine nanogel, and the micro-crosslinked sodium hyaluronate microgel are all measured by their dry matter mass. The cross-sectional outer diameter of the core-shell intermediate of the collagen microfiber-encapsulated oxidized dextran-ε-poly-L-lysine nanogel is 160-460 nm, the shell thickness is 20-80 nm, and the weight percentage of oxidized dextran in the shell is 20-50% based on the total dry matter mass of the shell. The preparation method of the core-shell intermediate of collagen microfiber bundles coated with oxidized dextran-ε-poly-L-lysine nanogel includes the following steps: A1. The collagen microfibril intermediate is dispersed in purified water at 4-10℃ to obtain a collagen microfibril suspension with a solid content of 0.5-2.0 wt% based on the total mass of the suspension. A2. The oxidized dextran-ε-poly-L-lysine nanogel intermediate is added to the collagen microfibril suspension, and the mass ratio is 0.5-3:1, based on the ratio of the mass of oxidized dextran in the dry matter of the nanogel intermediate to the mass of the dry matter of the collagen microfibril intermediate. A3. The pH value was controlled to 6.8-7.2 and allowed to stand for 10-30 minutes to obtain the core-shell intermediate of collagen microfibrils-encapsulated oxidized dextran-ε-poly-L-lysine nanogel; A4. The core-shell intermediate is kept in a suspended state or freeze-dried to obtain a powder intermediate; The preparation method of the oxidized dextran-ε-poly-L-lysine nanogel intermediate includes the following steps: B1. Dissolve oxidized dextran in an isotonic phosphate buffer solution, wherein the isotonic phosphate buffer solution is a phosphate buffer solution with a pH of 7.0-7.4, to prepare an oxidized dextran solution with a mass concentration of 20-40 mg / mL; B2. Dissolve ε-poly-L-lysine in the isotonic phosphate buffer solution described in step B1 to prepare an ε-poly-L-lysine solution with a mass concentration of 5-15 mg / mL; B3. Under conditions of 4-10℃ and pH value of 7.0-7.4, the ε-poly-L-lysine solution is added dropwise to the oxidized dextran solution over 10-30 min, so that the molar ratio of aldehyde group to primary amine group is 0.6-1.0∶1; B4. Stir the reaction for 20-40 minutes to form a nanogel; B5. Remove free small molecules and uncrosslinked ε-poly-L-lysine by dialysis or ultrafiltration; B6. The solid content of the nanogel suspension was adjusted by ultrafiltration concentration or buffer dilution, and the particle size distribution was measured to confirm that the particle size D50 of the nanogel suspension was 80-150 nm and the PDI was not greater than 0.25; The preparation method of the collagen microfibril intermediate includes the following steps: D1. Dissolve bovine type I collagen in 0.01-0.05 mol / L dilute hydrochloric acid solution to obtain an acidic collagen solution with a concentration of 3-6 mg / mL; D2. Add 0.1-0.5 mol / L sodium hydroxide solution dropwise to the acidic collagen solution to adjust the pH value to 7.2-7.4, and incubate at 37±1℃ for 1-3 h to induce collagen fiber self-assembly; D3. Apply the resulting fiber suspension for 500-800 seconds. -1 Shear at a shear rate of 2-5 min to obtain a collagen microfiber suspension; D4. Freeze-dry the collagen microfiber bundle suspension to obtain a collagen microfiber bundle freeze-dried sponge.

2. The type I collagen composition for tissue repair as described in claim 1, characterized in that, The preparation method of oxidized dextran includes the following steps: C1. Dissolve dextran in purified water to obtain a dextran solution with a mass concentration of 50-150 g / L; C2. Under conditions of 0-10℃ and protection from light, add 20-100 g / L sodium periodate aqueous solution dropwise to the dextran solution, so that the molar ratio of sodium periodate to repeating glucose units in dextran is 0.1-0.5:1, adjust the pH value to 3.0-4.5, and stir the reaction for 1-3 hours. C3. After the reaction is complete, inorganic salts and small molecules are removed by dialysis or ultrafiltration; C4. The dextran oxide powder was obtained by freeze drying and its aldehyde oxidation degree was determined to be 15-30 mol.

3. The type I collagen composition for tissue repair as described in claim 1, characterized in that, The collagen microfibril intermediate is made from bovine type I collagen, and the diameter D50 of a single fiber is 50-300 nm, the fiber length is 5-30 μm, and the collagen triple helix structure retention rate is not less than 80%.

4. The type I collagen composition for tissue repair as described in claim 1, characterized in that, The oxidized dextran-ε-poly-L-lysine nanogel intermediate satisfies the following conditions: The oxidized dextran is an oxidized polysaccharide obtained by reacting dextran with sodium periodate, and its aldehyde oxidation degree is 15-30 mol%; The number-average molecular weight of the ε-poly-L-lysine is 2000-5000; The nanogel is a cross-linked particle formed by the interaction of oxidized dextran and ε-poly-L-lysine through Schiff base bonds and electrostatic interaction. The particle size D50 is 80-150 nm, the polydispersity index PDI is not greater than 0.25, and the zeta potential is +10 to +30 mV.

5. The type I collagen composition for tissue repair as described in claim 1, characterized in that, The degree of carboxymethyl substitution of the carboxymethyl dextran is 0.4-0.9, and the low molecular weight heparin salt is selected from one or more of enoxaparin sodium and dalteparin sodium.

6. The type I collagen composition for tissue repair as described in claim 1, characterized in that, The micro-crosslinked sodium hyaluronate microgel is derived from a crosslinked hyaluronic acid gel prepared by crosslinking sodium hyaluronate with 1,4-butanediol diglycidyl ether. The crosslinked hyaluronic acid gel is then subjected to mechanical shearing or microfluidic pulverization to form a dry powder or wet microgel. In the dry powder state, the particle size D50 is 1-50 μm. Furthermore, the composition is subjected to mechanical shearing at 25°C and a shear rate of 100 s⁻¹. -1 The viscosity under the specified conditions is 0.5-2.0 Pa·s. The hydrogel formed after the composition is injected into physiological conditions at 37°C has a storage modulus G′ of 800-2500 Pa at an oscillation frequency of 1 Hz. The weight ratio of monosaccharides and oligosaccharides in the low molecular weight products released during the degradation of the composition is not less than 80%.

7. The method for preparing the type I collagen composition for tissue repair as described in any one of claims 1-6, characterized in that, Includes the following steps: S1 provides collagen microfibril intermediates, oxidized dextran-ε-poly-L-lysine nanogel intermediates, carboxymethyl dextran, low molecular weight heparin salt, micro-crosslinked sodium hyaluronate microgel, and isotonic phosphate buffer solution. S2 was used to prepare the first and second components at 2-8℃. S3 fills the first component and the second component into a double-barrel syringe or a two-way needle device with a Y-type mixer, so that the contents of the two barrels are mixed at the tip of the needle during injection to form an injectable type I collagen composition. S4 injects the mixed system into a simulated physiological environment or ex vivo tissue model at 37±1℃ and completes in-situ gelation within 1-5 minutes.

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