An injectable amniotic membrane compounded gel, a preparation method and application thereof

Abstract

This invention relates to the field of bio-tissue engineering technology, and particularly to an injectable amniotic membrane compound gel, its preparation method, and its application. It addresses the problems of degradation and unadjustable mechanical properties of existing materials, easy displacement and sedimentation of amniotic membrane particles, insufficient cell loading, and short-lasting repair effects. The gel comprises a composite scaffold membrane and an aldehyde-based hyaluronic acid gel in a mass ratio of 1:3 to 10. The membrane is formed by freeze-drying and grinding the composite scaffold. The scaffold comprises a sequentially stacked upper electrospun fiber membrane layer, a decellularized amniotic membrane matrix intermediate layer, and a lower electrospun fiber membrane layer, cross-linked and bonded together. The electrospun fibers in the upper and lower electrospun fiber membrane layers have a core-shell structure. The shell layer raw material components include natural polymer materials, and the core raw material components include synthetic polymer materials. The preparation method includes preparing a decellularized amniotic membrane matrix, constructing a gradient fiber membrane layer through coaxial electrospinning, cross-linking with tannic acid, freeze-drying and grinding, and preparing and compounding the aldehyde-based hyaluronic acid gel.

Description

Technical Field

[0001] This invention relates to the field of bio-tissue engineering technology, and in particular to an injectable amniotic membrane compound gel, its preparation method, and its application. Background Technology

[0002] Soft tissue repair is a crucial aspect of surgery, especially vital for functional and morphological recovery after surgery or trauma. Currently, commonly used soft tissue repair materials mainly include natural polymers (such as collagen and silk fibroin), synthetic polymers (such as polylactic acid and polycaprolactone), metals, and ceramics. Natural polymers are widely used due to their excellent biocompatibility and cell affinity; synthetic polymers, on the other hand, also hold a place in the field of soft tissue repair due to their controllable degradation and mechanical properties.

[0003] However, existing soft tissue repair materials still have the following limitations: most materials are prepared using single components or simple composite structures, resulting in fixed degradation cycles and mechanical properties, making it difficult to achieve long-term repair effects that dynamically match the tissue regeneration process. Furthermore, some materials are prone to displacement, sedimentation, or premature degradation after implantation, leading to poor repair outcomes.

[0004] Human amniotic membrane (hAM), a surgical waste, is widely available, inexpensive, and exhibits extremely low immunogenicity after decellularization, almost never causing rejection reactions, making it a highly promising soft tissue repair material. Studies have shown that micronized amniotic membrane can retain and slowly release various growth factors, which is beneficial for tissue repair. However, plain amniotic membrane degrades relatively quickly, has a limited contact area with cells after micronization, resulting in low cell adhesion rates, and is prone to migration and sedimentation after injection, limiting its clinical application.

[0005] Therefore, there is an urgent need in clinical practice to develop a new type of soft tissue repair composite material that can provide a stable three-dimensional support structure and cell attachment microenvironment, and can match degradation behavior with tissue regeneration process, thereby improving repair effect. Summary of the Invention

[0006] The purpose of this invention is to provide an injectable amniotic membrane compound gel, its preparation method, and its application, in order to solve the technical problems of existing soft tissue repair materials, such as fixed degradation cycle and mechanical properties, easy degradation, easy displacement and sedimentation of amniotic membrane microparticles, low cell adhesion, and inability to achieve long-term stable tissue repair.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides an injectable amniotic membrane composite gel, wherein the raw material components of the injectable amniotic membrane composite gel include a composite scaffold membrane and an aldehyde-modified hyaluronic acid gel, and the mass ratio of the composite scaffold membrane to the aldehyde-modified hyaluronic acid gel is 1:3~10.

[0009] The composite scaffold membrane is formed by freeze-drying and grinding the composite scaffold; the composite scaffold comprises an upper electrospun fiber membrane layer, a decellularized amniotic matrix intermediate layer, and a lower electrospun fiber membrane layer stacked sequentially; the upper electrospun fiber membrane layer, the decellularized amniotic matrix intermediate layer, and the lower electrospun fiber membrane layer are cross-linked together; the particle size of the composite scaffold membrane is 100~1000µm;

[0010] The composite scaffold membrane is prepared by freeze-drying and grinding the composite scaffold; the composite scaffold includes an upper electrospun fiber membrane layer, a decellularized amniotic membrane matrix intermediate layer, and a lower electrospun fiber membrane layer that are stacked and combined into one piece in sequence.

[0011] The electrospun fibers in both the upper and lower electrospun fiber membrane layers have a core-shell structure. The shell material components of the core-shell structure include natural polymer materials, while the core material components of the core-shell structure include synthetic polymer materials.

[0012] Furthermore, the natural polymer material is a mixture selected from one or more of chitosan, collagen, silk fibroin and gelatin; the synthetic polymer material is a mixture selected from one or more of polylactic acid (PLA), polycaprolactone (PCL), polyvinyl alcohol (PVA) and polyethylene oxide (PEO).

[0013] Furthermore, in the upper electrospun fiber membrane layer and the lower electrospun fiber membrane layer, from the side near the intermediate layer of the decellularized amniotic membrane matrix to the side away from the intermediate layer, the flow rate of the shell spinning solution varies from high to low in the range of 1.5 mL / h to 1.0 mL / h to 0.5 mL / h, and the flow rate of the core spinning solution varies from low to high in the range of 0.5 mL / h to 1.0 mL / h to 1.5 mL / h.

[0014] A second aspect of this invention provides a method for preparing an injectable amniotic membrane compound gel, comprising the following steps:

[0015] (1) Preparation of decellularized amniotic membrane matrix;

[0016] (2) An upper electrospun fiber membrane layer and a lower electrospun fiber membrane layer are prepared sequentially on both sides of the decellularized amnion matrix by coaxial electrospinning process to obtain a three-layer composite scaffold precursor.

[0017] (3) The three-layer composite scaffold precursor is cross-linked to obtain a composite scaffold;

[0018] (4) The composite scaffold is freeze-dried and ground to obtain a composite scaffold membrane;

[0019] (5) Preparation of aldehyde-modified hyaluronic acid gel;

[0020] (6) The composite scaffold membrane and the aldehyde-based hyaluronic acid gel are mixed in proportion to obtain the injectable amniotic membrane composite gel.

[0021] Further, in step (1), the method for preparing the decellularized amniotic matrix includes:

[0022] (1a) Cleaning and enzymatic treatment of fresh amniotic membrane;

[0023] (1b) The amnion treated in step (1a) was placed in a supercritical carbon dioxide environment and a carrier was added for decellularization.

[0024] (1c) Perform virus inactivation and deep washing on the amnion after decellularization in step (1b);

[0025] (1d) The amnion treated in step (1c) is freeze-dried to obtain the decellularized amnion matrix.

[0026] Further, in step (1b), the conditions for the supercritical carbon dioxide decellularization treatment are: pressure 9.3~15MPa, temperature 33~37℃, carbon dioxide flow rate 0.08~0.15 L / min, and treatment time 1~2 h.

[0027] Further, in step (2), the preparation methods of the upper electrospun fiber film layer and the lower electrospun fiber film layer include:

[0028] (2a) Preparation of spinning solution: The natural polymer material is dissolved in the first solvent to prepare the shell spinning solution; simultaneously, the synthetic polymer material is dissolved in the second solvent to prepare the core spinning solution.

[0029] (2b) Perform coaxial electrospinning: The shell spinning solution and the core spinning solution are injected into the shell channel and the core channel of the coaxial electrospinning equipment, respectively, and spinning is performed under the action of a high voltage electrostatic field;

[0030] In the process of spinning to form the upper electrospun fiber membrane and the lower electrospun fiber membrane, the injection flow rate of the shell spinning solution and the core spinning solution is controlled so that the content of the shell raw material component and the content of the core raw material component change in a continuous gradient along the direction perpendicular to the membrane surface.

[0031] Further, in step (2a), the first solvent is a mixture selected from one or more of acetic acid, hexafluoroisopropanol and trifluoroethanol; the second solvent is a mixture selected from one or more of acetic acid, hexafluoroisopropanol, N,N-dimethylformamide and dichloromethane.

[0032] Further, in step (2b), the coaxial electrospinning meets the following conditions: the concentration of the spinning solution for both the shell and the core is 10 wt%~30 wt%; the spinning voltage is 15 kV~25 kV; the receiving distance is 8 cm~12 cm; the spinning humidity is 50%; and the spinning time is 3h.

[0033] Furthermore, in step (3), tannic acid is used for crosslinking in the crosslinking process. The concentration of tannic acid solution is 0.2~3wt%, the crosslinking time is 0.5~3h, and the crosslinking temperature is 37℃.

[0034] Further, in step (5), the preparation method of the aldehyde-modified hyaluronic acid gel includes:

[0035] (5a) Hyaluronic acid aqueous solution and sodium periodate aqueous solution are mixed and stirred in the dark at 20~40℃ for 3~6h for oxidation reaction. Then a reducing agent is added to terminate the reaction. After dialysis and freeze drying, aldehyde-modified hyaluronic acid is obtained.

[0036] (5b) The aldehyde-modified hyaluronic acid obtained in step (5a) is mixed with a pH adjuster and an isotonic adjuster to obtain the aldehyde-modified hyaluronic acid gel.

[0037] Further, in step (5a), the concentration of the hyaluronic acid aqueous solution is 1~5 wt%, the concentration of the sodium periodate aqueous solution is 50~150 mg / mL, and the reducing agent is ethylene glycol;

[0038] In step (5b), the pH adjuster is a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate; the content of sodium dihydrogen phosphate is 0.5~5.5wt%, and the content of disodium hydrogen phosphate is 0.5~3.5wt%; the isotonic adjuster is sodium chloride.

[0039] A third aspect of the present invention provides the application of injectable amniotic membrane compound gel in soft tissue repair.

[0040] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0041] This invention discloses an injectable amniotic membrane composite gel, its preparation method, and its application. Through a sandwich composite scaffold design of "core-shell fiber membrane layer - amniotic matrix - core-shell fiber membrane layer," combined with in-situ cross-linkable aldehyde-based hyaluronic acid gel, a composite repair system is constructed that combines stable three-dimensional support, dynamic degradation matching, and efficient cell loading. Specifically, the decellularized amniotic membrane matrix serves as the intermediate permeable layer, continuously providing active nutrients for cell growth; the gradient core-shell fiber membrane layers act as support layers, providing mechanical properties and cell attachment points, while the shell collagen gradually degrades with cell growth, forming a "cell reserve layer" with increasing porosity, expanding space for cell proliferation. The core fibers near the intermediate layer, due to their larger diameter, continuously maintain structural support, achieving a match between degradation behavior and tissue regeneration.

[0042] Furthermore, tannic acid is used as a key cross-linking agent, which forms reversible non-covalent bonds with collagen through hydrogen bonds and hydrophobic interactions, effectively enhancing interlayer interface bonding, improving the overall stability of the scaffold, and delaying collagen degradation.

[0043] When the composite scaffold is combined with aldehyde-modified hyaluronic acid, the aldehyde-modified hyaluronic acid competitively replaces tannic acid and collagen to form covalent cross-links through a Schiff base reaction. This maintains the mechanical properties of the scaffold while further enhancing the cohesion of the hydrogel network. The released tannic acid then cross-links with the aldehyde-modified hyaluronic acid, forming a secondary network within it. This "competitive-cooperative" cross-linking mechanism transforms the composite gel from dynamic non-covalent bonding to stable covalent bonding, ensuring that the composite scaffold membrane is uniformly dispersed and stably occupies its position in the gel over a long period, effectively preventing displacement and sedimentation.

[0044] Aldehyaluronic acid and composite scaffold membranes can be immediately reconstituted before use. At this time, the system has not yet formed a strong gel network, maintaining good injectability. After injection into the defect site, its aldehyde groups rapidly undergo Schiff base reactions with the amino groups in the tissue and the collagen in the scaffold, achieving in-situ cross-linking and curing, perfectly fitting different morphological tissue cavities, and improving repair fit and effect.

[0045] The decellularized amniotic membrane matrix retains natural active ingredients, leveraging their low immunogenicity and bioactivity; the shell layer uses natural polymers such as collagen to promote cell adhesion and migration; and the core uses synthetic polymers such as polylactic acid to provide controllable mechanical support and degradation cycle. This organic combination achieves a balance between biocompatibility, structural stability, and adjustable degradation at the material level. Attached Figure Description

[0046] The following sections will describe some specific embodiments of the invention in a detailed manner by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0047] Figure 1 This is a schematic diagram of the composite support structure provided in this invention;

[0048] Figure 2 This is a schematic diagram of the structure of the compound gel provided in this invention;

[0049] Figure 3 This is an electron microscope image of the upper and lower layer electrospinning structure provided in this invention;

[0050] Figure 4 This is an electron micrograph of the lyophilized composite hydrogel provided in this invention;

[0051] Figure 5 This is a statistical chart of cell proliferation test results provided in this invention;

[0052] Figure 6 This is a statistical chart showing the ratio of new blood vessels 4 weeks post-surgery, provided in this invention.

[0053] Figure 7 This is a statistical chart showing the ratio of new blood vessels 8 weeks post-surgery, provided in this invention.

[0054] Figure 8 This is a statistical chart showing the ratio of new blood vessels 12 weeks post-surgery, as provided in this invention. Detailed Implementation

[0055] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0056] This embodiment provides an injectable amniotic membrane compound gel and its preparation method, including the following steps:

[0057] I. Preparation of decellularized amniotic matrix

[0058] 1) Cleaning and bio-enzyme treatment

[0059] Fresh amnion membranes were peeled off and cleaned, and after filtering out the water, a mixture of trypsin and disodium EDTA was added. The mixture was then treated in a constant temperature water bath at 37°C for 3 hours, and then removed and washed with ultrapure water. The mass ratio of amnion membrane to the mixture was 1:5.

[0060] 2) Decellularization treatment

[0061] The cleaned amnion was placed in a supercritical carbon dioxide extraction vessel, and 1 ml of carrier (0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide and 0.8% (v / v) sterile water) was added. Carbon dioxide was pumped in, and the pressure was maintained at 9.3 MPa, the temperature was maintained at 37 °C, the flow rate was 0.08 L / min, and the process was carried out for 2 h. Finally, the pressure was reduced to atmospheric pressure at a rate of 0.4 MPa / 10 min.

[0062] 3) Virus inactivation and deep cleaning

[0063] Amnion membrane was removed from a supercritical carbon dioxide environment and treated with a virus inactivating agent at a volume ratio of 1:1 for 4 hours. The virus inactivating agent consisted of 0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide, and 8% (v / v) ethanol. After 4 hours of treatment, the amnion membrane was washed with ultrapure water.

[0064] 4) Drying

[0065] The amnion processed in the above steps is placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized amnion matrix.

[0066] II. Fabrication of Composite Scaffolds

[0067] 1) Preparation of spinning solution

[0068] Collagen was dissolved in acetic acid to prepare a 10wt% solution, which was used as the shell spinning solution.

[0069] Polylactic acid was dissolved in N,N-dimethylformamide to prepare a 30wt% solution, which was used as the core spinning solution.

[0070] 2) Preparation of gradient electrospun membrane

[0071] The prepared shell and core spinning solutions were used for coaxial electrospinning. Specific spinning conditions were as follows: spinning voltage of 15 kV, receiving distance of 8 cm, spinning humidity of 50%, a flow rate gradient of 1.5–1.0–0.5 ml / h (1 h–2 h–3 h) for the shell solution, and a flow rate gradient of 0.5–1.0–1.5 ml / h (1 h–2 h–3 h) for the core solution. The spinning time for each layer was 3 h. See the electron microscope image of the prepared electrospun fiber membrane for details. Figure 3 .

[0072] 3) Composite and cross-linked stents

[0073] After the upper membrane was electrospun, the middle layer of decellularized amniotic membrane matrix was immediately attached in a wet state. Electrospinning of the lower layer continued on the decellularized matrix. After spinning, the three-layer structured membrane was removed and placed in a tannic acid solution for crosslinking. The concentration of the tannic acid solution was 0.2 wt%, the crosslinking time was 3 hours, and the crosslinking temperature was 37°C. See the schematic diagram of the prepared composite scaffold. Figure 1 .

[0074] 4) Drying and pulverizing

[0075] The cross-linked membrane was freeze-dried, then ground in a liquid nitrogen mill, and sieved to obtain a matrix membrane with a particle size of 100µm.

[0076] III. Preparation of Injectable Amniotic Membrane Compound Gel

[0077] 1) Hyaluronic acid aldehyde modification

[0078] Sodium periodate aqueous solution was added dropwise to hyaluronic acid aqueous solution, and the oxidation reaction was carried out at 20°C with stirring in the dark for 6 hours. Ethylene glycol was added to terminate the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid. The concentration of the hyaluronic acid was 1 wt%, and the concentration of the sodium periodate aqueous solution was 50 mg / ml.

[0079] 2) Preparation of aldehyde-modified hyaluronic acid gel

[0080] Aldehyaluronic acid was weighed and mixed with a pH adjuster and an isotonic adjuster to obtain a viscoelastic injectable hyaluronic acid gel. The pH adjuster was a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, with sodium dihydrogen phosphate comprising 0.5 wt% and disodium hydrogen phosphate comprising 3.5 wt%. The isotonic adjuster was sodium chloride.

[0081] 3) Preparation of injectable amniotic membrane compound gel

[0082] The lyophilized membrane and aldehyde-modified hyaluronic acid gel were mixed evenly at a mass ratio of 1:3 and then injected into the damaged area. See the schematic diagram of the prepared compound gel. Figure 2 See the lyophilized electron microscope image. Figure 4 . Example 2

[0083] This embodiment provides an injectable amniotic membrane compound gel and its preparation method, including the following steps:

[0084] I. Preparation of decellularized amniotic matrix

[0085] 1) Cleaning and bio-enzyme treatment

[0086] Fresh amnion membranes were peeled off and cleaned, and after filtering out the water, a mixture of trypsin and disodium EDTA was added. The mixture was then treated in a constant temperature water bath at 37°C for 4 hours, and then removed and washed with ultrapure water. The mass ratio of amnion membrane to the mixture was 1:4.

[0087] 2) Decellularization treatment

[0088] The cleaned amniotic membrane material was placed in a supercritical carbon dioxide extraction vessel, and 1 ml of carrier (0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide and 0.8% (v / v) sterile water) was added. Carbon dioxide was pumped in, and the pressure was maintained at 12 MPa, the temperature was maintained at 35°C, the flow rate was 0.1 L / min, and the process was maintained for 1.5 h. Finally, the pressure was reduced to atmospheric pressure at a rate of 0.6 MPa / 10 min.

[0089] 3) Virus inactivation and deep cleaning

[0090] The amnion was removed from the supercritical carbon dioxide environment and treated with a virus inactivating agent at a volume ratio of 1:1 for 4 hours. The virus inactivating agent consisted of 0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide, and 8% (v / v) ethanol. After 5 hours of treatment, the amnion was washed with ultrapure water.

[0091] 4) Drying

[0092] The amnion processed in the above steps is placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized amnion matrix.

[0093] II. Fabrication of Composite Scaffolds

[0094] 1) Preparation of spinning solution

[0095] Collagen was dissolved in acetic acid to prepare a 15 wt% solution, which was used as the shell spinning solution.

[0096] Polylactic acid was dissolved in N,N-dimethylformamide to prepare a 15wt% solution, which was used as the core spinning solution.

[0097] 2) Preparation of gradient electrospun membrane

[0098] The prepared shell and core spinning solutions were used for coaxial electrospinning. The specific spinning conditions were as follows: spinning voltage of 20 kV, receiving distance of 10 cm, spinning humidity of 50%, flow rate gradient of shell solution of 1.5~1.0~0.5 ml / h (1h~2h~3h), flow rate gradient of core solution of 0.5~1.0~1.5 ml / h (1h~2h~3h), and spinning time of each layer of the upper and lower layers of the core solution of 3h.

[0099] 3) Composite and cross-linked stents

[0100] After the upper membrane is electrospun, the middle layer of decellularized amniotic membrane matrix is ​​immediately attached in a wet state. Electrospinning of the lower layer then continues on the decellularized matrix. After spinning, the three-layer structure membrane is removed and placed in a tannic acid solution for crosslinking. The concentration of the tannic acid solution is 2 wt%, the crosslinking time is 2 hours, and the crosslinking temperature is 37°C.

[0101] 4) Drying and pulverizing

[0102] The cross-linked membrane was freeze-dried, then ground in a liquid nitrogen mill and sieved to obtain a matrix membrane with a particle size of 500µm.

[0103] III. Preparation of Injectable Amniotic Membrane Compound Gel

[0104] 1) Hyaluronic acid aldehyde modification

[0105] Sodium periodate aqueous solution was added dropwise to hyaluronic acid aqueous solution, and the oxidation reaction was carried out at 30°C with stirring in the dark for 5 hours. Ethylene glycol was added to terminate the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid. The concentration of the hyaluronic acid was 3 wt%, and the concentration of the sodium periodate aqueous solution was 100 mg / ml.

[0106] 2) Preparation of aldehyde-modified hyaluronic acid gel

[0107] Aldehyaluronic acid was weighed and mixed with a pH adjuster and an isotonic adjuster to obtain a viscoelastic injectable hyaluronic acid gel. The pH adjuster was a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, with each component containing 3 wt% sodium dihydrogen phosphate and 3 wt% disodium hydrogen phosphate. The isotonic adjuster was sodium chloride.

[0108] 3) Preparation of injectable amniotic membrane compound gel

[0109] The freeze-dried film and aldehyde-modified hyaluronic acid gel were mixed evenly at a mass ratio of 1:6 and then injected into the damaged area. Example 3

[0110] This embodiment provides an injectable amniotic membrane compound gel and its preparation method, including the following steps:

[0111] I. Preparation of decellularized amniotic matrix

[0112] 1) Cleaning and bio-enzyme treatment

[0113] Fresh amnion membranes were peeled off and cleaned, and after filtering out the water, a mixture of trypsin and disodium EDTA was added. The mixture was then treated in a constant temperature water bath at 37°C for 5 hours, and then removed and washed with ultrapure water. The mass ratio of amnion membrane to the mixture was 1:3.

[0114] 2) Decellularization treatment

[0115] The cleaned amniotic membrane material was placed in a supercritical carbon dioxide extraction vessel, and 1 ml of carrier (0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide and 0.8% (v / v) sterile water) was added. Carbon dioxide was pumped in, and the pressure was maintained at 15 MPa, the temperature was maintained at 33°C, the flow rate was 0.15 L / min, and the process was maintained for 1 h. Finally, the pressure was reduced to atmospheric pressure at a rate of 0.8 MPa / 10 min.

[0116] 3) Virus inactivation and deep cleaning

[0117] Amniotic membrane was removed from a supercritical carbon dioxide environment and treated with a virus inactivating agent at a volume ratio of 1:1 for 4 hours. The virus inactivating agent consisted of 0.02% (v / v) peracetic acid, 0.05% (v / v) hydrogen peroxide, and 8% (v / v) ethanol. After 6 hours of treatment, the amniotic membrane was washed with ultrapure water.

[0118] 4) Drying

[0119] The amnion processed in the above steps is placed in a vacuum dryer for freeze drying to obtain a highly bioactive decellularized amnion matrix.

[0120] II. Fabrication of Composite Scaffolds

[0121] 1) Preparation of spinning solution

[0122] Collagen was dissolved in acetic acid to prepare a 30wt% solution, which was used as the shell spinning solution.

[0123] Polylactic acid was dissolved in N,N-dimethylformamide to prepare a 10wt% solution, which was used as the core spinning solution.

[0124] 2) Preparation of gradient electrospun membrane

[0125] The prepared shell and core spinning solutions were used for coaxial electrospinning. The specific spinning conditions were as follows: spinning voltage of 25 kV, receiving distance of 12 cm, spinning humidity of 50%, the flow rate gradient of the shell solution was 1.5~1.0~0.5 ml / h (1h~2h~3h), the flow rate of the core solution was 0.5~1.0~1.5 ml / h (1h~2h~3h), and the spinning time for each layer was 3h.

[0126] 3) Composite and cross-linked stents

[0127] After the upper membrane is electrospun, the middle layer of decellularized amniotic matrix is ​​immediately attached in a wet state. Electrospinning of the lower layer then continues on the decellularized matrix. After spinning is complete, the three-layer structure membrane is removed and placed in a tannic acid solution for crosslinking. The concentration of the tannic acid solution is 3 wt%, the crosslinking time is 0.5 h, and the crosslinking temperature is 37 °C.

[0128] 4) Drying and pulverizing

[0129] The cross-linked membrane was freeze-dried, then ground in a liquid nitrogen mill, and sieved to obtain a matrix membrane with a particle size of 1000µm.

[0130] III. Preparation of Injectable Amniotic Membrane Compound Gel

[0131] 1) Hyaluronic acid aldehyde modification

[0132] Sodium periodate aqueous solution was added dropwise to hyaluronic acid aqueous solution, and the oxidation reaction was carried out at 40°C with stirring in the dark for 3 hours. Ethylene glycol was added to terminate the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid. The concentration of the hyaluronic acid was 5 wt%, and the concentration of the sodium periodate aqueous solution was 150 mg / ml.

[0133] 2) Preparation of aldehyde-modified hyaluronic acid gel

[0134] Aldehyaluronic acid was weighed and mixed with a pH adjuster and an isotonic adjuster to obtain a viscoelastic injectable hyaluronic acid gel. The pH adjuster was a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, with sodium dihydrogen phosphate comprising 5.5 wt% and disodium hydrogen phosphate comprising 0.5 wt%. The isotonic adjuster was sodium chloride.

[0135] 3) Preparation of injectable amniotic membrane compound gel

[0136] The freeze-dried film and aldehyde-modified hyaluronic acid gel were mixed evenly at a mass ratio of 1:10 and then injected into the damaged area.

[0137] Comparative Example 1

[0138] This embodiment provides an injectable amniotic membrane composite gel and its preparation method. The only difference between this embodiment and Embodiment 1 is the preparation of the composite scaffold. Specifically, after the upper membrane is electrospun, the middle layer of decellularized amniotic membrane matrix is ​​immediately attached in a wet state. The lower layer is then electrospun on the decellularized matrix. After spinning, the three-layer structure membrane is removed and crosslinked in a glutaraldehyde solution. The concentration of the glutaraldehyde solution is 2.5 wt%, the crosslinking time is 1 h, and the crosslinking temperature is 37 °C.

[0139] The remaining preparation steps are the same as in Example 1.

[0140] Comparative Example 2

[0141] This embodiment provides an injectable amniotic membrane compound gel and its preparation method, which differs from Example 1 only in that the hyaluronic acid is not aldehyde-treated.

[0142] The remaining preparation steps are the same as in Example 1.

[0143] Comparative Example 3

[0144] This embodiment provides an injectable amniotic membrane composite gel and its preparation method, which differs from Example 1 only in the preparation of the composite scaffold. Specifically:

[0145] 1) Preparation of spinning solution

[0146] Polylactic acid was dissolved in N,N-dimethylformamide to prepare a 30wt% solution, which was used as the spinning solution.

[0147] 2) Preparation of electrospun membranes

[0148] Take the prepared spinning solution. The specific spinning conditions are as follows: spinning voltage is 15kV, receiving distance is 8cm, spinning humidity is 50%, flow rate is 1ml / h, and spinning time for the upper and lower layers is 3h each.

[0149] The remaining preparation steps are the same as in Example 1.

[0150] In this invention, the injectable amniotic membrane compound gels prepared in the above embodiments and comparative examples were characterized and tested for performance, as detailed below:

[0151] 1. Cell proliferation assay

[0152] Samples prepared in Examples 1-3 and Comparative Examples 1-3 were used to detect cell proliferation at 37°C. Equal amounts of amniotic membrane compound gel were added sequentially to 24-well plates, followed by L929 cell suspension. The plates were incubated at 37°C, with the medium changed every other day for 7 days. Proliferation was then detected using the MTT assay.

[0153] Cell proliferation rate (%) = ×100%

[0154] Wherein OD0 represents the average absorbance of each well in the blank group at 570 nm; OD1 represents the average absorbance of each well in the test group at 570 nm.

[0155] Table 1. Cell proliferation rate (%)

[0156]

[0157] See Figure 5The experimental results show that Examples 1-3 not only have cell reserve layers that can accommodate more cells, but their stable gel networks also ensure that the composite scaffold is stably distributed in the gel without displacement, sedimentation, or stacking, resulting in good cell proliferation. Comparative Example 1 used glutaraldehyde for cross-linking, but the experimental results showed that glutaraldehyde is not suitable as a cross-linking agent for aldehyde-modified hydrogels, leading to poor gel stability. Furthermore, when glutaraldehyde binds tightly to collagen, it leaves no remaining amino groups in the collagen to bind with the aldehyde groups of aldehyde-modified hyaluronic acid, thus preventing the composite scaffold from stably occupying its position in the gel. Therefore, the overall stability of the composite scaffold is poor, leading to sedimentation, stacking, and poor cell proliferation rate. Comparative Example 2 did not undergo aldehyde-modification of the hyaluronic acid, which also resulted in sedimentation and stacking of the composite scaffold, thus leading to poor cell proliferation rate. Comparative Example 3 did not undergo gradient degradation treatment of the spun membrane, resulting in insufficient space within the spun membrane, leaving insufficient space for cell growth and poor cell proliferation rate.

[0158] 2. Gel mechanical property testing

[0159] Take 5g of samples prepared in Examples 1-3 and Comparative Examples 1-3, mix them together, and let them stand at room temperature for 1 hour to form a gel. Use a universal testing machine to test the mechanical properties of the mixed hydrogel. The thickness is 1mm, the width is 5mm, the tensile speed is 25mm / min, and there are 3 parallel tests per group.

[0160] Tensile strength (MPa) =

[0161] Table 2 Tensile strength test results (MPa)

[0162]

[0163] The experimental results show that the tensile strength of Examples 1-3 is good. The gels of Comparative Examples 1-2 failed to form a cross-linked network, so the tensile strength could not be tested. The spun membrane in Comparative Example 3 did not use collagen components, lacking the binding effect of collagen and aldehyde-modified hyaluronic acid. Furthermore, polylactic acid could not bind with tannic acid, resulting in a reduction in the overall tannic acid load on the composite scaffold. The amount of tannic acid that could cross-link with aldehyde-modified hyaluronic acid was limited, thus the cross-linked network strength was poor.

[0164] 3. In vitro degradation test

[0165] The samples prepared in Examples 1-3 and Comparative Examples 1-3 were freeze-dried and weighed, and the weight was recorded as W0. Water was added to fully swell the samples. The remaining solid samples were taken 1, 2, and 3 months after swelling, and the samples were freeze-dried and weighed as W1. The in vitro degradation rate was calculated.

[0166] Degradation rate (%) = ×100%

[0167] Table 3. Degradation percentage (%) for each group of samples

[0168]

[0169] The experimental results show that all samples began to degrade over time. Samples in Examples 1-3 maintained a relatively intact structure at the January time point, but still retained a gel morphology at the March time point. Comparative Examples 1-2 did not form a gel cross-linked network; at the January time point, only solid membrane material remained, and the subsequent process involved the degradation of the solid membrane material. Comparative Example 3, although forming a partial gel network, lacked collagen and sufficient tannins, resulting in poor gel network stability.

[0170] 4. Soft tissue repair test

[0171] Fifty-four healthy male New Zealand white rabbits were selected, with three rabbits in each group, for a total of six groups. The normal bladder area in each group served as a control. The abdominal cavity was opened, the bladder was exposed, and a 2cm × 2cm square defect was constructed on the posterior wall of the bladder. Materials from each group were injected into the defect area. The wound was sutured in the order of muscle layer, muscle fascia, subcutaneous fascia, and skin, and the abdominal cavity was closed. Animals were dissected at 4, 8, and 12 weeks post-surgery for histopathological observation, and the ratio of neovascularization in the surgical area to the number of blood vessels in the normal bladder area was calculated.

[0172] The experimental results show that Examples 1-3 significantly improved the bladder wall tissue repair effect. This indicates that by providing bioactive factors through the amniotic matrix and storing more cells through the special design of the material structure, tissue repair can be fully achieved. Furthermore, the stability of the entire gel system ensures sufficient contact between the material and cells, and the synergistic effect of each structure and component is evident. (See [reference needed]). Figures 6 to 8 The number of newly formed blood vessels was higher in the 4w, 8w, and 12w samples than in the control group, achieving the repair objective. Control groups 1-3, due to the lack of stability in the gel system and poor cross-linking effect, experienced rapid product degradation, fewer newly formed blood vessels, and poor repair effects.

[0173] In summary, this invention provides an injectable amniotic membrane composite gel, its preparation method, and its application. This gel system is composed of a composite scaffold membrane and aldehyde-based hyaluronic acid gel in a specific ratio. The composite scaffold membrane originates from a composite scaffold with a three-layer special structure. This scaffold consists of an upper electrospun fiber membrane layer, a decellularized amniotic membrane matrix intermediate layer, and a lower electrospun fiber membrane layer, which are sequentially stacked and tightly bonded together. Crucially, the fibers in the upper and lower electrospun fiber membrane layers have a core-shell structure. The shell layer is composed of natural polymer materials such as collagen, while the core layer is composed of synthetic polymer materials such as polylactic acid. Furthermore, the ratio of the shell layer to the core material changes continuously along the membrane thickness direction.

[0174] In terms of manufacturing process, this invention employs coaxial electrospinning technology, achieving a gradient distribution of fiber composition by precisely controlling the flow rate changes of the spinning solution between the shell and core layers. Subsequently, tannic acid is used to crosslink the three-layer composite scaffold. This step not only enhances the interlayer bonding strength but also lays the foundation for subsequent synergistic crosslinking with aldehyde-based hyaluronic acid. The crosslinked scaffold is freeze-dried, ground to a specific particle size, and then mixed with pre-prepared aldehyde-based hyaluronic acid gel to finally obtain a composite gel product with good injectability and in-situ molding capabilities.

[0175] Through systematic testing of the embodiments and comparative samples, the present invention demonstrates significant comprehensive advantages. Regarding cell compatibility, the cell proliferation rate of the materials in the embodiments all exceeded 105%, significantly superior to the comparative samples (all below 60%), proving that its three-dimensional structure can effectively support cell growth and prevent scaffold deposition. In terms of mechanical properties, the gels in the embodiments exhibited a tensile strength of approximately 0.6 MPa, forming a stable cross-linked network, while the comparative samples showed severely insufficient strength or even failure to form due to improper cross-linking or lack of key components. In vitro degradation experiments showed that the materials of the present invention exhibited a gradual and controllable degradation behavior (degradation rate 70-76%) within three months, perfectly matching the tissue regeneration cycle, while the comparative samples showed excessively rapid or irregular degradation. In animal models, the materials of the present invention significantly promoted angiogenesis and tissue repair at bladder defects, with sustained and stable effects.

[0176] This invention utilizes a multi-element synergistic approach combining a gradient core-shell fiber structure, an active amniotic membrane matrix, and a crosslinkable hyaluronic acid gel to construct a microenvironment that provides physical support, dynamically releases bioactive signals, and can be customized in situ. This overcomes the shortcomings of traditional materials in terms of degradation control, space maintenance, cell loading, and long-term repair, offering a novel, efficient, reliable, and clinically convenient solution for soft tissue regeneration and repair, demonstrating significant scientific value and application prospects.

[0177] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An injectable amniotic membrane compound gel, characterized in that, The raw material components of the injectable amniotic membrane compound gel include a composite scaffold membrane and an aldehyde-based hyaluronic acid gel, wherein the mass ratio of the composite scaffold membrane to the aldehyde-based hyaluronic acid gel is 1:3~10. The composite scaffold membrane is formed by freeze-drying and grinding the composite scaffold; the composite scaffold includes an upper electrospun fiber membrane layer, a decellularized amniotic membrane matrix intermediate layer, and a lower electrospun fiber membrane layer stacked sequentially. The upper electrospun fiber membrane layer, the decellularized amniotic membrane matrix intermediate layer, and the lower electrospun fiber membrane layer are cross-linked together and cross-linked with tannic acid. The electrospun fibers in both the upper and lower electrospun fiber membrane layers have a core-shell structure. The shell material component of the core-shell structure includes natural polymer materials, and the core material component of the core-shell structure includes synthetic polymer materials. The natural polymer material is a mixture of one or more selected from chitosan, collagen, silk fibroin, and gelatin; the synthetic polymer material is a mixture of one or more selected from polylactic acid, polycaprolactone, polyvinyl alcohol, and polyethylene oxide. In the upper electrospun fiber membrane layer and the lower electrospun fiber membrane layer, from the side near the intermediate layer of the decellularized amniotic membrane matrix to the side away from the intermediate layer, the flow rate of the shell spinning solution varies from high to low in the range of 1.5 mL / h to 1.0 mL / h to 0.5 mL / h, and the flow rate of the core spinning solution varies from low to high in the range of 0.5 mL / h to 1.0 mL / h to 1.5 mL / h.

2. The method for preparing the injectable amniotic membrane compound gel as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of decellularized amniotic membrane matrix; (2) An upper electrospun fiber membrane layer and a lower electrospun fiber membrane layer are prepared sequentially on both sides of the decellularized amnion matrix by coaxial electrospinning process to obtain a three-layer composite scaffold precursor. (3) The three-layer composite scaffold precursor is cross-linked to obtain a composite scaffold; (4) The composite scaffold is freeze-dried and ground to obtain a composite scaffold membrane; (5) Preparation of aldehyde-modified hyaluronic acid gel; (6) The composite scaffold membrane and the aldehyde-based hyaluronic acid gel are mixed in proportion to obtain the injectable amniotic membrane composite gel.

3. The method for preparing the injectable amniotic membrane compound gel according to claim 2, characterized in that, In step (1), the method for preparing the decellularized amniotic membrane matrix includes: (1a) Cleaning and enzymatic treatment of fresh amniotic membrane; (1b) The amnion treated in step (1a) was placed in a supercritical carbon dioxide environment and a carrier was added for decellularization. (1c) Perform virus inactivation and deep washing on the amnion after decellularization in step (1b); (1d) The amnion treated in step (1c) is freeze-dried to obtain the decellularized amnion matrix.

4. The method for preparing the injectable amniotic membrane compound gel according to claim 2, characterized in that, In step (2), the preparation method of the upper electrospun fiber film layer and the lower electrospun fiber film layer includes: (2a) Preparation of spinning solution: The natural polymer material is dissolved in the first solvent to prepare the shell spinning solution; simultaneously, the synthetic polymer material is dissolved in the second solvent to prepare the core spinning solution. (2b) Perform coaxial electrospinning: The shell spinning solution and the core spinning solution are injected into the shell channel and the core channel of the coaxial electrospinning equipment, respectively, and spinning is performed under the action of a high voltage electrostatic field; In the process of spinning to form the upper electrospun fiber membrane and the lower electrospun fiber membrane, the injection flow rate of the shell spinning solution and the core spinning solution is controlled so that the content of the shell raw material component and the content of the core raw material component change in a continuous gradient along the direction perpendicular to the membrane surface.

5. The method for preparing the injectable amniotic membrane compound gel according to claim 4, characterized in that, In step (2b), the coaxial electrospinning meets the following conditions: the concentration of the spinning solution for both the shell and the core is 10 wt%~30 wt%; the spinning voltage is 15 kV~25 kV; the receiving distance is 8 cm~12 cm; the spinning humidity is 50%; and the spinning time is 3h.

6. The method for preparing the injectable amniotic membrane compound gel according to claim 2, characterized in that, In step (3), tannic acid is used for crosslinking in the crosslinking process. The concentration of tannic acid solution is 0.2~3wt%, the crosslinking time is 0.5~3h, and the crosslinking temperature is 37℃.

7. The method for preparing the injectable amniotic membrane compound gel according to claim 2, characterized in that, In step (5), the preparation method of the aldehyde-modified hyaluronic acid gel includes: (5a) Hyaluronic acid aqueous solution and sodium periodate aqueous solution are mixed and stirred in the dark at 20~40℃ for 3~6h for oxidation reaction. Then a reducing agent is added to terminate the reaction. After dialysis and freeze drying, aldehyde-modified hyaluronic acid is obtained. (5b) The aldehyde-modified hyaluronic acid obtained in step (5a) is mixed with a pH adjuster and an isotonic adjuster to obtain the aldehyde-modified hyaluronic acid gel.

8. The use of the injectable amniotic membrane compound gel as described in claim 1 in the preparation of soft tissue repair products.

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