An asymmetrically reconstituted collagen anti-adhesion dressing and method of making same

Abstract

The application discloses an asymmetric recombinant collagen anti-adhesion dressing and a preparation method thereof. The anti-adhesion dressing comprises a first layer facing a non-wound surface and a second layer facing a wound surface; the first layer is composed of a photocrosslinked high molecular network containing polyethylene glycol bisacrylate, and the surface of the first layer has incompletely reacted unsaturated carbon-carbon double bonds; the second layer is composed of a directional porous network containing sulfhydrylated hyaluronic acid and recombinant humanized collagen, has vertical channels in the inside, and contains free sulfhydryl groups; the first layer and the second layer are interlocked by a Michael addition reaction of carbon-carbon double bonds and free sulfhydryl groups at a joint to form a covalent sulfide bond. The application can realize high-strength interlayer anti-peeling performance, excellent wet-state sutureless tissue adhesion, and liquid seepage active suction and isolation functions.

Description

Technical Field

[0001] This application relates to the fields of chemistry and materials, specifically to an asymmetric recombinant collagen anti-adhesion dressing for use in moist soft tissue wound covering, which has the dual functions of promoting healing through wet tissue adhesion and preventing adhesion and isolating the dorsal side, and its preparation method. Background Technology

[0002] In demanding medical settings such as abdominal and pelvic surgeries, extensive burns, and various deep tissue dissections, wound repair often faces conflicting physiological and engineering functional requirements. On one hand, the contact layer of the dressing adhering to the wound should serve as a temporary substitute for the extracellular matrix, requiring high hydrophilicity, permeability, and cell affinity to rapidly absorb large amounts of inflammatory exudate in the early postoperative period and induce fibroblast and vascular endothelial cell ingrowth through a biomimetic matrix. On the other hand, the outer layer of the dressing exposed on the non-wound side must exhibit extremely low cell adhesion and a high-density structural morphology to physically prevent abnormal adhesion and growth of adjacent normal tissues (such as intestinal serosa and muscle fascia). If the outer anti-adhesion barrier fails, it can easily lead to fatal mechanical intestinal obstruction postoperatively, or cause secondary tissue tearing and mechanical damage to new granulation tissue when removing the dressing during dressing changes.

[0003] Current composite medical dressings typically construct a two-layer structure by mechanically pressing an absorbent sponge and an anti-adhesion film together or by using medical bio-adhesives. However, in actual bodily fluid immersion environments, due to the significant differences in the absorbent swelling rates of different polymer network structures, the lower layer undergoes dramatic volume expansion after absorbing fluid, while the upper barrier film remains dimensionally stable. This results in significant shear stress at the interlayer interface. This stress concentration phenomenon causes the anti-adhesion film to completely delaminate from the absorbent layer within days of application. The delaminated, dense film loses the structural support of the underlying matrix, rapidly curling at the edges and even slipping or shifting anatomically, completely rendering its anti-tissue adhesion function ineffective.

[0004] Meanwhile, existing single-layer or composite dressings often rely on electrostatic interactions or non-specific hydrogen bonding on the surface of polymer materials to maintain initial tissue fixation. When faced with a moist wound surface covered by blood, tissue fluid, and a large amount of mucus, the hydration of interfacial water molecules can significantly shield electrostatic and hydrogen bonding interactions, resulting in a significant decrease in tissue adhesion. This makes the dressing extremely easy to peel off from the moist tissue around the wound, leading to wound exposure.

[0005] Existing double-layer composite dressings are prone to interlayer delamination and structural disintegration when exposed to complex moist body fluids, and their adhesion to the interface of wet soft tissue is severely insufficient. A novel interface bonding and structural forming mechanism is needed to solve this problem. Summary of the Invention

[0006] This application provides an asymmetric recombinant collagen anti-adhesion dressing and its preparation method, which solves the defects of existing composite dressings that are prone to interlayer peeling failure under liquid absorption and swelling conditions and have low adhesion in wet environments.

[0007] This application provides an asymmetric recombinant collagen anti-adhesion dressing, comprising a first layer and a second layer, which are connected at the interface to form a continuously transitioning asymmetric structure. The first layer is a dense barrier membrane facing the non-wound side, composed of a photocrosslinked polymer network containing polyethylene glycol diacrylate, and its surface has incompletely reacted unsaturated carbon-carbon double bonds. The second layer is a tissue-adhesive sponge facing the wound side, composed of a directional porous network containing thiolated hyaluronic acid and recombinant humanized collagen, with vertical channels extending through its thickness direction, and containing free thiol groups. The first and second layers are interlocked at the interface through a Michael addition reaction between the carbon-carbon double bonds of the first layer and the free thiol groups of the second layer to form covalent thioether bonds.

[0008] Furthermore, the molecular weight of polyethylene glycol diacrylate is configured to be 4000 Da.

[0009] Furthermore, the polyethylene glycol diacrylate contained in the first layer is formed by cross-linking and curing of an aqueous solution with a mass-volume ratio concentration of 5% by ultraviolet light, and the retention rate of unsaturated carbon-carbon double bonds in the first layer is configured to be 15%.

[0010] Furthermore, the dense barrier membrane of the first layer is configured as a physical barrier structure to prevent the invasion of exogenous fibroblasts and the diffusion of internal exudate to the non-wound side. The unsaturated carbon-carbon double bonds are configured to be excited by controlled ultraviolet light irradiation under the action of an initiator to form retention rates. The unsaturated carbon-carbon double bonds on the surface are configured to provide reserved covalent reactive sites for the second layer, and the interlocking connection structure at the interface is designed to resist the physical interlaminar separation that occurs between the first and second layers in the hydrated and swollen state.

[0011] Furthermore, the recombinant humanized collagen was configured as recombinant humanized type III collagen.

[0012] Furthermore, the degree of thiol substitution of the thiolized hyaluronic acid is configured to be between 35% and 45%, the pore size of the vertical channels is configured to be between 30 micrometers and 60 micrometers, and the vertical channels are configured as a microfluidic channel network that aspirates and circulates exudate in the second layer based on capillary action.

[0013] Furthermore, the remaining free thiol groups in the second layer that did not participate in the Michael addition reaction at the interface are distributed on the pore walls of the oriented porous network. These remaining free thiol groups are configured to undergo redox reactions with cysteine ​​residues contained in the surface tissue protein structure upon contact with the protein interface, forming disulfide bonds in situ for covalent anchoring. Based on the high specific surface area of ​​the oriented porous network and the chemical anchoring force of the disulfide bonds, the second layer constitutes a seamless adhesive that directly adheres to and is fixed to the surface of moist tissue covered by blood or tissue mucus.

[0014] This application also provides a method for preparing an asymmetric recombinant collagen anti-adhesion dressing, comprising the following steps: in a photocrosslinking and curing step, photocrosslinking a polyethylene glycol diacrylate solution to form a first layer, and controlling the photocrosslinking dosage to retain unsaturated carbon-carbon double bonds on the surface of the first layer; in a molding step, injecting a mixed solution containing thiolized hyaluronic acid and recombinant humanized collagen into a molding mold and covering the surface of the first layer to form a second layer precursor, wherein the mixed solution contains free thiol groups; in a freezing and interface grafting step, placing the molding mold carrying the first and second layer precursors on a freezing stage with a unidirectional heat conduction base plate, constructing a bottom-up unidirectional temperature gradient field in the mixed solution, and triggering an interlayer interface grafting reaction driven by the temperature gradient field between unsaturated carbon-carbon double bonds and free thiol groups to generate covalent sulfide bonds; and in a vacuum freeze-drying step, subjecting the reacted material to vacuum freeze-sublimation treatment to form vertical channels within the second layer precursor, thereby obtaining the asymmetric recombinant collagen anti-adhesion dressing.

[0015] Furthermore, in the freezing and interface grafting steps, the operating temperature of the freezing stage with the unidirectional heat conduction base plate is configured to be -78°C, and the material of the unidirectional heat conduction base plate is configured to be pure copper.

[0016] Furthermore, the unidirectional heat conduction base plate is configured to construct a unidirectional advancing temperature gradient field at -78℃ within the molding die. This temperature gradient field drives the solvent water molecules inside the second-layer precursor body to grow ice crystals vertically and oriented at a advancing rate of 2 mm / min to 4 mm / min. The ice crystal leading edge pushes and enriches the polymeric solute at the interface. Under extremely low temperature conditions, the growing ice crystals generate a strong local polymer chain concentration effect on the uncrystallized liquid phase region, increasing the local concentration of thiol groups and double bonds at the interface to more than 20 times the initial concentration. Simultaneously, the latent heat of crystallization released by ice crystal growth raises the actual temperature of the unfrozen liquid phase micro-region, and the local stress field generated by the mechanical expansion of the ice crystals reduces the apparent activation energy of the reaction. Under the synergistic effect of the freeze-concentration effect, local temperature rise, and mechanical stress, the local polymer chain concentration effect is configured to catalyze the covalent grafting interlocking of unsaturated carbon-carbon double bonds and free thiol groups on the surface of the first layer at the interface between the two layers based on the Michael addition reaction.

[0017] The beneficial effects of this application are as follows: The first and second layers form covalent thioether bonds through a Michael addition reaction between the reserved active double bonds and free thiol groups. This prevents the dressing from physically separating and disintegrating under external swelling forces, significantly enhancing the structural integrity during the coverage period. Simultaneously, the second layer's contact interface, with its vertically arranged micropores, exhibits the ability to directionally absorb exudate, preventing the risk of maceration of the perianal tissue. Furthermore, the retained free thiol groups can directly bond with macromolecules on the tissue surface, achieving robust wet fixation without the need for additional sutures. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the overall structure of the asymmetric recombinant collagen anti-adhesion dressing provided in the embodiments of this application.

[0019] Figure 2 This is a two-dimensional cross-sectional view of the asymmetric recombinant collagen anti-adhesion dressing provided in the embodiments of this application.

[0020] Figure 3 This is a partially enlarged view of the asymmetric recombinant collagen anti-adhesion dressing provided in the embodiments of this application.

[0021] Figure 4 This is a flowchart of the method for preparing asymmetric recombinant collagen anti-adhesion dressing provided in the embodiments of this application.

[0022] Explanation of reference numerals in the attached figures: 100-Asymmetric recombinant collagen anti-adhesion dressing, 101-First layer, 102-Second layer, 103-Boundary, 201-Vertical channel, 202-Dense barrier membrane, 301-Free thiol group, 302-Unsaturated carbon-carbon double bond. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0024] To address the mechanical failure issues of existing double-layer dressings, such as easy delamination and peeling upon swelling due to body fluid infiltration, and their poor fixation ability on moist wounds, this application provides a novel asymmetric recombinant collagen dressing structure. This structure utilizes a Michael addition chemical bonding mechanism between substrates of specific molecular weights, coupled with a specific temperature gradient freezing phase change process, to establish an in-situ covalent bridge between the dense polymer layer and the porous sponge layer, ensuring structural integrity and stability under extreme interfacial stress.

[0025] like Figure 1As shown, this embodiment provides an asymmetric recombinant collagen anti-adhesion dressing 100. From a macroscopic physical morphological perspective, the asymmetric recombinant collagen anti-adhesion dressing 100 possesses a Janus-like asymmetric structure with a continuous transition between upper and lower double-layer regions. The overall physical space of the asymmetric recombinant collagen anti-adhesion dressing 100 is divided into a first layer 101 and a second layer 102 tightly bonded to the surface of the first layer 101. The first layer 101 and the second layer 102 are solidly connected at their junction 103 to form an overall continuously transitioning structure.

[0026] In this embodiment, the first layer 101 is designed as a dense barrier membrane facing the non-wound side. In pathological applications, this layer primarily serves to prevent tissue adhesion and physically block external bacteria. The first layer 101 is composed of a photocrosslinked polymer network containing polyethylene glycol diacrylate. The polyethylene glycol diacrylate molecules have highly reactive acrylate groups attached to both ends, and its core polyethylene glycol segment can bind a large number of water molecules to form a dense hydrated outer layer. In this embodiment, the nominal molecular weight of polyethylene glycol diacrylate is strictly configured to 4000 Da. Too high a molecular weight would result in excessively large crosslinking node spacing and insufficient density; while too low a molecular weight would increase the rigidity and brittleness of the polymer network. A molecular weight of 4000 Da provides optimal steric hindrance while maintaining gel elasticity. Due to a specific controlled curing process, the crosslinked network on the surface and inside of the first layer 101 does not completely deplete the active monomers, and the surface of the first layer 101 contains unreacted unsaturated carbon-carbon double bonds 302. The unsaturated carbon-carbon double bonds 302 on this surface are configured to provide reserved covalent reactive sites to the second layer 102 below it. By employing a polyethylene glycol diacrylate crosslinking network with a molecular weight of 4000 Da, a strong repulsive effect against protein adsorption can be achieved by utilizing the hydration-stabilizing layer formed therein, thus constructing a physical barrier structure that can prevent the invasion of exogenous fibroblasts and the diffusion of internal exudate to the non-wound side.

[0027] The second layer 102 is designed as a tissue-adhesive sponge facing the wound. The second layer 102 is composed of a directional porous network composite containing thiolated hyaluronic acid and recombinant humanized collagen. Hyaluronic acid, as a natural component of the extracellular matrix, possesses excellent biocompatibility. In this embodiment, a hyaluronic acid backbone with a nominal molecular weight of 150-200 kDa was selected and chemically modified with 3,3'-dithiodipropionate dihydrazide to introduce active groups. The degree of thiol substitution of this thiolated hyaluronic acid is strictly configured to be between 35% and 45%. Simultaneously, the recombinant humanized collagen added to the second layer 102 is configured as recombinant humanized type III collagen. This recombinant humanized type III collagen provides a continuous, untruncation-free amino acid sequence of 164 amino acids. This untruncation-free sequence segment can fully expose integrin binding sites in the extracellular matrix. After the cross-linked network is formed, the second layer 102 contains a large number of free thiol groups 301 with protruding side chains. By combining highly thiol-substituted hyaluronic acid with recombinant humanized type III collagen containing a sequence of 164 consecutive amino acids, the high water absorption rate of hyaluronic acid and the specific receptor recognition pathway of collagen can be utilized to induce host fibroblasts and vascular endothelial cells to grow rapidly along a three-dimensional network, achieving excellent contact tissue affinity and wound healing promotion.

[0028] like Figure 2 As shown in the two-dimensional cross-sectional structure, the asymmetric recombinant collagen anti-adhesion dressing 100 exhibits a highly organized porous structure. Following a specific cryo-sublimation process, the second layer 102 has vertical channels 201 extending throughout its thickness. Under microscopic measurement, the pore size distribution of the vertical channels 201 is precisely configured within the range of 30 to 60 micrometers. These parallel arrays of vertical microducts form a vertically interconnected channel network, which is absolutely sealed off above by a dense barrier membrane 202. The vertical channels 201 are configured as a microfluidic channel network that aspirates and circulates exudate within the second layer 102 based on capillary action.

[0029] To elucidate the physical mechanism of the exudate suction in this structure, the Laplace pressure model from capillary fluid dynamics is introduced to analyze the liquid level lifting force inside the vertical channel. The capillary suction driving force within the micro-channels follows the analytical equation below:

[0030] in, This indicates the upward capillary suction pressure generated within the vertical channel. This indicates the surface tension of the exudate from the wound tissue. This represents the contact angle that occurs between the permeate system and the porous hyaluronic acid framework solid phase interface. r This represents the equivalent radius of the capillary pore size of a single microscopic vertical channel 201.

[0031] As can be seen from the above physical equations, with the support of a hydrophilic polymer network, an ideal micropore size range of 30 to 60 micrometers can generate strong [structures] at the liquid-gas-solid three-phase interface. Capillary suction with positive pressure. By employing this directional and size-controlled array of vertical channels 201, excess tissue exudate accumulated at the contact surface can be actively and rapidly drawn into the upper middle space of the second layer 102 using physical capillary suction. When the pressurized exudate rises along the microcatheter and touches the bottom surface of the dense barrier membrane 202 at the top, the highly dense cross-linked network forms a physical barrier that forces the liquid surface to stop seepage and instead stores and diffuses within the transverse structure. This process achieves adaptive flow and effective blockage of a large amount of exudate in the three-dimensional space inside the dressing without the aid of an external negative pressure device, eliminating the risk of maceration and erosion of normal tissue around the wound.

[0032] Combination Figure 3 As shown in the magnified view, the interface 103 between the first layer 101 and the second layer 102 is not mechanically pressed together, but rather anchored through an in-situ chemical reaction. Due to process control, a large number of unpolymerized unsaturated carbon-carbon double bonds 302 remain on the surface of the first layer 101, which are in close contact with the densely packed free thiol groups 301 on the macromolecular backbone of the second layer 102. During the directional freezing process, the mechanical compression caused by the upward growth of ice crystals pushes the polymeric solute in the second-layer precursor to the interface 103, resulting in an extremely high local concentration of free thiol groups 301 and unsaturated carbon-carbon double bonds 302 at the interface. Although both thiolated hyaluronic acid and recombinant humanized collagen are present in the second-layer precursor, the compliant molecular chains of thiolated hyaluronic acid are significantly more flexible than the triple-helix structure of collagen. During the mechanical pushing process of ice crystals, the compliant thiolated hyaluronic acid molecular chains preferentially diffuse to the front edge of the interface, while collagen molecules, due to their larger molecular size and rigid structure, are hindered in regions slightly further away from the interface. This self-separation effect driven by the difference in molecular chain flexibility ensures that the thiol groups enriched at the interface are mainly derived from thiolated hyaluronic acid, effectively avoiding steric interference from collagen in the multi-component mixture regarding the thiol-double bond reaction. Between the locally concentrated high concentration of thiol groups and double bonds at the interface, the thiol groups undergo nucleophilic attack on the unsaturated carbon-carbon double bonds, forming stable and robust covalent thioether bonds through Michael addition reactions. By generating covalent thioether bonds over a large area on the macroscopic contact surface, the extremely high chemical bond energy of covalent bonds can be utilized to promote the formation of strong molecular-level interlocking connections between the discontinuous two-layer network. This interlocking connection structure of the interface is designed to resist the physical interlayer tensile stress and peeling separation that occur when the first layer 101 and the second layer 102 are subjected to large-volume hydration and swelling.

[0033] Furthermore, in Figure 3In the implementation details, the remaining free thiol groups 301 in the second layer 102 that did not participate in the Michael addition reaction at the interface 103 are uniformly distributed on the broad pore wall surface of the oriented porous network. These remaining free thiol groups 301 are configured to rapidly undergo redox contact reactions with cysteine ​​residues contained in the macromolecular structure of the surface tissue proteins when the dressing contacts and covers the interface of the aqueous soft tissue containing natural proteins. This in-situ spontaneous oxidation process promotes the direct formation of disulfide bonds between the exogenous dressing matrix molecules and the endogenous human tissue macromolecules. By utilizing the high specific surface area and enriched thiol functional groups provided by the second-layer mesh architecture, a strong chemical anchoring force can be provided by the in-situ generated disulfide bonds, enabling the dressing to form a seamless adhesive that can directly adhere to and fix to the surface of extremely moist tissue covered by blood or tissue mucus, preventing physical slippage.

[0034] like Figure 4 As shown, this application also provides a method for preparing the above-mentioned asymmetric recombinant collagen anti-adhesion dressing. This preparation method is mainly based on controlling the curing mechanism and a directional freezing phase change process, and includes the following processing steps in sequence: In the photocrosslinking curing step, the polyethylene glycol diacrylate solution is photoinitiated to crosslink to form a first layer 101 with a certain morphological elasticity, and the ultraviolet light crosslinking exposure dose is precisely controlled to intentionally retain a certain proportion of unsaturated carbon-carbon double bonds 302 on the surface of the first layer 101.

[0035] In the specific solution preparation and operation stage, polyethylene glycol diacrylate core substrate with a molecular weight of 4000 Da was selected and fully dissolved in phosphate buffer solution with a pH of 7.4 at a mass-to-volume ratio of 5%. 0.1% (w / w) of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (initiator I2959) was added to the system and ultrasonically mixed thoroughly. The mixed and degassed precursor solution was then poured evenly into a specially designed molding base mold, and an excitation wavelength of 365 nm and an irradiation intensity precisely calibrated to 15 mW / cm² were used. 2 An ultraviolet light source is used to vertically irradiate the solution surface. In this embodiment, the ultraviolet light irradiation time is strictly controlled to 60 seconds.

[0036] Under ultraviolet light excitation at a specific wavelength and irradiation intensity, the photoinitiator component suspended within the first layer of the 101 precursor system undergoes a free radical cleavage reaction, initiating the continuous consumption of the acrylate double bonds at both ends of the polyethylene glycol diacrylate molecule and constructing a macromolecular crosslinked network. To establish the correspondence between the double bond retention rate and the irradiation parameters, a kinetic decay correlation formula for the crosslinking reaction stage is introduced. The reaction kinetic model of the system follows the equations below:

[0037] in, C t Indicates elapsed time t Real-time concentration of residual acrylate unsaturated carbon-carbon double bonds in the post-network. C 0 This represents the initial total concentration of double bonds in the solution system before ultraviolet light irradiation. k This represents the polymerization rate constant related to the initiator reaction efficiency. I This indicates the constant irradiance set by the ultraviolet light source. t This indicates the actual exposure reaction time.

[0038] By substituting the formula model into the calculations and combining it with infrared spectroscopy measurements, the 60-second light dose used in this embodiment just drove the system to consume approximately 85% of the acrylate double bonds. The macroscopic mesh achieved the complete mechanical and physical strength required for dense barrier, while the retention rate of unsaturated carbon-carbon double bonds 302 in the first layer 101 was controlled to 15% at the microscopic level. By employing the above-mentioned controlled photocrosslinking process and dose-limiting method, the terminateable nature of the photochemical reaction can be utilized to stably eject and retain active reaction sites at the interface of subsequent processing stages, while ensuring that the strength of the formed base film meets the requirements.

[0039] In the injection molding step, a mixed solution containing thiolized hyaluronic acid and recombinant humanized collagen is injected into the molding mold through a guide pipe, and is evenly spread on the surface of the first layer 101 which is in a semi-cured state, so as to initially form the second layer precursor in a physical form.

[0040] In the specific formulation preparation, thiolated hyaluronic acid with a nominal molecular weight of 150-200 kDa was selected, ensuring that its thiol substitution degree was within the active range of 35% to 45%. This thiolated hyaluronic acid powder was dissolved at a mass concentration of 3% in a phosphate buffer solution within the same system, followed by the simultaneous addition of 1% recombinant humanized type III collagen containing the complete sequence. Since no cross-linking agent was added in this step, the viscous liquid mixture contained completely free thiol groups 301, providing abundant nucleophilic donors for subsequent Michael addition interlocking. In this embodiment, the mass ratio of thiolated hyaluronic acid to recombinant humanized type III collagen is 3:1. This ratio is designed based on the following: on the one hand, a sufficient proportion of thiolated hyaluronic acid ensures that there are enough free thiols at the interface to participate in the Michael addition reaction with the unsaturated carbon-carbon double bonds on the first surface; on the other hand, the limited proportion of collagen added can provide cell recognition signals to promote wound healing without causing steric hindrance to the thiol-double bond reaction due to excessive protein concentration.

[0041] In the freezing and interface grafting step, the molding mold carrying the first layer 101 and the second layer precursor is placed on a freezing table with a unidirectional heat conduction base plate. A temperature gradient field that advances unidirectionally from bottom to top is constructed in the mixed solution, and the interlayer interface grafting reaction between the unsaturated carbon-carbon double bond 302 and the free mercapto group 301 is triggered.

[0042] The specific physical parameters and boundary conditions were controlled to configure and maintain the operating temperature of the freezing stage with a unidirectional heat conduction base plate at an extremely low -78°C. The unidirectional heat conduction base plate was made of pure copper. Polytetrafluoroethylene (PTFE) sidewalls provided thermal insulation. The extremely high thermal conductivity of pure copper was configured to create a unidirectionally advancing temperature gradient field in the solution inside the molding die, starting at -78°C. This temperature gradient field was used to drive a phase transition in the solvent water molecules inside the second-layer precursor body, resulting in the vertically oriented growth of columnar ice crystals at a stable advancing rate of 2 mm / min to 4 mm / min. At the interface 103 between the two layers, the freezing front generated ice crystal nucleation points with a size of approximately 15 to 25 micrometers, which then penetrated the second-layer precursor body from bottom to top. It is noteworthy that during the bottom-up oriented growth of ice crystals, the solute concentration in the unfrozen region continuously increased as the ice crystal front advanced, producing a significant freeze-concentration effect. Because pure water molecules preferentially crystallize into the ice crystal phase, high molecular weight solutes such as thiolated hyaluronic acid and collagen are gradually repelled and enriched in the unfrozen liquid phase microregions. With the continued effect of the temperature gradient field, the local polymer concentration near the interface 103 can reach tens of times the initial concentration. This extreme concentration effect causes the effective collision frequency of thiol groups and double bonds at the interface to increase exponentially. Simultaneously, the latent heat of crystallization generated by ice crystal growth is released in the local microregions, making the actual temperature of the unfrozen liquid phase microregions significantly higher than the ambient temperature of -78℃, placing them within a thermodynamic window favorable for Michael addition reactions. Furthermore, the mechanical pressure and volume repulsion effect generated by ice crystal expansion produce a local micro-stress field at the interface 103. This stress field reduces the apparent activation energy required for the reaction between thiol groups and double bonds through piezoelectric effects and molecular chain conformational distortion, enabling efficient covalent grafting even under macroscopically low-temperature conditions.

[0043] During phase transition growth, the bottom-up directional growth of ice crystals does not simply push the polymeric solutes away from the interface as a whole. Instead, it forms a dynamic solute-enriched transition zone between the ice crystal front and the unfrozen liquid phase. Specifically, after nucleation near the interface 103, the ice crystals advance into the second-layer precursor. Water molecules are continuously incorporated into the ice crystal lattice, while polymeric solutes such as thiolated hyaluronic acid and collagen are repelled into the unfrozen liquid phase channels between the ice crystal boundaries. Because the ice crystals grow perpendicular to the interface from the interface 103 upwards, the repelled polymeric solutes are actually pushed into the liquid phase micro-regions close to the interface 103, rather than moving away from the interface. As the ice crystal front continues to advance, the local polymeric concentration near the interface 103 can reach more than 20 times the initial concentration, forming an interface transition layer with a thickness of only a few micrometers but a high concentration of solutes. In this concentrated layer, the local concentration of free thiol groups 301 reaches the millimolecular level, forming close contact with the unsaturated carbon-carbon double bonds 302 on the surface of the first layer 101. Regarding the low-temperature reactivity, although the ambient temperature of -78°C is far below the conventional thermodynamic window of the Michael addition reaction, this application utilizes the following three synergistic effects to achieve efficient grafting: First, the freeze-concentration effect increases the reactant concentration by one to two orders of magnitude. According to the law of mass action, the reaction rate is proportional to the product of the reactant concentrations, and the surge in collision probability brought about by concentration can significantly improve the reaction rate. Second, the latent heat of crystallization released during the ice crystal phase transition forms local hot spots in the unfrozen liquid phase micro-region, making the actual temperature of this micro-region much higher than the ambient temperature of -78°C, maintaining it within a temperature range favorable to the Michael addition reaction. Third, the mechanical compression effect generated by the expansion of ice crystals produces a local stress field at the interface. According to the transition state theory, mechanical stress can reduce the activation free energy of the reaction by stretching the conformation of reactant molecules. The synergistic effect of these three effects allows the thiol group and double bond at the interface 103 to undergo Michael addition reaction in a highly efficient manner under macroscopic conditions of -78℃, forming a covalent thioether bond and achieving interlocking connection between the two layers.

[0044] In the vacuum freeze-drying step, the material is transferred to a freeze-drying device for vacuum freeze-sublimation treatment of the reacted and formed laminated material system. Under a high vacuum environment below the triple point pressure, the low temperature is maintained to promote the direct sublimation of the columnar ice crystal array in the second-layer precursor from solid to gas. After the sublimated water molecules are completely removed, the physical space previously occupied by ice crystals evolves in situ into parallel channels, forming vertical channels 201 within the second-layer system, ultimately yielding a dry, asymmetric recombinant collagen anti-adhesion dressing 100 with a porous and dense asymmetric morphology. It is worth emphasizing that in the directional freezing process of this application, the recombinant humanized type III collagen remains in an unfrozen, concentrated liquid micro-region and is not incorporated into the ice crystal lattice, thus avoiding direct mechanical penetration and damage to the protein structure by ice crystal growth. Simultaneously, the extreme concentration of the solute lowers the freezing point of the liquid micro-region, allowing it to maintain an amorphous liquid phase or high-viscosity flow dynamic at -78°C. Furthermore, during directional freezing, ice crystals advance steadily at a rate of 2 mm / min to 4 mm / min. This controllable freezing rate avoids the intracellular ice crystal damage effect caused by rapid freezing, which is beneficial for maintaining the native conformation of protein molecules. The role of recombinant humanized type III collagen in this system is to provide integrin recognition sites to induce host cell ingrowth, rather than as a structural support material. Therefore, even if local conformational fine-tuning occurs during freezing, its exposed functional peptide sequences can still effectively perform cell recognition and adhesion functions.

[0045] It should be noted that for surgical dressing products ultimately intended for clinical use, the process chain in this embodiment is also equipped with a corresponding sterilization module. The obtained dried asymmetric recombinant collagen anti-adhesion dressing 100 is sterilized using an industrial electron beam with strong penetration and low heat generation, or by introducing an ethylene oxide sterilization chamber for thorough terminal sterilization. By employing non-high-temperature, non-destructive sterilization methods, residual pathogenic microorganisms can be eliminated using the killing mechanism of radiation or chemical reagents. This ensures that the recombinant humanized collagen remains in a non-frozen concentrated liquid phase micro-region during freezing, and that ice crystals only grow in the pure water region without directly contacting protein molecules. This avoids direct mechanical damage to the protein structure from ice crystals. Simultaneously, the high viscosity of the concentrated liquid phase suppresses conformational fluctuations in protein molecules, thereby maintaining its three-dimensional conformation and bioactivity while fully meeting the stringent aseptic medical safety standards for direct contact with implanted human tissue and wounds.

[0046] Based on the requirement for scalability of chemical material composition, the content range of the core material components of the second layer 102 defined in this application can be further refined and optimized in specific implementation scenarios. In one optional formulation, the degree of thiol substitution of the thiolized hyaluronic acid can be configured to a lower limit of 35%. This formulation reduces the crosslinking density of the polymer scaffold, allowing the second layer 102 of the dressing to exhibit softness and mechanical compliance. It is mainly configured for irregular superficial wounds requiring close adhesion, such as tendon dissection and nerve sheath coverage, where mechanical traction is frequent and close adhesion is required. In another alternative formulation, the degree of thiol substitution can be configured to an upper limit of 45%. This configuration significantly increases the free thiol 301 loading at the contact interface. Through this formulation adjustment, a high density of thiol functional groups can be utilized to generate high-frequency cysteine ​​disulfide bond binding when facing extremely harsh abdominopelvic tissue wounds such as large-area heavy exudation, accompanied by high-velocity blood flushing, or intestinal digestive fluid immersion, providing strong anti-flushing wet adhesion and fixation capabilities. Those skilled in the art can also achieve similar wound repair assistance functions by using the equivalent proportions of the above-mentioned structures that fall within the parameter range.

[0047] To further objectively verify and support the real effects of the aforementioned specific implementation methods provided in this application in solving physical failures and improving clinical indicators, the complete product of Example 1 was prepared in accordance with the specifications of steps S401 to S404, and two sets of comparative examples with completely different principles were set up simultaneously for parallel physical and cellular biological tests.

[0048] Comparative Example 1, as a conventional physical pressing group, used polyethylene glycol diacrylate to prepare the first layer solution 101 and thiolized hyaluronic acid to prepare the second layer solution 102, with the same components and proportions. Abandoning the monolithic casting and interfacial reactive processes, the first layer 101 and the second layer 102 were prepared, cured, frozen, and dried independently. The two-layer entities were then extracted and physically bonded together using medical-grade fibrin glue, followed by gravity compaction.

[0049] Comparative Example 2, as a uniform crosslinked non-porous group, involves forcibly mixing thiolized hyaluronic acid and polyethylene glycol diacrylate precursor in the same container according to a certain ratio, and then directly immersing it in an ultraviolet light device for overall full-transmission crosslinking and curing. Ice crystals and the resulting micropores are generated by extremely low temperature directional gradient freezing without passing through a unidirectional heat conduction substrate.

[0050] The test results for the three groups of samples are summarized in Table 1.

[0051] Table 1 Parallel Validation Table of Dressing Material Science and Biological Test Data

[0052] The differences in the measured data reveal the irreplaceable advantages of the dual-track asymmetric structure of this application. In the 180-degree tensile peel failure test of interlayer peel strength in the hydrated state, the interlayer peel strength of the first layer 101 and the second layer 102, constructed by covalent sulfide bonds in a large amount of physiological saline hydration state, is defined as greater than or equal to 185.4 N / m. The interlayer bonding strength of up to 185.4 N / m in Example 1 confirms the existence of interfacial chemical interlocking; conversely, Comparative Example 1, which relies solely on the physical entanglement and adhesion of macromolecular fibrin glue, only has a test value of 12.6 N / m under the tension of swelling internal stress, resulting in rapid collapse and peeling separation, which cannot meet the anti-separation requirements of implantation in the body fluid environment for several days.

[0053] In the simulated bench test of wet porcine skin adhesion shear strength, which tests the bonding performance, Example 1 achieved an extremely high shear strength retention threshold of 48.2 kPa. This strongly supports the aforementioned mechanism. Because Example 1 retains a high vertical porosity of 78.3%, the huge porosity surface area fully exposes the pre-placed free thiol 301 to the cysteine ​​network of the porcine skin tissue to generate disulfide bonds. In contrast, although Comparative Example 1 has pores, the interface is covered by glue, and Comparative Example 2 lacks a porous framework, resulting in a sharp reduction in surface area. Therefore, the adhesion strength of both examples dropped to less than 12 kPa.

[0054] Regarding fluid management capabilities, Example 1 recorded a lateral aspiration swelling ratio as high as 16.5 g / g over 48 hours, demonstrating excellent capillary aspiration and exudate drainage capabilities. Comparative Example 2, lacking the vertical channel network 201 formed by directional cryotherapy with a pure copper base plate, became a completely impermeable homogeneous colloidal clot with an aspiration capacity of only 2.1 g / g. If applied to the wound, this would cause irreversible accumulation of inflammatory mediators. Simultaneously, all experimental groups exhibited ultra-high levels of extracellular cell arrest rates based on the dense barrier layer, confirming the anti-protein adsorption barrier effect of the first layer 101.

[0055] This application utilizes a Michael addition crosslinking of polyethylene glycol diacrylate and thiolated hyaluronic acid to construct an asymmetric fusion of the first layer 101 and the second layer 102. Combined with the vertical channels 201 provided by unidirectional cryoablation, this not only solves the frequent interlayer swelling, disintegration, and separation problem through macroscopic chemical covalent thioether bond interlocking, but also achieves a wet, seamless fixation effect triggered by free thiol groups 301. This dressing can intelligently absorb and seal excess exudate and prevent malignant adhesion to adjacent tissues using its own structure, achieving a synergistic unity of wound microenvironment management and anatomical isolation functions.

[0056] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An asymmetric recombinant collagen anti-adhesion dressing, characterized in that, It includes a first layer and a second layer, which are connected at the junction to form an asymmetric structure with a continuous transition; The first layer is a dense barrier membrane facing the non-wound side. The dense barrier membrane of the first layer is configured as a physical barrier structure to prevent the invasion of exogenous fibroblasts and to prevent the diffusion of internal exudate to the non-wound side. The first layer is composed of a photocrosslinked polymer network containing polyethylene glycol diacrylate. The surface of the first layer has unreacted unsaturated carbon-carbon double bonds. The second layer is a tissue-adhesive sponge facing the wound. The second layer is composed of a directional porous network containing thiolized hyaluronic acid and recombinant humanized collagen. The interior of the second layer has vertical channels running through its thickness direction. The second layer contains free thiol groups. The first layer and the second layer are interlocked at the interface by forming covalent thioether bonds through a Michael addition reaction between the unsaturated carbon-carbon double bonds of the first layer and the free mercapto groups of the second layer.

2. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 1, characterized in that, The molecular weight of the polyethylene glycol diacrylate is configured to be 4000 Da.

3. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 2, characterized in that, The polyethylene glycol diacrylate contained in the first layer is formed by cross-linking and curing of an aqueous solution with a mass-volume ratio of 5% by ultraviolet light, and the retention rate of the unsaturated carbon-carbon double bonds in the first layer is configured to be 15%.

4. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 3, characterized in that, The unsaturated carbon-carbon double bonds are configured to be excited by controlled ultraviolet light irradiation under the action of an initiator to form the retention rate. The unsaturated carbon-carbon double bonds on the surface are configured to provide reserved covalent reactive sites to the second layer. The interlocking connection structure at the interface is designed to resist physical interlayer peeling and separation between the first layer and the second layer in the hydration and swelling state.

5. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 1, characterized in that, The recombinant humanized collagen is configured as recombinant humanized type III collagen.

6. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 5, characterized in that, The degree of thiol substitution of the thiolized hyaluronic acid is configured to be between 35% and 45%, the pore size of the vertical channel is configured to be between 30 micrometers and 60 micrometers, and the vertical channel is configured as a microfluidic channel network that aspirates and circulates exudate within the second layer based on capillary action.

7. The asymmetric recombinant collagen anti-adhesion dressing as described in claim 6, characterized in that, The remaining free thiol groups in the second layer that did not participate in the Michael addition reaction at the interface are distributed on the pore walls of the oriented porous network. These remaining free thiol groups are configured to undergo a redox reaction with cysteine ​​residues contained in the surface tissue protein structure and generate disulfide bonds in situ for covalent anchoring when in contact with the interface of aqueous soft tissue containing proteins. Based on the high specific surface area of ​​the oriented porous network and the chemical anchoring force of the disulfide bonds, the second layer constitutes a seamless adhesive that is directly bonded and fixed to the surface of wet tissue covered by blood or tissue mucus.

8. A method for preparing the asymmetric recombinant collagen anti-adhesion dressing as described in any one of claims 1 to 7, characterized in that, In order, they include: A polyethylene glycol diacrylate solution is subjected to ultraviolet light crosslinking to form a first layer, and the ultraviolet light crosslinking dose is controlled to retain unsaturated carbon-carbon double bonds on the surface of the first layer; A mixed solution containing thiolized hyaluronic acid and recombinant humanized collagen is injected into a molding die and applied to the surface of the first layer to form a second layer precursor. The mixed solution contains free thiol groups. The molding die carrying the first and second layer precursors is placed on a freezing table with a unidirectional heat conduction base plate. A temperature gradient field that advances unidirectionally from bottom to top is constructed in the mixed solution, and the interlayer interface grafting reaction driven by the temperature gradient field occurs between the unsaturated carbon-carbon double bond and the free thiol group to generate covalent thioether bonds. The reacted material is subjected to vacuum freeze-sublimation to form vertical channels within the second layer precursor, thereby obtaining the asymmetric recombinant collagen anti-adhesion dressing.

9. The method as described in claim 8, characterized in that, In the freezing and interface grafting steps, the operating temperature of the freezing stage with the unidirectional heat conduction base plate is configured to be -78°C, and the material of the unidirectional heat conduction base plate is configured to be pure copper.

10. The method as described in claim 9, characterized in that, The unidirectional heat conduction base plate is configured to construct a unidirectional advancing temperature gradient field at -78°C within the molding die. This temperature gradient field drives the solvent water molecules inside the second layer precursor body to grow ice crystals vertically and oriented at a advancing rate of 2 mm / min to 4 mm / min, while simultaneously extruding the polymer solute. The growing ice crystals generate a local polymer chain concentration effect under extremely low temperature conditions. This local polymer chain concentration effect is configured to catalyze the covalent grafting interlocking of the unsaturated carbon-carbon double bonds and the free thiol groups on the surface of the first layer at the interface between the two layers based on the Michael addition reaction.

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