A hydrogel loaded with mesenchymal stem cells and a preparation method and application thereof

Abstract

This invention provides a hydrogel loaded with mesenchymal stem cells (MSCs), its preparation method, and its application, relating to the field of tissue repair technology. The method includes dissolving maleiminated hyaluronic acid in a complete cell culture medium containing MSCs to obtain precursor solution A; dissolving amino-modified dextran in the same medium to obtain precursor solution B; preparing an upper anti-fouling and anti-adhesion layer; using this upper anti-fouling and anti-adhesion layer as a template; placing the template with its bottom side facing up, adding precursor solution A followed by precursor solution B, and gently pressing to form a lower adhesion layer, thus obtaining the MSC-loaded hydrogel. This method retains the inherent physical protective advantages of the hydrogel, effectively addressing the problem of MSCs easily being lost with exudate as a cell carrier, ensuring cell colonization and survival on the wound surface, and ultimately producing a synergistic healing effect of physical barrier and cell therapy. It solves the technical problem in existing stem cell delivery carriers that cannot simultaneously achieve strong tissue adhesion and retention while maintaining efficient anti-fouling and anti-adhesion.

Description

Technical Field

[0001] This invention relates to the field of tissue repair technology, and in particular to a hydrogel loaded with mesenchymal stem cells, its preparation method, and its application. Background Technology

[0002] Mesenchymal stromal cells (MSCs) are a type of adult stem cells derived from connective tissues such as bone marrow, adipose tissue, umbilical cord, and placenta. Due to their ease of isolation and expansion, low immunogenicity, lack of ethical controversy, and multi-lineage differentiation potential and strong paracrine regulatory capacity, they have become the core therapeutic carrier in the field of tissue repair and regenerative medicine.

[0003] Currently, clinical delivery strategies for MSCs mainly include three categories: local injection, biological scaffold loading delivery, and exosome delivery. Among them, local injection is simple to operate and highly targeted, but it has the problem that the cells stay at the lesion site for a short time (the in vivo half-life is usually less than 24 hours), and are easily lost due to blood flow, immune clearance and mechanical stress, resulting in insufficient therapeutic dose, poor efficacy, and difficulty in supporting the long-term tissue regeneration needs. Biological scaffold loading and delivery: Physically embedding MSCs in three-dimensional hydrogels or sponge-like scaffolds can prolong their residence time and mimic the natural extracellular matrix (ECM) to provide mechanical support and a biochemical signaling microenvironment. However, existing biological scaffolds generally suffer from functional homogeneity. For example, for postoperative wounds in vivo (such as after abdominal or thoracic surgery), it is necessary to inhibit fibrin deposition, excessive fibroblast infiltration, and abnormal collagen deposition to achieve anti-tissue adhesion. For chronic wounds on the body surface (such as diabetic foot ulcers and burn wounds), it is necessary to block the invasion of external microorganisms and contaminants and maintain a moist healing environment to achieve anti-infection and physical isolation. Both anti-tissue adhesion and anti-infection / physical isolation rely on the formation of a stable hydration layer on the material surface to maximally inhibit the non-specific adsorption of proteins, bacteria, and fibroblasts. However, the efficient loading, residence, and function of MSCs depend on the appropriate hydrophobicity and specific adhesion sites of the material to promote MSC anchoring and proliferation. Single-structure hydrogels cannot simultaneously integrate the two contradictory but essential functions of "anti-fouling / anti-adhesion" and "cell loading / adhesion" in tissue repair. While exosome delivery can avoid the potential tumorigenesis and immune rejection risks of live cell transplantation, its therapeutic suitability is limited and its preparation is complex.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a hydrogel loaded with mesenchymal stem cells, its preparation method and application, so as to solve the technical problem that existing stem cell delivery carriers cannot simultaneously achieve strong tissue adhesion and retention and efficient anti-fouling and anti-adhesion.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing a hydrogel loaded with mesenchymal stem cells, comprising the following steps: A. Preparation of the lower adhesion layer precursor solution: Maleimide-modified hyaluronic acid was dissolved in a complete cell culture medium containing MSCs to obtain precursor solution A; amino-modified dextran was dissolved in a complete cell culture medium containing MSCs to obtain precursor solution B.

[0007] B. Preparation of the upper antifouling and anti-adhesion layer: Methacrylamide gelatin and sulfobetaine methacrylate are dissolved in phosphate buffer PBS in a water bath, a thermal initiator is added, and the mixture is sonicated until the solute is uniformly dissolved. Photopolymerization is then carried out in a container for 30 min to obtain the upper antifouling and anti-adhesion layer. C. In situ assembly of hydrogel loaded with mesenchymal stem cells: The upper antifouling and anti-adhesion layer obtained in B is used as the upper template; the bottom surface of the upper template is placed upward, the precursor solution A is added dropwise, and then the precursor solution B is added. The mixture is gently pressed for at least 10 minutes to form the lower adhesion layer, thus obtaining the hydrogel loaded with MSCs.

[0008] The purpose of applying light pressure is to ensure that the lower adhesive layer makes full contact with the skin wound and achieves chemical adhesion. Applying light pressure for at least 10 minutes is sufficient to achieve adhesion at the wound site. The application time can be extended according to the actual situation, such as preferably 30 minutes.

[0009] Furthermore, the molar ratio of maleimide groups in precursor solution A to amino groups in precursor solution B is 1~2:1. Furthermore, the concentration of maleimide-modified hyaluronic acid in precursor solution A is 115~155 mg / ml; furthermore, the concentration of amino-modified dextran in precursor solution B is 100~200 mg / ml.

[0010] Furthermore, the preparation method of the maleimide-modified hyaluronic acid includes dissolving hyaluronic acid in MES buffer of 2-(N-aminoethyl)maleimide hydrochloride, reacting, and then dialyzing and freeze-drying to obtain maleimide-modified hyaluronic acid; further, the molar ratio of hyaluronic acid to N-(2-aminoethyl)maleimide hydrochloride is 1:2~3; further, the grafting degree of the maleimide group on the hyaluronic acid is 20%~40%; Furthermore, the preparation method of the amino-modified dextran includes using dextran as a backbone, activating its hydroxyl groups with 1,1'-carbonyldiimidazole, and then reacting it with ethylenediamine in a nucleophilic substitution reaction, followed by dialysis and freeze-drying to obtain the product; further, the degree of substitution of amino groups in the amino-modified dextran is 30%~50%.

[0011] Furthermore, in step B, the hydrogel contains 1-5 wt% methacrylated gelatin, 18-22 wt% sulfobetaine methacrylate, and 0.3-0.6 wt% thermal initiator; preferably, the water bath heating temperature is 50°C; preferably, the container includes a bottom with thermal conductivity and a removable seal.

[0012] Furthermore, the method for preparing the methacrylated gelatin includes adding methacrylic anhydride dropwise to a gelatin buffer solution while stirring, and then dialyzing and freeze-drying the mixture to obtain the methacrylated gelatin; further, the molar ratio of methacrylic anhydride to lysine residues in the gelatin is 0.9~1.2:1; further, the rate of adding methacrylic anhydride is 0.3~0.6 mL / min, preferably 0.5 mL / min.

[0013] Secondly, the present invention provides a hydrogel loaded with mesenchymal stem cells, which is prepared by the above-described preparation method.

[0014] Furthermore, it includes: an upper antifouling and anti-adhesion layer, which comprises a three-dimensional network formed by photopolymerization of methacrylamide gelatin and sulfobetaine methacrylate; a lower adhesive layer preceding the lower adhesive layer, which comprises a porous network formed by crosslinking maleimide hyaluronic acid and amino-modified dextran via Michael addition reaction, with mesenchymal stem cells encapsulated within the porous network; the upper antifouling and anti-adhesion layer and the lower adhesive layer exhibit an interpenetrating network structure at the interface.

[0015] Thirdly, the present invention provides the application of the hydrogel prepared by the above-described preparation method or the hydrogel prepared by the above-described method in the preparation of mesenchymal stem cell delivery products.

[0016] Fourthly, the present invention provides the use of the hydrogel prepared by the above-described preparation method or the above-described hydrogel in the preparation of products that promote wound healing.

[0017] This invention provides a method for preparing a hydrogel loaded with mesenchymal stem cells (MSCs). The lower adhesion layer rapidly gels upon contact with wound exudate and covalently anchors to collagen fibers, prolonging the residence time of the encapsulated MSCs in a porcine skin model. Simultaneously, the formed porous network provides ample nutrient exchange channels for the cells, successfully maintaining the biological function of the stem cells. The upper antifouling and anti-adhesion layer maintains a low static water contact angle, reducing the adsorption of bovine serum albumin and the adhesion density of Staphylococcus aureus and Escherichia coli. The MSC-loaded hydrogel not only retains the physical protective advantages of hydrogels themselves but also effectively solves the problem of MSCs easily being lost with exudate as a cell carrier, ensuring cell colonization and survival on the wound surface, ultimately producing a synergistic healing-promoting effect of physical barrier and cell therapy. This solves the technical problem in existing stem cell delivery carriers that cannot simultaneously achieve strong tissue adhesion and residence while maintaining efficient antifouling and anti-adhesion. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The HA-mal provided in the embodiments of the present invention 1 H NMR spectrum; Figure 2 The Dex-NH2 provided in the embodiments of the present invention 1 H NMR spectrum; Figure 3 The results of screening the adhesion properties of different HD gels provided in the embodiments of the present invention are shown, where a is the adhesion strength trend and b is the adhesion strength statistics. Figure 4 The XPS spectrum of HD gel after it is bonded to the surface of pigskin is provided in the embodiment of the present invention, wherein a is the N1s spectrum of the treated pigskin, b is the C1s spectrum of the treated pigskin, c is the N1s spectrum of the untreated pigskin, and d is the C1s spectrum of the untreated pigskin. Figure 5 The following are the verification results of the adhesion mechanism of HD gel provided in the embodiments of the present invention. Among them, a is the XPS full spectrum of HD gel after contact with pigskin, b and c are the comparison of adhesion strength between formaldehyde-sealed and unsealed treatments, and d is a schematic diagram of the adhesion mechanism. Figure 6The test results of the water absorption and swelling performance of HD gel provided in the embodiments of the present invention are shown in the figure. In the figure, a is the curve of water absorption and swelling rate changing with time, and b is the curve of volume change changing with time. Figure 7 Gel-MA provided for embodiments of the present invention 1 1H NMR spectrum, a is the internal standard peak of phenylalanine, b is the characteristic peak of methacryl double bond; Figure 8 The following are the test results of the SG gel protein adhesion performance provided in the embodiments of the present invention, wherein a is the BCA protein adhesion standard curve and b is the BSA adhesion amount statistics; Figure 9 The hydrophilicity test results of the SG gel surface provided in the embodiments of the present invention are shown in the figure, where a is the static water contact angle test and b is the water contact angle statistics. Figure 10 The results of the SG gel anti-Escherichia coli adhesion performance test provided in the embodiments of the present invention are shown in the figure, where a is a CLSM fluorescence image and b is the bacterial adhesion density statistics. Figure 11 The results of the SG gel anti-Staphylococcus aureus adhesion performance test provided in the embodiments of the present invention are shown in the figure. In the figure, a is the CLSM fluorescence image and b is the bacterial adhesion density statistics. Figure 12 The results of the SG gel water absorption swelling rate test provided in the embodiments of the present invention; Figure 13 The cell adhesion results of SG gel with 3% Gel-MA provided in the embodiments of the present invention; Figure 14 The macroscopic adhesion results of SG gel with 3% Gel-MA provided in the embodiments of the present invention; Figure 15 SGHD gel scanning electron microscope images provided in embodiments of the present invention; Figure 16 The mechanical property test results of SGHD gel provided in the embodiments of the present invention are shown in the figure, where a is an adhesion demonstration diagram, b is an adhesion curve, and c is an adhesion strength statistics. Figure 17 The results of SGHD gel cytotoxicity MTT assay provided in this embodiment of the invention are shown, where a represents L929 cells and b represents MSCs cells. Figure 18 The results of direct contact toxicity between SGHD gel and MSCs provided in the embodiments of the present invention; Figure 19 The results show the effect of HDgel on MSC proliferation, where a is the staining image and b is the absorbance data; Figure 20 The Transwell results of SGHD gel-regulated unidirectional migration of MSCs provided in this embodiment of the invention are shown in the figure. In the figure, a is a CLSM migration image and b is the number of cells migrating out from the bottom surface. Figure 21 The in vitro antibacterial experiment of SGHD gel provided in this embodiment of the invention includes an observation graph of the antibacterial experiment and a statistical graph. Figure 22 The following are the in vivo antibacterial results of SGHDgel provided in the embodiments of the present invention, wherein a is an in vivo antibacterial image and b is a statistical representation of the antibacterial ratio; Figure 23 The images provided in this embodiment of the invention show the effect of SGHD gel on promoting the healing of skin defects in mice, where a is a wound healing image and b is a statistical representation of the wound healing area. Figure 24 The following are the HE staining results of wounds provided in the embodiments of the present invention, wherein a is an HE section image, b is the epidermal thickness statistics, and c is the wound gap width statistics. Figure 25 The Masson staining results of wounds provided in the embodiments of the present invention are shown in the figures, where a is a Masson stained section image and b is a collagen deposition statistics. Figure 26 The following are the wound Doppler blood flow imaging results provided in the embodiments of the present invention, wherein a is a Doppler image and b is a blood perfusion volume statistics; Figure 27 The images show the results of CD31 & α-SMA immunofluorescence double staining of wounds in various groups provided in the embodiments of the present invention, where a is an immunofluorescence image, b is the α-SMA positive area, and c is the CD31 positive area. Figure 28 The results of the safety evaluation of different organs using the SGHD gel provided in the embodiments of the present invention. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials in the embodiments are prepared according to existing methods or purchased directly from the market.

[0022] The cell conditioned culture medium was complete culture medium for human umbilical cord mesenchymal stem cells, purchased from Pronosai, 100ml, catalog number CM-CL11.

[0023] Example 1: Preparation and performance characterization of the lower adhesive layer (HD gel) of Janus hydrogel I. Synthesis and Preparation of Lower Adhesion Layer (HD gel) Material 1. Synthesis and characterization of HA-mal Based on the amidation reaction mechanism, N-(2-aminoethyl)maleimide hydrochloride (NEM) is reacted with the carboxyl site of HA. Amide bonds are formed between HCl, and maleimide functional groups are grafted onto them.

[0024] The specific synthesis steps are as follows: Weigh 2g of HA and dissolve it in 200 mL of 0.1M MES buffer (pH=5.5~6.0), stirring until completely dissolved to prepare a homogeneous solution with a mass fraction of 1% (w / v). Add NEM to the homogeneous solution. HCl (feed ratio HA:NEM) The reaction mixture (HCl molar ratio 1:2) was stirred continuously for 24 hours under light-protected, room temperature (25℃) conditions. After the reaction, the product was transferred to a dialysis bag with a molecular weight cutoff of 7000 Da. It was dialyzed against 0.1M NaCl solution for 3 days, followed by dialyzed against deionized water for 2 days to remove unreacted small molecules and salt ions. After dialysis, the sample was freeze-dried to obtain a white, flocculent HA-mal product, which was stored at -20℃ in the dark.

[0025] A 400 MHz nuclear magnetic resonance spectrometer was used. 1 The chemical structure of HA-mal was characterized by ¹H NMR (hydrogen spectroscopy) using deuterated water (D₂O) as the solvent. Spectra were acquired at room temperature, and the grafting rate of the maleimide group was calculated by integrating characteristic peaks. Results are as follows: Figure 1 As shown, with the methyl peak (δ=2.00 ppm) on HA as the baseline and the double bond (δ=7.00 ppm) on the maleimide group as the characteristic peak, the grafting degree of the maleimide group can be calculated to be 30%.

[0026] 2. Synthesis and Characterization of Dex-NH2 Based on the hydroxyl-nucleophilic substitution reaction mechanism of 1,1'-carbonyldiimidazole (CDI), the hydroxyl groups on the Dex molecular chain are first activated by CDI to form a highly reactive imidazole carbonate intermediate, which then undergoes a nucleophilic substitution reaction with ethylenediamine (ED) to graft the amino group onto the dextran backbone.

[0027] The specific synthesis steps are as follows: Weigh 1g of Dex and dissolve it in 8mL of anhydrous dimethyl sulfoxide (DMSO), stirring thoroughly until completely dissolved; separately weigh 0.8g of CDI and dissolve it in 5mL of DMSO. Slowly add the CDI solution dropwise to the Dex solution, mix thoroughly, and then purge the system with nitrogen for 10min to eliminate interference from oxygen and moisture; subsequently, activate the reaction under nitrogen protection, in the dark, and at room temperature with continuous stirring for 4h. After activation, add 3mL of ED to the reaction system and continue stirring under the same conditions for 48h. After the reaction is complete, transfer the product to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze with deionized water for 3 days, and freeze-dry the dialyzed product to obtain Dex-NH2 solid.

[0028] The characterization method is the same as HA-mal, and the results are as follows: Figure 2 As shown, the signal peak at chemical shift δ=5.00 ppm is attributed to the methylene proton at the a-position O-CH-O structure, while the characteristic peak at δ=3.27 ppm corresponds to the b-position methylene proton directly attached to the amino group, indicating a substitution degree of approximately 40%.

[0029] 3. Preparation of the lower adhesive layer HD gel Take the maleimide-modified hyaluronic acid HA-mal-01, HA-mal-02, and HA-mal-03 prepared in step 1, and the amino-modified dextran (Dex-NH2) prepared in step 2, and prepare them according to the following steps: Maleimide-modified hyaluronic acid (HA-mal) and amino-modified dextran (Dex-NH2) were dissolved separately in PBS buffer with a total solids content of 14%. The HA-mal solution was poured into the lower layer, and then the Dex-NH2 solution was added. After 30 min, the lower adhesion layer HD gel was formed. The ratio of maleimide groups to amino functional groups was controlled to be 1:1, 1.5:1 and 2:1, respectively, and were denoted as HD gel-01 (1:1), HD gel-02 (1.5:1) and HD gel-03 (2:1).

[0030] II. HD gel performance testing 1. HD gel in vitro adhesion performance testing and formulation screening HD gel-01, HD gel-02 and HD gel-03 were prepared as HD gel samples, and their tissue adhesion properties were evaluated according to ASTM F2255 standard. Thawed pig skin tissue was used as a biomimetic tissue model.

[0031] The testing procedure is as follows: After thawing, the subcutaneous fat layer of the pig skin tissue was removed, and the tissue was cut into uniformly sized blocks. The surface was treated according to ASTM F2255 standards, and a double-layered overlap sample with an adhesion area of ​​1cm × 1cm was prepared. To simulate the in vivo blood environment, 35μL of fresh rat blood was evenly applied to the adhesion interface of the pig skin tissue. Then, 50μL of HD gel sample was evenly spread on this interface, and a constant pressure of 5N was applied for 1 minute to ensure sufficient contact and initial curing of the HD gel with the tissue interface. Subsequently, an axial tensile force was applied at a constant tensile speed of 5 mm / min until the HD gel was completely separated from the pig skin tissue interface. The maximum tensile force (F) during the tensile process was recorded. max Shear strength (τ) is expressed by the formula τ = F. max / A is calculated, where A is the area of ​​the adhesion region (1 cm²). 2 Each test group has 5 parallel samples.

[0032] The results are as follows Figure 3 As shown, the adhesion strength of HD gel-01 (1:1) after gelation for 30 minutes can be stably maintained at about 30 kPa, which is significantly higher than that of HD gel-02 (1.5:1, adhesion strength about 20 kPa) and HD gel-03 (2:1, adhesion strength about 10 kPa). Moreover, HD gel-01 has the best adhesion stability and uniform gelation rate. Therefore, HD gel-01 (1:1) has the best performance and is the optimal formulation for HD gel.

[0033] 2. Study on the adhesion mechanism of HD gel Using HD gel-01 (1:1) as the sample, X-ray photoelectron spectroscopy (XPS, model ESCALAB Xi+, Thermo Fisher) was used to characterize the surface chemical composition of pig skin tissue after HD gel treatment, in order to investigate the interfacial interaction between the material and the tissue.

[0034] The test conditions are as follows: Al Kα ray source was used to perform full-spectrum scanning and high-resolution narrow-scan analysis on the pigskin samples treated with HD gel. The chemical state and content changes of amino (-NH2) on the tissue surface were analyzed by peak fitting to clarify the effect of the treatment method on the functional groups on the pigskin surface.

[0035] The results are as follows Figure 4As shown in Figures a and c, the N1s peak of the blank porcine skin appears at 399.8 eV. This peak corresponds to the N atom of the amide bond (-CONH-) in the collagen fibers of porcine skin, which is a typical characteristic of nitrogen-containing groups in skin tissue. The peak is broadened and the signal intensity is low, consistent with the structural characteristics of porcine skin tissue, which has limited nitrogen content and uneven distribution of functional groups. After HD gel treatment, the N1s characteristic peak of porcine skin shifts to 399.6 eV, with a sharper peak shape, significantly increased signal intensity, and a more stable baseline. The 0.2 eV negative shift in the N1s binding energy indicates that a new covalent bond is formed between HD gel and porcine skin tissue, altering the chemical microenvironment of the N atom. The significant increase in peak intensity confirms that HD gel has formed a stable functional coating on the porcine skin surface. Its nitrogen-containing groups (secondary amine bonds formed by HA-mal / Dex-NH2 crosslinking) are successfully enriched at the tissue interface. The -mal of HD gel undergoes a Michael addition covalent reaction with the -NH2 of porcine collagen, thus achieving strong adhesion in wet tissue.

[0036] The high-resolution C1s spectra and peak fitting results of pig skin tissue before and after treatment verified the interfacial binding effect, such as... Figure 4 As shown in Figures b and d, the C1s spectrum of the blank porcine skin can be fitted with five characteristic peaks. Among them, the CC / CH bond at 284.8 eV is the absolute main peak, accounting for over 60%, while the remaining peaks at 285.3 eV (CN), 285.7 eV (CO), 287.2 eV (C=O), and 289.3 eV (O=CO) are weak peaks, consistent with the structural characteristics of skin tissue, which is mainly composed of proteins and lipids with a low proportion of polar groups. After HD gel treatment, the peak shape and peak area proportion of the porcine skin C1s spectrum changed significantly: the dominance of the CC / CH bond main peak at 284.8 eV weakened, and its proportion decreased significantly; while the proportions of characteristic peaks of polar groups such as 285.4 eV (CN), 286.5 eV (CO), 288.2 eV (C=O), and 289.3 eV (O=CO) all increased significantly. The significant increase in the proportion of the CN bond peak corresponds to the secondary amine bond CN formed by the covalent bonding of HA and porcine skin in the HD gel, which is consistent with the characterization results of the N1s spectrum. The substantial enhancement of the CO bond peak is attributed to the enrichment of a large number of hydroxyl groups in the HA and Dex polysaccharide matrix at the tissue interface in the HD gel, which can form hydrogen bonds with porcine collagen, providing additional non-covalent forces for wet adhesion. In summary, XPS characterization clarifies that the interfacial binding mechanism between the HD gel and wet tissue is that the HD gel forms covalent bonds with porcine collagen-NH2 through a Michael addition reaction of -mal, providing the core binding force for adhesion.

[0037] The validation was performed using an amino-blocking treatment combined with an overlap-shear test. The specific procedure followed ASTM F2255: After thawing and treating, the subcutaneous fat layer of the pigskin was removed, and the samples were cut into double-layered overlap samples with an adhesion area of ​​1cm × 1cm. The pigskin was immersed in formaldehyde solution for 1 hour, achieving amino-blocking through the interaction between formaldehyde and the amino groups on the tissue surface, thus blocking the amino-mediated chemical bonding between HD gel and the tissue. A control group of pigskin was not treated with amino-blocking and only underwent the same physical cutting. 35 μL of fresh rat blood was evenly applied to the adhesion interface of both treated and untreated pigskin to simulate the in vivo environment. Then, 100 μL of HD gel was evenly spread on the interface, and a constant pressure of 5N was applied for 30 minutes to ensure that the HD gel was fully cross-linked and adhered to the tissue interface. The adhesion strength of the two treatments was tested using a tensile testing machine (using the same method as the HD gel in vitro adhesion performance test).

[0038] The results are as follows Figure 5 In cases b and c, the formaldehyde-treated pigskin reacts with excess HCHO, consuming the -NH2 on the skin surface, leaving only hydrogen bonds to interact with HD gel, thus leading to a sharp decrease in adhesion. This indicates that HD gel adhesion primarily occurs through a Michael addition reaction between the -MA on its surface and the -NH2 on the skin. Figure 5 d. Figure 5 As shown in Figure a, compared with normal skin tissue, the nitrogen content of pig skin treated with HD gel was significantly increased, which originated from Dex-NH2 and enhanced the cohesive energy of the hydrogel.

[0039] 3. HD gel water absorption and swelling rate test HD gel-01 (1:1) was used as the HD gel sample. The degradation trend and morphological stability of HD gel were investigated by water absorption swelling rate test. During the test, a 1.5cm×1.5cm HD gel sample was placed in a buffer solution simulating the physiological environment in vivo. The mass and volume changes of the sample were recorded at different time points (0h, 1h, 3h, 6h and 12h) at a constant temperature of 25℃. The water absorption swelling rate and volume changes were calculated.

[0040] The results are as follows Figure 6 As shown, the HD gel exhibited a stable and slow degradation trend during the testing period, with no obvious sudden degradation. Its water absorption and swelling rate remained within a reasonable range, and its overall shape changed gradually without any severe swelling or collapse. This indicates that the HD gel not only possesses good adhesive strength and structural stability, but its slow degradation characteristics also synchronize with the tissue repair process, avoiding adhesion failure caused by rapid material degradation. Furthermore, its minimal shape change reduces mechanical stimulation to surrounding tissues, demonstrating that this HD gel formulation can provide crucial performance assurance for in vivo implantation of MSCs-based systems.

[0041] Example 2: Preparation and performance characterization of the Janus hydrogel top antifouling and anti-blocking layer (SG gel) I. Synthesis and Preparation of Upper Hydrogel (SG Gel) Material 1. Synthesis and Characterization of Gel-MA The synthesis of Gel-MA is achieved by amidation of methacrylic anhydride (MA) with the amino groups of lysine residues on the gelatin molecular chain.

[0042] The specific steps are as follows: Weigh 5g of type A gelatin and add it to an appropriate amount of phosphate buffered saline (PBS, pH=7.4). Place the solution in a 50℃ constant temperature water bath and stir until completely dissolved, preparing a 10% (w / v) homogeneous gelatin solution. After the solution temperature stabilizes, slowly add 0.5mL of methacrylic anhydride (MA to lysine residues in gelatin in a 1:1 molar ratio) to the gelatin solution at a rate of 0.5mL / min, continuously stirring during the addition process to ensure uniform mixing. Maintain the reaction in a 50℃ constant temperature water bath with continuous stirring for 3 hours. After the reaction is complete, dilute the product 5-fold with PBS solution to terminate the reaction. Then transfer the product to a dialysis bag with a molecular weight cutoff of 7000 Da and dialyze with deionized water for 3 days (changing the dialysate 3 times daily) to thoroughly remove unreacted MA monomers and small molecule impurities. After dialysis, freeze-dry the sample to obtain a white porous gel-MA solid, which is then sealed and stored at -20℃ in the dark for later use.

[0043] use 1 The degree of substitution of Gel-MA was characterized by 1H NMR to verify the effect of its chemical structure modification. For example... Figure 7 As shown, compared with the spectrum of gel, the spectrum of gel-MA shows the addition of a characteristic absorption peak b (δ=5.4~5.6 ppm) corresponding to the methacrylic acid double bond, while this characteristic peak is absent in the gelatin spectrum. This confirms that the methacryloyl group has been successfully grafted onto the gelatin molecular chain, indicating the successful synthesis of gel-MA. To quantitatively calculate its degree of substitution (i.e., the number of methacryloyl groups grafted onto each 100 gelatin repeating units), the characteristic peak a (δ=7.1~7.4 ppm) of phenylalanine in the gelatin molecule was selected as an internal standard peak. By comparing the integrated area of ​​this internal standard peak with the characteristic peak of the methacrylic acid double bond, the degree of substitution of gel-MA was calculated to be 51.9%. This indicates that the amino groups on the gelatin molecular chain have successfully undergone a grafting reaction with the methacryloyl group, and the grafting efficiency is good, which can meet the functional requirements of the material in subsequent experiments.

[0044] 2. Preparation of the upper hydrogel (SG gel) The upper hydrogel, SG gel, was constructed by in-situ photopolymerization of sulfobetaine methacrylate (SBMA) and methacrylamide gelatin (Gel-MA) in a mold. First, the prepared Gel-MA was dissolved in PBS solution and heated and stirred at 50°C until completely dissolved (Gel-MA solid content 0~20wt%). Then, the zwitterionic monomer SBMA was added and stirred continuously until fully dissolved (SBMA solid content 0~30wt%). 0.5wt% of the thermal initiator ammonium persulfate (APS) was introduced into the system and dispersed evenly by ultrasonication. The mixture was then transferred to a mold and reacted at a constant temperature of 60°C for 30 minutes to obtain SG gel hydrogel with anti-blocking properties.

[0045] SG gel hydrogel samples were prepared according to the above method, as shown in Table 1.

[0046] Table 1

[0047] II. SG gel performance testing The hydrogels shown in Table 1 and commercially available anti-adhesion gels (polylactic acid anti-adhesion gel from Hebei Ruinuo Medical Device Co., Ltd., production batch number: 20240321, specification: 2.5ml / tube) were used as samples for performance testing and were designated as 1% Gel-MA group, 3% Gel-MA group, 5% Gel-MA group, commercially available anti-adhesion group, SBMA group, and Gel-MA group, for a total of 6 groups.

[0048] 1. SG gel anti-protein adhesion test and formulation screening The protein anti-adhesion properties of SG hydrogels were evaluated using the BCA method, with bovine serum albumin (BSA) as the model protein. SG gels with different formulations were cut into 1cm × 1cm samples, rinsed with deionized water, and then swollen in PBS (pH 7.4) at 37°C for 2 hours. The samples were then transferred to PBS containing 5 mg / mL BSA and incubated at 37°C for 2 hours. After incubation, the samples were gently washed three times (5 min each time) with PBS to remove unadsorbed free protein. The samples were then transferred to centrifuge tubes containing 1 mL of 2% sodium dodecyl sulfate (SDS) solution and sonicated at 100 W for 10 min to completely elute the adsorbed BSA. 50 μL of the eluent was mixed with 1 mL of freshly prepared BCA working solution (reagent A:B = 50:1, v / v), incubated in a water bath at 37°C for 30 min, and the absorbance was measured at 562 nm. A standard curve was plotted using 2 mg / mL BSA standard serially diluted with PBS (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL and 0.125 mg / mL), and the amount of protein adhering to the sample surface was calculated.

[0049] The results are as follows Figure 8 The protein adhesion concentrations of the SBMA group, 1% Gel-MA and 3% Gel-MA were significantly lower than those of the 5% Gel-MA group, the Gel-MA group and the commercially available anti-adhesion group, and the anti-adhesion effect was significant; among them, the SBMA group had the best anti-adhesion performance.

[0050] The hydrophilicity of different formulation materials was evaluated using static water contact angle testing, and the influence of different formulation components on the hydrophilicity and hydrophobicity of SG gel surfaces was further investigated. The tests were conducted entirely in a constant temperature and humidity environment, and multiple parallel controls were set up for each sample group. Results are as follows: Figure 9 The SBMA group exhibited the smallest water contact angle and the best hydrophilicity, consistent with the optimal anti-adhesion effect observed in the BCA bovine serum albumin adhesion test. Among different SG gels, the water contact angle gradually increased with the introduction and concentration of Gel-MA components, indicating that the introduction of Gel-MA significantly increases the hydrophobicity of the material surface. While 1% Gel-MA and 3% Gel-MA introduced Gel-MA components, their water contact angles remained low, maintaining strong hydrophilicity, and their anti-adhesion effects were similar to those of the SBMA group. Although the SBMA group demonstrated extremely strong anti-adhesion and hydrophilic properties, its extremely poor water absorption and swelling rate made it highly susceptible to degradation, thus failing to meet practical application requirements.

[0051] 2. SG gel bacterial adhesion test and formulation screening Staphylococcus aureus and Escherichia coli were used as model pathogens to evaluate the antibacterial adhesion properties of SGgel. Frozen Staphylococcus aureus and Escherichia coli were inoculated into LB liquid medium and cultured at 37°C with shaking at 150 rpm until the logarithmic growth phase, then transferred to 4°C for later storage. Different hydrogel samples were cut into 1cm × 1cm pieces and subjected to alternating ultrasonic cleaning with anhydrous ethanol and deionized water (100W, 5 min / time, 5 times in total) to remove impurities. The samples were then placed in PBS buffer and swollen at 37°C for 2 h. The bacterial concentration was adjusted to 1E5 CFU / mL using the plate count method, and the swollen samples were immersed in centrifuge tubes containing 5 mL of the above bacterial solution and statically incubated at 37°C for 24 h to simulate bacterial adhesion. After incubation, the samples were removed and gently rinsed three times (3 min each time) with sterile saline to remove non-specifically adhered free bacteria. Adhering bacteria were stained using the SYTO 9 / propidium iodide (PI) live / dead bacteria staining kit, and five randomly selected fields of view were scanned using a laser confocal microscope (CLSM, Zeiss LSM 880). Quantitative analysis of the fluorescence images was performed to determine the total bacterial count, live / dead ratio, and bacterial adhesion density (cells / mm²).2 To evaluate the antibacterial adhesion properties of SG gel.

[0052] The results are as follows Figure 10 As shown, a large number of stained bacteria were visible on the surface of the Gel-MA group. The number of bacteria adhering to the surface of the 5% Gel-MA group and the commercially available anti-adhesion group was reduced, indicating a certain degree of anti-adhesion ability. However, only a small number of scattered stained bacteria were observed on the surface of the 3% Gel-MA group and the 1% Gel-MA group, with significantly lower bacterial adhesion density than the other groups, indicating excellent anti-adhesion effect, consistent with the results of protein adhesion and water contact angle tests. The results of the Staphylococcus aureus adhesion test are shown below. Figure 11 As shown, the 1% Gel-MA group and the 3% Gel-MA group still maintained good anti-adhesion effect, and the adhesion density was significantly lower than that of the 5% Gel-MA group, the commercially available anti-adhesion group and the Gel-MA group, which is consistent with the E. coli adhesion test results.

[0053] 3. SG gel water absorption swelling rate test and formula screening The experimental method was the same as described in "Water Absorption Swelling Rate Test in Example 1". The results are as follows: Figure 12 As shown, SG gel samples with different Gel-MA contents exhibit significant differences in swelling characteristics. The 1% Gel-MA group expands nearly ninefold in volume upon reaching swelling equilibrium. This excessive expansion and insufficient structural stability lead to significant slippage during wound application, hindering stable adhesion to the wound tissue and impacting both anti-adhesion efficacy and clinical safety. In contrast, the 3% and 5% Gel-MA groups show moderate swelling, doubling in volume only upon reaching equilibrium. This ensures a tight fit to the wound tissue, allowing for synchronized movement with wound activity without causing significant mechanical strain or additional pain to the patient. Therefore, the 3% Gel-MA group demonstrates superior anti-adhesion properties, good hydrophilicity, and antibacterial adhesion, making SG gel-02 (3% Gel-MA) the appropriate formulation for SG gel.

[0054] 4. SG gel cell adhesion test Using SG gel-02 (3% Gel-MA) as the SG gel sample and mouse fibroblast L929 cells as the model, the anti-cell adhesion properties of SG gel were systematically evaluated. Logarithmically growing cells were seeded at 1E5 cells / well in 6-well plates pre-coated with SG gel and co-cultured at 37°C and 5% CO2 for 24 h. The supernatant was then discarded, and the cells were gently washed three times with sterile PBS to remove non-adhering cells and impurities. Viable cells were then labeled using calcein-AM / propidium iodide (PI) double staining and multi-slice imaging was performed using a laser confocal microscope (CLSM).

[0055] The results are as follows Figure 13 As shown, the surface of the SG gel had almost no intact adherent cells, only scattered cell fragments, and no typical cell colony formation was observed. This indicates that the SG gel can effectively inhibit cell adhesion and has a significant cell repulsion effect, effectively blocking the abnormal adhesion of fibroblasts.

[0056] 5. SG gel blood adhesion test Using SG gel-02 (3% Gel-MA) as the SG gel sample, we investigated the adhesion of SG gel to blood and its macroscopic morphological changes, simulating its actual performance in a wound-blood contact environment.

[0057] The experimental method was as follows: Take an appropriate amount of fresh rat blood and drop it evenly onto the surface of SG gel material. Incubate it in a simulated in vivo physiological environment (37℃, 5% CO2) for a certain period of time. After incubation, gently remove the excess blood that has not adhered to the surface and observe the macroscopic state of the material and the blood adhesion.

[0058] The results are as follows Figure 14 As shown, after incubation with rat blood, the SG gel surface showed no blood coagulation patches or obvious blood adhesion, and the material maintained good transparency throughout, without becoming cloudy, discolored, or structurally damaged due to blood contact. This macroscopic test result further confirms that the 3% Gel-MA SG gel not only possesses excellent microscopic anti-adhesion (antibacterial, anti-cellular) properties, but also effectively resists blood adhesion while maintaining good transparency. This is beneficial for subsequent observation of wound healing and avoids material failure or wound adhesion caused by blood adhesion.

[0059] Example 3: Preparation and performance characterization of Janus hydrogel (SGHD gel) I. Preparation of Janus hydrogel (SGHD gel) Based on the screening results of Examples 1 and 2, a Janus hydrogel (SGHD gel) was prepared by selecting an HD gel with a maleimide group to amino functional group ratio of 1:1 for the lower layer and an SG gel containing 3% Gel-MA for the upper layer.

[0060] The upper antifouling and anti-adhesion layer SG gel was prepared according to the method in Example 2. Subsequently, precursor solutions for the lower adhesion layer were prepared: HA-mal was dissolved in a buffer solution (cell culture medium, pH 7.2-7.5) containing mesenchymal stem cells (MSCs) to prepare precursor solution A; Dex-NH2 was dissolved in cell culture medium to prepare precursor solution B. After the SG gel solidified, precursor solution A was first dropped onto its surface, followed by rapid and uniform drop-down of precursor solution B onto the surface of precursor solution A, and then quickly applied to the wound. After gentle pressure for 30 min, cross-linking was achieved, thereby forming a bilayer hydrogel (SGHDgel) with a Janus structure in situ at the wound interface, in which MSCs were encapsulated in the lower adhesion layer for subsequent performance testing.

[0061] II. SGHD gel performance determination 1. Microstructure characterization of SDHD gel The following methods were used for characterization: First, the prepared bilayer hydrogel was frozen and stored in a refrigerator to fix its morphology. Then, it was freeze-dried to avoid dehydration and deformation. Next, it was quenched at low temperature with liquid nitrogen to obtain a fresh fracture surface, which was then pasted onto conductive adhesive. After being treated with gold plating by vacuum ion sputtering, the HD gel, SG gel and the transition region between the two were observed at multiple magnification using a JSM-7500F scanning electron microscope at an accelerating voltage of 5kV. The focus was on analyzing the pore structure characteristics of each layer and the bonding state of the interface.

[0062] The results are as follows Figure 15 As shown, the SGHD gel bilayer hydrogel exhibits a clear three-layer microstructure. The HD gel layer displays a uniformly distributed macroporous structure, providing ample space and channels for stem cell colonization, nutrient exchange, and metabolic waste removal, perfectly aligning with its core function as a cell carrier. The SG gel layer exhibits a dense microporous structure; this dense microporous network effectively enhances the material's hydrophilicity and anti-adhesion properties, while ensuring structural stability in the swollen state. At the transition interface, the network structures of the HD and SG layers intertwine and interlock tightly, with no obvious delamination or separation observed. This robust interlocking bonding confirms the integrity and bonding stability of the SGHD gel bilayer hydrogel, effectively mitigating the risk of interlayer separation that may occur in practical applications.

[0063] 2. Mechanical testing and characterization of SDHD gel To ensure the SGHD bilayer hydrogel does not easily detach in practical applications, a maximum tensile strength test was conducted. First, SGHD gel samples with a diameter of 8 mm were synthesized according to the method described in step one, strictly controlling the thickness uniformity of the bilayer structure to ensure a tight bond between the HD gel and SG gel without delamination. The two ends of the SGHD gel were fixed to the upper and lower clamps of a universal tensile testing machine. The clamp spacing was adjusted to ensure the sample was in a naturally straight state with uniform force, avoiding experimental errors caused by force deviation during testing. The tensile testing machine was then started, and a tensile test was performed at a constant rate until the sample broke. The maximum tensile force was recorded, and the maximum tensile strength was calculated.

[0064] The results are as follows Figure 16 As shown, the maximum tensile strength of this SGHD gel is nearly 10 kPa, which can effectively ensure that the material will not easily break or fall off when moving around the wound site, meeting the actual application requirements of medical wound dressings and ensuring the stability and application safety of the hydrogel-carried stem cell system.

[0065] 3. Biocompatibility testing and characterization of SGHD gel Using mouse fibroblasts (L929) and mesenchymal stem cells (MSCs) as research subjects, the cell compatibility of SGHD gel was comprehensively evaluated from multiple dimensions, including its effects on cell viability, morphology, and direct contact toxicity, using the MTT assay and LIVE / DEAD fluorescence staining.

[0066] 1) Preparation and grouping of extracts: The upper (SG gel) and lower (HD gel) layers of sterilized SGHD gel were incubated with cell conditioned medium for 24 h to prepare extracts, which were then filtered through a 0.22 μm filter membrane for later use. The experiment included gradient concentration (2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL) extract groups and a blank control group (containing only culture medium).

[0067] 2) Indirect contact toxicity (MTT method): L929 cells and MSCs were seeded in 96-well plates at a rate of 1E5 cells / well. After the cells adhered to the plates, different concentrations of extract prepared in step 1) were added, and the plates were co-cultured at 37°C and 5% CO2 for 24 hours. Sterile water was set as a negative control and conditioned medium was set as a positive control. Cell viability was detected by the MTT method, and cytotoxicity was calculated by absorbance value.

[0068] The calculation formula is: ; Wherein, ODt represents the absorbance of the experimental group SGHD gel material, ODn represents the absorbance of the blank group conditioned medium group, and ODp represents the absorbance of the sterile water group.

[0069] The results of the MTT method are as follows: Figure 17 The results showed that SG gel and HD gel extracts exhibited excellent biocompatibility with L929 fibroblasts and MSCs in the concentration range of 2.5~20 mg / mL. The cell survival rate of each concentration group was the same as that of the blank control group, and no significant decrease was observed. These results suggest that SGHD gel has no obvious cytotoxicity and has excellent biocompatibility.

[0070] 3) Direct Contact Toxicity: The upper (SG gel) and lower (HD gel) layers of the SGHD gel were cut to appropriate sizes and laid flat on the bottom of a cell culture plate. After aseptic treatment, MSCs were seeded into the culture plate, ensuring that the stem cells were evenly distributed on and around the material surface. The culture plate was then placed in a cell culture incubator at 37°C and 5% CO2 for direct co-culture for 24 hours. After co-culture, cell morphology, quantity, and viability were observed using a Thunder fluorescence microscope. Specific staining was used to distinguish between live and dead cells to determine whether the material had an abnormal effect on cell morphology.

[0071] The results are as follows Figure 18 As shown, after co-culturing MSCs with SG gel and HD gel for 24 h, the materials exhibited excellent biocompatibility. Thunder fluorescence microscopy revealed that the experimental group cells maintained a typical spindle-shaped extended state with clear outlines, without any pathological morphological changes such as cell shrinkage, deformation, or rupture. Cell counting showed no significant difference between the experimental group and the blank control group, and no fluorescent signals of dead cells were observed, suggesting that SGHD gel did not induce apoptosis or necrosis. These results not only ruled out the direct contact toxicity of SGHD gel but also confirmed that it can provide a suitable physicochemical microenvironment for the long-term adhesion, growth, and proliferation of MSCs, supporting the cells to perform normal physiological functions.

[0072] 4. Effect of SGHD gel on MSC proliferation test Since the lower layer of HD gel serves as the scaffold layer for MSC loading and attachment, the effect of HD gel on MSC proliferation was specifically evaluated to clarify whether SGHD gel can provide a stable and suitable microenvironment for MSC proliferation. After uniformly spreading sterile HD gel at the bottom of the wells, MSCs were inoculated. A blank control (MSCs inoculated only, without HD gel) was set up. Cell morphology and number were observed on days 3 and 6 of culture using phalloidin / DAPI staining, and the absorbance value of proliferation was detected using a microplate reader. The results are as follows: Figure 19As shown, the cells in the experimental group with added HD gel had normal morphology, exhibited typical spindle-shaped extension, and had uniform nuclear staining. Their proliferation data showed no significant difference from the blank control group and gradually increased with the extension of culture time, suggesting that SGHD gel can provide a good microenvironment for the growth and proliferation of MSCs.

[0073] 5. Effect of SGHD gel on MSC migration The purpose of this test is to verify the regulatory ability of SGHD gel on unidirectional migration of MSCs, and to clarify the blocking effect of SG gel on cell migration and the guiding effect of HD gel on cell migration.

[0074] Three experimental groups were set up using Transwell chambers: in experimental group 1, a lower layer of HD gel loaded with MSCs and an upper layer of SG gel were sequentially layered in the chamber; in experimental group 2, a lower layer of SG gel and an upper layer of HD gel loaded with MSCs were sequentially layered in the chamber; and a blank control group (without hydrogel in the chamber, only an equal volume of MSC suspension was added) was also added. After the bilayer hydrogels were completely solidified, culture medium was added to the top of each chamber, and the chambers were co-cultured at 37°C and 5% CO2 for 24 h. Subsequently, the number of cells migrating from the bottom of the Transwell chamber was counted by staining for live and dead cells to quantitatively evaluate the migration of MSCs from the HD gel layer in each group.

[0075] The results are as follows Figure 20 As shown in Figure a, stem cells in the blank control group could migrate freely. In experimental group 2, the SG gel was located in the lower layer, effectively blocking the migration of MSCs; staining revealed that very few cells migrated. In contrast, in experimental group 1, the HD gel loaded with MSCs was located in the lower layer, effectively guiding the migration of MSCs; staining revealed that a large number of cells migrated. Statistical analysis showed that... Figure 20 As shown in Figure b, approximately 50% of the cells migrated out of the HD gel layer after 24 hours of co-culture. These results indicate that the HD gel not only supports MSCs, providing a microenvironment for their survival and proliferation, but also guides their migration in a suitable direction. Meanwhile, the SG gel blocks cell migration. The two work synergistically to achieve precise regulation of unidirectional MSC migration, further meeting the core requirement of hydrogel-loaded stem cell systems in tissue repair: directional guidance of stem cell homing and prevention of abnormal migration.

[0076] 6. Functional study of SGHD gel 1) Evaluation of the in vitro antimicrobial barrier efficacy of SGHD gel in a porcine skin defect model In vitro antibacterial experiments used porcine skin tissue to construct a full-thickness wound model. Before the experiment, *Staphylococcus aureus* was inoculated onto LB liquid medium and cultured at 37°C with shaking at 150 rpm until the logarithmic growth phase, then stored at 4°C for later use. Simultaneously, LB solid medium (100 mL deionized water + 4 g tryptone soy agar) was prepared, autoclaved for 30 min, poured onto plates, cured at room temperature, and refrigerated at 4°C for later use. In a clean bench, circular full-thickness porcine skin wounds with a diameter of 1 cm were prepared on agar plates, divided into a control group (no gel / stem cells), a stem cell group (MSCs resuspended in complete medium and inoculated), a pure gel group (2 cm × 2 cm SGHD gel covering the wound), and an experimental group (2 cm × 2 cm SGHD gel loaded with MSCs covering the wound). Subsequently, 50 μL of 1E8 CFU / mL S. aureus bacterial suspension was evenly added to the wound area and incubated at 37°C for 24 h. The wound surface was then gently wiped with a sterile cotton swab moistened with PBS. After serial dilution, the solution was plated and incubated for 24 h to count the number of colonies and analyze the bacterial growth of each group.

[0077] The results are as follows Figure 21 As shown in Figure a, after 24 hours of bacterial culture, a large number of bacteria proliferated at the wound site in the control group and stem cell group due to the lack of a protective barrier; while in the pure gel group and experimental group, due to the physical coverage of SGHD gel, the upper SBMA layer played a protective role for the lower adhesion layer and the wound site, and no obvious bacterial growth was observed in the wound area; the plate-laying results also showed that the plates of the control group and stem cell group were covered with dense colonies, while the plates of the pure gel group and experimental group had very few colonies or even no colony growth. Figure 21 As shown in Figure b, the bacterial inhibition rate was significantly higher in the blank group and the stem cell group than in the pure gel group and the experimental group (p<0.05). This indicates that the anti-adhesion layer (SG gel) of SGHD gel can effectively block bacterial invasion.

[0078] 2) Antibacterial evaluation of SGHD gel in a mouse model of skin lesions combined with bacterial infection The in vivo antibacterial properties of SGHD gel were evaluated using a Balb / c mouse skin defect + bacterial infection model. Male Balb / c mice (6-7 weeks old) were anesthetized with isoflurane, and a circular skin defect with a diameter of 1 cm was created using a punch. Different intervention methods were then applied.

[0079] Control group: 50 μL of S. aureus suspension with a density of 1E8 CFU / mL was injected into the wound; Experimental group: Wounds were sealed using SGHD gel patches loaded with MSCs, and S. aureus suspension was dropped onto the patch surface; Stem cell group: MSCs culture medium was directly injected into the wound surface, followed by the addition of S. aureus suspension to the culture medium; Pure gel group: SGHD gel patches without MSCs were applied directly to the wound, and S. aureus suspension was dropped onto the surface of the patch.

[0080] After bacterial treatment, mice were kept in a standard feeding environment. On the third day after infection, the bacterial status of the wound, the presence of abnormal secretions, and the accumulation of white colonies were observed. Subsequently, the mice were sacrificed and wound and surrounding tissue samples were taken under aseptic conditions. The tissue homogenate was then processed, serially diluted, and evenly spread on bacterial culture plates. The plates were incubated at 37°C for 12-18 hours. The degree of bacterial infection at the wound site was quantitatively assessed by the number of colony-forming units.

[0081] The results are as follows Figure 22 As shown in the results, wound morphology observation revealed that on day 3 post-infection, both the blank control group and the pure stem cell group showed a distinct white, tightly adhered substance on the wound surface, which could not be detached even after gentle scraping. Tissue homogenate smears also showed a significant increase in CFU, confirming severe infection. In contrast, the wound surfaces of the pure gel group and the experimental group were clean, without any white film or abnormal secretions, and the wound edges were regular. No bacterial colony growth was observed on the smears, indicating no obvious signs of infection, and the bacterial barrier rate reached 100%. This demonstrates that SGHD gel can completely block bacterial colonization and effectively prevent infection regardless of whether it is loaded with MSCs.

[0082] 3) Evaluation of the healing efficacy and safety of SGHD gel in a mouse model of full-thickness skin defects. To systematically verify the healing-promoting effect of MSCs loaded in SGHD gel on acute skin defects, healthy Balb / c male mice aged 6-7 weeks were selected. After acclimatization for 3 days, they were anesthetized with isoflurane inhalation. A full-thickness skin defect was prepared in the center of the back using a 1 cm diameter sterile punch. The mice were randomly divided into a blank group (wound treatment only, no other intervention), a stem cell group (MSCs), a pure gel group (SGHD gel), and an experimental group (SGHD gel loaded with MSCs). The wounds in all groups were not sutured but covered with sterile gauze. The mice were housed individually after surgery and their general condition was monitored daily. Wound photographs were analyzed on postoperative days 0, 3, 7, and 14 to calculate the closure rate. Doppler blood flow imaging was used to quantitatively assess local blood supply and angiogenesis. At each time point, 3 mice from each group were randomly selected and sacrificed. The back skin tissue containing the wound was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and stained histologically to evaluate the synergistic effect of this cell-loaded system compared to material or cell therapy alone.

[0083] Wound healing results as follows Figure 23As shown, there were significant differences in wound healing effects among the groups. From the 3rd day post-surgery, the experimental group (SGHD gel loaded with MSCs) showed the fastest healing speed, with a significant reduction in wound size and active granulation tissue growth. The remaining wound area was significantly smaller than that of the control group and the pure stem cell group. The therapeutic effect remained optimal until the 7th day post-surgery, with the wound basically closed and the newly formed skin smooth. Complete healing was achieved by the 14th day post-surgery, with the best skin appearance and smoothness. The pure gel group (SGHD gel) showed the second fastest wound healing speed, with basic healing by the 14th day post-surgery, but the color was slightly red, which was better than the control group and the stem cell group. The pure stem cell group, lacking carrier support, had cells lost with the exudate and could not colonize, and its healing progress was no different from that of the control group. The results indicate that SGHD gel can promote healing through physical barriers and a stable microenvironment, and can also serve as a carrier to ensure cell colonization and survival in the wound; loaded with MSCs, it has both barrier and cell therapy synergistic effects in promoting healing.

[0084] Figure 24 and Figure 25 The H&E and Masson staining results for each group of wounds are shown. Significant differences were found among the treatment groups: the blank group showed severe inflammatory cell infiltration, sparse and disordered collagen, with a coverage rate of only about 25%, resulting in delayed healing; the pure gel group, while providing some moisturizing and supportive effects and promoting collagen deposition to about 50%, had a thinner epidermis with larger gaps, limiting regenerative capacity; the stem cell group improved epidermal integrity through the differentiation and paracrine effects of MSCs, achieving a collagen coverage rate of about 35%, but the effect was not as good as the experimental group. The experimental group performed best, with moderate epidermal thickness, minimal gaps (nearly closed), neatly arranged dermal collagen, mild inflammation, and a collagen coverage rate of about 70%, with the defect essentially closed. The results indicate that hydrogel or stem cell therapy alone has limited effect on promoting collagen maturation; while SGHD gel combined with MSCs not only provides physical support but also more effectively leverages the advantages of cell therapy. By activating collagen synthesis pathways, it increases collagen deposition while optimizing its arrangement, demonstrating a synergistic effect that significantly promotes tissue repair and functional reconstruction.

[0085] Doppler blood supply results as follows Figure 26 As shown, on postoperative days 3 and 7, the wound blood perfusion in the experimental group was significantly higher than that in the other groups, indicating active angiogenesis and providing oxygenation for rapid healing. The pure gel group showed limited blood flow increase; due to the lack of paracrine regulation by MSCs, early rapid angiogenesis was difficult to achieve solely through physical barriers and a moist environment. The blood flow signals in the control group and the pure stem cell group remained consistently low with no significant difference, confirming that stem cells without carrier support cannot engraft and are unlikely to improve local microcirculation.

[0086] The effects of different interventions on wound angiogenesis and maturation were evaluated using a double immunofluorescence staining (CD31 & α-SMA) system. The results are as follows: Figure 27As shown, the positive signals of CD31 and α-SMA in the blank group were both approximately 1.5%, with weak vascular response. In the stem cell group, these signals increased to approximately 2.0% and 5.0%, respectively, demonstrating the pro-angiogenic potential of MSCs' paracrine effect. In the pure gel group, relying on a physical scaffold, the signals of CD31 and α-SMA were approximately 5.5% and 6.5%, respectively. The experimental group performed best, with positive areas of CD31 and α-SMA reaching approximately 7.0% and 8.0%, respectively. Morphological observation results were consistent with the quantitative data above. The blank group showed only sporadic fragmented signals; while the stem cell and pure gel groups showed a small amount of newly formed vascular endothelium, the lumen was immature and co-localized; whereas the experimental group formed a large number of mature co-localized tubular vessels with a dense network and intact lumen. It can be concluded that Janus hydrogel, by providing a colonization microenvironment for MSCs and synergistically combining material properties and cell activity, simultaneously enhances the quantity and maturity of wound angiogenesis.

[0087] Histopathological effects on major organs: To further comprehensively evaluate the in vivo biosafety of the SGHD gel-loaded MSCs system and rule out potential damage to mouse metabolism and vital organs, histopathological examinations were performed on the heart, liver, spleen, lungs, and kidneys of all surviving mice after the experiment to determine the presence of abnormal changes such as inflammatory infiltration, cell degeneration, and necrosis. H&E staining results are shown below. Figure 28 As shown, the heart, liver, spleen, lung, and kidney tissues of mice in each experimental group exhibited normal morphology, with well-organized cell arrangement and no obvious abnormalities such as inflammatory cell infiltration, cell degeneration, or necrosis. There were no significant differences in organ tissue morphology compared to the blank control group. This indicates that the SGHD gel vector and its loaded MSCs did not cause significant systemic toxicity or pathological damage to major organs, demonstrating good tissue compatibility and biosafety, thus validating its safety as a novel biomaterial scaffold for clinical application.

[0088] The above embodiments successfully constructed a two-sided functional partitioned SGHDgel consisting of an "anti-adhesion protective layer and a stem cell delivery adhesion layer." The top SG gel is composed of SBMA and Gel-MA cross-linked composites. The highly hydrating layer formed by the zwitterionic ions of SBMA achieves efficient antifouling, effectively inhibiting protein adsorption and non-specific cell adhesion. Its anti-adhesion effect is significantly superior to traditional homogeneous hydrogels. Combined with the excellent biocompatibility and mechanical regulation capabilities of Gel-MA, it endows the material with anti-swelling, anti-slip, and structural stability. The bottom HD gel is constructed in situ from HA-mal and Dex-NH2 via a Michael addition reaction. It rapidly gels upon contact with wound exudate, exhibiting excellent interfacial adhesion strength while effectively maintaining the adhesion efficiency and activity of MSCs. The SGHDgel assembled from these two components has a stable connection between the upper and lower layers, effectively promoting unidirectional migration of MSCs. It also possesses excellent biocompatibility, with a hemolysis rate below 5% and cell viability maintained above 90%. Furthermore, it effectively degrades subcutaneously by day 14. Animal experiments showed that the SGHD gel-loaded MSCs system significantly accelerated wound healing, achieving a wound closure rate of over 90% by day 14. Simultaneously, it demonstrated good tissue re-epithelialization and collagen deposition, improved blood perfusion and angiogenesis, and effectively enhanced wound healing quality. Furthermore, SGHD gel could isolate over 99% of bacterial invasion, preventing wound infection. In summary, the Janus hydrogel-loaded stem cell system constructed in this study, through structural biomimicry and functional synergy, provides a new material and method for wound repair with both theoretical value and clinical translational potential. It effectively overcomes many bottlenecks of traditional repair methods and lays a solid foundation for the development of precise and intelligent stem cell-based wound repair technologies.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a hydrogel loaded with mesenchymal stem cells, characterized by, Includes the following steps: A. Preparation of the lower adhesion layer precursor solution: Maleimide-modified hyaluronic acid was dissolved in a complete cell culture medium containing MSCs to obtain precursor solution A; amino-modified dextran was dissolved in a complete cell culture medium containing MSCs to obtain precursor solution B. B. Preparation of the upper antifouling and anti-adhesion layer: Methacrylamide gelatin and sulfobetaine methacrylate are dissolved in phosphate buffer PBS in a water bath, a thermal initiator is added, and the mixture is sonicated until the solute is uniformly dissolved. Photopolymerization is carried out in a container for 30 minutes to obtain the upper antifouling and anti-adhesion layer. C. Hydrogel for in situ assembly of mesenchymal stem cells: The upper antifouling and anti-adhesion layer obtained in B is used as the upper template; Place the upper template with the bottom side facing up, add precursor solution A, then add precursor solution B, and press gently for at least 10 minutes to form the lower adhesion layer, thus obtaining the hydrogel loaded with MSCs.

2. The production method according to claim 1, characterized by, The molar ratio of maleimide groups in precursor solution A to amino groups in precursor solution B is 1~2:1; Preferably, the concentration of the precursor solution A, maleimide-modified hyaluronic acid, is 115~155 mg / ml; Preferably, the concentration of amino-modified dextran in the precursor solution B is 100~200 mg / ml.

3. The preparation method according to claim 1, characterized in that, The method for preparing maleimide-modified hyaluronic acid includes dissolving hyaluronic acid in MES buffer of 2-(N-aminoethyl)maleimide hydrochloride, and then dialyzing and freeze-drying the solution to obtain maleimide-modified hyaluronic acid. Preferably, the molar ratio of hyaluronic acid to N-(2-aminoethyl)maleimide hydrochloride is 1:2~3; Preferably, the grafting degree of the maleimide group onto the hyaluronic acid is 20% to 40%; Preferably, the preparation method of the amino-modified dextran includes using dextran as a backbone, activating its hydroxyl groups with 1,1'-carbonyldiimidazole, and then reacting it with ethylenediamine in a nucleophilic substitution reaction, followed by dialysis and freeze-drying to obtain the product; Preferably, the degree of substitution of amino groups in the amino-modified dextran is 30% to 50%.

4. The preparation method according to claim 1, characterized in that, In step B, the hydrogel contains 1-5 wt% methacrylamide gelatin, 18-22 wt% sulfobetaine methacrylate, and 0.3-0.6 wt% thermal initiator. Preferably, the water bath heating temperature is 50°C; Preferably, the container includes a bottom with heat-conducting function and a removable seal.

5. The preparation method according to claim 1, characterized in that, The method for preparing the methacrylamide gelatin includes adding methacrylic anhydride dropwise to a gelatin buffer solution while stirring, and then dialyzing and freeze-drying the mixture to obtain the methacrylamide gelatin.

6. The preparation method according to claim 5, characterized in that, The molar ratio of methacrylic anhydride to lysine residues in gelatin is 0.9~1.2:1; Preferably, the rate of adding methacrylic anhydride is 0.3~0.6 mL / min, and more preferably 0.5 mL / min.

7. A hydrogel loaded with mesenchymal stem cells, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The hydrogel according to claim 7, characterized in that, include: The upper anti-fouling and anti-adhesion layer comprises a three-dimensional network formed by photopolymerization of methacryloyl gelatin and sulfobetaine methacrylate; Before the lower adhesion layer, there is a porous network formed by cross-linking maleimide-modified hyaluronic acid and amino-modified dextran via Michael addition reaction, and mesenchymal stem cells are encapsulated within the porous network; The upper antifouling and anti-adhesion layer and the lower adhesive layer have a network interpenetrating structure at the interface.

9. The use of the hydrogel prepared by the method according to any one of claims 1 to 6 or the hydrogel according to claim 7 or 8 in the preparation of mesenchymal stem cell delivery products.

10. The use of the hydrogel prepared by the method according to any one of claims 1 to 6 or the hydrogel according to claim 7 or 8 in the preparation of products that promote wound healing.

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