A transient hydrogel microparticle scaffold system and its use in promoting tissue regeneration repair

Abstract

The present application belongs to the technical field of biomedical materials, and particularly relates to a transient hydrogel microparticle scaffold system and application thereof in promoting tissue regeneration and repair. The present application provides a transient hydrogel microparticle scaffold system, which is composed of a plurality of hydrogel microparticles, and any two of the hydrogel microparticles are not cross-linked. The particle size of the hydrogel microparticle is 50-100 mu m. The transient hydrogel microparticle scaffold system of the present application exists in a dispersed state at a tissue defect site after being implanted in vivo without persistent cross-linking between particles or cell-mediated persistent cross-linking, and can be expelled and removed under the action of physiological tissue movement, so that the controlled transient retention of the scaffold is realized. Compared with a long hydrogel microparticle scaffold, the transient hydrogel microparticle scaffold system of the present application has a significantly better endometrial structure reconstruction, fibrosis inhibition and fertility recovery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a transient hydrogel microparticle scaffold system and its application in promoting tissue regeneration and repair. Background Technology

[0002] Damage to the endometrium often leads to the formation of intrauterine adhesions (IUA) due to the destruction of the basal layer. This seriously affects embryo implantation and pregnancy outcomes, and is one of the important causes of infertility and recurrent miscarriage.

[0003] Hydrogel microparticle scaffolds have shown broad application prospects in the field of tissue regeneration and repair due to their injectability, high specific surface area, and controllable physicochemical properties. However, the uterine cavity is a dynamic open environment connected to the outside world through the cervix. Implanted materials can be naturally expelled by uterine peristalsis, which is quite different from the closed defect models (such as subcutaneous or skin wounds) used in most studies. Under the dynamic expulsion of the uterine cavity, durable scaffolds are difficult to achieve uniform degradation. The differences in material retention in different areas further amplify the spatial unevenness of immune activation and regeneration, which is not conducive to the full-thickness endometrial function recovery. The persistent volume of durable materials in the small dynamic space of the uterine cavity may interfere with normal uterine peristalsis and the periodic shedding and regeneration of the endometrium, increasing the risk of foreign body-related complications. Summary of the Invention

[0004] The purpose of this invention is to provide a transient hydrogel microparticle scaffold system and its application in promoting tissue regeneration and repair, thereby improving the regeneration and repair effect of tissues, especially the endometrium.

[0005] This invention provides a transient hydrogel microparticle scaffold system, which is composed of a plurality of hydrogel microparticles, and there is no cross-linking between any two hydrogel microparticles; The particle size of the hydrogel microparticles is 50~100μm; The raw materials for preparing the non-crosslinked hydrogel microparticles include photoinitiators, photosensitive gel matrices, biodegradable crosslinking agents, and cell adhesion peptides. The degradable crosslinking agent is a matrix metalloproteinase-degradable peptide; The photosensitive gel matrix is ​​any one of the following: (1) one or more of polyethylene glycol, hyaluronic acid, gelatin and alginate; (2) a derivative or copolymer of the compound in (1).

[0006] Preferably, the matrix metalloproteinase-degradable peptide is the polypeptide shown in SEQ ID NO:1 or a functionally equivalent variant thereof; The cell adhesion peptide contains an RGD sequence; The photoinitiator is the photoinitiator LAP; The polyethylene glycol is an octagonal polyethylene glycol norbornene.

[0007] This invention provides a method for preparing the transient hydrogel microparticle scaffold system described above, comprising the following steps: A photopolymerization reaction was carried out by mixing the aqueous phase and the oil phase to obtain in-situ solidified hydrogel microparticles; The oil phase component in the in-situ solidified hydrogel microparticles is removed to obtain a transient hydrogel microparticle scaffold system; The aqueous phase includes a photoinitiator, a photosensitive gel matrix, a degradable crosslinking agent, cell adhesion peptides, and PBS buffer. The oil phase comprises sorbitan monooleate and mineral oil.

[0008] Preferably, when the photosensitive gel matrix is ​​octagonal polyethylene glycol norbornene, the molar ratio of thiol groups in the MMP degradable peptide crosslinking agent to norbornene in the octagonal polyethylene glycol norbornene is 0.6:1; The concentration of the octagonal polyethylene glycol norbornene in the aqueous phase is 5-10 wt%.

[0009] Preferably, the concentration of cell adhesion peptide in the aqueous phase is 1-5 mM; The concentration of the photoinitiator in the aqueous phase is 1~10mM; The concentration of sorbitan monooleate in the oil phase is 10-20%.

[0010] Preferably, the photopolymerization reaction step includes: co-flowing an aqueous phase and an oil phase to form microdroplets, and applying ultraviolet light to the microdroplets; The ultraviolet light irradiation has a wavelength of 365~405 nm and a light intensity of 10~40 mW / cm. 2 .

[0011] This invention provides the application of the transient hydrogel microparticle scaffold system described above in the preparation of medical products.

[0012] Preferably, the medical product is a medical product that promotes tissue regeneration and repair.

[0013] Preferably, the tissue includes the endometrium.

[0014] Preferably, the medical product further includes mesenchymal stem cells.

[0015] Beneficial effects: This invention provides a transient hydrogel microparticle scaffold system, composed of several hydrogel microparticles, with no cross-linking between any two hydrogel microparticles; the particle size of the hydrogel microparticles is 50~100μm; the raw materials for preparing the non-cross-linked hydrogel microparticles include a photoinitiator, a photosensitive gel matrix, a degradable cross-linking agent, and a cell adhesion peptide; the degradable cross-linking agent is a matrix metalloproteinase-degradable peptide; the photosensitive gel matrix is ​​any one of the following: (1) one or more of polyethylene glycol, hyaluronic acid, gelatin, and alginate; (2) a derivative or copolymer of the compound in (1). After implantation in vivo, the transient hydrogel microparticle scaffold system of this invention does not undergo persistent cross-linking between particles or mediated by cells, existing in a dispersed state at the site of tissue defect, and can be expelled and cleared under physiological tissue movement (such as peristalsis and contraction), achieving controlled transient retention of the scaffold. The results of the embodiments of this invention show that, after effective removal, the transient hydrogel microparticle scaffold system provided by this invention completes the activation of lymph node B cells and transitions them towards plasma cell differentiation, subsequently migrating to the tissue repair area. In contrast, the persistent hydrogel microparticle scaffold, due to its inability to be effectively removed, results in the continued activation of germinal centers in the lymph nodes, but it cannot effectively transform into local reparative immunity. Compared to the persistent hydrogel microparticle scaffold, the transient hydrogel microparticle scaffold system of this invention exhibits significantly superior endometrial structure reconstruction, fibrosis inhibition, and fertility restoration. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 Bright-field images of the transient HMP stent of Example 1 and fluorescence images of the hMSC-assembled persistent HMP stent of Example 2 are shown. Figure 2 A comparison of the AFM Young's modulus of the transient HMP stent of Example 1 and the hMSC assembled durable HMP stent of Example 2; Figure 3 The retention of stents in the uterine cavity at different time points; where A and C are the H&E staining results of uterine tissue pathological sections; and B is the quantitative analysis result of the retention rate of uterine cavity materials. Figure 4 The results of the finite element simulation of uterine peristalsis are shown below; where A is a schematic diagram of cross-sectional stress simulation; and B is a violin statistical chart. Figure 5This diagram illustrates how transient HMP scaffolds promote endometrial regeneration and functional recovery. A shows the experimental procedure design; B shows H&E histopathologically stained sections; C shows the quantitative statistical results of the number of endometrial glands; D shows the quantitative statistical results of endometrial thickness; E shows photographs of fertility testing, with a gross photograph of the mouse uterus at the top and isolated embryos at the bottom; F shows the quantitative statistical results of the number of implanted embryos. Figure 6 Results of macrophage recruitment for HMP implantation and its necessity in repair were presented. A shows spatial transcriptome sections and UMAP dimensionality reduction plots; B shows the quantitative statistical results of the proportion of various cell types in uterine samples at different time points; C shows bright-field sections of uterine tissue and macrophage F4 / 80 fluorescent staining images; D shows the statistical results of the average fluorescence intensity of macrophage F4 / 80; E shows the fluorescence image of the macrophage clearance control; F shows the flowchart and quantitative bar chart of the macrophage depletion experiment; G shows tissue staining images; and H shows a multi-index quantitative bar chart. Figure 7 This diagram illustrates the restorative immune microenvironment promoted by B cells after transient scaffold clearance. Specifically, A is a scatter plot of UMAP-reduced clustering of macrophage subsets; B represents the percentage of each of the three macrophage subsets; C shows the dynamic change curve of B cell percentage; D shows the dynamic change curve of fibroblast percentage; E is a heatmap of cell-cell interactions; F is a ligand-receptor bubble diagram; G is a scatter plot of UMAP-reduced clustering of B cell subsets; H represents the percentage of each of the three B cell subsets; I is a visualization of spatial transcriptome slices; and J is a schematic diagram and bar chart of multiple cytokine detection. Figure 8 This diagram illustrates the role of B cells in the activation of draining lymph nodes and scaffold clearance. A shows the results of a macrophage depletion experiment; B shows the results of an experiment in Rag1-KO T / B cell-deficient mice; C shows the material retention rate within the mouse uterine cavity; D shows a schematic diagram of the proximal-distal uterine region and HE sections; E shows the material retention rate, the number of endometrial glands, and the endometrial thickness; F shows a schematic diagram of a flow cytometry experiment; G shows a schematic diagram of an open uterine wound model; H shows the flow cytometry results of B cell subsets; I shows a schematic diagram of a closed skin wound model; and J shows a flow cytometry quantitative bar chart of uterine and skin tissue controls. Figure 9The results show the detection of SPP1-ITGAV signal and epithelial regeneration in macrophages. A represents representative immunofluorescence images (scale bar: 50 μm) of EdU (green) and DAPI nuclear counterstaining in organoids cultured in the control or Macro-CM culture medium. B shows the corresponding EdU positivity rate and quantitative statistics of cell viability. C represents representative immunofluorescence images (scale bar: 100 μm) of JC-1 (red, mitochondrial membrane potential) and cleaved Caspase-3 (green, apoptosis) in the control and Macro-CM groups. D shows the quantitative analysis of the fluorescence intensity of JC-1 and Caspase-3. E represents bright-field images (scale bar: 100 μm) of epithelial organoids cultured on days 5 and 7 after culture, using Macro-CM alone or Macro-CM combined with the integrin inhibitor cilengitide. μm); F represents the statistical quantification of organoid number and average organoid area; G represents the dynamic CCK-8 proliferation curves (OD450 value) of the control group, recombinant SPP1 group, Macro-CM group and Macro-CM+Cilengitide group within 5 days of culture. Figure 10 This section presents the dynamic changes of uterine epithelial subsets and Ki67 expression changes during uterine repair in a spatial transcriptome dataset. Specifically, A shows the UMAP projections of epithelial subsets (Epi1, Epi2, Epi3, Epi4) in the PBS group, transient group, and persistent group at days 3, 7, and 14 post-injury; B shows the UMAP maps of epithelial subsets in different groups (PBS group, transient group, and persistent group) at different time points (days 3, 7, and 14 post-injury); C shows... Stacked bar charts show the changes in the proportion of epithelial subpopulations over time and in different treatment groups, showing a gradual transition from early inflammatory / proliferative Epi1 / Epi2 to later regenerative / transformative populations. The transient group showed the highest proportion of newly regenerated Epi3 on day 14 after injury. D is a dot plot showing the average expression level (color intensity) and percentage of expressing cells (dot size) of classic marker genes in the four epithelial subpopulations. E is a representative Ki67 immunohistochemical staining of a transverse section of the uterus. Brown Ki67+ cell nuclei represent proliferative endometrial cells. A magnified view of the local area is shown below the whole section image. F is a quantitative statistical chart of Ki67 positive cells. Detailed Implementation

[0018] This invention provides a transient hydrogel microparticle scaffold system, which is composed of a number of hydrogel microparticles, and no two hydrogel microparticles are cross-linked; the particle size of the hydrogel microparticles is 50~100μm; the raw materials for preparing the non-cross-linked hydrogel microparticles include photoinitiator, photosensitive gel matrix, degradable cross-linking agent and cell adhesion peptide; the degradable cross-linking agent is matrix metalloproteinase degradable peptide; the photosensitive gel matrix is ​​any one of the following: (1) one or more of polyethylene glycol, hyaluronic acid, gelatin and alginate; (2) derivatives or copolymers of the compounds in (1).

[0019] In one embodiment, the hydrogel microparticles of the present invention have a particle size of 75~85μm. The transient hydrogel microparticle scaffold system of the present invention does not undergo interparticle cross-linking treatment (such as not co-incubating with hMSCs to form cell assemblies, and not adding chemical cross-linking agents such as DBCO / azide), and exists in a dispersed state at the tissue defect site, which can be expelled and cleared under the action of physiological tissue movement, thereby achieving controlled transient retention of the scaffold.

[0020] In one embodiment, the matrix metalloproteinase-degradable peptide of the present invention is the polypeptide shown in SEQ ID NO:1 or a functionally equivalent variant thereof. The present invention utilizes matrix metalloproteinase-degradable peptides to enable the internal network of individual hydrogel microparticles (HMPs) to be degraded in vivo by MMPs secreted by cells, and because there are no covalent or persistent cross-links between HMP particles, the overall dispersibility and removability of the scaffold are maintained.

[0021] In one embodiment, the polyethylene glycol of the present invention is an eight-arm polyethylene glycol norbornene. In another embodiment, the eight-arm polyethylene glycol norbornene of the present invention has a molecular weight of 20 kDa.

[0022] In one embodiment, the photoinitiator of the present invention is the photoinitiator LAP.

[0023] In one embodiment, the cell adhesion peptide of the present invention contains an RGD sequence. In another embodiment, the amino acid sequence of the cell adhesion peptide of the present invention is shown in SEQ ID NO:2. RGD modification endows HMP cells with adhesion ability, enabling implanted immune cells, especially macrophages, to adhere and aggregate on the HMP surface.

[0024] This invention provides a method for preparing the transient hydrogel microparticle scaffold system described above, comprising the following steps: A photopolymerization reaction was carried out by mixing the aqueous phase and the oil phase to obtain in-situ solidified hydrogel microparticles; The oil phase component in the in-situ solidified hydrogel microparticles is removed to obtain a transient hydrogel microparticle scaffold system; The aqueous phase includes a photoinitiator, a photosensitive gel matrix, a degradable crosslinking agent, cell adhesion peptides, and PBS buffer. The oil phase comprises sorbitan monooleate and mineral oil.

[0025] In another embodiment, the molar ratio of thiol groups in the MMP degradable peptide crosslinking agent and norbornene in the octagonal polyethylene glycol norbornene is 0.6:1. This invention limits the molar ratio of thiol groups to norbornene groups, which has the advantage of maintaining the structural stability of the hydrogel microspheres. Furthermore, the present invention has a higher proportion of norbornene groups than thiol groups, using the excess norbornene groups as reaction sites for subsequent biomolecule grafting, facilitating the functionalization modification of the microspheres.

[0026] In one embodiment, the concentration of octagonal polyethylene glycol norbornene in the aqueous phase of the present invention is 5-10 wt%; in another embodiment, the concentration of octagonal polyethylene glycol norbornene in the aqueous phase of the present invention is 7.5 wt%.

[0027] In one embodiment, the concentration of cell adhesion peptide in the aqueous phase of the present invention is 0-5 mM; in another embodiment, the concentration of cell adhesion peptide in the aqueous phase of the present invention is 1 mM.

[0028] In one embodiment, the concentration of the photoinitiator in the aqueous phase of the present invention is 1~10mM; in another embodiment, the concentration of the photoinitiator in the aqueous phase of the present invention is 10mM.

[0029] In one embodiment, the concentration of sorbitan monooleate in the oil phase of the present invention is 10-20%; in another embodiment, the concentration of sorbitan monooleate in the oil phase of the present invention is 15%.

[0030] In one embodiment, the photopolymerization reaction step of the present invention includes: co-flowing an aqueous phase and an oil phase to form microdroplets, and applying ultraviolet light irradiation to the microdroplets. In one embodiment, the wavelength of the ultraviolet light irradiation is 365~405 nm; in another embodiment, the wavelength of the ultraviolet light irradiation is 365 nm. In one embodiment, the light intensity of the ultraviolet light irradiation is 10~40 mW / cm². 2 As another embodiment, the intensity of the ultraviolet light irradiation described in this invention is 20 mW / cm². 2In one embodiment, the flow rate of the aqueous phase in this invention is 30~100 μL / h; in another embodiment, the flow rate of the aqueous phase is 60 μL / h. In one embodiment, the flow rate of the oil phase is 120~300 μL / h; in another embodiment, the flow rate of the oil phase is 200 μL / h. In this invention, the aqueous and oil phases flow together, and at the convergence point of the flow focus, the aqueous phase is sheared into uniform droplets by the shear force of the oil phase. When ultraviolet light is applied, a thiol-olefin photopolymerization reaction occurs in the irradiated area, resulting in in-situ solidification to form HMP.

[0031] As one embodiment, after the photopolymerization reaction of the present invention is completed, the crosslinking product is collected, centrifuged and washed to remove mineral oil and sorbitan monooleate (Span 80), sterilized with 30% ethanol, fully swollen with sterile PBS, and stored at 4°C.

[0032] The preparation method of the transient hydrogel microparticle scaffold system of this invention differs from that of the durable HMP scaffold. This invention does not perform interparticle cross-linking treatment before or during implantation (e.g., it does not co-incubate with hMSCs to form cell assemblies, and does not add chemical cross-linking agents such as DBCO / azide). The HMP is used directly for injection implantation in a dispersed state.

[0033] This invention provides the application of the transient hydrogel microparticle scaffold system described above in the preparation of medical products.

[0034] In one embodiment, the medical product of this invention is a medical product that promotes tissue regeneration and repair. In another embodiment, the tissue described in this invention is a tissue with a natural opening (open tissue defect), i.e., a tissue environment in which the implanted material can be physically removed from the body; in yet another embodiment, the tissue described in this invention includes the endometrium (with a cervical opening). In another embodiment, the medical product of this invention is a medical product for the prevention and / or treatment of iatrogenic endometrial injury. In yet another embodiment, the iatrogenic endometrial injury described in this invention includes iatrogenic endometrial injury caused by one or more of intrauterine adhesions (IUA), endometrial injury, thin endometrium, and intrauterine procedures. In principle, any tissue with a natural opening or drainage channel (such as nasal cavity, oral cavity, intestinal mucosa, etc.) is applicable. In yet another embodiment, the medical product of this invention also includes mesenchymal stem cells.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a transient hydrogel microparticle scaffold system and its application in promoting tissue regeneration and repair, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 Construction of transient HMP scaffold 1. Preparation of non-crosslinked hydrogel microparticles (HMPs) (1) Preparation of aqueous precursor solution: 8-arm PEG-norbornene with a molecular weight of 20 kDa, MMP degradable peptide crosslinking agent with amino acid sequence KCGPQGIWGQCK (SEQ ID NO:1), cell adhesion peptide with amino acid sequence CGRGDS (SEQ ID NO:2) and photoinitiator LAP were dissolved in PBS buffer to obtain aqueous precursor solution; The aqueous precursor solution contained 7.5 wt% 8-arm PEG-norbornene, MMP degradable peptide crosslinking agent at a molar ratio of 0.6:1 (thiol to norbornene), 1 mM cell adhesion peptide, and 10 mM photoinitiator LAP.

[0037] (2) Oil phase: light mineral oil containing 15% sorbitan monooleate.

[0038] (3) Using a PDMS flow focusing microfluidic chip (PDMS-FF-100), the aqueous phase flow rate was set to 60 μL / h and the oil phase flow rate to 200 μL / h, under 365 nm ultraviolet light (20 mW / cm²). 2 Under the conditions of cross-linking by irradiation, after centrifugation at 10000 rpm for 5 min, the cells were washed with 0.1% poloxamer 407 (Pluronic F127), centrifuged again, and then washed twice with PBS. After centrifugation, PBS was added and the cells were stored at 4°C. Before cell experiments, the cells were removed and sterilized twice by washing with 30% ethanol. After ethanol washing, the cells were washed twice with sterile PBS. No interparticle cross-linking treatment was applied to obtain transient HMP scaffolds for later use.

[0039] Store at 4°C.

[0040] Example 2 Construction of hMSC-assembled durable HMP stent The HMP obtained in Example 1 was dissolved in PBS and mixed with human umbilical cord mesenchymal stem cells (hMSCs, 1×10⁻⁶) at a dose of 10 μL / well. 5 The cells were incubated in a 96-well plate with ultra-low adsorption for 2 hours, allowing the cells to adhere and bridge multiple HMPs through integrin-RGD to form assemblies, thus obtaining an assembled persistent HMP scaffold for hMSCs.

[0041] Example 3 Construction of chemically cross-linked durable HMP scaffolds 1. The HMP obtained in Example 1 was subjected to DBCO functionalization to obtain DBCO-functionalized HMP. Specifically, the HMP obtained in Example 1 was incubated with methyltetrazine-dibenzocyclooctyne (Methyltetrazine-DBCO) dissolved in anhydrous DMSO at 37°C for 1 h. Through a tetrazine-norbornene bioorthogonal reaction, DBCO groups were covalently introduced onto the surface of the HMP.

[0042] 2. Under physiological conditions, DBCO-functionalized HMP was mixed with four-arm polyethylene glycol azide (4-arm PEG-azide). The cross-linking assembly between HMP microspheres was achieved by utilizing the click chemical reaction between DBCO and azide, resulting in a chemically cross-linked durable HMP scaffold.

[0043] Test Example 1 The transient HMP scaffolds (transient HMPs) obtained in Example 1 and the hMSC-assembled persistent HMP scaffolds (hMSC-assembled HMPs) obtained in Example 2 were respectively subjected to SEM scanning electron microscopy and fluorescence confocal microscopy. The results showed that... Figure 1 and Figure 2 As shown, the average particle size of transient HMPs was 77.90 ± 2.83 μm, and the Young's modulus measured by AFM was 39.5 kPa. The Young's modulus of hMSC-assembled HMPs measured by AFM was 43.57 kPa.

[0044] Test Example 2 1. Animal Model Construction according to Figure 5 Animal models were established using the A-procedure, with the following steps: Female C57BL / 6J mice (8-10 weeks old) were anesthetized via intraperitoneal injection of 1.25% tribromoethanol. A midline abdominal incision was made to expose the left uterine horn. The cervix and fallopian tube ends were temporarily ligated. 50 μL of 95% ethanol was injected into the uterine cavity and left for 15 seconds to induce endometrial damage. After rinsing with PBS, the mice were randomly divided into 5 treatment groups and subjected to the following treatments: Damage control (PBS) group: 50 μL of PBS was injected into the uterine cavity once after modeling. Hyaluronic acid (HA) group: After modeling, clinical hyaluronic acid gel was injected into the uterine cavity once, with an injection volume of 50 μL; Transient HMP group: After modeling, the transient HMP stent obtained in Example 1 was injected into the uterine cavity once, with an injection volume of 50 μL; hMSC assembled (crosslinked) HMP group: After modeling, the hMSC assembled durable HMP scaffold obtained in Example 2 was injected into the uterine cavity once, with an injection volume of 50 μL; Chemically cross-linked HMP group: After modeling, the chemically cross-linked durable HMP scaffold obtained in Example 3 was injected into the uterine cavity once, with an injection volume of 50 μL.

[0045] 2. Stent Removal Dynamics Testing (1) Stent retention assessment Uterine tissue was harvested on days 3, 6, and 7 post-surgery, and H&E staining was used to assess scaffold retention. Results were as follows: Figure 3 As shown, in the transient HMP group, only trace amounts of material (area ratio <5%) remained after day 3, and it was almost completely cleared by day 7. In the hMSC-assembled HMP group, significant stent occupancy and dilation of the uterine cavity were still visible on day 6, and partial clearance occurred by day 7, but the uterine cavity morphology was nearly normal. In the chemically cross-linked HMP group, stents still remained and dilated the uterine cavity on day 7, consistent with the trend in the hMSC-assembled HMP group. Figure 3 ).

[0046] (2) Finite element simulation of uterine peristalsis A two-dimensional axisymmetric model was established using COMSOL Multiphysics, and the Mooney-Rivlin hyperelastic constitutive model was used for the uterine wall to simulate the propagation of physiological peristaltic waves (contraction amplitude 0.3 mm, wavelength 15 mm) from proximal to distal. A continuous material model predicted directional material expulsion. Stress analysis of discrete HMPs showed that transient HMPs experienced higher contractile forces and were more easily expelled, while cross-linked HMP assemblies had reduced force exposure due to interparticle connections. Figure 4 ).

[0047] 3. Tissue repair and fertility assessment (1) Histological assessment On postoperative day 7, mouse uterine samples were collected for H&E staining. Results showed that the endometrial thickness in the transient HMP group (285±32 μm) was significantly higher than that in the PBS group (142±23 μm), HA group (185±31 μm), and hMSC-crosslinked HMP group (210±28 μm). In terms of glandular number, the transient HMP group (7.2±1.6 glands / section) was significantly better than all control groups. Masson staining showed that the fibrosis area ratio in the transient HMP group (10.5±2.8%) was significantly lower than that in the hMSC-crosslinked HMP group (18.3±4.1%). Figure 5 (B~D). The results of the chemically cross-linked HMP group were consistent with the trend of the hMSC cross-linked HMP group, with intima thickness and gland number being significantly worse than those of the transient group.

[0048] (2) Fertility assessment On postoperative day 14, animals were housed together at a female-to-male ratio of 2:1. On day 28 (day 14 of gestation), the difference in live fetus count between the healthy and damaged sides (ΔEmbryo Number) was counted. The results showed that the Δ value in the transient HMP group was 1.2±0.5, significantly lower than that in the PBS group (3.8±0.9), HA group (3.1±0.8), and hMSC-crosslinked HMP group (2.5±0.7). P<0.05%. There were no significant differences in placental weight and morphology among the groups, suggesting no developmental toxicity. Figure 5 (E~F).

[0049] 4. Macrophage recruitment and immune cascade mechanisms (1) Uterine tissue was collected on postoperative days 3, 7, and 14, embedded in FFPE, and spatial transcriptome sequencing was performed using the 10x Visium platform. Data quality control (UMI > 50% median), LogNormalize normalization, Harmony batch integration, PCA dimensionality reduction, and UMAP visualization were performed using R (v4.5.2) and Seurat (v5). Based on spatial transcriptomics, UMAP clustering identified 12 major cell types. In the endometrial and pericavitary repair areas, the five most prominent cell types were fibroblasts (Aldh1a2, Col6a4, Wnt5a), epithelial cells (Krt8, Krt19, Prom1), endothelial cells (Pecam1, Egfl7, Eng), B cells (Igkc, Igha, Jchain), and macrophages (Gpnmb, Lgals3, Lyz2). Figure 6 (A~B)

[0050] (2) Spatial distribution of macrophages Uterine tissue was collected on the 3rd day after surgery and subjected to F4 / 80 immunofluorescence staining. The results showed that macrophages were highly enriched in the area surrounding the implanted HMP, regardless of whether it was transient HMP or hMSC-crosslinked HMP. Figure 6 (C~D).

[0051] (3) Macrophage clearance experiment 48 hours preoperatively, 200 μL of clodronate liposomes (CL) were injected intraperitoneally. During surgery, HMP was resuspended in 200 μL of CL solution and injected intraperitoneally. 24 hours postoperatively, another 200 μL of CL was injected intraperitoneally. The clearance efficiency was verified as 85–90% using F4 / 80 immunofluorescence. After macrophage clearance, the repair effect of transient HMP was significantly blocked: endometrial thickness decreased to 158 ± 30 μm (vs. 285 ± 32 μm in the control group), glandular number decreased to 2.5 ± 1.2 per slice (vs. 7.2 ± 1.6), and Ki67... + Cell density decreased to 35% of the control group, CD31 + The vascular area decreased to 40% of the control group. Figure 6 (E~H).

[0052] 5. The central role of B cells in transient HMP-mediated repair (1) B cell enrichment: Uterine tissue was collected on days 3, 7, and 14 post-surgery and quantitatively detected using spatial transcriptomics. The results showed that the abundance of B cells in the transient HMP group on days 7 and 14 was significantly higher than that in the PBS group and the hMSC-crosslinked HMP group. The abundance of fibroblasts in the hMSC-crosslinked HMP group was increased ( Figure 7 (C~D).

[0053] (2) Macrophage and B cell subsets: Uterine tissue was collected on the 7th day after surgery, and PCA subset cluster analysis was performed to identify three macrophage subsets (MF1 / MF2 / MF3) and three B cell subsets. MF2 was enriched on the 7th day in the transient HMP group, and its expression characteristics included repair macrophage markers (Gpnmb) and B cell-related transcripts (Igkc, Igha). MF1 was enriched on the 7th day in the hMSC-crosslinked HMP group, characterized by antigen presentation and immune activation genes (Cd74, H2-Aa, H2-Eb1). B_plasma (high expression of Igkc, Igha), B_resident (Ltf, Lcn2, Cd24a), and B_effector (H2-Aa, H2-Eb1, Cd74) were also identified. B_plasma was significantly enriched on the 7th day in the transient HMP group, while B_effector was more prominent in the hMSC-crosslinked HMP group. Figure 7 (A~B and G~H).

[0054] (3) B cell-macrophage interaction: Based on the conclusions of step (2), CellChat analysis was performed. The results showed that the signal transduction from B cells to macrophages was the strongest in the transient HMP group (significantly higher than in the persistent and PBS groups), involving the Gas6-Axl and App-Cd74 pathways. Subpopulation-level analysis further revealed that the signal transduction from B cells to MF2 was mainly mediated by Gas6. MF2 macrophages were spatially located in the interface region between MF3 and B_plasma ( Figure 7 (E~F).

[0055] (4) Multiplex cytokine analysis: Uterine tissue was collected on the 7th day after surgery for multiplex cytokine analysis. The results showed that the levels of IL-1α (3.5-fold), MIP-1α (2.8-fold), and MIP-1β (2.5-fold) in the uterine tissue of the hMSC-crosslinked HMP scaffold group were significantly higher than those in the transient HMP group (P<0.05). These inflammatory factors were strongly correlated with the MF1 macrophage subset in the spatial transcriptome, suggesting that local B cells may support the repair microenvironment by reducing the production of MF1-related inflammatory factors. Figure 7 (I~J).

[0056] 6. Open tissue environment and stent removal (1) B cell recruitment is independent of macrophages: On the 7th day after surgery, macrophage clearance was performed according to step 4. After macrophage clearance, B220 immunofluorescence detection was performed. The results showed that B cells were still recruited normally on the 7th day in the transient HMP group, indicating that the recruitment of B cells and macrophages is through an independent mechanism ( Figure 8 (A)

[0057] (2) Rag1- / - mouse experiment: On the 7th day after surgery, the purchased Rag1 knockout mice (lacking T / B cells) were subjected to F4 / 80 immunofluorescence detection. The results showed that transient HMPs could still recruit macrophages, indicating that macrophage recruitment was independent of adaptive immunity. Figure 8 (B) However, HMP clearance was significantly impaired in Rag1- / - mice, with 43% of the slides from the distal uterine region still showing scaffold residue on day 7, while wild-type mice showed near-complete clearance in both the proximal and distal regions. The endometrial thickness in the distal region (125±28 μm) was significantly lower than that in the proximal region (255±35 μm). P <0.001, indicating that adaptive immunity is involved in scaffold clearance and incomplete clearance leads to impaired repair. Figure 8 (C~E).

[0058] (3) Comparison between closed wound model and open (IUA) uterine cavity model Eight- to ten-week-old female C57BL / 6 mice were selected. After anesthesia, the dorsal skin was disinfected sequentially with povidone-iodine and 70% ethanol. Two symmetrical full-thickness skin defects were created along the dorsal midline using a 5 mm biopsy punch. To inhibit wound contraction, each wound was fixed around a rubber splint (tissue adhesive + non-absorbable sutures for reinforcement) to construct a closed skin wound model. Transient HMP or hMSC-assembled HMP scaffolds were directly implanted into the closed skin wound model, and covered with Tegaderm dressing and elastic bandages. Postoperatively, the mice were housed individually to prevent wound disturbance. On postoperative day 7, inguinal drainage lymph nodes were harvested for flow cytometry analysis. Unlike the IUA iliac lymph node results, HMP in the skin model did not induce germinal center B cells (CD95). + GL7 + Activation of plasmablasts and plasma cells. In the IUA model, an increase in plasmablasts and plasma cells was observed as early as day 3, and a specific increase in germinal center B cells was observed in the persistent group on day 7. Figure 8 (F~J). This demonstrates that adaptive immune activation is triggered by a state of scavengable foreign matter in an open tissue environment, rather than by the material composition itself.

[0059] 7. Macrophage SPP1-ITGAV signaling and epithelial regeneration To investigate the molecular mechanism by which macrophages promote epithelial regeneration, macrophages (RAW264.7) were co-cultured with HMP for 24 h, and the supernatant was collected to prepare conditioned medium (Macro-CM), which was then used to culture endometrial organoids. The results showed that the EdU incorporation rate in the Macro-CM treatment group was 1.8 times that of the control group. P <0.01%, and cell viability as detected by CCK-8 increased by 1.5 times ( P <0.05; Under oxidative stress (H2O2) conditions, Macro-CM can reduce cleaved caspase-3-positive apoptotic cells and maintain mitochondrial membrane potential (JC-1 red / green ratio). The growth-promoting effect of Macro-CM was significantly eliminated upon the addition of the ITGAV inhibitor Cilengitide (10 μM); while recombinant SPP1 protein (100 ng / mL) could mimic the growth-promoting effect of Macro-CM, and this effect could also be blocked by Cilengitide, indicating that the SPP1-ITGAV axis is a key pathway for macrophage paracrine support of epithelial regeneration. Figure 9 ).

[0060] 8. Epithelial cell proliferative regeneration status Building upon step 7, the effects of different scaffold conditions on epithelial subpopulations were further analyzed. Four epithelial subpopulations (Epi1-Epi4) were identified by PCA clustering. Epi2, a proliferative regeneration subpopulation (highly expressing Stmn1 and Clu), was selectively maintained in the transient HMP group on day 14, but decreased in the hMSCs-crosslinked HMP group. Epi3, a neonatal / mature epithelial subpopulation (highly expressing Alpl and Add2), was also enriched in the transient group on day 14. Epi4, an immune-activated / mucosal defense state subpopulation (highly expressing Agr2, Lpo, Ccl28, and Fcgbp), was enriched in the hMSCs-crosslinked HMP group on day 14. Ki67 immunostaining of tissue sections further validated this trend; the number of proliferating epithelial cells in the transient HMP group was significantly higher than that in the PBS group and the hMSCs-crosslinked HMP group. P <0.01, highly consistent with the results of spatial transcriptomic epithelial subset analysis. Transient scaffolds selectively preserve proliferative and regenerative subsets by maintaining SPP1-ITGAV signaling-mediated macrophage-epithelial interactions, thereby providing a favorable regenerative microenvironment for endometrial injury repair, while persistent scaffolds tend to induce the epithelium to a state of immune activation. Figure 10 ).

[0061] This invention provides the first systematic demonstration that the pro-repair adaptive immune activation of biomaterials does not depend on the specific chemical composition or physical properties of the material, but rather on the host immune system's recognition of a foreign substance that can be cleared. This invention fully elucidates the causal chain from scaffold transient clearance – drainage lymph node B cell activation – local B cell infiltration and plasma cell differentiation – B cell-macrophage interaction (Gas6-Axl / App-Cd74) – reparative macrophage polarization → SPP1-ITGAV-driven epithelial regeneration. This invention allows for the conversion of the same HMP material between durable and transient scaffolds using an extremely simple method without applying interparticle crosslinks, without altering the material's chemical composition. Through control experiments using the same material in open (IUA) and closed (skin wound) models, this invention demonstrates for the first time that an open tissue environment is a necessary condition for implanted materials to trigger adaptive immunity, providing important guidance for material selection in different clinical applications. Rag1 knockout experiments revealed that adaptive immunity participates in scaffold clearance, demonstrating for the first time that B / T cells are not only downstream effectors activated by the scaffold, but also actively participate in the scaffold clearance process, forming a positive feedback loop of clearance → immunity → clearance. Compared with persistent cross-linked scaffolds, the transient HMP scaffold provided by this invention achieves significantly better endometrial structure reconstruction, fibrosis inhibition, and fertility restoration.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A transient hydrogel microparticle scaffold system, characterized in that, It consists of several hydrogel microparticles, and there is no cross-linking between any two hydrogel microparticles; The particle size of the hydrogel microparticles is 50~100μm; The raw materials for preparing the non-crosslinked hydrogel microparticles include photoinitiators, photosensitive gel matrices, biodegradable crosslinking agents, and cell adhesion peptides; The degradable crosslinking agent is a matrix metalloproteinase-degradable peptide; The photosensitive gel matrix is ​​any one of the following: (1) one or more of polyethylene glycol, hyaluronic acid, gelatin and alginate; (2) a derivative or copolymer of the compound in (1).

2. The transient hydrogel microparticle scaffold system according to claim 1, characterized in that, The matrix metalloproteinase-degradable peptide is the polypeptide shown in SEQ ID NO:1 or a functionally equivalent variant thereof; The cell adhesion peptide contains an RGD sequence; The photoinitiator is the photoinitiator LAP; The polyethylene glycol is an octagonal polyethylene glycol norbornene.

3. The method for preparing the transient hydrogel microparticle scaffold system according to claim 1 or 2, characterized in that, Includes the following steps: A photopolymerization reaction was carried out by mixing the aqueous phase and the oil phase to obtain in-situ solidified hydrogel microparticles; The oil phase component in the in-situ solidified hydrogel microparticles is removed to obtain a transient hydrogel microparticle scaffold system; The aqueous phase includes a photoinitiator, a photosensitive gel matrix, a degradable crosslinking agent, cell adhesion peptides, and PBS buffer. The oil phase comprises sorbitan monooleate and mineral oil.

4. The preparation method according to claim 3, characterized in that, When the photosensitive gel matrix is ​​an octagonal polyethylene glycol norbornene, the molar ratio of thiol groups in the MMP degradable peptide crosslinking agent to norbornene in the octagonal polyethylene glycol norbornene is 0.6:

1. The concentration of the octagonal polyethylene glycol norbornene in the aqueous phase is 5-10 wt%.

5. The transient hydrogel microparticle scaffold system according to claim 3, characterized in that, The concentration of cell adhesion peptides in the aqueous phase is 1-5 mM; The concentration of the photoinitiator in the aqueous phase is 1~10mM; The concentration of sorbitan monooleate in the oil phase is 10-20%.

6. The transient hydrogel microparticle scaffold system according to claim 3, characterized in that, The steps of the photopolymerization reaction include: co-flowing an aqueous phase and an oil phase to form microdroplets, and applying ultraviolet light to the microdroplets; The ultraviolet light irradiation has a wavelength of 365~405 nm and a light intensity of 10~40 mW / cm. 2 .

7. The application of the transient hydrogel microparticle scaffold system according to claim 1 or 2 or the transient hydrogel microparticle scaffold system obtained by the preparation method according to any one of claims 3 to 6 in the preparation of medical products.

8. The application according to claim 7, characterized in that, The medical product in question is a medical product that promotes tissue regeneration and repair.

9. The application according to claim 8, characterized in that, The tissue in question is the uterine lining.

10. The application according to any one of claims 7 to 9, characterized in that, The medical products also include mesenchymal stem cells.

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