Use of a hydrogel loaded with anti-ifn-gamma antibodies for the preparation of a medicament for the treatment of bisphosphonate-associated osteonecrosis of the jaw

Abstract

The application discloses application of an anti-IFN-gamma antibody loaded hydrogel in preparation of a drug for treating bisphosphonate-related osteonecrosis of the jaw. The research result of the application shows that ZOL treatment can promote secretion of IFN-gamma by gamma delta T cells, enhance glycolysis of osteoclasts, induce histone H3K18 lactic acidification of lactic acid generated by glycolysis, activate transcription factor IKZF1 and downstream effector molecule ZBP1, finally drive necrotic apoptosis of osteoclasts and damage bone remodeling function. By locally applying GelMA@ab at a BRONJ lesion, controllable release of the anti-IFN-gamma antibody can be realized, IFN-gamma activity can be effectively blocked, necrotic apoptosis of osteoclasts and local inflammatory reaction can be significantly reduced, and a rat BRONJ-like lesion can be improved. Therefore, the application provides an effective strategy for treatment of BRONJ and provides experimental feasibility support for clinical conversion application of the composite hydrogel.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of hydrogels loaded with anti-IFN-γ antibodies in the preparation of medicaments for the treatment of bisphosphonate-related osteonecrosis of the jaw. Background Technology

[0002] Bisphosphonate-associated osteonecrosis of the jaw (BRONJ) is a specific type of osteonecrosis caused by bisphosphonates, with a cumulative incidence of 2.8% to 16.3%. Clinical manifestations of BRONJ include gingival ulceration, jawbone necrosis, and bone exposure, causing significant suffering for patients. However, according to the American Academy of Oral and Maxillofacial Surgery (AAOMS) guidelines and domestic expert consensus, current clinical treatments primarily rely on potent antibiotic therapy and surgical debridement, with limited efficacy. Currently, there are no drugs targeting the underlying cause of BRONJ. Therefore, elucidating the pathogenesis of BRONJ and finding effective treatment strategies are urgent and challenging issues in current clinical practice.

[0003] Studies have shown that oral local immune dysregulation and bone metabolic dysfunction are key factors in the pathogenesis of BRONJ. Among immune cells, γδ T cells are the core effector cells maintaining oral mucosal immune homeostasis. Previous studies have reported that γδ T cells are heavily recruited in lesioned tissues and are closely related to the development of BRONJ. Previous research has confirmed that the commonly used bisphosphonate drug zoledronate (ZOL) can significantly enhance the proliferation and effector function of γδ T cells. ZOL tends to accumulate in bone tissue, is taken up by monocytes and accumulates intracellularly, subsequently being converted into a phosphate antigen specifically recognized by γδ T cells, thereby promoting their activation. However, the specific mechanism by which γδ T cells affect the pathogenesis of BRONJ remains unclear.

[0004] Therefore, a deeper understanding of the pathogenesis of BRONJ and the development of a novel, targeted, and controlled-release treatment strategy are crucial to overcoming the limitations of existing BRONJ treatment strategies. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of an anti-IFN-γ antibody in the preparation of a drug for treating bisphosphonate-related osteonecrosis of the jaw.

[0006] A second objective of this invention is to provide the use of hydrogels loaded with anti-IFN-γ antibodies in the preparation of medicaments for the treatment of bisphosphonate-related osteonecrosis of the jaw.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] This invention primarily explores the role of γδ T cell-derived interferon-γ (IFN-γ) in the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BRONJ) and evaluates a local treatment strategy. This invention establishes BRONJ models in mice and rats using zoledronic acid (ZOL) combined with maxillary tooth extraction. Bulk RNA-seq, single-cell RNA sequencing (scRNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) analyses were employed to reveal the molecular pathways of osteoclast dysfunction. Mechanistic experiments (including γδ T cell isolation, co-culture experiments, drug inhibition, and gene knockdown) were used to investigate how interferon-γ (IFN-γ) regulates glycolysis, H3K18 lactation (H3K18la), and activates the IKZF1-ZBP1 signaling axis, thereby inducing osteoclast necrotizing apoptosis. Finally, methacrylamide gelatin (GelMA@ab) hydrogel loaded with anti-IFN-γ antibody was applied topically to the BRONJ lesions in rats, and its therapeutic effect was evaluated by micro-CT, histological and immunofluorescence analysis.

[0009] The results of this study indicate that γδ T cell-mediated immune dysregulation is a core driving factor in BRONJ. Single-cell RNA sequencing revealed significant activation of γδ T cells, with monocytes (as osteoclast precursors) undergoing necrotizing apoptosis. Mechanistically, activated γδ T cells secrete IFN-γ, which enhances osteoclast glycolysis and promotes the accumulation of reactive oxygen species (ROS). The accumulation of lactate from glycolysis induces histone H3K18 lactation, activating transcription factor IKZF1 and its downstream effector molecule ZBP1, ultimately driving osteoclast necrotizing apoptosis and impairing bone remodeling function. On the other hand, tumor necrosis factor-α (TNF-α) and IFN-γ are both known necrotizing apoptosis inducers. The classical pathway is initiated by the activation of receptor-interacting protein kinase 1 (RIPK1) and RIPK3, which then phosphorylates mixed kinase domain-like proteins (MLKL). Phosphorylated MLKL forms necrosomes, which bind to and disrupt the cell membrane, thereby triggering and amplifying the inflammatory cascade. To explore therapeutic potential, this invention developed a methacrylamide gelatin (GelMA) hydrogel loaded with anti-IFN-γ antibodies (GelMA@ab). Topical application to BRONJ lesions showed that GelMA@ab could achieve controlled release of anti-IFN-γ antibodies, effectively blocking IFN-γ activity, significantly reducing osteoclast necrosis and apoptosis and local inflammation, and significantly promoting bone repair and regeneration in BRONJ defects, thus improving BRONJ-like lesions in rats. This research reveals that the H3K18la-dependent IKZF1-ZBP1 pathway links γδ T cell-derived IFN-γ to the pathogenesis of BRONJ, and demonstrates that targeting IFN-γ to block osteoclast necrosis and apoptosis is a promising therapeutic strategy for BRONJ, providing experimental feasibility support for the clinical translation of composite hydrogels.

[0010] Therefore, the present invention provides the use of anti-IFN-γ antibody in the preparation of a medicament for treating bisphosphonate-related osteonecrosis of the jaw.

[0011] The basic components of GelMA hydrogel are widely used in tissue engineering due to their biodegradability and biocompatibility. Its porous structure enables efficient loading and sustained release of anti-IFN-γ antibodies. GelMA hydrogel can form a gel at the lesion site, significantly increasing the local drug concentration while avoiding the potential risks of systemic administration, resulting in high safety. This hydrogel has optimal fluidity at 4°C and rapidly gels after light exposure, making it ideal for injecting and filling irregular BRONJ defects, suitable for clinical procedures. After degradation, GelMA@ab is replaced by new bone islands, promoting osteoclast differentiation and reducing necrotic bone formation in vivo, providing a scaffold for new bone tissue.

[0012] Therefore, the present invention also provides the use of hydrogels loaded with anti-IFN-γ antibodies in the preparation of medicaments for treating bisphosphonate-related osteonecrosis of the jaw.

[0013] Furthermore, the drug achieves treatment by inhibiting the secretion of IFN-γ by γδ T cells.

[0014] Furthermore, the drug achieves therapeutic effects by inhibiting osteoclast glycolysis and / or inhibiting osteoclast necrotizing apoptosis.

[0015] Furthermore, the drug achieves its therapeutic effect by inhibiting histone H3K18 lactation.

[0016] Furthermore, the drug achieves therapeutic effects by inhibiting the activation of transcription factor IKZF1 and its downstream effector molecule ZBP1.

[0017] Preferably, the drug achieves treatment by inhibiting the H3K18-IKZF1-ZBP1 axis.

[0018] Furthermore, the hydrogel loaded with anti-IFN-γ antibody is a methacrylamide gelatin hydrogel loaded with anti-IFN-γ antibody.

[0019] Furthermore, the final concentration of anti-IFN-γ antibody in the hydrogel loaded with anti-IFN-γ antibody is 4–6 μg / mL.

[0020] Preferably, the final concentration of anti-IFN-γ antibody in the hydrogel loaded with anti-IFN-γ antibody is 5 μg / mL.

[0021] Furthermore, the method for preparing the methacrylamide gelatin hydrogel loaded with anti-IFN-γ antibody is to obtain it by photoinitiated polymerization of methacrylamide gelatin, photoinitiator and anti-IFN-γ antibody.

[0022] Preferably, the photoinitiator is LAP. LAP is the most commonly used visible light initiator, with an excitation wavelength of 365–405 nm, and has extremely low cytotoxicity, making it suitable for hydrogel polymerization.

[0023] Furthermore, the dosage form of the drug includes oral, injectable, or patch formulations.

[0024] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0025] The present invention also provides the use of 2-deoxy-D-glucose in the preparation of a medicament for treating bisphosphonate-associated osteonecrosis of the jaw.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides the application of a hydrogel loaded with anti-IFN-γ antibodies in the preparation of a drug for treating bisphosphonate-related osteonecrosis of the jaw. The results of this invention indicate that ZOL treatment promotes the secretion of IFN-γ by γδ T cells, which enhances the glycolysis level of osteoclasts and promotes the accumulation of reactive oxygen species. The accumulation of lactate produced by glycolysis induces histone H3K18 lactation, activating transcription factor IKZF1 and its downstream effector molecule ZBP1, ultimately driving osteoclast necroptosis and impairing bone remodeling function. Furthermore, the results of this invention show that the local application of methacrylamide gelatin hydrogel loaded with anti-IFN-γ antibodies (GelMA@ab) to BRONJ lesions allows for the controlled release of anti-IFN-γ antibodies, effectively blocking IFN-γ activity, significantly reducing osteoclast necroptosis and local inflammatory response, and improving BRONJ-like lesions in rats. Therefore, this invention provides an effective strategy for the treatment of BRONJ, provides experimental feasibility support for the clinical translation of composite hydrogels, and could potentially be extended to other bone diseases caused by immune metabolic abnormalities. Attached Figure Description

[0028] Figure 1 Necrosis and apoptosis of cells are characteristic of BRONJ lesions in mice. Figure 1 In the diagram, A represents an intraoral photograph of mice in the ZOL group 4 weeks after tooth extraction; B shows the H&E staining results of the extraction socket (scale bar, 400 µm); C shows the Masson staining results of the extraction socket; and D shows the TRAP staining results (scale bar, 250 µm). The bar charts below B and D quantify the area of ​​the hollow socket, bone filling, and TRAP. + Cells; E represents KEGG analysis; F represents GO analysis; GI represents immunohistochemical analysis. Scale bar, 200 µm. Data are presented as mean ± SD; **p < 0.01.

[0029] Figure 2 The pathological process of BRONJ is driven by necrosis and apoptosis of osteoclast precursor cells. Among them, Figure 2In the figures, A shows intraoral photographs of the gingival mucosa of mice treated with ZOL and Fer-1 or Nec-1, respectively, 4 weeks after tooth extraction; B shows the H&E staining results of the extraction socket (scale bar: 400 µm); C shows the Masson staining results of the extraction socket (scale bar: 400 µm); D shows the immunohistochemical staining results (scale bar: 200 µm); E shows the quantitative analysis of the area of ​​the empty bone lacunae, the area of ​​blue bone tissue, and the proportion of 4-HNE and p-MLKL positive cells (*p<0.05, **p<0.01); FG shows the UMAP analysis of single-cell RNA-seq; H shows the TRAP staining results (scale bar: 250 µm, **p<0.01); I shows the immunofluorescence staining results of CTSK (osteoclasts, red) and Ki67 (proliferation marker, green) in the extraction socket (scale bar: 250 µm, **p<0.01).

[0030] Figure 3 γδ T cells are a key factor in promoting osteoclast necrosis and apoptosis in BRONJ. Among them, Figure 3 A in the figure is a violin plot of the T cell activation module score; B is the GO enrichment analysis and KEGG analysis of differentially expressed genes (DEGs) in T cell clusters; C is the UMAP visualization of IFN-γ and TNF module scores in single cells of BRONJ lesions. IFN-γ expression is mainly enriched in T cells, indicating its major contribution to IFN-γ production. The gene sets used to calculate IFN-γ and TNF scores are from GO:0032609 (interferon-γ production) and GO:0032640 (tumor necrosis factor production), respectively; D is the immunofluorescence staining showing γδT cells (red) and IFN-γ (green) and CTSK in the extraction sockets of ZOL-treated mice. + (Osteoclasts, red) and Ki67 + (Green) expression and its effect on IFN-γ + γδ T cells and Ki67 + Quantitative analysis of CTSK osteoclast count was performed, **p<0.01; E represents the number of osteoclasts after TCRδ. - / - Intraoral photograph of BRONJ gingival mucosa in mice; F represents TCRδ. - / - H&E staining results of extraction sockets in mice, scale bar: 400 µm; G represents TCRδ. - / - TRAP staining results in mouse extraction sockets, scale bar: 200 µm; H represents TCRδ. - / - Immunohistochemical staining results of p-MLKL and 4-HNE in mouse extraction sockets, scale bar: 200 µm; I represents the local IFN-γ level in the extraction socket as detected by ELISA, *p<0.05; J represents TCRδ. - / -Quantitative analysis of the area of ​​hollow bone lacunae, the number of TRAP-positive osteoclasts, and the ratio of 4-HNE and p-MLKL-positive cells in mouse tooth extraction sockets, **p<0.01.

[0031] Figure 4 The relevant signaling pathways in ZOL-treated γδ T cells. Among them, Figure 4 A in the figure is a volcano plot comparing ZOL-treated γδ T cell supernatant (SPN(ZOL)) with the untreated group (SPN); SPN: γδ T cell supernatant; SPN(ZOL): ZOL-treated γδ T cell supernatant; B is the GSEA result comparing ZOL-treated γδ T cell supernatant (SPN(ZOL)) with the untreated group (SPN), *p<0.05; C is the intracellular lactate level after IFN-γ treatment, **p<0.01; D is the protein level of total protein lactation (Pan-Kla) and H3K18 lactation (H3K18la) in osteoclast precursors detected by Western blotting, **p<0.01; E is the ChIP-seq analysis, and the gray histogram shows the results across 200... The log2 (IP / Input) fold change within the bp interval is shown, with significantly enriched regions (log2>0.5) highlighted in pink. Red lines indicate transcription start sites, and yellow shading marks the promoter region (TSS±2 kb). F represents the intracellular lactate level, **p<0.01, ns indicates no significant difference. G represents the effect of 2-DG treatment on the protein levels of Pan-Kla and H3K18la. H is an immunofluorescence image showing the effect of TCRδ... - / - Colocalization of Pan-Kla (red) and CTSK (osteoclasts, green), and H3K18la (red) and CTSK (green) in tooth extraction socket tissues of ZOL mice and wild-type WT ZOL mice, scale bar 250 µm.

[0032] Figure 5 The GelMA@ab hydrogel continuously releases anti-IFN-γ antibodies to alleviate BRONJ lesion formation. Among other things, Figure 5In the table, A represents scanning electron microscopy (SEM) images of GelMA and GelMA@ab hydrogels, scale bar: 100 µm; B represents Fourier transform infrared (FTIR) spectra of GelMA and GelMA@ab hydrogels; C represents X-ray photoelectron spectroscopy (XPS) spectra of GelMA and GelMA@ab hydrogels; D represents AO / PI staining images of RAW264.7 cells cultured on GelMA@ab hydrogels, with live / dead cells marked in green / red, scale bar: 100 μm; E represents intraoral photographs of the gingival mucosa of rats 4 weeks after tooth extraction; F represents Micro-CT images showing bone regeneration in the extraction sockets of the control group, GelMA group, and GelMA@ab treatment group, with quantitative analysis of bone volume fraction (BV / TV) and bone mineral density (BMD), **p<0.01, ns indicates no significant difference; G represents H&E staining results of the extraction sockets, scale bar: 400 µm. µm, **p<0.01, ns indicates no significant difference; H represents TRAP staining results, and the number of TRAP-positive cells was quantitatively analyzed. Scale bar: 200 µm, **p<0.01, ns indicates no significant difference; IK represents the expression of ZBP1, p-MLKL and IKZF1 in ZOL-induced rat tooth extraction sockets detected by immunohistochemical staining. Scale bar: 500 µm, **p<0.01, ns indicates no significant difference; L represents the expression of NDUFA9 in ZOL-induced rat tooth extraction sockets detected by immunohistochemical staining. Scale bar: 500 µm, **p<0.01, ns indicates no significant difference. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] I. Experimental Methods

[0036] 1. Preparation of the mouse BRONJ model

[0037] All animal experiments were conducted in accordance with the Laboratory Animal Care and Use Guidelines and approved by the Animal Care and Use Committee. Eight-week-old male C57BL / 6J mice and TCRδ mice were used. - / - Mice were purchased from Huawei Testing Technology Co., Ltd. The absence of γδ T cells in the spleen was analyzed by PCR and flow cytometry, verifying the TCRδ... - / -The effectiveness of gene knockout in mice. Eight-week-old Sprague Dawley (SD) rats (weighing 180-200 g) were purchased from Vital River Laboratory Animal Technology Ltd. All animals were housed in a specific pathogen-free (SPF) facility with a controlled 12-hour light / 12-hour dark light cycle.

[0038] Establishment of a mouse BRONJ (bisphosphonate-associated osteonecrosis of the jaw) model: TCRδ - / - Mice and wild-type (WT) mice were randomly assigned to two groups: an untreated control group (control mice were intraperitoneally injected with an equal volume of sterile PBS) and a ZOL (zoledronic acid) treatment group. The ZOL treatment group received intraperitoneal injections of ZOL (500 μg / kg, 200 μL, Sigma-Aldrich, catalog number Z0158) once weekly for 3 weeks prior to extraction of the maxillary first molar. The maxillary first molar was then extracted using a microsurgical dental probe, and ZOL treatment continued for 4 weeks after extraction.

[0039] The establishment of the rat BRONJ model was the same as above (the rat and mouse BRONJ models showed almost the same phenotype).

[0040] 2. micro-CT

[0041] The microstructural characteristics of the jawbone were analyzed using a micro-computed tomography (micro-CT) system (Scanco Medical AG, Switzerland). The simplified procedure is as follows: The jawbone was scanned at a high resolution of 10 μm, with scanning parameters set to 55 kV voltage, 456 μA current, and an integration time of 200 ms. The scan resolution was 1024 × 1024 × 148 pixels. Three-dimensional images of the jawbone were reconstructed using MicroView software, and bone volume (BV, mm²) was quantitatively analyzed. 3 Total volume (TV, mm) 3 Bone volume fraction (BV / TV, %) and tissue mineral density (BMD, g / cm³) 3 ).

[0042] 3. Histological, immunohistochemical and immunofluorescence staining

[0043] Maxillary bone tissue from mice and rats was fixed, decalcified, embedded in paraffin, and sectioned (8 μm thick), followed by hematoxylin and eosin (H&E) staining. Using ImageJ software, areas of alveolar bone containing ≥5 aggregated necrotic osteocytes were defined as osteonecrosis. Tissue sections were stained with Masson's trichrome and tartrate-resistant acid phosphatase (TRAP). Mature osteoclasts were fixed in 4% paraformaldehyde (PFA) for 15 minutes and stained using the TRAP staining kit (387A-1KT, Sigma) according to the manufacturer's instructions. Osteoclast formation was assessed using an optical microscope; cells with three or more positively stained nuclei were defined as mature osteoclasts.

[0044] For immunofluorescence staining, sections were treated with 1% Triton X-100 for 15 minutes, blocked with goat serum at room temperature for 30 minutes, and then incubated with primary antibodies overnight at 4°C. The primary antibodies used included: c-Caspase-3 (1:200, Asp175, Affinity), p-MLKL (1:200, Ser358, Affinity), 4-HNE (1:200, bs-6313R, Bioss), ZBP1 (1:200, 13285-1-AP, Proteintech), IKZF1 (1:200, DF6659, Affinity), and NDUFA9 (1:200, ...). Sections were prepared using 20312-1-AP (Proteintech), CTSK (1:200, 11239-1-AP, Proteintech), Ki67 (1:200, 11F6, Biolegend), TCRγδ (1:100, 118108, BioLegend), pan-kla (1:200, PTM-1401RM, PTMBIO), and H3K18la (1:200, sc-517576, Santa Cruz). Sections were then incubated at room temperature for 2 hours with HRP-labeled or Alexa Fluor488 / 594-labeled secondary antibodies. Isotype-matched control antibodies were used as negative controls. Fluorescence images were acquired using a microscope (Stellaris LAS X, Leica), and the percentage of positive cells per unit area (mean ± standard deviation) was calculated using ImageJ software.

[0045] 4. Bulk RNA sequencing (Bulk RNA-seq)

[0046] From wild-type (WT) mice, ZOL-treated WT mice, and ZOL-treated TCRδ - / - Total RNA was extracted from extraction sockets of mice. Library construction and sequencing were performed by Novogene (Beijing). TruSeq was used. TMA next-generation sequencing library was constructed using an RNA sample preparation kit (Illumina, USA) according to the manufacturer's instructions. Differential expression analysis was performed using the DESeq2 software package (v3.40.6) in R, defining genes with an absolute log2 folding change >1.5 as differentially expressed genes (DEGs). KEGG and gene ontology (GO) enrichment analyses were performed, with pathways showing a corrected P < 0.05 considered significantly enriched. Gene set enrichment analysis (GSEA) was performed using GSEA software (v3.0).

[0047] 5. Single-cell RNA sequencing (scRNA-seq)

[0048] Public scRNA-seq data were obtained from the GEO database (accession number GSE193110). Preprocessing and quality control were performed using Seurat (v4.0.3), retaining high-quality cells with 500-2500 detected genes and mitochondrial gene content <20%. Data from qualified cells were normalized using NormalizeData, and hypervariable genes were identified using FindVariableFeatures. Dimensionality reduction was performed using PCA, and the top 20 principal components were selected for downstream analysis. Cell clustering was performed using the Louvain algorithm combined with FindNeighbors and FindClusters. Two-dimensional visualization maps were generated using UMAP. Cell types were annotated based on known marker genes and FindAllMarkers results. Differential expression of cell subpopulations under different conditions was assessed using FindMarkers, and GO and KEGG pathway enrichment analysis was performed using ClusterProfiler (v4.0.5) (p<0.05 was considered significant). Finally, pathway activity was assessed using GSEA (v4.0.3).

[0049] 6. ChIP-seq Data Acquisition and Visualization

[0050] H3K18la ChIP-seq publicly available data (accession number GSE156675) from human samples were downloaded from the Gene Expression Comprehensive Database (GEO). This dataset contains two H3K18la immunoprecipitation (IP) biological replicates and their matched input controls. Genome alignment and signal orbitals were acquired in bigWig format and analyzed based on the hg38 human genome reference sequence. The Gviz R package (v1.44.0) was used to visualize H3K18la enrichment at the IKZF1 gene locus. Signal intensities of IP and input orbitals were imported using the rtracklayer package, limiting the analysis to a ±25 kb region upstream and downstream of the transcription start site (TSS). Replicated sample data were overlaid to compare IP with input signals. Log2(IP / Input) folding changes were calculated within non-overlapping 200 bp intervals, and intervals with log2(IP / Input) > 0.5 were marked as significantly enriched. Promoter regions were defined as ±2 kb upstream and downstream of the TSS, with the TSS marked with a red vertical line.

[0051] 7. Isolation and in vitro culture of mouse γδ T cells

[0052] γδ T cells were isolated from the spleen of 6-8 week old mice using tissue homogenization and erythrocyte removal. CD3 cells were sorted using a FACSAria flow cytometer. + γδ TCR + or CD3 +T lymphocytes were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco) and IL-2 (20 ng / mL, #210-21, PeproTech). For the first 48–72 hours of culture, anti-mouse γδ TCR antibody (10 μg / mL, 107502, BioLegend), anti-mouse CD28 antibody (1 μg / mL, 102116, BioLegend), and ZOL (0, 0.5, 5, or 15 μM) were added. After replacing the medium with fresh medium and culturing for 2 days, the cell supernatant was collected. γδ T cell proliferation, purity, and IFN-γ expression were detected by flow cytometry after 3 and 6 days of ZOL treatment. After three washes, collected cells were stained with Live / Dead fixation dye (565388, BD Biosciences), CD3 (145-2C11, BioLegend), γδ TCR (GL3, 118108, BioLegend), IFN-γ (50709-R373-F, SinoBiological), or PI (C2015M, BioLegend). IgG was used as an isotype control. Samples were analyzed by flow cytometry (Beckman, USA) using FlowJo software (BD Biosciences).

[0053] The score was calculated using the Seurat software (AddModuleScore function) on the gene ontology dataset (GO:0042110, T cell activation). The higher the value, the stronger the pathway enrichment.

[0054] 8. In vitro osteoclast differentiation and IFN-γ treatment

[0055] Mouse RAW264.7 cells were induced into osteoclast precursors for 2 days using M-CSF (10 ng / mL, R&D Systems), followed by 3 days of further culture with RANKL (50 ng / mL, R&D Systems) and M-CSF (50 ng / mL, R&D Systems). The culture medium was changed every two days for all experiments. Osteoclast differentiation was assessed by counting TRAP-positive multinucleated cells (≥3 nuclei), using the same method as before. After osteoclast differentiation, IFN-γ treatment was used to detect gene expression in the osteoclasts.

[0056] 9. Proteomics detection

[0057] RAW264.7 cells were induced into osteoclast precursors. The supernatant of γδ T cells treated with ZOL (SPN(ZOL)) was mixed with the untreated group (SPN) and then sent to the company (Guangke Ande) for proteomics analysis.

[0058] 10. Quantitative PCR and Western blot analysis of proteins

[0059] RAW264.7 cells were induced into osteoclast precursors by mixing γδ T cell supernatant with culture medium or treating with IFN-γ (200 ng / mL) for 24 hours. Some experiments involved cell transfection or treatment with the glycolysis inhibitor 2-DG (5 mM). Total RNA was extracted from cells using RNA extraction kits (AG21022, AG21023, Abbio), and cDNA was generated by reverse transcription using a kit (AG11707, Abbio). Quantitative RT-PCR analysis was then performed (SYBR Green PCR Master Mix, AG11701, Abbio). Each experiment was independently repeated at least three times.

[0060] Cells were lysed using RIPA buffer (Beyotime) containing a protease inhibitor (Beyotime), and total protein concentration was determined using the BCA method (Solepro). An equal volume of denatured protein was loaded onto a PVDF membrane, separated by SDS-PAGE, and then transferred to the membrane. The membrane was incubated with primary antibodies and corresponding HRP-labeled secondary antibodies. Primary antibodies used included: anti-GAPDH (GB15004, Saive Biotech), anti-β-actin (GB15003, Saive Biotech), anti-IKZF1 (DF6659, Affinity), anti-p-MLKL (Ser358, Affinity), anti-MLKL (D6W1K, CST), anti-H3 (sc-517576, Santa Cruz), anti-H3K18la (PTM-1406RM, PTM BIO), and anti-pan-kla (PTM-1401RM, PTM BIO). ECL substrate (SQ20, Yisheng Biotechnology) was used for color development, and the bands were analyzed and quantified using ImageJ software. Protein band intensity was normalized to GAPDH / β-actin.

[0061] 11. IFN-γ detection

[0062] To measure the IFN-γ content in extraction socket tissue, an ELISA kit (E-EL-M0048, Elite) was used according to the manufacturer's instructions. The procedure was as follows: After tissue homogenization, the supernatant was collected, blocked with 3% BSA for 1 hour, and then incubated with primary antibody at 37°C for 2 hours to allow antigen-antibody binding. After washing, the tissue was incubated with secondary antibody at room temperature for 1 hour. After adding the substrate, the absorbance was measured at 450 nm using an ELISA reader, and the antigen concentration was quantified using a standard curve.

[0063] 12. Lactic acid colorimetric method for detection

[0064] RAW264.7 cells were induced into osteoclast precursors and treated with IFN-γ (200 ng / mL) for 24 hours. Some experiments also included treatment with the glycolysis inhibitor 2-DG (5 mM). Lactate concentration was assessed using a lactate assay kit (S0208S, Beyotime). Brief procedure: The reaction solution was added to the cell supernatant according to the manufacturer's instructions, and the lactate concentration was determined by measuring the absorbance at 570 nm using a microplate reader.

[0065] 13. ROS testing

[0066] Intracellular ROS levels in osteoclast precursors were detected using a fluorescent probe (KGAF018, Kaiji Biotechnology). Osteoclast precursors were treated with γδT cell supernatant for 24 hours, followed by incubation with the ROS fluorescent probe DCFH-DA for 20 minutes. After washing with PBS and replacing with fresh culture medium, ROS fluorescence intensity was quantified by fluorescence microscopy or flow cytometry.

[0067] 14. Statistical Analysis

[0068] Data are expressed as mean ± standard deviation. Intergroup comparisons were performed using unpaired two-tailed Student's t-test or one-way ANOVA combined with Dunnett's test or Tukey's multiple comparison test. p < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7.0.

[0069] Example 1: Necrotic apoptosis is a characteristic of BRONJ lesions in mice.

[0070] Compared with the control group (C57BL / 6J mice), intraoral photographs of mice in the ZOL-treated group 4 weeks after tooth extraction showed delayed wound healing, persistent wound opening (black circles), and gingival swelling, confirming the successful establishment of the mouse BRONJ model. Figure 1 In the A section, H&E staining of the extraction socket showed immune cell infiltration, epithelial cell proliferation, and osteonecrosis in the ZOL-treated group of mice. Black arrows indicate infiltrative inflammatory cells, and white arrows indicate necrotic bone cells with vacuolated nuclei. Figure 1 (B in the text). Masson staining showed that in ZOL-treated mice, the amount of newly formed bone collagen (blue) was reduced and the amount of fibrous collagen (red) was increased in the extraction sockets. Figure 1 C). TRAP staining showed a significant reduction in osteoclasts in the extraction sockets of ZOL-treated mice (C). Figure 1 (D in the middle).

[0071] Transcriptome sequencing results showed that, compared with the control group, KEGG analysis of the ZOL-treated group enriched signals including osteoclast differentiation, necrosis and apoptosis, and T-cell immune-related signals. Figure 1E in the text). GO analysis results showed that ZOL treatment increased the expression of genes related to immune and cell death programs, such as complement activation, positive regulation of T cell activation, positive regulation of inflammatory responses, apoptosis, and necroptosis. Figure 1 (F in the middle).

[0072] Immunohistochemistry showed that, compared with the control group, ZOL-treated mice had significantly higher levels of p-MLKL ( Figure 1 G in 4-HNE Figure 1 H) and c-Caspase-3 ( Figure 1 The expression of I) in BRONJ was increased, with p-MLKL showing the most significant increase, indicating significant necrosis and apoptosis in BRONJ.

[0073] In summary, ZOL-induced BRONJs exhibit characteristics of immune dysregulation and necrotizing apoptosis activation.

[0074] Example 2: Osteoclast precursor cell necrosis and apoptosis drive the pathological process of BRONJ.

[0075] To investigate whether necroptosis is involved in the development of BRONJ lesions, mice were treated with ZOL injection for 3 weeks followed by tooth extraction. Then, mice with BRONJ lesions were treated with ZOL or, respectively, with the addition of the ferroptosis inhibitor Fer-1 (1 mg / kg) and the necroptosis inhibitor Nec-1 (1 mg / kg). Four weeks after tooth extraction, compared to the ZOL-treated group (control group), the gingival mucosa of BRONJ mice treated with Fer-1 and Nec-1 showed that Nec-1 treatment significantly promoted wound healing. Figure 2 (A, E in the text). H&E staining of extraction sockets showed that, compared with the ZOL control group and the Fer-1 treatment group, Nec-1 treatment reduced immune cell infiltration, inhibited abnormal epithelial proliferation, and osteonecrosis. Black arrows indicate vacuolated necrotic bone cells (…). Figure 2 B and E in the text). Masson staining of extraction sockets showed that collagen formation (blue) was increased in the Nec-1 treatment group compared to the ZOL and Fer-1 groups. Figure 2 C and E in the text). Immunohistochemical staining showed that the necroptosis inhibitor Nec-1 specifically and effectively inhibited the expression of the necroptosis-executing protein p-MLKL in BRONJ lesions, significantly reducing the proportion of 4-HNE and p-MLKL positive cells (C and E in the text). Figure 2 (D and E in the text). These results collectively indicate that Nec-1 alleviates the pathological progression of BRONJ by inhibiting necroptosis.

[0076] Furthermore, UMAP analysis of single-cell RNA-seq of BRONJ lesions showed that, through annotation of classical markers, the major cell populations (epithelial cells, fibroblasts, endothelial cells, macrophages, neutrophils, erythrocytes, B cells, and T cells) were significantly affected. Figure 2 The F in the text indicates that macrophages and neutrophils had significantly higher necrosis and apoptosis scores than other cell types, suggesting that these two immune cell types are the main contributors to necrosis and apoptosis in BRONJ lesions. Figure 2 (G in the middle).

[0077] TRAP staining showed that ZOL treatment significantly reduced osteoclast activity, while Nec-1 treatment significantly restored TRAP activity. + Number of positive osteoclasts ( Figure 2 The H in the image. Immunofluorescence staining results of CTSK (osteoclasts, red) and Ki67 (proliferation marker, green) in extraction sockets showed that Nec-1 treatment could increase osteoclast activity / proliferation in BRONJ mice. Figure 2 (I) significantly reduced BRONJ lesions.

[0078] In summary, these results collectively indicate that necrotizing apoptosis of osteoclast precursor cells impairs their osteoclast differentiation function, thereby promoting the pathological process of BRONJ.

[0079] Example 3: γδ T cells are a key factor in promoting osteoclast necrotizing apoptosis in BRONJ.

[0080] Osteoclast dysfunction and immune dysregulation are two core pathological drivers of BRONJ. However, the upstream regulatory mechanisms of osteoclast necrosis and apoptosis in BRONJ remain unclear.

[0081] The T cell activation module score of ZOL-treated wild-type mice is as follows: Figure 3 As shown in Figure A, T cells in BRONJ lesions exhibit a highly activated state. GO and KEGG enrichment analyses of differentially expressed genes (DEGs) in T cell clusters revealed significant alterations in pathways related to T cell differentiation and IFN-γ production. Figure 3 B). UMAP visualization of IFN-γ and TNF module scores in single cells of BRONJ lesions showed that IFN-γ expression was mainly enriched in T cells, indicating that IFN-γ is mainly produced by T cells. Figure 3 (C). Immunofluorescence staining results showed that γδ T cells (red) and IFN-γ (green) and CTSK cells were present in the extraction sockets of ZOL-treated mice. + (Osteoclasts, red) and Ki67 + (Green) expression indicates that ZOL promotes IFN-γ expression. +The generation of γδ T cells inhibits the proliferation of osteoclasts. Figure 3 (D in the middle).

[0082] Based on this, using TCRδ - / - Investigating the effect of γδ T cells on the pathogenesis of BRONJ in mice. ZOL-treated TCRδ cells... - / - Mice showed good epithelial healing after tooth extraction with no bone exposure, which was superior to ZOL-treated wild-type mice. Figure 3 (E in the text). H&E staining of mouse extraction sockets showed that ZOL-treated TCRδ - / - Reduced immune cell infiltration, decreased necrotic bone cells, and epithelial hyperplasia in mouse tooth extraction sockets. Figure 3 F and J), while the number and activity of osteoclasts were significantly increased (in the F and J), Figure 3 (G and J in the text). Immunohistochemical staining showed that TCRδ - / - The number of p-MLKL-positive and 4-HNE-positive cells in mice also decreased significantly. Figure 3 (H and J in the text). ELISA detection also confirmed the TCRδ of ZOL treatment. - / - The level of IFN-γ in the extraction sockets of mice was significantly reduced. Figure 3 (I in the middle).

[0083] In summary, the results of this invention indicate that ZOL-induced release of ZOL from bone tissue after tooth extraction in wild-type mice induces γδ T cell activation and recruitment; conversely, ZOL-induced TCRδ... - / - Mice lacking γδ T cells exhibited reduced necrotizing apoptosis in bone tissue and increased osteoclast activity and number. That is, ZOL-derived phosphonate antigens can be sensed and recognized by γδ T cells, and activated γδ T cells, acting as local immune coordinators, drive BRONJ progression by regulating osteoclast differentiation and cell death.

[0084] Example 4: Mechanism of γδ T cell-induced osteoclast necrotizing apoptosis

[0085] To investigate the mechanism by which γδ T cells induce necrotizing apoptosis in osteoclasts, proteomic analysis was performed on the supernatant of γδ T cells treated with ZOL (SPN(ZOL)) and osteoclasts treated with untreated ZOL (SPN). The results are as follows: Figure 4 As shown. Figure 4The volcano plot in Figure A shows that, compared with the untreated group (SPN), osteoclast precursors from ZOL-treated γδ T cell supernatant (SPN(ZOL)) exhibited the following changes in metabolism-related proteins: most mitochondrial complex I subunit proteins (NDUFA / NDUFS family) were downregulated, while glycolysis-related proteins (HKs, PKs) were significantly upregulated. This indicates that osteoclasts underwent metabolic reprogramming after induction with ZOL-treated γδ T cell supernatant (SPN(ZOL)), transitioning from oxidative phosphorylation to glycolysis. Gene set enrichment analysis (GSEA) results are as follows... Figure 4 As shown in Figure B, after co-incubation with supernatant from ZOL-treated γδ T cells, the oxidative phosphorylation level of osteoclasts decreased, while the glycolytic pathway was significantly enhanced. Cells with active glycolysis are often accompanied by the accumulation of mitochondrial ROS (mtROS), which drives necrosome formation and mediates the metabolic-necrotizing-apoptotic signaling cycle.

[0086] The results in Example 3 show that ZOL-treated γδ T cells mainly express IFN-γ, and direct treatment of osteoclasts with IFN-γ significantly increases intracellular lactate levels. Figure 4 (C) The protein levels of total protein lactation (Pan-Kla) and H3K18 lactation (H3K18la) in osteoclast precursors were detected by Western blotting. The results showed that IFN-γ treatment increased the expression of both Pan-Kla and H3K18la (both normalized with histone H3 as an internal reference). Figure 4 (D in the middle).

[0087] ChIP-seq analysis results are as follows Figure 4 As shown in E, H3K18la is significantly enriched in the promoter and upstream regions of the IKZF1 gene. This suggests that H3K18la may play a regulatory role in the transcription of IKZF1. IKZF1 is a zinc finger transcription factor, mainly reported to regulate lymphocyte differentiation, proliferation, and survival, and has recently been identified as a driver of T cell exhaustion. Secondly, the study found that IKZF1 is a transcription factor of ZBP1. ZBP1 is a Z-DNA sensor with two Zα domains and a RIP isomorphic interaction motif (RHIM). IFN-β and IFN-γ-induced necroptosis both depend on ZBP1 activation. ZBP1 recruits RIPK3 through protein-protein interaction via homodimerization of its N-terminal domain and its C-terminal RHIM, triggering a cascade phosphorylation of RIPK1, RIPK3, and MLKL, ultimately leading to necrosome-mediated necroptosis. The results of lactate level detection in osteoclasts are shown below. Figure 4As shown in F and G, 2-DG (2-deoxy-D-glucose) can reduce lactate levels, Pan-Kla and H3K18la protein levels, and further reduce IKZF1 and p-MLKL expression levels, thereby weakening osteoclast necrosis and apoptosis, while NAC (N-acetylcysteine) cannot.

[0088] Immunofluorescence image results as follows Figure 4 As shown in H, it indicates the TCRδ - / - Colocalization of Pan-Kla (red) with CTSK (osteoclast marker, green) and H3K18la (red) with CTSK (green) in extraction socket tissues of ZOL mice and wild-type (WT) ZOL mice. In TCRδ - / - In ZOL mice, Pan-Kla + CTSK + and H3K18la + CTSK + The number of osteoclasts in the mice was far less than in the WT ZOL mice.

[0089] In summary, ZOL-treated γδ T cells secrete IFN-γ, and IFN-γ-mediated enhanced glycolysis and lactate accumulation significantly increase the level of H3K18 lactation (H3K18la) in osteoclasts. H3K18la is highly enriched in the IKZF1 promoter region, inducing ZBP1-dependent necrotizing apoptosis in osteoclasts. Inhibition of glycolysis with 2-DG reduces the expression levels of H3K18la and IKZF1, thereby attenuating osteoclast necrotizing apoptosis. H3K18la, as a metabolic-epigenetic link, promotes BRONJ progression by activating the IKZF1-ZBP1 signaling pathway in osteoclasts.

[0090] Example 5: Preparation of methacrylamide gelatin (GelMA) hydrogel loaded with anti-IFN-γ antibody and in vivo efficacy assay

[0091] I. Experimental Methods

[0092] 1. Preparation of methacrylamide gelatin (GelMA) hydrogel loaded with anti-IFN-γ antibody

[0093] Using FDA-approved pharmaceutical excipient GelMA hydrogel as a carrier, a 5% (w / v) GelMA (catalog number: 105671, XFNANO) solution was prepared, and a 0.5% (w / v) LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphine, photoinitiator LAP) solution was added to the mixture. Anti-IFN-γ (catalog number: 606853, Biolegend, final concentration 5 μg / mL) antibody was added or not added to the mixture. After gentle mixing, the solution was transferred to cell culture plates for cell experiments or into a 1 mL syringe for in vivo injection. The mixture was cross-linked to form a hydrogel by irradiation with 405 nm blue light for 10–20 seconds. After 24 hours of freeze-drying, the porous structure of GelMA@ab was observed using a scanning electron microscope (SEM, SU3800, Hitachi, Japan); the surface elemental composition, chemical state, and functional group information of GelMA@ab were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA); and Fourier transform infrared spectroscopy (FTIR, VERTEX 33, Bruker, Germany, spectral range 4000-400 cm⁻¹) was performed. -1 )Analyze its chemical functional groups.

[0094] 2. Live / dead cell staining and cell viability analysis

[0095] RAW264.7 cells were cultured on GelMA@ab hydrogels for 24 hours, then incubated with AO / PI dye (Beyotime, China) at 37°C for 15 minutes. Live / dead cell images were captured using a Stellaris LAS X (Leica, Germany). After 24 hours of cell culture on the hydrogel, cell viability was assessed using the CCK-8 assay kit according to the manufacturer's instructions: 10 μL of CCK-8 solution was added, and the reaction was carried out for 2 hours. The absorbance at 450 nm was then measured using a microplate reader. Each experiment was independently repeated at least three times.

[0096] 3. In vivo efficacy testing of GelMA@ab hydrogel

[0097] To evaluate the in vivo efficacy of GelMA@ab hydrogel, a rat BRONJ model was established (using the same method and phenotype as the mouse BRONJ model), and GelMA@ab hydrogel was locally injected into irregular extraction sockets. After ZOL injection and extraction, rats were randomly divided into three groups (n=6 per group): ZOL group: sterile PBS (30 μL) was injected into the extraction socket only; GelMA group: GelMA hydrogel (30 μL) was injected into the extraction socket; GelMA@ab group: 30 μL of GelMA hydrogel containing anti-rat IFN-γ neutralizing antibody was injected into the extraction socket. The GelMA hydrogel solution was slowly injected into the extraction socket using a 1 mL syringe (with a 30 G needle), ensuring complete filling of the bone cavity. Subsequently, the gel was cross-linked by in situ irradiation with 405 nm blue light for 10–20 seconds. The extraction was gently repositioned and the overlying soft tissue was sutured. All animals continued ZOL treatment for 4 weeks post-surgery.

[0098] II. Experimental Results

[0099] Scanning electron microscopy (SEM) revealed that the GelMA@ab hydrogel possesses a uniform porous structure, enabling efficient loading and sustained release of anti-IFN-γ antibodies, which facilitates cell migration and infiltration. Figure 5 (A) Fourier transform infrared spectroscopy (FTIR) analysis showed that GelMA and GelMA@ab hydrogels exhibited high activity in the 1500-1750 cm⁻¹ range. -1 A characteristic peak of the C=C functional group appears at 3000 cm⁻¹. -1 and 2750 cm -1 The peak for CH stretching vibration is located at 3000-3500 cm⁻¹. -1 The range represents the stretching vibrations of OH and NH ( Figure 5 B in the text). X-ray photoelectron spectroscopy (XPS) shows that O, C, N, and S elements are present in the GelMA hydrogel. Figure 5 (C in the text). The above results collectively verify the successful construction of the GelMA@ab hydrogel.

[0100] Biocompatibility assessment of RAW264.7 cells using Live / Dead staining and CCK-8 assay showed that the GelMA@ab hydrogel exhibited extremely low cytotoxicity and high safety. Figure 5 (D in the text). GelMA@ab hydrogel has good flowability at room temperature and gels rapidly after light exposure, effectively filling irregular bone defects in the alveolar bone.

[0101] Macroscopic assessment showed that the GelMA@ab group exhibited significantly improved wound healing 4 weeks after extraction, with only mild swelling, while the control group and GelMA group showed unhealed wounds and persistent fistulas (marked by black circles). Figure 5 (E in the text).

[0102] Micro-CT analysis showed significant new bone formation in the GelMA@ab group, while the control and GelMA groups showed very little or no bone regeneration. Quantitative analysis showed that the bone volume fraction (BV / TV) in the GelMA@ab treatment group was approximately four times that of the control group, and the tissue mineral density (BMD) was three times higher. Figure 5 (F in the middle).

[0103] HE staining showed that the GelMA@ab group had good mucosal healing with limited necrotic cells and inflammatory infiltration; the control group showed typical oral epithelial dysplasia extending to the bone surface, accompanied by open fistulas; the GelMA group showed unhealed mucosa with significant inflammatory cell infiltration. Figure 5 (G in the middle).

[0104] TRAP staining further showed that the number and activity of osteoclasts in the GelMA@ab group were significantly higher than those in the other two groups. Figure 5 The H in the text). Immunohistochemical analysis showed that the expression of necrosis-associated markers ZBP1, p-MLKL, and IKZF1 in the lesions of the GelMA@ab treatment group was downregulated, while the expression of the glycolysis marker Ndufa9 was upregulated. Figure 5 These results collectively demonstrate that the sustained release of anti-IFN-γ antibody from GelMA@ab hydrogel effectively blocks IFN-γ-mediated necrotizing apoptosis and promotes BRONJ tissue regeneration.

[0105] In summary, this invention demonstrates that ZOL activates γδ T cells to secrete IFN-γ, thereby enhancing H3K18 lactation modification. This modification triggers the IKZF1-ZBP1 pathway to induce osteoclast necrosis and apoptosis. Local delivery of anti-IFN-γ antibodies via GelMA@ab hydrogel can inhibit osteoclast necrosis and apoptosis and accelerate the repair of BRONJ defects. This treatment strategy provides a clinically feasible solution for the prevention and treatment of BRONJ and lays the mechanistic foundation for clinical translation.

Claims

1. The application of hydrogels loaded with anti-IFN-γ antibodies in the preparation of drugs for treating bisphosphonate-related osteonecrosis of the jaw, characterized in that, The hydrogel loaded with anti-IFN-γ antibody is a methacrylamide gelatin hydrogel loaded with anti-IFN-γ antibody; The method for preparing the methacrylamide gelatin hydrogel loaded with anti-IFN-γ antibody is to obtain it by photoinitiated polymerization of methacrylamide gelatin, photoinitiator and anti-IFN-γ antibody; the photoinitiator is LAP.

2. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by inhibiting the secretion of IFN-γ by γδ T cells.

3. The application according to claim 1, characterized in that, The drug achieves therapeutic effects by inhibiting osteoclast glycolysis and / or inhibiting osteoclast necrosis and apoptosis.

4. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by inhibiting histone H3K18 lactation.

5. The application according to claim 1, characterized in that, The drug achieves therapeutic effects by inhibiting the activation of transcription factor IKZF1 and its downstream effector molecule ZBP1.

6. The application according to claim 1, characterized in that, The dosage form of the drug includes injections or patches.

7. The application according to claim 1, characterized in that, The drug also includes other pharmaceutically acceptable excipients.

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