Composite material loaded with carbonic anhydrase III

Abstract

The invention discloses a composite material loaded with carbonic anhydrase III. The catalytic activity of the CAR3 is obviously lower than that of other CA subtypes, and analysis finds that the CAR3 has an obvious high expression level and specific distribution characteristics in osteoblasts, so that the composite material prepared by loading the CAR3 with the I-type collagen scaffold can be used for preparing osteoblasts. The composite material loaded with carbonic anhydrase III has a functional structure with good scaffold performance and degradation characteristics, when the composite material is transplanted to a bone defect area, CAR3 can modify collagenous fibers, nucleation sites are formed, crystallization in the collagenous fibers is driven, an ideal repairing environment is achieved, and the composite material is used for repairing bone defects. The composite material loaded with the carbonic anhydrase III, disclosed by the invention, has important significance on repairing bone defects.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a composite material loaded with carbonic anhydrase III. Background Technology

[0002] Bone defects, typically caused by surgical procedures, trauma, tumor resection, or inflammation, severely impact patients' quality of life and represent a significant clinical challenge requiring urgent solutions in craniofacial surgery, plastic surgery, and orthopedics. While autologous bone grafting is currently considered the "gold standard" for treating bone defects, its clinical application is severely limited by factors such as limited donor availability, significant surgical trauma, and donor site complications. Therefore, developing alternative materials with good biocompatibility and osteoinductive activity has become a key research focus in the field of bone tissue engineering.

[0003] In recent years, the development of biomimetic bone graft materials has provided new insights into bone tissue regeneration. These materials, by precisely mimicking the multi-level structure and mineralized organic composite characteristics of natural bone tissue, can not only achieve good integration with host tissue but also effectively promote bone tissue regeneration and repair. It is worth noting that collagen fibers, as the main organic component of mineralized tissues such as bone and dentin, have a unique confined structure that significantly increases the difficulty of biomimetic mineralization. Therefore, how to achieve the directional penetration and crystal transformation of mineralization precursors within the fibers and precisely control this process has become a key scientific issue in biomimetic mineralization research. Currently, the academic community generally believes that the regulatory role of organic components on intrafiber crystallization may be the key breakthrough in solving this problem. This understanding stems from a deep analysis of the nature of biomineralization. Studies have shown that during natural bone formation, various proteins secreted by osteoblasts play a crucial role in the crystallization of inorganic minerals (such as hydroxyapatite) within the fibers by regulating crystal nucleation, growth, and orientation. For example, osteocalcin secreted by osteoblasts binds to calcium ions through its carboxylic acid groups, inhibiting the disordered growth of hydroxyapatite crystals, limiting crystal size (nanoscale), and promoting the formation of plate-like crystals. In addition, osteopontin secreted by osteoblasts is rich in acidic amino acids, which adsorb onto the surface of hydroxyapatite crystals through strong negative charge, blocking further crystal growth or aggregation and inhibiting extrafibrous ectopic mineralization. Therefore, in recent years, the research direction of organic components in biomimetic mineralization has gradually shifted towards the study of proteins secreted by osteoblasts in living organisms that have mineralization-inducing functions, in order to better simulate the natural bone formation process.

[0004] Carbonic anhydrase (CA) is widely involved in mineralization processes in various organisms. Phylogenetic studies have shown that in metazoans where calcium carbonate is the primary mineralized form, the α-CA subtype contributes HCO3- by providing these minerals. -CA plays an important role by promoting mineral nucleation or acting as a structural protein. In marine organisms such as corals, mollusks, and echinoderms, CA can catalyze the conversion of CO2 produced by metabolism into HCO3. - or CO3 2- CAR3 provides the necessary ionic basis for the formation of the shell and skeleton. This mechanism reflects a conserved carbon enrichment strategy in marine organisms, namely, maintaining sustainable biomineralization by optimizing the use of metabolic CO2. However, the composition of human bone tissue differs significantly from that of marine organisms. Human bone is mainly composed of orderly arranged organic components such as calcium phosphate crystals and collagen fibers. This structural feature suggests that CAR3 may have a unique mechanism of action in human bone formation. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a composite material loaded with carbonic anhydrase III.

[0006] This invention utilizes single-cell sequencing analysis of 17.5-day-old mouse cranial bone tissue to successfully identify multiple progenitor cell populations using classic biomarkers and reveals a series of osteogenic-related genes specifically highly expressed during early cranial development. Among these, carbonic anhydrase III (CAR3) exhibits significantly high expression levels and specific distribution characteristics. This invention found that CAR3 is expressed in osteoblasts, but its role in bone formation may differ from that of marine calcification. CAR3 catalytic activity is significantly lower than other CA subtypes. Therefore, CAR3's promotion of mammalian bone tissue formation may involve different mechanisms. The results of this invention demonstrate that CAR3 is secreted into the extracellular matrix, facilitating research into its potential role in collagen mineralization.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides the use of carbonic anhydrase III in the preparation of products for treating bone defects.

[0008] A second aspect of the present invention provides a bone repair composite material comprising carbonic anhydrase III and a load scaffold for surface loading of carbonic anhydrase III.

[0009] In some embodiments of the present invention, the load support has a porous mesh structure.

[0010] In some embodiments of the present invention, the load scaffold comprises at least one selected from collagen, gelatin, chitosan, and hyaluronic acid.

[0011] In some embodiments of the present invention, the load holder is a collagen-containing load holder.

[0012] In some embodiments of the present invention, the collagen includes at least one of type I collagen, type II collagen, and type III collagen.

[0013] A third aspect of the present invention provides a method for preparing the bone repair composite material described above, the method comprising the following steps: immersing a load scaffold in carbonic anhydrase III to obtain the bone repair composite material.

[0014] A fourth aspect of the present invention provides the use of the bone repair composite material described above in at least one of the following (I) to (III): (I) To prepare products for the treatment or repair of bone injuries; (II) Preparation of products for promoting stem cell migration; (III) Prepare products that induce stem cells to differentiate into osteocytes.

[0015] In some embodiments of the present invention, the stem cells include at least one of adipose-derived stem cells and bone marrow mesenchymal stem cells.

[0016] A fifth aspect of the present invention provides the use of the bone repair composite material described above in the preparation of products for treating bone defects.

[0017] A sixth aspect of the invention provides the use of a substance that promotes the synthesis of carbonic anhydrase III in the preparation of a medicament for treating bone defects.

[0018] In some embodiments of the invention, the treatment of bone defects includes promoting osteogenesis.

[0019] In some embodiments of the present invention, the promotion of osteogenic formation includes promoting the formation of mineralization.

[0020] In some embodiments of the present invention, the substance that promotes the synthesis of carbonic anhydrase III includes a substance that promotes the expression of the carbonic anhydrase III gene.

[0021] In some embodiments of the present invention, the substance that promotes the expression of the carbonic anhydrase III gene includes: (1) A vector that promotes the expression of carbonic anhydrase III; (2) Recombinant cells containing the expression vector described in (1).

[0022] In some embodiments of the present invention, the vector includes a plasmid or a viral vector.

[0023] In some embodiments of the present invention, the vector includes at least one of a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0024] A seventh aspect of the invention provides the use of a reagent for detecting carbonic anhydrase III in the preparation of products for evaluating bone formation.

[0025] The beneficial effects of this invention are: This invention utilizes a type I collagen scaffold loaded with CAR3 to form a composite material. CAR3 is a biologically derived protein, while type I collagen is the main organic component of the extracellular matrix of bone cells, providing a topological template for mineral deposition. CAR3 stably interacts with the assembled collagen fibers, controlling nucleation within the collagen interstitial regions and synergistically regulating the orientation of hydroxyapatite crystals within the fibers. This composite material possesses a functional structure with excellent scaffold performance and degradation characteristics. When transplanted into bone defect areas, CAR3 can modify collagen fibers, forming nucleation sites and driving intrafibrillation, achieving an ideal repair environment, which is of great significance for bone repair. Attached Figure Description

[0026] Figure 1 UMAP plot of single-cell RNA sequencing data from E17.5 mouse skull tissue.

[0027] Figure 2 A heatmap of lineage-specific gene expression during cranial osteogenesis in E17.5 mice.

[0028] Figure 3 A heatmap of differential gene expression after the branching point of the osteogenic lineage.

[0029] Figure 4 This is a pseudo-time trajectory analysis diagram of cells in the late embryonic stage of the skull of E17.5 mice.

[0030] Figure 5 Images show the results of Von Kossa staining and immunofluorescence analysis of craniofacial tissues from E13.5–E16.5 mice. a) Von Kossa staining results of skulls at different embryonic stages (scale bar = 1 mm); b) Immunofluorescence images of skulls at different embryonic stages (scale bar = 1 mm).

[0031] Figure 6 Figure 1 shows the results of detecting the dynamic expression of CAR3 during the mineralization process of BMSCs. Figure 2a shows the distribution of CAR3 in BMSCs and osteoblasts on day 7 of mineralization, scale bar = 200 μm; Figure 3b shows the expression analysis results of osteogenic-related proteins and CAR3 protein in BMSCs on days 0, 7, and 14 of mineralization; Figure 4c shows the qPCR results analysis of osteogenic-related genes and CAR3 expression in BMSCs on days 0, 7, and 14 of mineralization.

[0032] Figure 7The images shown are: A) Detection results of extracellular vesicles isolated in the example; A is a transmission electron microscope image with a scale bar of 250 μm; B is a magnified view of A with a scale bar of 100 μm.

[0033] Figure 8 This is a graph showing the results of the detection of extracellular vesicle size.

[0034] Figure 9 Figure 1 shows the analysis results of CAR3 localization in osteoblasts. Figure 2 shows the Western blot results of CAR3 and CD9 proteins in osteoblasts and their secreted matrix vesicles (MVs); Figure 3 shows the immunofluorescence staining results of matrix vesicles secreted by osteoblasts. Scale bar = 5 μm.

[0035] Figure 10 Immunoelectron microscopy image of CAR3 distribution in skull tissue, scale bar = 100 μm.

[0036] Figure 11 Figure 1 shows the effect of CAR3 on osteogenic mineralization. Figure 2 shows the results of alizarin red staining and quantitative analysis of BMSCs in the control group and CAR3 knockout group of the example; Figure 3 shows the results of Western blot analysis of mineralization-related markers in BMSCs in the control group and CAR3 knockout group of the example; Figure 4 shows the results of alizarin red staining and quantitative analysis of BMSCs in the control group and CAR3 overexpression group of the example; Figure 5 shows the results of Western blot analysis of mineralization-related markers in BMSCs in the control group and CAR3 overexpression group of the example.

[0037] Figure 12 The following figures illustrate the effects of CAR3 on collagen. a) shows the co-localization of CAR3, COL1A1, and calcium ions during in vitro collagen mineralization (scale bar = 100 μm); b) shows the immunoprecipitation results of COL1A1-CAR3 interaction; c) shows a high-resolution transmission electron microscope image of polyacrylic acid (PAA)-mediated intrafiber mineralization (scale bar = 100 μm); d) shows the EDS spectra of collagen fibers mineralized using PAA-ACP with and without recombinant CAR3 (scale bar = 100 μm).

[0038] Figure 13 X-ray energy dispersive spectroscopy (EDS) of polyacrylic acid (PAA)-mediated mineralization within collagen fibers; A is the X-ray energy dispersive spectroscopy of type I collagen fibers after 6 hours of mineralization; B is the X-ray energy dispersive spectroscopy of type I collagen fibers with added recombinant CAR3 protein after 6 hours of mineralization; C is the X-ray energy dispersive spectroscopy of type I collagen fibers after 12 hours of mineralization; D is the X-ray energy dispersive spectroscopy of type I collagen fibers with added recombinant CAR3 protein after 12 hours of mineralization.

[0039] Figure 14The figures show the verification results of the collagen scaffold functionalized with recombinant CAR3 protein of this invention. a. Schematic diagram of the in vivo bone defect model of the example and comparison of bone volume fraction (BV / TV) between the CAR3 functionalized scaffold and the unmodified control group, scale bar = 3 mm; b. Von Kossa staining of the CAR3 functionalized scaffold and the unmodified scaffold in the skull, scale bar = 100 μm; c. Distribution of Prx1+ osteoblast progenitor cells after transplantation of different collagen biomembranes in the skull defect of mice with dual-color fluorescence, scale bar = 100 μm; d. Cell percentage analysis of Prx1+ osteoblast progenitor cells after transplantation of different collagen biomembranes. Detailed Implementation

[0040] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0041] Example 1 This embodiment provides a screening of specific biomarkers for osteoblasts with functional characteristics. Single-cell sequencing technology was used to detect the cell community in the cranial bone tissue microenvironment of C57BL / 6J mouse embryos on day 17.5 and the key gene information of the corresponding cells. The specific experimental steps are as follows.

[0042] To investigate marker genes in osteoblast differentiation, this embodiment analyzed single-cell RNA sequencing data (GSE174716) from the skull tissue of E17.5 mice (day 17.5 of embryonic development). UMAP dimensionality reduction analysis (Angelozzi M, daSilva RP, Gonzalez MV, Lefebvre V. Single-cell atlas of craniogenesis uncovers SOXC-dependent, highly proliferative, and myofibroblast-like osteodermal progenitors. Cell reports. 2022 Jul 12;40(2)) was used. The subpopulation gene classification parameters in this embodiment were: Cranio.markers.all <- Seurat::FindAllMarkers(CranioSoxc_Angelozzi_WT_small, only.pos=TRUE, min.pct=0.25, logfc.threshold=0.25) to identify eight transcriptionally distinct cell populations. Figure 1Based on lineage-specific markers, these cell populations were labeled as meningeal cells, osteoblastic skin progenitor cells, chondrocytes, pre-osteoblasts, skin cells, subcutaneous cells, mature osteoblasts, and chondrocytes. Figure 1 and Figure 2 ).

[0043] KEGG pathway enrichment analysis showed that pathways related to "ossification" and "osteoblast differentiation" were significantly enriched in preosteoblasts and osteoblasts. Figure 2 Among them, CAR3 ranks among the top ten differentially expressed genes (DEGs) in osteoblast populations. Figure 2 Based on the pseudo-time-sequencing function in Monocle 3, starting with osteoblast progenitors, we revealed their differentiation into two lineages: osteoblast progenitors and osteoblasts. Figure 3 CAR3 expression is dynamically upregulated after branching of the osteogenic differentiation trajectory. Figure 3 The activation of CAR3 specifically in osteoblast lineages was confirmed by validating Runx2 (early differentiation), Sp7 (mid-differentiation), and Bglap / Ibsp (late differentiation) using stage-specific markers. Figure 4 Through a series of bioinformatics analyses, this embodiment confirmed CAR3, a functional and specific biomarker expressed by osteoblasts, and validated it in subsequent experiments.

[0044] Example 2 The Prx1-Cre-tdTomato fluorescently labeled mice (the offspring of Prx1-Cre tool mice and LSL-tdTomato red fluorescent reporter mice, both of which were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd.) provided in this embodiment are used to label Prx1-positive cells during bone formation.

[0045] Skulls of Prx1-Cre-tdTomato fluorescently labeled mouse embryos at days 13.5, 14.5, 15.5, and 16.5 of development were collected, fixed with paraformaldehyde, and the expression and co-localization of CAR3 protein in the skull were semi-quantitatively analyzed by frozen section analysis and immunofluorescence staining. The specific experimental steps are as follows.

[0046] (1) Frozen tissue sections of the skull of Prx1-Cre-tdTomato fluorescently labeled mice at embryonic days 13.5, 14.5, 15.5 and 16.5 were rinsed in PBS for 15 min.

[0047] (2) Sealing treatment: Gently shake off the liquid on the tissue section, add goat serum (Zhongshan Jinqiao), incubate in a constant temperature incubator at 37°C for 1 hour, do not wash with water, directly use absorbent paper to remove excess serum; (3) Antibody incubation treatment: 60 μL of rabbit-derived anti-CAR3 antibody dilution solution (Proteintech, dilution ratio 1:200) was added to each tissue section, and the tissue sections were then placed in a humidified culture box and incubated overnight at 4°C. The next day, the tissue sections were washed three times with PBS solution for 5 min each time. Then, 60 μL of goat anti-rabbit Alex488 fluorescent secondary antibody IgG dilution solution (Invitrogen, dilution ratio 1:200) was added to each tissue section, and the tissue sections were incubated at 37°C in the dark for 1 hour, followed by washing three times with PBS solution for 10 min each time. (4) Complete the mounting and take pictures: Gently shake off the liquid on the tissue section, use mounting solution containing DAPI (Shanghai Beyotime Biotechnology Co., Ltd.) to complete the mounting operation, and then place it under an upright fluorescence microscope to take pictures and record.

[0048] Von Kossa staining was performed on craniofacial tissues from mouse embryos at days 13.5, 14.5, 15.5, and 16.5 of development. The specific experimental steps were as follows: Tissue sections were baked in a 60°C oven for 30-60 min, then dewaxed with xylene I / II (5-10 min per step), rehydrated with a gradient of ethanol (100%→95%→80%→70%) (3-5 min per step), and finally soaked in distilled water for 5 min. The sections were then transferred to a 5% (w / v) silver nitrate solution (in a brown staining jar or a light-proof box) and irradiated under UV light for 10 min until the calcium salt areas turned brownish-black. The reaction was then terminated by soaking in 5% (w / v) sodium thiosulfate solution for 5-10 min, followed by rinsing with distilled water three times (1-2 min each time). The sections were then placed in hematoxylin staining solution for 5-10 min to counterstain the cell nuclei. After rinsing with distilled water, the sections were dehydrated by gradient ethanol (70%→80%→95%→100%) (3-5 min each step), cleared with xylene I / II (5-10 min each step), and finally mounted with neutral resin. The sections were then dried at room temperature or in an oven at 37°C before observation.

[0049] Von Kossa staining and immunofluorescence analysis results of craniofacial tissues from mice at days 13.5, 14.5, 15.5, and 16.5 are as follows: Figure 5 As shown, CAR3 protein levels are positively correlated with the mineralization process; CAR3 expression levels gradually increase with the development of craniofacial bone tissue. Figure 5 (a and b).

[0050] Example 3 This embodiment provides the detection of CAR3 expression during the mineralization process of human bone marrow mesenchymal stem cells (BMSCs). The dynamic spatiotemporal expression pattern of CAR3 during BMSCs mineralization was detected using super-resolution immunofluorescence microscopy, qPCR, and Western blot. The specific experimental steps are as follows.

[0051] Human bone marrow mesenchymal stem cells (BMSCs) were cultured in osteogenic medium (a-MEM medium (Gibco) containing 10 mM β-glycerophosphate, 50 μg / mL vitamin C, and 1 nM dexamethasone) for 7 and 14 days to induce osteogenic differentiation. After 7 and 14 days of culture, staining was performed using Alizarin Red reagent. Gene and protein expression levels were assessed using qRT-PCR and Western blot, respectively. The expression levels of osteogenic-related genes (Ifitm5, Runx2, and Bglap) and CAR3 were determined by quantitative reverse transcription polymerase chain reaction. To analyze the target protein levels in BMSCs, Western blot analysis was performed after 14 days of culture.

[0052] Cells induced to osteogenic differentiation for 7 days were subjected to immunofluorescence and confocal imaging analysis, with BMSCs used as a control. The specific steps are as follows: (1) Discard the original supernatant of the cells and wash twice with PBS; (2) The cells were fixed with 4% paraformaldehyde and then treated with 0.2% (v / v) Triton-X-100 for 10 min to increase cell membrane permeability; (3) After washing three times with PBS, the sample was sealed in a 37°C incubator for 1 hour with 1% goat serum. (4) Add rabbit-derived anti-CAR3 antibody (Proteintech, diluted 1:200 in PBS solution) and incubate overnight at 4°C; (5) Wash 3 times with PBS for 10 min each time, and incubate with diluted Star Red-labeled goat anti-rabbit secondary antibody IgG (diluted 1:200 in PBS solution, purchased from Abberior) at 37°C for 1 hour. (6) Wash 3 times with PBS for 10 minutes each time, using Acti-stain TM 555 fluorescein-labeled phalloidin for cytoskeleton labeling; (7) The cell nuclei were restained with the fluorescent dye DAPI, treated at room temperature for 10 min, and washed 3 times with PBS; (8) Imaging using a confocal microscope.

[0053] qPCR experiments: Total RNA was extracted using TRIzol reagent (Takara, Japan) according to the manufacturer's instructions. RNA concentration and purity were determined using a Nanodrop 2000 (Thermo Fisher Scientific, USA). PrimeScript was used for analysis. TM cDNA was synthesized using an RT kit (Takara, Japan). qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, China). The relative expression level of the target gene was determined using a 2-1T / T kit. -ΔΔCt The method is used for quantification, in order to Gapdh As an internal control gene. All experiments were performed in triplicate. The primers used in this embodiment are shown in Table 1 below: Table 1 Primer sequences used for qPCR detection

[0054] Western blot experiment: (1) Sample loading: Prepare a gradient gel with concentrations of 4%-12%. Remove the comb, add the protein marker and protein sample in sequence, and record the sample order; (2) Electrophoresis: The electrophoresis was initially performed at 80 V for 30 min, and then increased to 120 V for 60 min. (3) Transfer: After electrophoresis, the PVDF membrane was activated by methanol treatment, and then transferred in an ice bath for 2 hours at a constant current of 200 mA. (4) Blocking: The protein bands were rapidly blocked for 20 minutes at room temperature using a rapid blocking solution (purchased from Bio-Rad Laboratories); (5) Primary antibody incubation: Cut the band at the corresponding position and incubate the corresponding primary antibody dilution solution (rabbit-derived anti-CAR3 antibody (Proteintech), anti-IFITM5 antibody (Abcam), anti-OPN antibody (Abcam), anti-OSX antibody (Abcam), anti-HSP90α / β antibody (Abclonal), with a volume ratio of 1:1000) with the band overnight at 4°C; (6) Secondary antibody incubation: After collecting the primary antibody dilution solution, wash the bands 3 times with TBST solution for 10 min each time, add secondary antibody dilution solution (HRP-labeled goat anti-rabbit secondary antibody, Shanghai Beyotime Biotechnology Co., Ltd., dilution ratio of 1:6000), incubate on a shaker at room temperature for 1 hour, discard the secondary antibody dilution solution, wash 3 times with TBST solution for 10 min each time; (7) Development: Chemiluminescence detection was performed using the ECL-HRP luminescent reagent kit (Millipore) according to the instructions. After incubating the band in a dark environment for 2 minutes, the image was recorded and captured in the imaging system.

[0055] Experimental results are as follows Figure 6 As shown, on day 7 of mineralization, CAR3 expression was significantly upregulated compared to day 0. Figure 6 (a). Western blot and qPCR analyses further validated that the expression trends of CAR3 were consistent with those of osteogenic markers (OPN, OSX, and IFITM5). Figure 6 (b and c).

[0056] Example 4 This embodiment provides the detection of CAR3 secretion in osteoblasts.

[0057] Extracellular vesicles (MVs) were collected from the supernatant 14 days after osteogenic induction using an ultracentrifuge. The size and shape of the MVs were analyzed using a nanoparticle tracking analyzer (NTA) and transmission electron microscopy. The expression levels of MV markers CD9 and CAR3 were detected by Western blot. The MVs secreted by osteoblasts were labeled with the lipophilic dye PKH26 (red fluorescence), and CAR3 was simultaneously fluorescently labeled (green fluorescence) using immunofluorescence staining. The co-localization of both was observed dynamically in real time using super-resolution confocal microscopy. Details are as follows.

[0058] Extracellular vesicles were isolated from cell culture supernatant using differential ultracentrifugation. The specific steps were as follows: Human bone marrow mesenchymal stem cells were cultured in osteogenic medium containing 10 mM β-glycerophosphate, 50 μg / mL vitamin C, and 1 nM dexamethasone. After 14 days of induced osteogenic differentiation, the cells were centrifuged at 300×g for 10 min at 4°C to remove floating cells, followed by centrifugation at 2000×g for 20 min to remove dead cells and large debris. The supernatant was filtered through a 0.22 µm pore size polyethersulfone (PES) membrane to remove residual particulate matter. The clarified supernatant was then ultracentrifuged at 140,000×g for 70 min at 4°C using a Type 70 Ti fixed-angle rotor (Beckman Coulter, USA) to concentrate the extracellular vesicles. The obtained extracellular vesicles were washed with phosphate-buffered saline (PBS) and then subjected to a second ultracentrifugation under the same conditions to remove contaminating proteins. The final exosome-rich precipitate was resuspended in 100 µL PBS and stored at -80°C until further use.

[0059] The size and shape of MVs were detected by NTA and transmission electron microscopy, respectively, and the expression levels of MV markers CD9 and CAR3 were detected by Western blot. The experimental procedures were the same as those in the Western blot experiment in Example 3, except that the primary antibodies used were rabbit-derived anti-CAR3 antibody (Proteintech), anti-β-Tubulin (abcam), and anti-CD9 antibody (abcam).

[0060] The CAR3 fluorescent labeling procedure is the same as the cell immunofluorescence procedure in Example 3. The specific experimental procedure for labeling osteoblast-secreted MVs with the lipophilic dye PKH26 is as follows: (1) Add exosome red fluorescent dye (PKH26) (Shanghai Yumeibo Biotechnology Co., Ltd.) to exosomes. The final concentration of exosome red fluorescent dye is 5 μM (which can be adjusted in the range of 2~20 μM). (2) After adding the dye working solution, tighten the centrifuge tube, mix it with a vortex shaker for 1 min, and then let it stand and incubate for 10 min. (3) Add an appropriate amount of 1×PBS to the incubated exosome dye complex and mix well; (4) Extract exosomes again using the above-mentioned exosome extraction method (differential ultracentrifugation) to remove free dye; (5) Add 200 μL of 1×PBS to resuspend the precipitate, which is the stained exosome.

[0061] The cranial bone tissue of E16.5 embryonic mice was labeled with colloidal gold and then observed by transmission electron microscopy. The specific experimental steps are as follows: The cranial bone tissue of E16.5 embryonic mice was initially fixed with 2.5% (v / v) glutaraldehyde, washed with PBS, and post-fixed with 1% (w / v) osmium tetroxide. It was then dehydrated by gradient ethanol (30%-100%), and after gradient infiltration with resin and acetone (resin to acetone volume ratio 1:2, 1:1, 2:1), it was infiltrated with pure resin and polymerized at 60°C for 48 hours to form resin blocks for embedding for subsequent colloidal gold labeling.

[0062] Colloidal gold labeling: First, cut the resin block into 60-80 nm thin slices using an ultramicrotome and transfer them to a copper mesh. Place them in 1% (v / v) H2O2 for 30 min; wash three times with double-distilled water for 10 min each time; drop them onto normal sheep serum (volume ratio 1:100, Zhongshan Jinqiao); incubate at room temperature for 60 min; then incubate on anti-CAR3 antibody (Proteintech) serum, pre-incubate at room temperature for 1 h, then place at 4℃ for 24-36 h; wash three times with PBS for 3 min each time; place in PBS (containing 1% BSA, pH 8.2) for 5 min; finally, incubate with colloidal gold-labeled antibody solution (purchased from Jackson ImmunoResearch Laboratories). Inc.) Volume dilution ratio 1:50, light red is the appropriate dilution, incubate at room temperature for 1 hour; wash with double distilled water for 3 minutes, 3 times; stain with 5% (w / v) uranium acetate (prepared with double distilled water) for 5 minutes; wash with double distilled water for 3 minutes, 3 times; stain with lead citrate (or lead acetate) for 5 minutes, wash with double distilled water, and finally observe under an electron microscope.

[0063] Experimental results are as follows Figure 7-10 As shown, Figure 7 Electron micrographs of cellular matrix vesicles and Figure 8 The image shows the vesicle size distribution. Experimental results indicate that the extracellular vesicles isolated in this embodiment exhibit typical vesicular structures, and their size falls within the typical range for extracellular vesicles. (Exosome Western blot results) Figure 9 (a) and co-localization studies with the exosome marker PKH26 ( Figure 9 (b) confirmed that CAR3 is present in the stromal vesicles of these cells.

[0064] Transmission electron microscopy combined with colloidal gold labeling further localized CAR3 not only in secreted exosomes, but also on extracellular collagen fibers of the skull. Figure 10 ).

[0065] Example 5 This embodiment explores the effect of CAR3 on osteogenic mineralization. The specific experimental steps are as follows.

[0066] A CAR3 overexpression lentiviral vector (purchased from General Biotechnology Co., Ltd., using pLV3-CMV-EF1a-Puro) was constructed and transfected into BMSCs according to the instructions. Samples were collected on days 0, 7, and 14 of osteogenic induction culture of BMSCs, following the same osteogenic induction culture procedure as in Example 3. The expression levels of mineralization-related markers were detected by Western blot. Alizarin Red staining was performed on days 7 and 14, and the degree of Alizarin Red staining was quantified. The CAR3 gene sequence used was 5'-3' (SEQ ID NO:11).

[0067] Simultaneously, bone marrow stem cells (BMSCs) from CAR3 knockout mice (purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd.) were used for osteogenic induction culture and detection, following the same procedures as for BMSCs transfected with CAR3-overexpressing lentiviral vectors. The steps for obtaining CAR3 knockout mouse BMSCs are as follows: The femurs of the mice were severed at both ends (hip and knee joints), and any remaining muscle on the bone surface was rinsed with PBS. The BMSCs were then placed in a culture dish containing a small amount of low-glucose DMEM medium (to prevent bone marrow drying). Take a 1mL syringe, draw in preheated a-MEM medium, and insert the needle into the medullary cavity from one end of the femur; slowly inject the medium to flush the bone marrow out from the other end and collect it into a 50mL centrifuge tube; filter the collected bone marrow suspension through a 70μm cell filter into a new 50mL centrifuge tube to remove bone fragments and muscle debris; centrifuge at 1000rpm for 5min, discard the supernatant, add 5-8mL of complete medium (a-MEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution) to the cell pellet, and gently pipette 3-5 times to prepare a single-cell suspension; evenly seed the cell suspension into 10cm cell culture dishes and incubate in a 37℃, 5% CO2 incubator. After 48-72 hours of culture, BMSCs begin to adhere to the culture dish (growing in a spindle or polygonal shape). At this point, gently rinse the culture dish twice with sterile PBS (adding it slowly along the dish wall to avoid washing away the adhered cells) to remove non-adhered red blood cells, white blood cells, etc. Add fresh complete culture medium and continue culturing. Change the medium every 3-4 days until the cell confluence reaches 80%-90% and start passage. By passage to P3, the cells are basically purified (BMSC ratio >90%), and subsequent osteogenic induction culture and detection can be performed.

[0068] The Western blot procedure is the same as in Example 3, except that the antibodies used to detect BMSCs from CAR3 knockout mice are anti-CAR3 antibody (Proteintech), anti-IFITM5 antibody (Abcam), anti-OPN antibody (Abcam), and anti-ACTIN antibody (Abclona). For BMSCs transfected with CAR3 overexpression vector, the primary antibodies used are anti-CAR3 antibody (Proteintech), anti-OPN antibody (Abcam), anti-OSX antibody (Abcam), anti-OCN antibody (Abmart), and anti-ACTIN antibody (Abclona).

[0069] Experimental results are as follows Figure 11 As shown, Alizarin Red S quantitative analysis revealed that CAR3 knockdown cells exhibited reduced calcium deposition, while CAR3 overexpression cells showed significantly enhanced mineralization capacity compared to the control group. Figure 11(a and c). Western blot analysis of osteogenic markers further confirmed these findings, showing reduced protein levels in CAR3 knockout cells (a and c). Figure 11 (b), while overexpression cells showed accelerated osteogenic process ( Figure 11 (d).

[0070] Example 6 This embodiment provides the effect of CAR3 on the biomimetic mineralization of collagen fibers. To clarify the relationship between CAR3 and collagen, an immunofluorescence staining experiment was conducted on type I collagen (containing COL1A1 protein) co-incubated with CAR3 protein and calcium phosphate solution, as well as an immunoprecipitation experiment on COL1A1 and CAR3 in the cell lysate of BMSCs after 7 days of mineralization. In order to reproduce collagen mineralization in vitro, this embodiment simulated the polymer-induced liquid precursor (PILP) process and used transmission electron microscopy (TEM), high-resolution TEM and selected area electron diffraction (SAED) for observation and analysis. The CAR3 protein used in the examples was purchased from Kangti Biotechnology Co., Ltd., and its sequence is MAKEWGYASHNGPDHWHELYPIAKGDNQSPIELHTKDIKHDPSLQPWSASYDPGSAKTILNNGKTCRVVFDDTYDRSMLRGGPLSGPYRLRQFHLHWGSSDDHGSEHTVDGVKYAAELHLVHWNPKYNTFGEALKQPDGIAVVGIFLKIGREKGEFQILLDALDKIKTKGKEAPFTHFDPSCLFPACRDYWTYHGSFTTPPCEECIVWLLLKEPMTVSSDQMAKLRSLFSSAENEPPVPLVGNWRPPQPVKGRVVRASFK (SEQ ID NO:12).

[0071] The specific experimental steps for the immunofluorescence staining experiment of co-incubating type I collagen (containing COL1A1 protein) with CAR3 protein and calcium phosphate solution are as follows: Take a 14mm aminated circular glass coverslip and add 50μL of assembly solution (prepared with 50mM glycine and 200mM potassium chloride, pH adjusted to 9.2±0.1) containing type I collagen (final type I collagen concentration of 75μg / mL, purchased from Corning). Place the coverslip face up in a 6cm petri dish lined with 200-300μL of moistened neutral filter paper and ultrapure water. Cover the petri dish, seal with sealing film, and incubate at 37℃ for 12h. Add 0.05% (v / v) glutaraldehyde solution and incubate at 37℃ for 1h. Rinse with ultrapure water for 5 min, shake on a shaker at 50 rpm (while floating on water), gently blot away water from the side of the round glass coverslip with filter paper, place the round glass coverslip face up on neutral filter paper, and dry at room temperature (about 15-20 min). Place it face up in a 24-well plate, add 0.1 mg / mL recombinant CAR3 protein and incubate for 2 hours. Then, place the incubated round glass coverslip in a 12-well plate, add mineralization solution (PAA-ACP solution (calcium phosphate solution containing polyacrylic acid), by first mixing equal volumes of CaCl2 and Na2HPO4 solution, and then adding polyacrylic acid (PAA, 225kJ) (final concentration: Ca...). 2+ 4.5 mM, HPO4 2- The solution was prepared by dissolving 2 mL of PAA (polyacrylic acid, 225kJ, 50 μg / mL, calcium chloride dihydrate (4.5mM), disodium hydrogen phosphate dodecahydrate (2.1mM)) in TBS solution. The well plates were sealed with a sealing film and incubated at 37°C for 1 day. The immunofluorescence procedure was the same as the staining procedure in Example 3: After washing with PBS, the cells were blocked in a 37°C incubator for 1 hour with 1% goat serum. Then, anti-CAR3 antibody (Proteintech) diluted 1:100 (v / v) was added and incubated overnight at 4°C. The cells were washed three times with PBS for 5 minutes each time. The cells were then incubated with goat anti-rabbit IgG H&L (Alexa Fluor 594, Abcam) secondary antibody diluted 1:200 (v / v) at 37°C for 1 hour, followed by three washes with PBS for 5 minutes each time. The cells were then blocked again in a 37°C incubator with 1% goat serum for 1 hour. Finally, anti-COL1A1 antibody (Abcam) diluted 1:100 (v / v) was added and incubated overnight at 4°C. The cells were washed three times with PBS for 5 minutes each time. The cells were then incubated with goat anti-rabbit IgG H&L (Alexa Fluor 594) secondary antibody diluted 1:200 (v / v) with PBS. The cells were incubated with 405 (Abcam) secondary antibody at 37°C for 1 hour, washed three times with PBS for 5 minutes, and then stained with calcein (2 mg / mL, Solarbio) to stain calcium ions. The images were then captured using a confocal microscope.

[0072] Immunoprecipitation assay: BMSCs at 7 days of osteogenic differentiation were cultured as in Example 3. After lysis using rapid cell / tissue lysis buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd.), the cells were centrifuged at 4°C at high speed (12000×g, 15 minutes) to remove cell debris and unlysed nuclei. An appropriate amount of supernatant was collected, and anti-COL1A1 specific antibody (Abcam) was added. The mixture was incubated slowly at 4°C for 12 hours. Protein A / G agarose beads (which specifically bind to the Fc fragment of the antibody, Thermo Fisher) were added to the incubation system and incubated slowly at 4°C for another 2 hours to allow the antibody-protein complex to be captured by the binding of the antibody to the agarose beads, forming an agarose bead-antibody-protein A-protein B precipitate. After incubation, the agarose bead precipitate was collected by low-temperature, low-speed centrifugation (3000×g, 5 minutes) and washed with ice-cold washing buffer (containing 140 mM... The precipitate was gently washed 3-5 times with NaCl and PBS. After each wash, the supernatant was discarded by centrifugation to remove unbound proteins. Finally, SDS-PAGE loading buffer was added to the washed precipitate and heated at 95-100°C for 5 minutes to denature and dissociate the protein complex from the agarose beads and antibody. After centrifugation, the supernatant was used for Western blot experiments, following the same procedure as in Example 3. Anti-CAR3 antibody (Proteintech) and anti-COL1A1 specific antibody (Abcam) were used for detection.

[0073] A monolayer type I collagen fiber model was prepared by collagen self-assembly and glutaraldehyde cross-linking on a TEM nickel mesh. Recombinant CAR3 protein (Kangti Biotechnology Co., Ltd.) and PBS solution (control) were co-incubated with the monolayer type I collagen fiber model at 37°C for 2 hours. Then, the nickel mesh loaded with collagen fibers was floated in calcium phosphate solutions containing and without 50 μg / mL polyacrylic acid (PAA, a non-collagenous protein (NCP) mimic) for mineralization. This verified that CAR3 and the non-collagenous protein mimic jointly drive the biomimetic mineralization of collagen fibers. The specific experimental steps are as follows.

[0074] 20 μL of type I collagen stock solution (5 mg / mL, provided by Coring Biotech) derived from rat tail was mixed with 500 μL of assembly solution (containing 50 mM glycine and 300 mM KCl, pH adjusted to 9.3). 2.5 μL of the collagen solution was carefully added dropwise to each 200-mesh nickel grid. The grids were incubated in a humidified incubator at 37°C for 12 hours. Subsequently, the collagen fibers on the grids were crosslinked using 0.05% (v / v) glutaraldehyde solution at 37°C for 1 hour. The nickel grids were then thoroughly rinsed with deionized water and air-dried at room temperature.

[0075] 0.1 mg / mL recombinant CAR3 protein (Kangti Biotechnology Co., Ltd.) and PBS solution (control) were co-incubated with a monolayer type I collagen fiber model at 37°C for 2 hours. The mesh was then mineralized and incubated with PAA-ACP solution at 37°C for 6 hours and 12 hours, respectively. After incubation, the nickel mesh was rinsed with deionized water, air-dried, and analyzed using transmission electron microscopy (TEM, HT7700, Hitachi, Japan) and high-resolution transmission electron microscopy (HRTEM, JEM-F200, JEOL, Japan).

[0076] Experimental results are as follows Figure 12-13 As shown, this confirms that CAR3-mediated collagen fiber biomimetic mineralization is achieved. Figure 12 (a). Further immunoprecipitation (Co-IP) experiments revealed that a COL1A1-CAR3 complex (a) was formed in the late stage of BMSC mineralization. Figure 12 (b) Transmission electron microscopy (TEM) analysis showed that the reconstructed type I collagen fibers exhibited characteristic 67 nm periodic bands, confirming that the procollagen molecules were correctly interlaced and assembled into a fiber structure. Figure 12 c and Figure 13 (AD). High-resolution TEM and selected area electron diffraction (SAED) showed that collagen fibers treated with CAR3 (0.1 mg / mL CAR3) exhibited increased fiber diameter and complete mineralization coverage compared to the control group. Figure 12 (c and d).

[0077] Example 7 Based on the crucial role of CAR3-collagen interaction in the mineralization process, this embodiment provides a collagen scaffold functionalized with recombinant CAR3 protein for the treatment of bone defects, and the specific preparation is as follows: Type I collagen 3D scaffold (Geistlich Bio-Gide) ® Cut the CAR3 protein into small round pieces with a diameter of 3 mm, soak them in 0.1 mg / mL recombinant CAR3 solution, and incubate at 37°C for 2 hours to obtain the collagen scaffold functionalized with recombinant CAR3 protein of the present invention.

[0078] Example 8 This embodiment provides the application of the recombinant CAR3 protein-functionalized collagen scaffold prepared in Example 7 in a skull defect model, as detailed below.

[0079] Six-week-old Prx1-Cre;Rosa-CAG-ZsGreen Stop-Tdtomato fluorescently labeled mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were injected intraperitoneally with an anesthetic drug (Shutai™ 50), and two 3 mm diameter bone defects were constructed on the parietal bone on both sides of the cranial midline of each animal using a saline-cooled dental trephine.

[0080] Experimental group: The recombinant CAR3 protein-functionalized collagen scaffold prepared in Example 7 was implanted at the bone defect site; Control group: Type I collagen membrane scaffolds soaked in PBS were implanted at the bone defect site.

[0081] All animals received penicillin treatment for three days after scaffold implantation. Eight weeks post-surgery, animals were euthanized under overdose anesthesia, and skull samples were collected and preserved in 4% paraformaldehyde. Frozen sections were used to observe mesenchymal stem cell recruitment in the bone defect area; MicroCT was used to assess new bone density, bone volume, bone surface area, trabecular bone morphology, and cortical bone morphology in the bone defect area to evaluate the level of new bone formation. Von Kossa staining and other procedures were the same as in Example 2. The specific steps for tissue frozen sections are as follows: (1) Dehydration: Wash the tissue of the maxillary molar area with running water for about 6 hours to remove excess paraformaldehyde. Immerse the tissue in 15% and 30% sucrose solutions overnight for dehydration treatment until the tissue completely sinks. (2) OCT embedding: Wipe away excess sucrose solution with a paper towel. First, slowly pour some OCT embedding agent into the embedding mold, then place the tissue in the OCT embedding agent, and continue to slowly pour in the OCT embedding agent until the entire tissue is submerged. During this process, care should be taken to avoid the formation of air bubbles. (3) OCT curing: Fix the mold filled with OCT embedding agent with clamps, quickly immerse its bottom in liquid nitrogen, and wait 5 to 10 seconds until the OCT embedding agent cures. At this time, the tissue and OCT embedding agent form a white solid. If long-term storage is required, wrap it with aluminum foil and store it in a freezer at -80°C. (4) Frozen sectioning: Before sectioning, adjust the room temperature of the microtome to -20℃, place the OCT-embedded tissue block into the microtome for sectioning, use Cryofilms (2C (10), 3.5 cm, Part # CFS 105), use the cryostat to cut the sample into 10 μm thin slices and attach them to the glass slide; (5) Imaging: Immerse the frozen section sample in PBS and wash at room temperature for 15 min. Add anti-fluorescence quenching mounting medium (containing DAPI, Shanghai Beyotime Biotechnology Co., Ltd.) to the tissue, cover with coverslip to avoid air bubbles, seal the edges of the coverslip with clear nail polish, and observe and photograph the tissue using a Leica Thunder high-speed imaging microscope.

[0082] Experimental results are as follows Figure 14 As shown, micro-CT analysis revealed that the new bone formation area and bone volume fraction in the CAR3 functionalized scaffold group were significantly higher than those in the unmodified scaffold control group. Figure 14 (a). Von Kossa staining showed that, 8 weeks after implantation, the CAR3-functionalized scaffold induced a higher calcium phosphate nucleation density in the collagen-guided bone matrix. Figure 14 (b) indicates enhanced osseointegration capacity. Furthermore, the CAR3 functionalized scaffold significantly promoted the recruitment of Prx1 lineage cells (red fluorescence) in the skull defect model. Figure 14 (c and d).

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of carbonic anhydrase III in the preparation of products for treating bone defects.

2. A bone repair composite material, characterized in that, The bone repair composite material includes carbonic anhydrase III and a load scaffold for surface loading of carbonic anhydrase III; preferably, the load scaffold has a porous mesh structure; preferably, the load scaffold comprises at least one of collagen, gelatin, chitosan and hyaluronic acid.

3. The bone repair composite material according to claim 2, characterized in that, The collagen includes at least one of type I collagen, type II collagen, and type III collagen.

4. The method for preparing the bone repair composite material according to any one of claims 2-3, characterized in that, The method includes the following steps: immersing the load scaffold in a carbonic anhydrase III solution to obtain the bone repair composite material.

5. The use of the bone repair composite material according to any one of claims 2-3 in at least one of the following (I) to (III): (I) To prepare products for the treatment or repair of bone injuries; (II) Preparation of products for promoting stem cell migration; (III) Prepare products that induce stem cells to differentiate into osteocytes.

6. The application according to claim 5, characterized in that, The stem cells include at least one of adipose-derived stem cells and bone marrow mesenchymal stem cells.

7. The use of the bone repair composite material according to any one of claims 2-3 in the preparation of products for treating bone defects.

8. Application of substances that promote the synthesis of carbonic anhydrase III in the preparation of drugs for treating bone defects.

9. The application according to claim 8, characterized in that, The substances that promote the synthesis of carbonic anhydrase III include substances that promote the expression of the carbonic anhydrase III gene. Preferably, the substance that promotes the expression of the carbonic anhydrase III gene includes: (1) A vector that promotes the expression of carbonic anhydrase III; (2) Recombinant cells containing the expression vector described in (1).

10. Application of reagents for detecting carbonic anhydrase III in the preparation of products for assessing bone formation.

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