A support for embedding in a bone defect area

Abstract

This invention belongs to the field of biomimetic regenerative medicine and relates to a support for insertion into a bone defect. The invention provides a support for insertion into a bone defect, exhibiting the following mechanical properties: a storage modulus of 10 to 5500 Pa and a loss modulus of 0 to 650 Pa at 1% strain. By constructing a support with specific viscoelastic properties, the support can be inserted into a bone defect to replace blood clots, thereby addressing the technical problem of difficult-to-heal bone injuries in individuals with dyscoagulability.

Description

Technical Field

[0001] The invention belongs to the technical field of bionic regenerative medicine and relates to a support for being embedded in a bone defect area. Background Art

[0002] Bones are the supporting structure of the human body and part of the human locomotor system. Their function is to move, support and protect the body, and they are of great significance to maintaining human health. Trauma, tumors, surgery, osteoporosis and other factors can lead to bone defects. Most defective bone tissues can rely on the body's own repair ability to heal the defect site and return to normal without forming scars. The first critical stage after bone injury is the formation of a blood clot, which is not only an important guarantee to prevent excessive bleeding, but also serves as a supporting structure to coordinate subsequent remodeling processes to promote bone repair and regeneration. Current research mainly focuses on promoting the formation of blood clots, or paying attention to the role of bone cells in the bone healing process. For individuals with coagulation difficulties, such as the elderly or patients with hypertension, there are many difficulties and risks in repairing bone injuries. Summary of the Invention

[0003] The purpose of the present invention is to solve at least one of the above-mentioned technical problems. It can solve the technical problem of difficult healing of bone injuries in individuals with coagulation difficulties in a simple and low-cost manner. To this end, the present invention provides a support for embedding in a bone defect area to solve this need in the art.

[0004] In one aspect, the present invention relates to a support for embedding in a bone defect area, wherein the support has a storage modulus of 10 to 5500 Pa and a loss modulus of 0 to 650 Pa at 1% strain.

[0005] Furthermore, in the support provided by the present invention for embedding in a bone defect area, the support is a gel, and the gel is formed by protein and a dispersion medium.

[0006] Furthermore, in the support for embedding in a bone defect area provided by the present invention, the protein is fibrin, and the dispersion medium is an aqueous solution.

[0007] Furthermore, in the support provided by the present invention for embedding in a bone defect area, the mass concentration of the fibrin is 0.1-1%.

[0008] Furthermore, in the support provided by the present invention for embedding in the bone defect area, the protein is Ca 2+ Chelating peptide, the dispersion medium is Ca-containing 2+ of solution;

[0009] The Ca 2+ Chelating peptides contain a gelatinous peptide motif and a calcium-binding motif.

[0010] Furthermore, in the support for embedding in a bone defect area provided by the present invention, the gelatinous peptide motif is (RADA)4, and the calcium binding motif is GSVLGYIQIR.

[0011] Furthermore, in the support provided by the present invention for embedding in the bone defect area, the Ca 2+ The mass concentration of the chelating peptide in the dispersion medium is 0.1-4%. 2+ Ca in solution 2+ The concentration is 0.02~1mg / mL.

[0012] Furthermore, in the support provided by the present invention for embedding in the bone defect area, the Ca 2+ The mass concentration of the chelating peptide in the dispersion medium is 3%, and the Ca-containing 2+ Ca in solution 2+ The concentration is 0.1 mg / mL.

[0013] Furthermore, in the support provided by the present invention for embedding in a bone defect area, the support activates integrin-mediated focal adhesion signaling and downstream force-sensitive transcription factor Runx2 to promote bone formation.

[0014] Furthermore, in the support provided by the present invention for embedding in a bone defect area, the support promotes bone formation in the bone defect area of ​​individuals with coagulation difficulties.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0016] (1) The present invention verifies that for individuals with bone damage, the formation of new bone in the bone defect area depends largely on the participation of blood clots. The time of blood clot formation greatly affects new bone formation, which is mainly manifested in the reduction of bone mineral density, the ratio of bone volume to total volume, the number of trabeculae, the thickness of trabeculae, osteogenic proteins, mesenchymal stem cell markers, and the increase of trabecular separation. The present invention also verifies that the formation of blood clots can promote osteogenesis by enhancing the focal adhesion signaling pathway mediated by integrin (Itgb) in MSCs, and further verifies that blood clots mainly promote osteogenesis by activating Itgb-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2 through their mechanical properties.

[0017] (2) The present invention unexpectedly discovered that the mechanical properties of the support in the bone defect area play a crucial role in its bone regeneration, and it mechanically affects the Itgb1 / Fak-mediated focal adhesion and the downstream Runx2 migration to the cell nucleus to promote the regulatory mechanism of bone regeneration. The technical solution proposed by the present invention is conducive to solving the technical problem of the difficulty in healing the bone defect area in individuals with coagulation difficulties. The present invention uses Ca 2+ Chelated peptides and fibrin were used as supports to verify their viscoelasticity and their effect in promoting bone formation in bone defect areas. 2+ Chelating peptides promoted bone formation by mechanically activating Itgb1-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2, significantly enhancing osteogenesis in the bone defect model of blood clot-deficient (BCD) rats. It was unexpectedly found that this mechanism of action (indicating that the transcription factor Runx2 promotes bone regeneration) is mechanosensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Figure 1. Validation trial results demonstrating that blood clot involvement is critical for new bone formation. (A) Meta-analysis using specific keywords related to osteogenesis and blood clots. (B) Comparison of coagulation time and blood loss between the Ctrl and BCD groups (rats) after tooth extraction. (C & D) Fibrin staining and blood clot quantification 3 hours, 6 hours, 24 hours, and 1 week after tooth extraction (D). (E) Masson trichrome and VG staining of the extraction area in the Ctrl and BCD groups 1 week after tooth extraction. (F) Micro-CT analysis of the extraction area in the Ctrl and BCD groups. (G) Quantification of micro-CT analysis, including bone mineral density (BMD), bone volume to total volume ratio (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). (H & I) Immunofluorescence staining and quantification of Runx2 and Alp (I). (J & K) Immunofluorescence staining of CD73 and CD105 analyzed by confocal laser scanning microscopy (CLSM). Figure K shows the statistics of double-positive signals. Data are shown as mean ± SD (n = 6). Unpaired two-tailed Student's t-test was used in B, G, and K; one-way or two-way analysis of variance (ANOVA) was used in D and I.

[0020] Figure 2Figure 1: Validation of a study demonstrating that clot formation promotes osteogenesis by enhancing integrin-mediated focal adhesion signaling. (A) t-SNE plot of a wide range of cell types within the alveolar and surrounding tissues in the Ctrl and BCD groups 24 hours after tooth extraction (n = 3). (B) Characteristic gene expression within each cluster was used as the clustering criterion. (C) Osteoblasts were further divided into four subclusters based on the dynamic changes of four genes: CD73, CD105, Alpl, and Ocn. (D) Proportions of subclusters within osteoblasts in quasi-temporal analysis. (E) Expression of osteoblast-related genes in the Ctrl and BCD groups. (FI) Single-cell gene set enrichment analysis (sc-GSEA) was performed on the four subclusters to identify specific signaling pathways between the Ctrl and BCD groups.

[0021] Figure 3 Figure 1: Validation of the experiment demonstrating that blood clots promote osteogenesis by mechanically activating Itgb-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2. (A) Immunohistochemical staining and quantification of Fak and Itgb1 in the Ctrl and BCD groups 1 week after tooth extraction. (B) Schematic diagram of mechanically activated Itgb-mediated focal adhesion signaling and osteogenesis in MSCs. (C) SEM images of fibrin and MSC morphology. (DG) CLSM analysis of immunofluorescence staining for Fak and Itgb5 (D) and Fak and Itgb1 (F) in MSCs incubated with or without fibrin for 24 hours, and quantification of double-positive signals (E and G). (H) Western blot analysis of Itgb5, Itgb1, Fak, activated Rhoa (ARhoa), Rock2, and c-Jun in MSCs incubated with or without 0.5% fibrin for 24 hours. (I) Immunofluorescence staining of Runx2 in MSCs incubated with different concentrations of fibrin for 24 hours. (J) Quantitative analysis of Runx2 in the nucleus and nucleoplasm based on fluorescence intensity. (K) Western blotting analysis of Runx2 expression in the nucleus and cytoplasm of MSCs incubated with different concentrations of fibrin. (Left to right) MSCs with or without Itgb1 knockdown were incubated with 0.5% fibrin for 24 hours, and Runx2 expression was detected by immunofluorescence staining (L) and quantification (M) as well as Western blotting (N). Data are shown as mean ± SD (n = 6). A, E, G, M, unpaired two-tailed Student's t-test; J, one-way analysis of variance (ANOVA).

[0022] Figure 4Experimental diagram for the design and preparation of blood clot biomimetic gel (BCgel). Among them, (A) The 3D structure of the interaction between Itgb1 and fibrin predicted by AlphaFold. (B) The binding interface and Gibbs free energy of two interactions: Itgb1 and fibrin (top); Itgb1 and BCgel monomer (bottom). (C) Fluorescence polarization (FP) analysis of the binding between Itgb1 and fibrin / BCgel monomer was performed using FITC-labeled fibrin or BCgel monomer mixed with equal dilutions of Itgb1. (D) The synthesized BCgel monomer was identified by LC-MS analysis. (E) The storage modulus (G') and loss modulus (G") of BCgel at different concentrations were measured using a rheometer under a low-frequency rotation mode of 0-4 Hz with 1% deformation. (F) The interaction between BCgel monomer and Ca was determined by measuring Alp activity. 2+ Schematic diagram of the high-throughput screening system for optimal concentration ratio. (GH) Heat map (G) and graph (H) depict the results of Alp activity obtained using the screening method (F). (I) BCgel monomer concentration was 1.5% or 3% and Ca 2+ (J) 3% BCgel and 0.1 mg / mL Ca 2+ Contact angle. (K) Storage modulus (G') and loss modulus (G") of BCgel were measured using a rotational rheometer. (L) SEM and TEM images of BCgel. (M&N) CLSM analysis (M) and field emission scanning electron microscopy (FESEM) analysis (N) of BCgel incubated with MSCs.

[0023] Figure 5Figures show the results of a validation experiment demonstrating that BCgel promotes osteogenesis by mechanically activating Itgb1-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2. (A & B) Immunofluorescence staining of Fak and Itgb1 in MSCs incubated with or without BCgel for 24 hours was analyzed by CLSM, and double-positive signals were quantified (B). (C) Western blot quantification of Itgb1, Fak, activated Rhoa (ARhoa), Rock2, and c-Jun in MSCs incubated with or without BCgel for 24 hours. (D & E) Single-sample gene set enrichment analysis (ss-GSEA) was performed on MSCs in the Ctrl and BCgel groups to examine the focal adhesion assembly signaling pathway (D), Itgb-mediated signaling pathway, cell adhesion and Itgb signaling pathway, osteoblast development signaling pathway, and Runx2 bone development signaling pathway (E). (F) Immunofluorescence staining of Runx2 in MSCs incubated with different concentrations of BCgel for 24 hours. (G&H) Quantification of Runx2 in the nucleus and nucleoplasm based on fluorescence intensity. (I&J) MSCs with or without Itgb1 knockdown were incubated with BCgel for 24 hours and immunofluorescence staining and quantification (I) were performed to detect Runx2 expression. J. (KM) The expression of Fak, active Rhoa (ARhoa), Rock2, and c-Jun was assessed by Alp staining (K), Alp activity analysis (L), and Western blotting (WB) on day 7 after osteogenic induction. (N) MSCs with or without Itgb1 knockdown were incubated with BCgel and WB analysis was performed on day 7 after osteogenic induction. Data are shown as mean ± SD (D&E: n = 3; other groups: n = 6). B, I, unpaired two-tailed Student's t-test; G, H, L, one-way analysis of variance (ANOVA).

[0024] Figure 6Figures show the validation results of a study demonstrating that BCgel significantly enhances osteogenesis in a rat model of blood clot deficiency (BCD). (A & B) Proteomic data from extraction sockets and surrounding tissues in the BCgel and Ctrl groups were obtained using the 4D label-free LC-MS method, and gene set enrichment analysis of osteogenic signaling pathways was performed. (C) Heat map illustrating key proteins involved in osteogenic signaling in the datasets shown in (A & B). (D) Micro-CT analysis and quantification of BCD rats treated with gelform (Ctrl) and BCgel 1 week and 1 month after tooth extraction. (E) H&E staining of extraction sockets 1 week and 1 month after Ctrl and BCgel treatment. (F & G) Immunohistochemical staining and quantification of Fak 1 week (F) and 1 month (G) after Ctrl and BCgel treatment. (H) Observation of new bone formation in the extraction socket using sequential fluorescent labeling (green: calcein, 1 week; red: alizarin red, 2 weeks; yellow: tetracycline, 4 weeks). (I & J) CLSM analysis of CD73 and Fak immunofluorescence staining and quantification of double-positive signals in the Ctrl and BCgel groups 1 week (I) and 1 month (J) after treatment. (K & L) Immunofluorescence staining and quantification of Runx2 and Alp in the Ctrl and BCgel groups 1 month after treatment (L). Data are shown as mean ± SD (AC: n = 3; other groups: n = 6). D, F, G, I, J, unpaired two-tailed Student's t-test; L, one-way analysis of variance (ANOVA).

[0025] Figure 7Figures show the results of a validation study demonstrating that BCgel effectively enhances osteogenesis in a beagle dog model of blood clot defect (BCD). (A) Photographs of extracted teeth in beagle dogs treated with gelform (Ctrl) and BCgel, along with comparisons of clotting times immediately after tooth extraction. (n = 7, including 4 beagle dogs treated for 2 months and 3 beagle dogs treated for 5 months). (B & C) Micro-CT analysis and quantitative results of the Ctrl and BCgel groups after 2 months of treatment. (D) H&E, Masson's, and VG staining of extraction sockets after 2 months of Ctrl and BCgel treatment. (EH) H&E staining of organs after 2 months of BCgel treatment (E) and immunotoxicity, hemotoxicity, hepatotoxicity, and nephrotoxicity in the Ctrl and BCgel groups (FH). (J & K) Micro-CT analysis and quantitative results of the Ctrl and BCgel groups after 5 months of treatment. (L) H&E, Masson's, and VG staining of extraction sockets after 5 months of treatment in the Ctrl and BCgel groups. (MP) H&E staining of organs after 5 months of BCgel treatment (M) and immunotoxicity, hematotoxicity, hepatotoxicity, and nephrotoxicity in the Ctrl and BCgel groups (MP). Data are shown as mean ± SD (2-month treatment: n = 4, 5-month treatment: n = 3). All experiments were compared using an unpaired, two-tailed Student's t-test.

[0026] Figure 8 Figures show the results of tooth extraction and blood clot formation experiments in rats. (A) Schematic diagram of tooth extraction and blood clot formation in Ctrl and BCD rats. (B) Static coagulation analysis in the Ctrl and BCD groups. (C) H&E staining of the extraction area in the Ctrl and BCD groups one week after tooth extraction. Data are shown as mean ± SD (n = 6). B, unpaired two-tailed Student's t-test.

[0027] Figure 9Figures show validation of the support-dependent role of Itgb-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2 in promoting osteogenesis. (A & B) Immunofluorescence staining for CD73 and Fak was analyzed by confocal laser scanning microscopy (CLSM) and double-positive signals were quantified (B). (C) Cell viability of MSCs treated with different concentrations of fibrin was measured using CCK-8. (D) Ki67 and Tunel staining was performed in MSCs incubated with or without 0.5% fibrin for 24 hours. (E) SEM images of 0.5% fibrin. (F) Western blot analysis of Itga5, Itgb1, Fak, activated Rhoa (ARhoa), Rock2, and c-Jun in MSCs incubated with or without 0.5% fibrin for 24 hours. (G) MSCs were transfected with si-Itga5 and si-Itgb1 and incubated with 0.5% fibrin for 24 h. The expressions of Fak, activated Rhoa (ARhoa), Rock2, and c-Jun were analyzed by Western blotting. (H) The storage modulus (G') and loss modulus (G") of different fibrin concentrations were measured using a rotational rheometer in a low-frequency rotation mode below 4 Hz. (I) Immunofluorescence staining of Runx2 in MSCs incubated with different concentrations of fibrin for 24 h, and cell nuclei were stained with DAPI. (J) MSCs with or without Itgb1 knockdown were incubated with 0.5% fibrin for 24 h and then immunofluorescence staining was used to detect Runx2 expression, and cell nuclei were stained with DAPI. (KO) Osteogenic differentiation of MSCs was assessed by Alp immunofluorescence staining and quantification (K&L), Alp activity assay (M), Alp staining (N), and Western blotting (WB) (O) on day 7 after osteogenic induction. (P) MSCs with or without Itgb1 knockdown were incubated with fibrin and subjected to osteogenic induction for day 7, and then WB analysis was performed. Data are shown as mean ± SD (n = 6). B, unpaired two-tailed Student's t-test; C, L, M, one-way analysis of variance (ANOVA).

[0028] Figure 10 The mechanical properties test diagram of BCgel. (A) The structural formula of BCgel monomer peptide. (B) The rotational rheometer was used to measure the mechanical properties of different concentrations of Ca in the low-frequency rotation mode of 0-4 Hz. 2+ Storage modulus (G') and loss modulus (G") of 3% BCgel at different monomer concentrations. (C) BCgel with 0.1 mg / mL Ca 2+ ions, and 3% BCgel with different concentrations of Ca 2+Photographs. (D) Cell viability of MSCs treated with different concentrations of BCgel was measured using CCK-8. (E) Ki67 and Tunel staining was performed in MSCs incubated with or without 0.5% BCgel for 24 hours. Data are shown as mean ± SD (n = 6). D, One-way analysis of variance (ANOVA).

[0029] Figure 11 Figures show the experimental results of BCgel's effect on promoting osteogenesis in bone defect areas. (A) Western blotting analysis of Itgb1, Fak, active Rhoa (ARhoa), Rock2, and c-Jun in MSCs incubated with or without BCgel for 24 hours. (B) Western blotting of Runx2 in the nuclei and cytoplasm of MSCs incubated with different concentrations of BCgel for 24 hours. (C) Immunofluorescence staining of Runx2 in MSCs incubated with different concentrations of BCgel for 24 hours, with nuclei stained with DAPI. (D-F) Runx2 expression was assessed by immunofluorescence staining (D) and Western blotting (E) after 24 hours of incubation of MSCs with or without Itgb1 knockdown with BCgel. (F) Western blotting of Fak, active Rhoa (ARhoa), Rock2, and c-Jun. (G & H) Osteogenic differentiation of MSCs was assessed by Alp immunofluorescence staining (G) and quantification (H) after 7 days of osteogenic induction. Data are shown as mean ± SD (n = 6). H, one-way analysis of variance (ANOVA).

[0030] Figure 12 Figure 1 shows the experimental results of BCgel's effect on promoting osteogenesis in rats after tooth extraction. (A) Comparison of clotting time in BCD rats treated with gelform (Ctrl) or BCgel immediately after tooth extraction (n = 12, including 6 rats treated for 1 week and 6 rats treated for 1 month). (B) Masson's trichrome and VG staining of extraction sockets in the Ctrl and BCgel groups 1 week and 1 month after treatment. (C) Immunofluorescence staining and quantification of Runx2 and Alp 1 week after Ctrl and BCgel treatment. Data are shown as mean ± SD (B, C: n = 6). A, unpaired two-tailed Student's t-test; C, one-way analysis of variance (ANOVA).

[0031] Figure 13Figures show the effects of BCgel on rat organs after tooth extraction. (A & B) H&E staining of organs in the Ctrl and BCgel groups after 1 week and 1 month of treatment. (C) Analysis of white blood cells (WBC), red blood cells (WBC), lymphocytes (LYMPH), hemoglobin (HGB), and platelets (PLT) in the blood of rats in the Ctrl and BCgel groups after 1 month of treatment. Data are shown as mean ± SD (n = 6). C, unpaired two-tailed Student's t-test.

[0032] Figure 14 The figures show the test results of BCgel toxicity in rats. (A) H&E staining of rats after subcutaneous transplantation of gelform (Ctrl) and BCgel 1, 3, and 7 days. (B&C) Immunohistochemical staining and quantification of TNF-α 1, 3, and 7 days after Ctrl and BCgel transplantation. (D&E) Immunohistochemical staining and quantification of IL-1β 1, 3, and 7 days after Ctrl and BCgel transplantation. (FH) Immunotoxicity (F), hepatotoxicity or nephrotoxicity (G), and hematotoxicity (H) in the Ctrl and BCgel groups. Data are shown as mean ± SD (n = 6). F, G, H, unpaired two-tailed Student's t-test; C, E, one-way analysis of variance (ANOVA).

[0033] Figure 15 Figures show the results of a BCgel toxicity study in beagle dogs. (A-C) Immunotoxicity (A), hematotoxicity (B), and cardiotoxicity (C) were measured in the Ctrl and BCgel groups after 2 months of treatment. (D) Cardiotoxicity and neutrophil counts (NEUT) were analyzed in the Ctrl and BCgel groups after 5 months of treatment. Data are presented as mean ± SD (A-C: n = 4; D: n = 3). All experiments were compared using an unpaired, two-tailed Student's t-test.

[0034] Figure 16 The changes in ALP activity of supports with different storage moduli after 7 days of osteogenic induction. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified.

[0036] Example 1

[0037] This example provides a verification experiment on the relationship between osteogenesis and blood clot (BC) in bone defect areas.

[0038] In this example, a blood clot defect (BCD) model was established by performing mandibular extraction injury on hypertensive rats with systolic and diastolic blood pressure exceeding 150 / 120 mmHg to provide further evidence ( Figure 8 A). In the case of hypertension, the increased bleeding rate caused by hypertension often leads to increased bleeding volume and prolonged bleeding time, which in turn partially affects the formation of blood clots. According to this theory, compared with the healthy rat control group (Ctrl), the coagulation time of hypertensive rats was prolonged by 47% and the blood loss increased by 137% ( Figure 1 B), while the static coagulation ability of the two groups of rats was similar ( Figure 8 B). In addition, by analyzing the blood clots 3h, 6h, 24h and 1 week after tooth extraction through fibrin staining, it was found that healthy rats could always form complete blood clots in the extraction socket after tooth extraction ( Figure 1 C), while the blood clotting defect rate of hypertensive rats was at least 30% ( Figure 1 The above data indicate that a blood clot-deficient (BCD) bone defect model was successfully established in hypertensive rats through tooth extraction injury.

[0039] Then, the bone regeneration capacity of the BCD group and the Ctrl group was compared 1 week after tooth extraction. Figure 1 E) and H&E staining ( Figure 8 C), new bone formation was observed in the extraction sockets of both groups. Importantly, quantification of new bone by micro-computed tomography (micro-CT) showed that compared with the control group, the BCD group had a 29% lower bone mineral density (BMD), a 48% lower bone volume to total volume ratio (BV / TV), a 49% lower trabecular number (Tb.N), a 51% lower trabecular thickness (Tb.Th), and a 71% higher trabecular separation (Tb.Sp). Figure 1 F&G). Further mechanistic investigation of Runt-related transcription factor 2 (Runx2) and osteoblast alkaline phosphatase (Alp), two key osteogenic proteins, by immunofluorescence staining showed that the BCD group had a significant decrease in osteogenic proteins ( Figure 1 H&I). Consistent with these findings, the expression of mesenchymal stem cell markers CD73 and CD105 was significantly decreased in the extraction sockets of the BCD group ( Figure 1 J&K). Together, these results demonstrate that BCD inhibits osteogenesis and suggest that blood clots play a crucial role in new bone formation. This example confirms that BC plays a crucial role in osteogenesis in the bone defect region, and that the timing of BC formation significantly influences the rate of osteogenesis in the bone defect region.

[0040] Example 2

[0041] This example provides an experiment on the mechanism by which blood clot (BC) affects osteogenesis in bone defect areas.

[0042] Single-cell sequencing analysis was used to elucidate the cellular and molecular characteristics of alveolar bone and surrounding tissue cells in the BCD and Ctrl groups 24 hours after tooth extraction. Figure 2 A and B) (n = 3 per group), a remarkable diversity of immune cells was found in two different alveolar socket regions and their surrounding tissues, including four different types (B lymphocytes, granulocytes, mononuclear phagocytes, and T lymphocytes), as well as four tissue cell populations (basal cells, endothelial cells, neurons, and osteoblasts). Further quasi-temporal analysis of the temporal dynamics of four genes, CD73, CD105, Alpl, and Ocn, during osteogenesis identified four distinct but continuous subpopulations: mesenchymal stem cells, osteogenic cells, preosteoblasts, and osteoblasts ( Figure 2 C and D). In theory, these four subpopulations appear sequentially throughout the osteogenesis process. Compared with the Ctrl group, the proportion of mesenchymal stem cells and osteoblasts in the BCD group increased significantly, while the proportion of pre-osteoblasts and osteoblasts decreased significantly, indicating that the osteogenic differentiation of poorly differentiated cells was stagnant. The expression levels of four key genes involved in osteogenesis (i.e., Alpl, Runx2, Ocn, and Col1a1) further confirmed these findings. Notably, a strong inhibition of gene expression was observed in both pre-osteoblasts and osteoblasts in the BCD group ( Figure 2 E). In addition, single-cell genomic enrichment comparative analysis (sc-GSEA) was performed on the four subclusters, and specific signaling pathways between the BCD group and the Ctrl group were identified. In terms of signaling pathways related to MSC osteogenesis, four signaling pathways related to Itgb-mediated focal adhesion were significantly downregulated in the BCD group compared with the Ctrl group, including Itgb cell surface interaction, focal adhesion, Itgb binding, and focal adhesion assembly ( Figure 2 F). In addition, signal transduction and adhesion mediated by Itgb are also downregulated in osteoblasts ( Figure 2 G). Moreover, the two most significantly downregulated pathways in preosteoblasts were the Runx2 expression and activity pathway and the Runx2 transcriptional regulation pathway ( Figure 2 H). In addition, the two most significantly downregulated pathways in osteoblasts were found to be related to osteoblast organogenesis and development ( Figure 2 I). All these findings suggest that BC formation promotes primary osteogenesis by enhancing Itgb-mediated focal adhesion signaling in MSCs, thereby ensuring normal osteogenesis in the later stages.

[0043] Example 3

[0044] This example provides an experiment on the effect of blood clot (BC) on the osteogenesis mechanism in bone defect area through mechanical mechanics.

[0045] To further demonstrate that the Itgb-mediated focal adhesion signaling pathway promotes osteogenesis at the protein level, semi-quantitative analysis of two key proteins in this pathway, Fak and Itgb1, was performed in the BCD and Ctrl groups. Notably, significant decreases of 49% and 51% in Fak and Itgb1 levels were observed in the BCD group, respectively. Figure 3 A). In addition, immunofluorescence analysis of CD73 (a marker of MSCs) and Fak co-staining showed that the double-positive cell population in the BCD group was significantly reduced by 83% ( Figure 9 A and B). This indicates that the Itgb-Fak focal adhesion signaling pathway in MSCs is significantly inhibited. Based on the above results, we hypothesize that fibrin, a key component of blood clots, activates the Itgb-mediated focal adhesion signaling pathway, thereby triggering the force-sensitive transcription factor Runx2 in MSCs to promote osteogenesis ( Figure 3 B). To explore this hypothesis and its mechanism, rat femur-derived bone marrow mesenchymal stem cells (MSCs) were treated with fibrin. Notably, fibrin concentrations of 0.1%, 0.2%, 0.5%, and 1% had negligible effects on cell viability ( Figure 9 C). Fibrin did not induce cell cycle arrest or apoptosis of MSCs until the concentration reached 0.5%. Figure 9 D). In addition, the pore structure in 0.5% fibrin ( Figure 9 E) has good biocompatibility with MSCs. Cells can attach to their surfaces and extend their tentacles into the pores ( Figure 3 C). Importantly, after incubation with 0.5% fibrinogen for 24 hours, the Fak / Itga5 double-positive signal in cells was significantly increased compared with the control group ( Figure 3 D), accompanied by a 99% increase in Fak-Itga5 focal adhesions ( Figure 3 E). Similar results were observed by co-staining of Fak / Itgb1 ( Figure 3 F), Fak-Itgb1 focal adhesions increased by 485% after incubation with fibrin ( Figure 3 G). Similarly, fibrin led to an upregulation of more than 40% of proteins involved in the Itgb-Fak focal adhesion signaling pathway, including Itga5, Itgb1, Fak, activated Rhoa (ARhoa), Rock2, and c-Jun ( Figure 3 H and Figure 9F). These data suggest that fibrin has the ability to enhance mechanical activation of Itgb-Fak focal adhesion signaling. Notably, Itgb1 plays a stronger role in activating this signaling pathway than Itga5, as si-Itgb1 significantly inhibited this pathway more than si-Itga5 ( Figure 9 G).

[0046] Due to the force sensitivity of Itgb-Fak focal adhesion signaling, it is hypothesized that fibrins with different mechanical properties will induce different responses through this pathway. The following example uses a low-frequency rotation of 0 to 4 Hz to apply 1% strain to the support to consider the effect of supports with different mechanical properties on osteogenic performance. Figure 9 As shown in Figure 5, adjusting the fibrin concentration from 0.1% to 1% under low-frequency rotation of 0-4 Hz resulted in a continuous change in the storage modulus (G') from 925 Pa to 5648 Pa and the loss modulus (G") from 54.9 Pa to 569 Pa. As expected, the use of different viscoelastic fibrin preparations resulted in different Runx2 nuclear translocation rates ( Figure 3 IK and Figure 9 I), among which Runx2 activity responded most significantly to 0.5% fibrin ( Figure 3 J&K). Furthermore, Runx2 nuclear translocation was significantly impaired in Itgb1 knockdown (Itgb1KD) MSCs, in which Itgb1 expression was stably suppressed by lentiviral-encapsulated shRNA ( Figure 3 LN and Figure 9 J). These findings indicate that fibronectin-mediated Runx2 nuclear translocation is dependent on Itgb1. In addition, Runx2 accumulation in the nucleus leads to the activation of its downstream protein Alp ( Figure 9 K&L), thereby promoting the osteogenic differentiation of MSCs ( Figure 9 Importantly, osteogenic differentiation was also inhibited in Itgb1-knockdown MSCs ( Figure 9 These results suggest that fibrin promotes osteogenesis by activating Itgb-mediated focal adhesion signaling and downstream Itgb1-dependent Runx2 force-sensitive nuclear translocation.

[0047] The results of Examples 1-3 demonstrate that BC plays a key role in bone regeneration during bone defect repair. BC formation promotes primary osteogenesis by enhancing Itgb-mediated focal adhesion signaling in MSCs, thereby ensuring normal healing of later osteogenesis. Furthermore, experiments using fibrin (which has a similar structure to BC) as a support material demonstrated its ability to enhance mechanical activation of Itgb-Fak focal adhesion signaling. The following work will examine whether other supports with similar mechanical properties can replace BC or fibrin to achieve the same effect.

[0048] Example 4

[0049] This example provides the design and preparation of blood clot biomimetic gel (BCgel).

[0050] The BCgel monomer peptide sequence is (RADA)4-GSVLGYIQIR, where (RADA)4 is the gel-forming peptide motif and GSVLGYIQIR is the calcium-binding motif. Based on the solid-phase synthesis technique for FMOC peptides, the monomeric peptide was synthesized on MBHA resin using an automated peptide synthesizer (CS Bio 336X) with HBTU as the condensing agent and DIEA as the catalyst. After cleavage and deprotection in a reagent mixture containing 88% TFA, 5% phenol, 5% H2O, and 2% TIPS, the peptide was precipitated with cold ether. Finally, the peptide was characterized by electrospray ionization mass spectrometry (ESI-MS) and high-performance liquid chromatography (HPLC) and purified to homogeneity using preparative C18 reverse-phase HPLC using acetonitrile and water containing 1 / 1000 TFA. BCgel concentrations ranged from 0 to 4% to meet different experimental requirements.

[0051] αFold 2.0 was used to analyze the topological structure of the interaction between Itgb1 and fibrin and to study the biological function of Itgb1-dependent fibrin. As expected, the N-terminus of fibrin contains an RGD motif that can bind to Itgb1 ( Figure 4 A), the bonding interface area is Gibbs free energy is -2.0 Kcal / mol ( Figure 4 B), indicating a strong affinity between the two molecules. FITC-labeled fibrin was mixed with an equal dilution of Itgb1, and the binding of Itgb1 to fibrin was quantitatively analyzed. The measured fluorescence polarization results were fitted using the standard equation for protein-ligand binding. This analysis showed that the binding affinity between Itgb1 and fibrin was 29.8 nM ( Figure 4 C). This discovery inspired the development of a biomimetic hydrogel that can mimic the biochemical and biomechanical microenvironment of fibrin, aiming to address the recurrent bone regeneration barrier caused by mechanical bone defects.

[0052] Therefore, it is proposed that the gelatinous peptide motif (RADA) 4 can mimic the effect of the RGD motif when it binds to Itgb1 ( Figure 4 A). αFold results show that the interaction area between (RADA)4 and Itgb1 is 300.4 and the Gibbs free energy is -1.3 Kcal / mol, which theoretically supports this hypothesis ( Figure 4 B). To further enhance the tunability of the hydrogel viscoelasticity of (RADA)4, a calcium-binding motif was attached to the C-terminal region ( Figure 10 A). Subsequently, BCgel monomer peptide was successfully synthesized using HBTU / HOBT coupled solid phase FMOC chemistry and purified by reversed phase high performance liquid chromatography (HPLC). The synthesized BCgel monomer molecular weight was close to the theoretical value of 2760.15 Da, and the purity was 95.6% ( Figure 4 D) In ​​addition, the binding affinity of the FP assay to Itgb1 was measured to be approximately 90.2 nM, and the K for fibrin was d The value is equivalent to ( Figure 4 C).

[0053] To investigate the viscoelastic properties of BCgel, it was dissolved in deionized water at concentrations ranging from 0.5% to 4%, and G' and G" were measured using a rotational rheometer. Figure 4 As shown in E, when the concentration of BCgel was changed under low-frequency rotation of 0-4 Hz with 1% deformation, G' changed continuously from 62 Pa to 4973 Pa and G" from 14 to 607 Pa. In addition, Ca 2+ The addition of expands the viscoelastic range ( Figure 10 B&C). These results indicate that BCgel is a two-dimensional hydrogel with adjustable viscoelasticity. In order to determine the optimal viscoelasticity of BCgel in promoting osteogenesis, a high-throughput screening system was established to evaluate the effects of Ca 2+ concentration and 2D parameters related to BCgel monomer concentration ( Figure 4 F). In this system, MSCs were mixed with BCgels of different viscoelasticity and added to a 96-well plate together with serum-containing cell culture medium ( Figure 4 F) After 24 h of incubation, the Alp activity in the culture medium was measured, and the obtained heat map and cumulative curve were shown in Figure 2. Figure 4 G and Figure 4 The results showed that 3% BCgel monomer concentration and 0.1 mg / mL Ca 2+ The Alp activity was highest under the combination of Figure 4 H).

[0054] like Figure 16As shown, the present invention further tested the changes in ALP activity of supports with different storage moduli (i.e., different viscoelasticity) after 7 days of osteogenic induction. Among them, Ctrl is a support without addition of support, and supports with storage moduli of 10Pa, 2500Pa, and 5500Pa are prepared by adjusting the BCgel monomer concentration to 0.1-4% and the calcium ion concentration to 0.02-1mg / mL, corresponding to loss modulus of 0Pa, 100Pa, and 600Pa. In addition, for comparison, supports with a storage modulus of 5Pa and a loss modulus of 0Pa are prepared from BCgel and water, and supports with a storage modulus of 6000Pa and a loss modulus of 800Pa are prepared from high-concentration cellulose. By Figure 16 It can be seen that when the viscoelasticity of the support is low or high, it has no promoting effect on osteogenesis. When the storage modulus of the support is 10-5500Pa and the loss modulus is 0-650Pa, it has a significant promoting effect on osteogenesis in the bone defect area.

[0055] Under these conditions, BCgel exhibited excellent injectable hydrogel properties ( Figure 4 I&J) and good viscoelasticity, G' is significantly greater than G" ( Figure 4 K), indicating that it has excellent elastic behavior and efficient force transmission performance. It is worth noting that the viscoelasticity of BCgel is comparable to that of 0.5% fibrin. In addition, at concentrations of 3% and 0.1 mg / mL Ca 2+ Under the condition of 0.5% fibrin, BCgel showed a spatial network structure similar to that of 0.5% fibrin ( Figure 4 L). Importantly, this BCgel not only has the same biocompatibility as 0.5% fibrin, promoting MSCs adhesion and extension into its pores, but also promotes the three-dimensional arrangement of stem cells, reflecting better environmental adaptability ( Figure 4 M&N, Figure 10 D&E). The preparation of blood clot biomimetic gel BCgel has significant potential in simulating the biological function of fibrin.

[0056] Example 5

[0057] This example provides that BCgel promotes bone formation by mechanically activating Itgb1-mediated focal adhesion signaling and the downstream force-sensitive transcription factor Runx2.

[0058] To investigate the biological function of BCgel, we first co-stained Itgb1 and Fak and investigated Itgb1-mediated focal adhesions based on confocal laser scanning spectroscopy (CLSM) images. As expected, the Itgb1-mediated focal adhesions in the BCgel group increased significantly by 10.7-fold compared to the control group ( Figure 5A and B). Therefore, BCgel significantly enhanced the activity of Itgb1-mediated focal adhesion signaling. The upregulation of Itgb1, Fak, ARhoa, Rock2, and c-Jun in WB ( Figure 5 C and Figure 11 A) and enrichment analysis results of focal adhesion assembly-related genes using RNA-seq ( Figure 5 D) is consistent with the above conclusion. In addition, GSEA analysis also showed that the Itgb-mediated signaling pathway, the cell adhesion signaling pathway related to Itgb function, the osteoblast development signaling pathway, and the Runx2-mediated bone development signaling pathway were significantly upregulated in the BCgel group ( Figure 5 E). This indicates that BCgel can enhance Itgb-mediated focal adhesion signaling, thereby promoting Runx2-dependent bone formation. To further verify the force sensitivity of Runx2, Ca 2+ 0.1 mg / mL, BCgel concentrations ranging from 0.1% to 4.0%. Subsequently, these BCgels were co-cultured with MSCs, and Runx2 expression levels were analyzed by immunofluorescence staining and WB. As expected, different concentrations of BCgel showed different degrees of Runx2 nuclear translocation, among which 3% BCgel showed the highest level, with more than 90% of Runx2 localized in the cell nucleus ( Figure 5 FH and Figure 11 B&C). These findings not only confirmed the force-sensitivity of Runx2, but also reaffirmed that Runx2 was activated in 3% BCgel and 0.1 mg / mL Ca 2+ is the best choice. In addition, in Itgb1 knockdown MSCs, BCgel-induced Runx2 ( Figure 5 I and Figure 11 D&E) nuclear translocation and the corresponding Itgb-mediated activation of focal adhesion signaling ( Figure 5 J& Figure 11 F) were inhibited, indicating that the process depends on Itgb1. In addition, the accumulation of Runx2 in the nucleus also leads to the activation of its downstream protein Alp ( Figure 11 G&H), thereby promoting MSCs ( Figure 5 KM) osteogenic differentiation. In addition, this osteogenic differentiation process was also inhibited in Itgb1 knockdown MSCs ( Figure 5 In conclusion, BCgel enhances the osteogenic differentiation of MSCs by mechanically activating Itgb1-mediated focal adhesion signaling and downstream Runx2 mechanosensitive nuclear translocation.

[0059] BCgel exhibits significant in vitro bioactivity, prompting investigation of its in vivo efficacy. To this end, a comparative study was conducted using the BCD tooth extraction model in hypertensive rats to compare BCgel with a commercially available tooth extraction hemostatic gel, gelform (Ctrl group). Interestingly, BCgel also exhibited hemostatic activity, with a significant 55% reduction in clotting time compared to gelform ( Figure 12 A). One week after the animal BCD model was established and treated, proteomic results were obtained using a 4D label-free LC-MS method. Gene set enrichment analysis (GSEA) of these results showed that Itgb1 / Fak-mediated focal adhesion signaling and Runx2-mediated bone regeneration signaling pathways were activated ( Figure 6 AC), indicating the effectiveness of BCgel at the protein level. In addition, micro-CT scans also showed that one week after treatment, the BCgel group had formed more new bone on the inner wall of the extraction socket. The quantitative analysis results of micro-CT showed that compared with the Ctrl group, the bone mineral density (BMD) of the BCgel group increased by 73%, the bone volume to total volume ratio (BV / TV) increased by 84%, and the trabecular spacing (Tb.Sp) decreased by 47%, indicating that the amount of new bone in the BCgel group increased significantly. In addition, in the tissue sections one month after treatment, a large amount of new bone was observed in the extraction socket of the BCgel group, which was significantly more than that of the Ctrl group ( Figure 6 D). Consistent with this result, micro-CT scans one month after treatment also showed that the BCgel group had more new bone formation in the extraction socket ( Figure 6 D). One week and one month after treatment, the mandibular extraction sockets were decalcified, sectioned, and histologically stained with H&E, Masson's trichrome, and VG. The results showed that new bone formation was observed on the medial wall of the extraction socket in the BCgel group one week after treatment (see Figure 6 E& Figure 12 B), and it became more obvious one month after treatment. In addition, immunohistochemical staining showed that the Fak level in the BCgel group was significantly higher than that in the Ctrl group one week and one month after treatment ( Figure 6 F&G). This further supports the view that BCgel promotes Itgb1 / Fak-mediated focal adhesion signaling activation. In addition, to further observe the formation of new bone, calcein (green) was injected through the tail vein one week after treatment, alizarin red (red) was injected two weeks later, and tetracycline (yellow) was injected four weeks later. Hard tissue sections were taken three days after tetracycline injection. Confocal images showed that the depth of the three colors in the BCgel group significantly exceeded that of the Ctrl group, indicating that the rate of new bone formation in the BCgel group was accelerated ( Figure 6H). This finding was further confirmed by immunofluorescence co-staining of CD73 and Fak, which showed that the double-positive cells increased significantly by 31-fold after one week of BCgel treatment and by 15-fold after one month of treatment ( Figure 6 I&J). These results indicate that BCgel enhanced Itgb1 / Fak-mediated focal adhesion signaling in MSCs to a greater extent. Immunofluorescence staining and quantitative analysis results were consistent with these findings. After one week and one month of treatment, the expression levels of Runx2 and Alp in the BCgel group were increased compared with the Ctrl group ( Figure 12 C and Figure 6 K&L). Taken together, these results indicate that BCgel enhances osteogenic signaling pathways in the BCD rat model.

[0060] In addition, histopathological analysis was performed on samples of heart, liver, spleen, lung, and kidney collected after one week and one month of treatment ( Figure 13 A&B) and blood test ( Figure 13 C), BCgel treatment did not show any signs of systemic toxicity compared to the control group. To further evaluate its biosafety, BCgel was subcutaneously implanted into healthy SD rats, and the commercial gel gelform was used as a control. As expected, BCgel showed a lower inflammatory response than gelform, as evidenced by H&E staining of the implantation site collected 1, 3, and 7 days after implantation ( Figure 14 A) and TNF-α ( Figure 14 B&C) and IL-1β( Figure 14 This finding was supported by immunohistochemical staining of D&E. In addition, this finding was also supported by serum inflammatory markers including TNF-α, IL-1β, IL-6, IL-8, IFN-γ, and IL-17 levels ( Figure 14 Importantly, neither BCgel nor gelform caused any liver or kidney dysfunction ( Figure 14 G), nor did it have any effect on hematological parameters ( Figure 14 H). Taken together, these findings provide strong evidence for the excellent biosafety profile of BCgel and highlight its potential for clinical translation.

[0061] To further enhance the bioactivity of BCgel in promoting bone formation in vivo, another clot-deficient bone defect model was established using beagle dogs, and heparin was injected to disrupt clot formation after mandibular lateral incisor extraction. The extraction sockets were then treated with BCgel or gelform (Ctrl) for hemostasis. The clotting time in the BCgel group was significantly shortened by 69% ( Figure 7 A). Two months after tooth extraction, micro-CT ( Figure 7B and Videos 4, 5) and the corresponding BMD, BV / TV and Tb.Sp ( Figure 7 C) Quantitative data showed that alveolar bone regeneration was significantly enhanced in the BCgel group. H&E, Masson staining, and VG staining of decalcified tissue sections further supported this result ( Figure 7 D). In addition, no visible immunotoxicity was observed after two months of continuous BCgel treatment ( Figure 7 F& Figure 15 A) Blood toxicity ( Figure 7 G& Figure 15 B) Hepatotoxicity Figure 7 E&H), nephrotoxicity ( Figure 7 E&I) and cardiotoxicity ( Figure 7 E& Figure 15 C), indicating that BCgel has good biosafety.

[0062] Five months after tooth extraction, the new alveolar bone trabeculae were completely ossified in the BCgel group ( Figure 7 J). In addition, quantitative analysis of micto-CT results showed that compared with the Ctrl group, the BCgel group had a significant increase in BMD by 105%, an increase in BV / TV by 143%, an increase in Tb.N by 56%, and a decrease in Tb.Sp by 60% ( Figure 7 K). These findings strongly demonstrate the strong bioactivity of BCgel in promoting osteogenesis. Consistent with these results, histological staining of decalcified tissue sections, including H&E, Masson staining, and VG staining, showed that the extraction sockets in the BCgel group had almost complete bone healing, while the healing level in the gel-treated control group was less than half ( Figure 7 Importantly, no signs of physiological toxicity, including hematologic toxicity ( Figure 7 N), hepatotoxicity ( Figure 7 M&O), nephrotoxicity ( Figure 7 M&P) and cardiotoxicity ( Figure 7 M& Figure 15 D). Overall, BCgel exerted a potent osteogenesis-enhancing effect in the beagle dog model of blood clot deficiency (BCD) while maintaining good biosafety.

[0063] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. Application of a support in preparing a blood clot biomimetic gel, characterized in that: The blood clot biomimetic gel activates integrin-mediated focal adhesion signaling and downstream force-sensitive transcription factor Runx2 to promote bone formation; The blood clot biomimetic gel promotes bone formation in bone defect areas in individuals with clotting difficulties; At 1% strain, the support has a storage modulus of 10 to 5500 Pa and a loss modulus of 0 to 650 Pa; The support is a gel, and the gel is formed by protein and a dispersion medium; The protein is Ca 2+ Chelating peptide, the dispersion medium is Ca-containing 2+ of solution; The Ca 2+ The chelating peptide comprises a gelatinous peptide motif and a calcium binding motif; the gelatinous peptide motif is (RADA) 4, the calcium binding motif is GSVLGYIQIR; the Ca 2+ The mass concentration of the chelating peptide in the dispersion medium is 0.1-4%. 2+ Ca in solution 2+ The concentration is 0.02~1mg / mL.

2. The use according to claim 1, characterized in that The Ca 2+ The mass concentration of the chelating peptide in the dispersion medium is 3%, and the Ca-containing 2+ Ca in solution 2+ The concentration is 0.1 mg / mL.

Images