Pure inorganic waxy polyphosphate and application thereof in preparation of bone repair material

Abstract

The invention provides pure inorganic waxy polyphosphate and application thereof in preparation of a bone repair material, and belongs to the technical field of medical materials.The preparation method of the pure inorganic waxy polyphosphate comprises the following steps that a sodium polyphosphate solution and a divalent cation solution are mixed and chelated to obtain waxy co-doped polyphosphate CoPPW; the divalent cation solution is a solution containing any one, two or three of Ca < 2 + >, Mg < 2 + > and Sr < 2 + >. The pure inorganic waxy active material provided by the invention has certain strength, can be molded and degraded, and has the effects of resisting bacteria, stopping bleeding, repairing bone defects and the like.

Description

[0001] The present application is a divisional application of Chinese Patent Application No. 202411414465.1, entitled "A Pure Inorganic Waxy Active Material and Its Preparation Method and Application", filed on October 11, 2024. TECHNICAL FIELD

[0002] The present application belongs to the technical field of medical materials, and particularly relates to a pure inorganic waxy polyphosphate and its application in preparing bone repair materials. BACKGROUND

[0003] Bone defect refers to a situation in which the integrity of the structure of bone is destroyed due to congenital or acquired reasons. There are various reasons for bone defect, including acute bone loss, debridement after bone infection, and nonunion or bone loss of blood supply after radiotherapy or bone tumor resection. At present, effectively solving the key bone defects caused by different reasons is a major challenge in clinical treatment. Bone, as a hard calcified tissue mainly composed of a biomaterial phase and an organic matrix, is considered to be difficult to self-repair, so external intervention is needed to promote its repair and regeneration. At present, the main method is to use synthetic bone substitutes for repair. Generally speaking, in the field of tissue engineering, materials replicating the structure, mechanics and biological characteristics of natural bone are widely used to fill bone defects and promote in situ bone regeneration. Two different materials, hard materials and soft biomaterials, have been widely studied, namely bioactive ceramics and gels. Their applications are mainly in the form of particles, porous scaffolds, putty and injectable forms (cement and hydrogel), and deformable forms attract more attention due to easy handling.

[0004] Sodium polyphosphate (NaPP) is a highly water-soluble inorganic polymer commonly used to prepare polyphosphate solutions. Polyphosphate solutions have strong chelating ability for multivalent cations in water. According to the difference in ionic radius, the addition of multivalent cations will cause the phase separation of the solution, i.e. larger cations (Ba 2+ , Sr 2+ and Ag + ) will cause flocculation, while smaller cations (Ca 2+ , Mg 2+ and Zn 2+) will lead to coacervation. The definition of a polyphosphate coacervate (CPP) is a separation consisting of a dense colloid phase containing mainly long polyphosphate chains chelated with metal ions and a balanced liquid phase called supernatant. Polyphosphate coacervation precipitation is the result of electrostatic repulsion forces and van der Waals attractive interactions with polynuclear multivalent cation chelation. Therefore, by adjusting the solution pH, precursor concentration, multivalent cation type and average degree of polymerization, the coacervation process can be controlled. Polyphosphate coacervates have great potential as guided matrices for bone repair because they meet the typical requirements for bone regeneration, such as biodegradability to stimulate calcium-phosphate mineralization and leave enough space for new bone formation; osteogenesis and osteo-compatibility to promote bone regeneration; and controllable biological properties and injection ability through chelation with the desired cations. These properties are similar to synthetic hydrogels such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and polyacrylamide (PAM), which are currently commonly used for bone repair and regeneration. Although there is much research on the development of CPPs as biomaterials, there are few reports on the use of CPPs as hydrogel-like structures for regeneration, and there is no research on wax-like structures.

[0005] Although both hydrogels and waxes can be reformed, they are two different types of materials, hydrogels are biphasic materials containing a polymer matrix and a medium, mainly water. While waxes are solid materials with less internal medium, higher hardness and higher viscosity, which can be used for occlusion and as a physical barrier. A typical wax is composed of beeswax, salicylic acid and almond oil, which is completely organic and hydrophobic. Due to the composition and properties of the wax, the biological application of the wax is limited, one of which is hemostasis in orthopedic surgery. However, the wax is not degradable, has no biological activity, has no osteogenic effect, and will cause more infection. It is still a challenge to develop a new generation of wax-like materials with multiple functions such as biological activity, degradability and antibacterial properties. Further problems and challenges are whether a wax-like material can be made based on inorganic materials rather than polymers. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a pure inorganic wax-like polyphosphate and its application in the preparation of bone repair materials, which has certain hardness, formability, degradability, antibacterial, hemostatic, bone defect repair and other functions.

[0007] The present application provides a preparation method of a pure inorganic wax-like active material, comprising the following steps: Mixing a sodium polyphosphate solution with a divalent cation solution to obtain a wax-like co-doped polyphosphate CoPPW by chelation; The divalent cation solution is a solution containing any one or several of Ca 2+ , Mg 2+ and Sr 2+ .

[0008] Preferably, the concentration of the sodium polyphosphate solution is 0.1-1.5 mol / L, and the average polymerization degree of the sodium polyphosphate is 10-100.

[0009] Preferably, the solution containing Ca 2+ is a calcium chloride solution or a calcium nitrate solution, the solution containing Mg 2+ is a magnesium chloride solution or a magnesium nitrate solution, the solution containing Sr 2+ is a strontium chloride solution or a strontium nitrate solution, and the total concentration of the divalent cations in the divalent cation solution is 0.1-1.5 mol / L.

[0010] Preferably, the molar ratio of the sodium polyphosphate solution to the total divalent cations in the divalent cation solution is 1:1-1:5.

[0011] Preferably, the chelation is accompanied by stirring, the chelation time is 0.15-2 h, and the chelation temperature is 10-60℃.

[0012] The application provides a wax-like co-doped polyphosphate CoPPW prepared by the preparation method, which comprises sodium polyphosphate, divalent cations and water, the molar ratio of the divalent cations to the sodium polyphosphate is 5:1-1:1, and the water content of the wax-like co-doped polyphosphate CoPPW is 25%-50%.

[0013] The application also provides application of the wax-like co-doped polyphosphate CoPPW in preparation of a hemostatic material.

[0014] The application also provides application of the wax-like co-doped polyphosphate CoPPW in preparation of a material for repairing bone defects.

[0015] The application also provides application of the wax-like co-doped polyphosphate CoPPW in preparation of an agent for promoting cell proliferation, promoting expression of osteogenesis genes, inducing new bone formation or inducing extracellular matrix formation.

[0016] Compared with the prior art, the application has the following beneficial effects: the pure inorganic wax-like active material provided by the application is prepared by chelating sodium polyphosphate with divalent cations, by limiting the concentration and molar ratio of the sodium polyphosphate and the divalent cations and by selecting the specific type of the divalent cations, the prepared pure inorganic wax-like active material has certain hardness, can be formed, is degradable, has antibacterial, hemostatic and bone defect repairing effects.

[0017] According to the description of the embodiments of the present application, the CoPPW prepared by the present application has ductility and a faster degradation rate. In vitro studies show that CoPPW can better promote cell proliferation and osteogenic differentiation than beeswax, which may be due to the release of polyphosphate, magnesium and strontium ions. By analyzing the mRNA expression level of osteogenic related genes and the expression of bone related proteins through RT-PCR and transcriptome sequencing, it is found that CoPPW significantly up-regulates the expression of transcription factors BGLPA, OGN, OPN and COL1A1. In addition, the up-regulation of the PI3K / AKT signaling pathway confirms that CoPPW has osteogenic effect, while the expression of the coagulation cascade pathway indicates that CoPPW can effectively stop bleeding. In vivo studies show that CoPPW is more effective than bone wax in promoting bone regeneration and hemostasis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Schematic diagram of a waxy co-doped polyphosphate CoPPW material for repairing bone defects, wherein (A) is that by chelating sodium polyphosphate with various divalent cations, the material CoPPW is transformed from a hydrogel into a wax; (B) is that after implantation in a rabbit model, CoPPW shows excellent hemostatic and osteogenic effects.

[0019] Figure 2 Appearance of CoWPP with different compositions, the photo is taken after the sample is pressed once by a punch and lifted.

[0020] Figure 3 Performance comparison of CoWPP with different compositions, wherein (A) is a representative photo of CaSr and MgSr samples; (B) is the dependence of viscosity on shear rate of CoPPW; (C) is the viscosity-shear rate of MgSr2.5 and bone wax measured after 1 day and 4 days; (D) is a scanning electron microscope-EDX element map of the MgSr2.5 sample, showing the distribution of strontium, phosphorus and magnesium elements; (E) is the cumulative release of magnesium, strontium and phosphorus ions in the MgSr2.5 sample; the dependence of weight loss percentage on time is shown in the image; (F) is the applied pressure-stroke curve of the bone wax and MgSr2.5 sample (insert image: in vivo blood occlusion test).

[0021] Figure 4 Experimental schematic diagram of hemostatic test, A is to insert about 2 mL of CoWPP gel into the needle, B is to apply load to the needle by a mechanical tester.

[0022] Figure 5 Viscoelasticity of CoWPP sample showing loss modulus and storage modulus.

[0023] Figure 6 Viscoelasticity of commercial bone wax.

[0024] Figure 7Viscosity-shear rate for CaMg samples after 4 days of preparation.

[0025] Figure 8 Appearance of CaSr samples after 4 days of preparation showed that the samples solidified.

[0026] Figure 9 Cumulative ion concentration of Ca, Mg and P ions released from CaMg2.5 samples in PBS solution.

[0027] Figure 10 Representative XRD pattern of CoWPP, showing that CaMg and CaSr crystallized after drying at 120 °C, forming Ca(H2PO4)2H2O crystals (PDF: 96-231-0631); while MgSr2.5 remained amorphous.

[0028] Figure 11 Representative SEM morphology of CoWPP powder after drying at 120 °C for 72 h.

[0029] Figure 12 In vitro biocompatibility of CoPPW-MSP with bone wax, where (A) is the CCK-8 results in different groups, showing the dependence of OD value on culture days; (B) is the EDU staining of the blank group; (C) is the EDU staining of high concentration CoPPW-MSP (H-CoPPW); (D) is the EDU staining of low concentration CoPPW-MSP (L-CoPPW-MSP); (E) is the EDU staining of high concentration bone wax (H-bone wax); (F) is the EDU staining of low concentration bone wax (L-bone wax), where green dots represent cells that are dividing and proliferating, and blue dots represent normal cell nuclei.

[0030] Figure 13 Osteogenic properties of CoPPW-MSP, where (A) is the ALP staining of hBMSCs cultured with normal cell medium (blank group), L-bone wax group, L-CoPPW-MSP group, H-bone wax group and H-CoPPW-MSP group at day 14; (B) is the alizarin red S staining of each of the above groups; (C) is the gene expression (BGLAP, OGN, OPN and COL1A1) of h-BMSCs cultured with blank group, L-bone wax group, L-CoPPW-MSP group, H-bone wax group and H-CoPPW-MSP group evaluated by q-PCR, where p < 0.05, p < 0.01, p < 0.001.

[0031] Figure 14Transcriptome analysis of h-MSC function and mechanism regulated by CoPPW-MSP, wherein (A) is a heat map distribution of CoPPW-MSP group and control group, up-regulated genes are marked with red, and down-regulated genes are marked with blue; (B) is a volcano plot of CoPPW-MSP group and control group; (C) is gene GO enrichment of CoPPW-MSP group and control group; (D) is KEGG analysis of CoPPW-MSP group and control group; (E) is a related gene showing PI3K-Akt and ECM-receptor interaction signaling pathways (q value < 0.05) enriched in KEGG metabolic pathways.

[0032] Figure 15 Comparison of implantation effects of CoPPW-MSP and bone wax, wherein (A) is a micro-CT image of CoPPW-MSP and bone wax groups after implantation for 4 weeks and 8 weeks; (B) is a quantitative analysis of bone volume of CoPPW-MSP group and bone wax group after implantation for 4 weeks and 8 weeks; (C) is H&E staining of bone defect areas of bone wax group and CoPPW-MSP group after implantation for 4 weeks and 8 weeks; (D) is Masson staining of bone defect areas of bone wax group and CoPPW-MSP group after implantation for 4 weeks and 8 weeks.

[0033] Figure 16 CoPPW-MSP and bone wax hemostatic effect, A: New Zealand white rabbit femur drilling, more bleeding in the medullary cavity; b: CoPPW is used for hemostasis at the drilling site, and no fresh blood flows out after waiting for 1 minute after filling, and the hemostatic effect is satisfactory. c: New Zealand white rabbit femur drilling, more bleeding in the medullary cavity. d: Johnson bone wax is used for hemostasis at the drilling site, and no fresh blood flows out after waiting for 1 minute after filling, and the hemostatic effect of CoPPW-MSP and bone wax is the same. DETAILED DESCRIPTION

[0034] The application provides a preparation method of a pure inorganic waxy active material, which comprises the following steps: mixing a sodium polyphosphate solution and a divalent cation solution to obtain a waxy co-doped polyphosphate CoPPW by chelation; the divalent cation solution is a solution containing any two of Ca 2+ , Mg 2+ and Sr 2+ .

[0035] In the application, the sodium polyphosphate solution is first mixed with the divalent cation solution. In the application, the concentration of the sodium polyphosphate solution is 0.1-1.5 mol / L, preferably 0.3-0.8 mol / L, further preferably 0.4-0.7 mol / L, and more further preferably 0.5-0.6 mol / L; and in the application, the average polymerization degree of the sodium polyphosphate is preferably 10-100, and further preferably 20-50. In the application, the sodium polyphosphate is preferably mixed with water to prepare the sodium polyphosphate solution, and the water is preferably deionized water.

[0036] In the present application, the divalent cation solution is a solution comprising any one or several of Ca 2+ , Mg 2+ and Sr 2+ , the solution comprising Ca 2+ is a calcium chloride or calcium nitrate solution, the solution comprising Mg 2+ is a magnesium chloride or magnesium nitrate solution, and the solution comprising Sr 2+ is a strontium chloride or strontium nitrate solution, and the total concentration of divalent cations in the divalent cation solution is preferably 0.1-1.5 mol / L, further preferably 0.3-0.8 mol / L, and more further preferably 0.5 mol / L; when the divalent cation solution preferably comprises Ca 2+ and Mg 2+ , the molar ratio of Ca 2+ to Mg 2+ is preferably 1:5-5:1; when the divalent cation solution preferably comprises Ca 2+ and Sr 2+ , the molar ratio of Ca 2+ to Sr 2+ is preferably 1:5-5:1; when the divalent cation solution preferably comprises Mg 2+ and Sr 2+ , the molar ratio of Mg 2+ to Sr 2+ is preferably 1:5-5:1; and when the divalent cation solution preferably comprises Ca 2+ , Mg 2+ and Sr 2+ , the molar ratio of Ca 2+ to Mg 2+ to Sr 2+ is (1-5):(5-1):(1-5). In the present application, the solution comprising Ca 2+ is a calcium chloride or calcium nitrate solution, the solution comprising Mg 2+ is a magnesium chloride or magnesium nitrate solution, and the solution comprising Sr 2+ is a strontium chloride or strontium nitrate solution.

[0037] In the present application, after mixing the sodium polyphosphate solution with the divalent cation solution, chelation is performed, and the chelation is preferably accompanied by stirring, the stirring speed is preferably 100-500 rpm, the chelation time is preferably 0.15-2 h, and the chelation temperature is preferably 10-60°C. After the chelation is completed, the obtained coagulum is precipitated, and the wax-like co-doped polyphosphate CoPPW is obtained by solid-liquid separation.

[0038] The application further provides the wax-like co-doped polyphosphate CoPPW prepared by the preparation method, which comprises sodium polyphosphate, divalent cations and water; the molar ratio of the divalent cations to the sodium polyphosphate is preferably 5:1-1:1; and the water content of the wax-like co-doped polyphosphate CoPPW is preferably 25%-50%.

[0039] The application further provides application of the wax-like co-doped polyphosphate CoPPW in preparation of a hemostatic material.

[0040] The application further provides application of the wax-like co-doped polyphosphate CoPPW in preparation of a material for repairing bone defects.

[0041] The application further provides application of the wax-like co-doped polyphosphate CoPPW in preparation of an agent for promoting cell proliferation, promoting expression of osteogenesis genes, inducing new bone formation or inducing extracellular matrix formation.

[0042] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0043] Examples

[0044] The average polymerization degree (Mn) of the commercial sodium polyphosphate (NaPP) used is 20 (Thermo Fisher Scientific Inc., USA). Calcium chloride (calcium chloride, Thermo Fisher Scientific Inc., USA), magnesium chloride (magnesium chloride, Sigma-Aldrich, USA) and strontium nitrate (Sr(NO3)2, Sigma-Aldrich, USA) are used as divalent cation (M 2+ ) chelation sources. Two of the calcium chloride, the magnesium chloride and the strontium nitrate are dissolved in deionized water to prepare cation solutions with different concentrations. The cation solution is added to the NaPP solution, and stirred for 0.5 h until the coagulum precipitates at the bottom of the solution. Different molar ratios of divalent cations to phosphorus (M 2+ / P) are shown in Table 1. For double-ion chelation, three different molar ratios of M / / P are prepared, i.e. 3, 2.5 and 1.5.

[0045] Table 1 Sample composition and water content of the wax-like co-doped polyphosphate CoPPW

[0046] Material characterization: Chemical composition and water content of CoPPW: The CoPPW obtained above was washed with deionized water and then... The powder was dried in an oven at a specific temperature for 72 hours. X-ray diffraction (XRD, D8advance, Bruker, USA) was used to determine the polarization of the dried powder between 10° and 8°. Within the range, the scan step size is 0.2 s / step, and the step size is 0.0. The microstructure and elemental distribution of CoPPW were analyzed using a scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (EDS) (SEM, Merlin, Zeiss, Germany). The water content of CoPPW was calculated using the formula: Water content (%) = (W 湿 W 干 ) / W 湿 ×100%, where W 湿 and W 干 The weights are wet and dry CoPPW, respectively.

[0047] The results are as follows Figure 2 As shown, the image of the newly synthesized CoPPW is as follows: Figure 2 and Figure 3 As shown in A, the CaSr sample appears white and opaque, while the CaMg and MgSr samples appear grayish-white. The MgSr sample is more translucent than the other samples. Elemental composition was analyzed using SEM-EDS, as shown in... Figure 3 As shown in Figure D, the EDS elemental diagram of the dried MgSr2.5 shows a uniform distribution of Mg, Sr, and P elements. The experimentally obtained molar ratio of cations to phosphorus (Mⅱ / P) is lower than the theoretical value, indicating that some cations were not chelated to NaPP during preparation but remained in the supernatant. As shown in Table 1, the water percentage in CoPPW ranges from 30 wt% to 43 wt%.

[0048] The concentration of divalent cations plays a crucial role in determining chelating ability. In divalent cation-phosphate systems, the occupancy of metal ions depends on the concentration of the divalent cations; at low concentrations, cations are trapped within the polyphosphate cages, while at higher concentrations, the cages become saturated, and thus the cations are chelated to the external polyphosphate chains 15 and 16. Therefore, the precipitation of the condensate layer depends on van der Waals attraction and electrostatic repulsion. The critical concentration (M) leading to precipitation... The concentration and the average degree of polymerization of NaPP are determined by the concentration.

[0049] Rheological properties of CoPPW: The rheological properties of CoPPW were evaluated using a rheometer (Discovery HR10, TA instruments, USA). The dependence of viscosity on shear rate was measured in the range of 0.1-150 s -1 and 3 , respectively. Rheological measurements were performed after one hour of coacervation. All samples were measured after 1 day and 4 days to determine the change in viscosity over time.

[0050] The effect of divalent cation complexation on the rheological properties of CoPPW was investigated in dynamic frequency sweep. All CoPPW exhibited shear thinning behavior, with the viscosity decreasing as the shear rate increased (B in FIG. 1). The viscosity of the CaSr sample was higher than that of the other samples, except for the MgSr3 sample. The viscoelastic behavior of CoPPW varied with different types and amounts of cation complexation. In the MgSr3 and MgSr2.5 samples, the loss modulus (G'') was higher than the storage modulus (G'), and both (G'') and (G') increased with increasing frequency, indicating that CoPPW exhibited more viscous fluid properties. In the CaSr3 and CaMg samples, (G') was higher than (G''), indicating solid-like behavior. As shown in FIG. 2, the change in viscosity of CoPPW samples was measured after 1 day and 4 days. It can be seen that after 4 days, only the CaMg sample and the MgSr2.5 sample were moldable, while the other samples were solidified (as shown in FIG. 3). Specifically, the comparison of the viscosity of the MgSr2.5 sample with that of bone wax is shown in C of FIG. 4. After 1 day, the MgSr2.5 was still in a liquid-like gel structure, with a lower viscosity than the wax; after 4 days, the MgSr2.5 became solidified and could be shaped. Figure 3 Figure 9 Figure 10 Figure 3

[0051] CoPPW had very high viscosity, which was consistent with its waxy appearance. This could be due to the multiple effects of double cation complexation. In polyelectrolyte solutions, the addition of cations has opposite effects on viscosity. At low concentrations, cation complexation reduces viscosity due to chain breakage caused by charge shielding effects, while at high concentrations, cation complexation increases viscosity due to increased interaction between chains. The water content of CoPPW was between 32% and 43%, indicating that the samples were highly concentrated. Therefore, the higher the cation content, the higher the viscosity. The viscosity of MgSr and CaSr was also higher than that of CaMg, which could be due to the fact that Sr 2+ is more effective in cross-linking polyphosphate chains. ​​​​​

[0052] In vitro degradation behavior and ion release: By immersing the sample in 3 The in vitro degradation behavior of CoPPW was evaluated in PBS solution at temperature C. The ratio of PBS volume (mL) to sample weight (g) was 10 mL: 1 g. PBS solution was collected on days 1, 7, 14, 21, 28, 35, and 46 to measure weight loss. The percentage of weight loss was determined by the ratio of (original weight - residual weight) to original weight. Ion release was also investigated. At each time interval, 1 mL of supernatant was collected and replaced with fresh PBS solution. The collected supernatant was analyzed by inductively coupled optical emission spectrometry (Inductor, Inc., USA) to determine Sr. 2+ Mg 2+ and P 4+ Cumulative concentration of ion release. Solutions were collected on days 1, 2, 4, 6, 8, 14, and 21.

[0053] The degradation of MgSr2.5 and CaMg2.5 was investigated by immersing samples in PBS solution. The percentage weight loss of MgSr2.5 over time is shown in the figure. Figure 3 As shown in Figure E. During the initial week, the MgSr2.5 sample absorbed water, increasing in weight by 10%, followed by a gradual decrease in weight over the next week. Significant weight loss was observed between days 14 and 28, with a 37% weight loss on day 28. At the end of the 46-day measurement period, the weight loss of MgSr2.5 was approximately 41%. Furthermore, the release of Mg from MgSr2.5 was analyzed using ICP-OES. 2+ 、Sr 2+ and P 5+ Ion concentration. Consistent with the results of weight loss, the ions released from MgSr2.5 were released slowly over the first week, without any sudden release. P 5+ The release of ions was higher than that of divalent cations, indicating a high Mⅱ / P ratio in the remaining MgSr₂.₅; after 21 days, Mg 2+ 、Sr 2+ and P 5+ The cumulative release concentrations of ions were 2.57, 2.34, and 7.89 × 10⁻⁶, respectively. 3 mg·L -1Due to the presence of water, CPPs are prone to degradation, with P-O-P bonds dissociating to form trimetaphosphate and orthophosphate. The presence of cations catalyzes this process by neutralizing the charge on the chain. In previous studies, CPPs remained stable for the first two days, then rapidly degraded, resulting in significant weight loss and a sudden release of ions. In contrast, in the present invention, MgSr2.5absorbed moisture in the first week and slowly degraded over the next week, indicating its potential for long-term use in the body. 2+ and Mg 2+ The slow dissolution of ions can be the reason for the slow degradation of MgSr2.5.

[0054] Evaluation of hemostatic ability: To compare the hemostatic ability of CoPPW with commercial bone wax (B Braun Surgical SA, Spain), the maximum force to initiate water leakage was measured using a universal mechanical testing machine (Autograph AGS-H; Shimadzu, Japan). The bone wax consists of 75% beeswax, 15% paraffin wax, and 10% isopropyl palmitate. The experimental setup is shown in Figure 4 Specifically, about 2 mL of sample was inserted into a syringe and 5 mL of water was injected. Then, this force was applied to the syringe until water dripped down and the test stopped.

[0055] The results are shown in F of Figure 3 When the load was applied, the sample was pushed until the maximum pressure was reached, after which water started to leak and the pressure dropped. The maximum pressure applied to MgSr2.5to initiate water leakage was about twice that of the wax, indicating that the MgSr2.5sample had excellent occlusion ability. Figure 3 The images inserted in F of

[0056] Evaluation of biocompatibility of CoPPW

[0057] In the present invention, MgSr2.5-CoPPW (strontium magnesium polyphosphate, CoPPW-MSP) was chosen to continue the biological evaluation, taking into account the viscosity and potential ion effects.

[0058] Human bone marrow mesenchymal stem cells (h-BMSCs; ScienCell, #7500, USA) were cultured in h-MSC basal medium (Cyagen, HUXMA-90, 011, China) containing 10% fetal bovine serum (Cyagen, HUXMA-90, 011, China), 1% antibiotics (Cyagen, HUXMA-90, 011, China), 37°C, 95% air, and 5% CO2. 1 g of MgSr2.5-CoPPW was added to 10 ml of cell culture medium and mixed at 37°C with 95% air and 5% CO2 for 24 hours. The supernatant was collected and filtered three times through a 0.22 μm filter membrane (Merck Millipore, USA) to obtain the biomaterial extract. Finally, the supernatant was diluted 5-fold and 10-fold with cell culture medium to obtain high-concentration and low-concentration biomaterial extract culture media, respectively.

[0059] The effects of the biomaterial MgSr2.5-CoPPW on the proliferation and cytotoxicity of h-MSCs were evaluated using a CCK-8 assay (CCK-8, purchased from Dojindo, Japan). In short, h-MSCs were cultured at 2 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 1.5 × 10⁶ cells / well in 96-well plates and cultured in h-MSC medium and MgSr2.5-CoPPW extraction medium, respectively. After 1, 2, 3, 5, and 7 days of culture, CCK-8 solution was added to the wells and reacted for 2 hours. The absorbance at 450 nm was measured for each well. h-MSCs were then cultured at a density of 1.5 × 10⁶ cells / well. 5 Cells were seeded at a density per well in 6-well plates. The blank group was cultured in normal cell culture medium, while the control and experimental groups were cultured in high- and low-concentration extracts, respectively. On day 7 of culture, edu (5-ethynyl-2'-deoxyuridine) staining was performed using the EDU kit (Beyotime, China) according to the manufacturer's protocol, and photographs were taken.

[0060] h-MSCs were 2×10 5 Cells were seeded at a density of 12-well plates and stimulated for 14 days with cell culture medium and biomaterial extraction buffer. Total RNA was extracted from BMSCs using Trizol reagent (Invitrogen, California, USA). cDNA was synthesized using a one-step SYBR Primescript qPCR kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed using thermal cycling conditions on a Bio-Rad CFX96 real-time PCR system with SYBR Premix Ex Taq (TaKaRa).

[0061] The following specific primers were used to amplify cDNA fragments via real-time PCT.

[0062] Table 2 Specific primer sequences

[0063] Results are shown in A of Figure 12 , the optical density (OD) values of bone wax and CoPPW-MSP gradually increased after 1, 2, 3, 5 and 7 days of culture, indicating an increase in the number of living cells. After 5 days, the cell viability of the low and high concentration CoPPW-MSP groups was higher than that of the bone wax and blank groups. To further confirm the CCK-8 results, on the 7th day of h-BMSC culture, different concentration treatment groups were subjected to edu (5-ethynyl-2'-deoxyuridine) staining Figure 12 B-F of ). In the low concentration CoPPW-MSP group, the mesenchymal stem cells undergoing replication and division were significantly higher than in the other groups. The cell proliferation rate of the low concentration CoPPW-MSP group was 29.3% Figure 12 A), higher than that of the blank group (20.8%) and the low concentration bone wax group (21.7%).

[0064] Alizarin red staining and alkaline phosphatase staining

[0065] h-BMSCs were seeded at 2 x 10 5 cells / well in 6-well plates and treated with cell culture medium containing or not containing biomaterial extract. After 14 days of culture, alizarin red staining (Cyagen, HUXMA-90, 021, China) was used to detect calcium deposition by h-BMSCs. Inverted microscopy (Nikon, Japan) was used to observe mineralized particles. After 14 days of culture in MSCs treated with cell culture medium containing or not containing biomaterial extract, alkaline phosphatase staining (Beyotime, China) was performed on all groups and photographs were taken under a microscope (Nikon, Japan).

[0066] The ions released from CoPPW-MSP material can stimulate cell proliferation and replication at a certain concentration, and there is no significant difference between CoPPW-MSP and bone wax in the first 3 days Figure 12 A), which is attributed to the slow release of Mg 2+ and Sr 2+ ions (as shown in E of Figure 3 ). After 5 days, the release of Mg 2+ and Sr 2+ ions from CoPPW-MSP increased, promoting cell proliferation and division.

[0067] In vitro osteogenic performance of CoPPW-MSP

[0068] After 14 days of stimulation of the culture medium with CoPPW-MSP and bone wax, further investigation of the osteogenic properties induced by CoPPW-MSP and bone wax was performed by alkaline phosphatase (ALP) and alizarin red S staining. As shown in Figure Figure 13 , there were more ALP-positive cells in the high and low concentration CoPPW-MSP groups compared to the bone wax and blank groups. In addition, the ALP-positive rate of the high concentration CoPPW-MSP group was higher than that of the low concentration CoPPW-MSP group. As shown in Figure Figure 13 , calcium deposition, as an indicator of the late stage of osteogenesis, was evaluated by alizarin red staining of the h-MSCs extracellular matrix. The calcium nodule staining in the CoPPW-MSP group was more intense and obvious compared to the blank and bone wax groups. In addition, increasing the concentration of CoPPW-MSP further promoted the formation of calcium nodules. Quantitative real-time polymerase chain reaction (q-PCR) was used to explore the potential osteogenic mechanism of CoPPW-MSP at the gene expression level. As shown in Figure Figure 13 , the gene expression of osteoglycin (OGN), osteopontin (OPN), collagen type I alpha 1 (col1a1), and osteocalcin (BGLAP) in the CoPPW-MSP group was significantly higher than that in the control and bone wax groups. In addition, high concentration CoPPW-MSP showed the most significant effect on the expression of these osteogenesis-related genes, including OGN, OPN, COL1A1, and BGLAP, indicating that CoPPW-MSP has excellent osteogenic ability. The typical bone formation process includes cell proliferation, extracellular maturation, and mineralization. ALP activity and the formation of mineralized nodules are used to evaluate the early stage of osteogenesis. In the present invention, CoPPW may promote osteogenic differentiation and mineralization because it shows a better expression effect on osteogenesis-related genes (Figure Figure 13 C). This may be due to the release of Sr 2+ and Mg 2+ ions. The osteogenic and angiogenic ability of strontium-doped calcium polyphosphate bioceramics (Sr-CaPP) has been studied, however, their hardness does not meet the clinical requirements of waxiness and stickiness. In the present invention, CoPPW-MSP shows excellent osteogenic ability while maintaining excellent waxiness, indicating its good application prospects in bone repair and regeneration.

[0069] Potential mechanisms of CoPPW-MSP osteogenesis and coagulation

[0070] Transcriptome sequencing and data analysis

[0071] The samples were divided into three groups: a control group (undifferentiated h-BMSCs), a bone wax group, and a CoPPW-MSP group. h-BMSCs from all three groups were cultured in 6-well Trans plates for 14 days to allow for stimulated differentiation. Each group had three replicates. Cells were isolated using trypsin, suspended to remove the supernatant, and stored in liquid nitrogen. RNA sequencing analysis was performed at the Beijing Genomics Institute. Furthermore, network and pathway analysis was performed using the IPA analysis software.

[0072] Further transcriptome sequencing of h-BMSCs was performed to explore the potential osteogenic mechanism induced by CoPPW-MSP. The heatmap revealed differentially expressed genes in h-BMSCs ( Figure 14 In the group A), upregulated genes are marked in red, and downregulated genes are marked in blue. Compared with the bone wax group, the total number of differentially expressed genes (p-value < 0.05 and |log2-fold change| > 0) was 3226, including 1881 upregulated genes and 1345 downregulated genes (e.g., A). Figure 14 (As shown in B). GO enrichment was applied to analyze these differentially expressed genes, most of which are involved in osteogenic and extracellular matrix processes, including extracellular matrix and structural tissue, collagen metabolism, cell adhesion molecule binding, growth factor binding, and collagen binding. Figure 14 (C in the text). Based on these significantly differentially expressed genes, key osteogenic signaling pathways were analyzed using the KEGG metabolic pathway, such as... Figure 14 As shown in D in the diagram. KEGG signaling pathway analysis revealed a high enrichment of the PI3K / Akt signaling pathway and the ECM receptor interaction signaling pathway, which play important roles in promoting osteogenic differentiation of h-BMSCs. The PI3K / Akt signaling pathway is an important lipid kinase activation pathway that significantly regulates osteogenic differentiation, proliferation, and apoptosis of MSCs. AKt phosphorylation can upregulate the Runx2 gene and induce the expression of bone repair and remodeling-related genes, such as BGLAP, COL1, OPN, and ALP26. In this invention, gene sequencing ( Figure 14 The results (E) show that CoPPW-MSP effectively activates the PI3K / Akt signaling pathway, further affecting the mTOR signaling pathway through REDD1 overexpression. The activated AKT-mTOR signaling pathway promotes MSC differentiation into osteoblasts and increases mRNA translation rate, thereby increasing cellular protein synthesis. Furthermore, the overexpression of the downstream target protein p53, activated by AKT, can promote cell proliferation and cell cycle progression. Figure 14 E in CoPPW-MSP releases Mg. 2+ and Sr 2+ Ions enhance the activation of the PI3K / Akt signaling pathway, thereby upregulating the expression of osteogenic genes and proteins and promoting osteoogenesis. Simultaneously, CoPPW-MSP also activates the ECM-receptor interaction signaling pathway (…).Figure 14 D) in Figure 1. This signaling pathway affects collagen metabolism and synthesis in bone and plays an important role in regulating cell adhesion, thus promoting the formation of extracellular matrix. The upregulation of ECM-receptor interaction signaling pathway is consistent with the significant increase in collagen type I expression in CoPPW-MSP in QT-PCR Figure 13 C) in Figure 1. On the other hand, CoPPW-MSP activates complement and coagulation cascade signaling pathways Figure 14 E) in Figure 1, which plays an important role in subsequent cellular responses that affect anti-inflammatory and tissue repair. In the coagulation cascade response of CoPPW-MSP, due to activated coagulation factor VIII and coagulation factor X, the expression of PAR1, PAR3, and PAR4 is upregulated, leading to the release of anti-inflammatory mediators and increased endothelial permeability. Polyps have been shown to accelerate blood clotting, with increased thrombin generation in the case of polyp-activated factor xi32. In addition, polyps slow down fibrinolysis, as they regulate the structure of fibrin clots, making them more resistant to fibrinolysis.

[0073] In vivo study: rabbit femoral defect model surgery

[0074] An improved femoral defect model was constructed to evaluate the repair of rabbit bone based on the method reported in the reference (Sun, Y., Helmholz, H. & Willumeit-Romer, R. Surgical Classification for Preclinical Rat Femoral Bone Defect Model: Standardization Based on Systematic Review, Anatomical Analysis and Virtual Surgery. Bioengineering 9, (2022).). First, the rabbits were anesthetized with 1% pentobarbital (50 mg / kg) by ear marginal vein injection. After the surgical area was shaved and sterilized, the animals were placed in a fixed position on the left side of the operating table. Next, a 5-mm incision was made around the femoral shaft. After the superficial fascia and deep fascia were dissected, the protuberance was identified as the drilling target. A Kirschner wire with a diameter of 5 mm was successively drilled into the target area to form a 5-mm-diameter, 10-mm-deep cavity defect from the lateral to the medial side in a direction perpendicular to the longitudinal axis of the femoral shaft. Critical defect sizes were created in 12 rabbits. Bone wax was used for the cavity defect in the left femoral shaft, while CoPPW-MSPs were implanted into the cavity defect of the right femoral shaft. All procedures strictly followed the aseptic principle, and each subject received a muscle injection of antibiotics on the first day after surgery. Rabbits were allowed to move freely after surgery without immobilization. At 4 and 8 weeks after surgery, the rabbits were sacrificed, and the bilateral femurs were collected for evaluation.

[0075] Micro-CT evaluation

[0076] To assess bone volume and density at specific Hu values, the lateral region of the femur was scanned using micro-CT (Siemens, Germany). The micro-CT scan had a resolution of 10.3 μm, a voltage of 70 kV, and a current of 400 mA. Images obtained using Dslicer software (Inveon Research Workplace) were used to construct a 3D model of the femur. Bone volume was calculated by identifying regions of interest using Hu signals ranging from 700 to 2100. The bone volume / total bone volume (BV / TV) ratio was then calculated using the bone volume of the selected regions. Histological analysis involved decalcifying the femoral samples and cutting them into 4 μm thick paraffin sections using a rotary microtome (RM2255, Leica, Germany). The sections were then stained with hematoxylin-eosin (HE) (607317-0100 & E607321-0100, Sangon Biotech, China) and Masson'strichrome (G1340, Solarbio, China) and examined under a microscope (Ci-S, Nikon, Japan) to detect bone formation.

[0077] Figure 15 Figure A shows μ-CT images of bone wax and CoPPW-MSP at 4 and 8 weeks post-implantation. At week 4, new bone formation was evident in the CoPPW-MSP group, while the femoral defect remained hollow in the bone wax group. Over time, the femoral defect gradually healed, with newly formed tissue partially filling the defect site. By week 8, the femoral defect was no longer visible in the CoPPW-MSP group, while in the bone wax group, only a small amount of new bone tissue was observed at the defect periphery. Quantitative micro-CT analysis of bone volume is shown below. Figure 15 As shown in Figure B, at weeks 4 (p=0.0055) and 8 (p=0.0151) post-implantation, the bone volume density (BV / TV) in the CoPPW-MSP group was significantly higher than that in the bone wax group. Histological analysis was performed using hematoxylin-eosin (H&E) staining and Masson staining to assess the degree of defect recovery. Figure 15 (C, D in the original text). In the bone wax group, the defect remained hollow 4 weeks after implantation, and only a small amount of fibrous tissue had formed beneath the defect by week 8. In contrast, the CoPPW-MSP group showed more newly formed bone towards the center of the defect at week 4, and by week 8, almost the entire defect was filled with newly formed bone, indicating that CoPPW-MSP has better bone regeneration capacity.

[0078] To observe the blood occlusion, the femur of New Zealand white rabbits was drilled to cause a large amount of blood to flow out of the defect. CoPPW-MSP and bone wax were used to stop bleeding, respectively. Both groups showed good occlusion effect Figure 16 ) In orthopedic surgery, hemostasis at bone defects is challenging. It is usually necessary to fill the defect with a bioadhesive waxy material such as beeswax to physically occlude the defect. However, current waxy materials cannot induce new bone formation. The pure inorganic waxy material CoPPW provided by the present application can quickly stop bleeding while inducing bone formation.

[0079] In summary, the present application first developed a pure inorganic waxy material based on ion-regulated polyphosphate formulations; by controlling the type and concentration of ions, the viscosity and modulus of CoPPW can be adjusted from gel-like to waxy with flowability and desired moldability. In vitro studies have confirmed that CoPPW has good biocompatibility and can promote cell proliferation. The synergistic release of magnesium and strontium ions can effectively promote the expression of osteogenic genes and exhibit regulation of the PI3K / AKT signaling pathway and ECM-receptor interaction, thereby inducing extracellular matrix and new bone formation. At the same time, CoPPW can activate the complement and coagulation cascade signaling pathways to play a hemostatic function. The proximal femoral defect model of New Zealand white rabbits verified its hemostatic effect, which is comparable to the hemostatic effect of existing bone wax. In addition to hemostasis, the CoPPW provided by the present application also promotes bone regeneration through in vivo self-degradation.

[0080] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A process for the preparation of a pure inorganic waxy polyphosphate salt, characterized in that, The method comprises the following steps: mixing a sodium polyphosphate solution with a divalent cation solution to obtain a waxy co-doped polyphosphate CoPPW by chelation; The divalent cation solution is any two of Ca 2+ , Mg 2+ , and Sr 2+ ; the concentration of the sodium polyphosphate solution is 0.1-1.5 mol / L, and the average polymerization degree of the sodium polyphosphate is 10-100; the total concentration of divalent cations in the divalent cation solution is 0.1-1.5 mol / L; the molar ratio of the sodium polyphosphate solution to the total divalent cations in the divalent cation solution is 1:1-1:5; the chelation is accompanied by stirring, the chelation time is 0.15-2 h, and the chelation temperature is 10-60℃; the water content of the waxy co-doped polyphosphate CoPPW is 25%-50%.

2. The production method according to claim 1, characterized by, when the divalent cation solution comprises Ca 2+ and Mg 2+ , the molar ratio of Ca 2+ , Mg 2+ and sodium polyphosphate is 2:1 :1, 2:0.5:1 or 1 :0.5:1 ; When the divalent cation solution comprises Ca 2+ and Sr 2+ , the molar ratio of Ca 2+ , Sr 2+ and sodium polyphosphate is 2:1:1, 2:0.5:1 or 1:0.5:

1.

3. The preparation method according to claim 1, characterized in that, including Ca 2+ The solution including Mg 2+ The solution including Sr 2+ The solution including Sr 4. The process according to any one of claims 1 to 3, characterized in that the pure inorganic waxy polyphosphate salt is obtained by the process according to any one of claims 1 to 3, wherein the process is carried out in the presence of a base. The waxy co-doped polyphosphate CoPPW comprises sodium polyphosphate, divalent cations and water, the molar ratio of the divalent cations to the sodium polyphosphate is 3:1, 2.5:1 or 1.5:1, and the water content of the waxy co-doped polyphosphate CoPPW is 25%-50%.

5. Use of the waxy co-doped polyphosphate CoPPW of claim 4 in the preparation of a material for repairing bone defects.

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