A pure inorganic waxy active material, its preparation and use

Abstract

The application provides a kind of pure inorganic waxy active material and its preparation method and application, belong to medical material technical field, the preparation method of the pure inorganic waxy active material includes the following steps: the solution of sodium polyphosphate is mixed with divalent cation solution, chelation obtains waxy co-doped polyphosphate CoPPW;The divalent cation solution is the solution of any one of Ca 2+ , Mg 2+ And Sr 2+ Two or three kinds.The pure inorganic waxy active material provided by the application has certain strength, can be formed, can be degraded, has antibacterial, hemostatic, repair bone defect and the like effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a pure inorganic waxy active material and a preparation method and application thereof. BACKGROUND

[0002] 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 causing bone defects, 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 biological mineral phase and an organic matrix, is considered to be difficult to self-repair, and thus requires external intervention 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.

[0003] Sodium polyphosphate (NaPP) is a highly water-soluble inorganic polymer, which is often used to prepare polyphosphate solutions. Polyphosphate solutions have a 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 polyphosphate solution, that is, larger cations (Ba 2+ , Sr 2+ and Ag + ) will cause flocculation, while smaller cations (Ca 2+ , Mg 2+ and Zn 2+) will lead to condensation. The definition of a polyphosphate condensate (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 condensate 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 condensation process can be controlled. Polyphosphate condensates 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 extensive research on 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.

[0004] 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. 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 can cause more infections. 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

[0005] Therefore, the purpose of the present application is to provide a pure inorganic wax-like active material and a preparation method and application thereof, which has certain hardness, can be formed, is degradable, has antibacterial, hemostatic, and bone defect repair effects.

[0006] The present application provides a preparation method of a pure inorganic wax-like active material, comprising the following steps:

[0007] The sodium polyphosphate solution is mixed with a divalent cation solution to obtain a wax-like co-doped polyphosphate CoPPW through chelation;

[0008] The divalent cation solution comprises Ca 2+ , Mg 2+ , and Sr2+ solution of any one or several of the above.

[0009] 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.

[0010] Preferably, 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, the solution comprising Sr 2+ is a strontium chloride or strontium nitrate solution, and the total concentration of the divalent cations in the divalent cation solution is 0.1-1.5 mol / L.

[0011] Preferably, the molar ratio of the sodium polyphosphate solution to the total divalent cations in the divalent cation solution is 1:1-1:5.

[0012] Preferably, the chelation is accompanied by stirring, the chelation time is 0.15-2 h, and the chelation temperature is 10-60℃.

[0013] The present 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%.

[0014] The present application also provides the use of the wax-like co-doped polyphosphate CoPPW in the preparation of a hemostatic material.

[0015] The present application also provides the use of the wax-like co-doped polyphosphate CoPPW in the preparation of a material for repairing bone defects.

[0016] The present application also provides the use of the wax-like co-doped polyphosphate CoPPW in the preparation of an agent for promoting cell proliferation, promoting the expression of osteogenic genes, inducing new bone formation or inducing the formation of extracellular matrix.

[0017] Compared with the prior art, the present application has the following beneficial effects: the pure inorganic wax-like active material provided by the present application is prepared by chelating sodium polyphosphate with divalent cations, and by limiting the concentration and molar ratio of the sodium polyphosphate and the divalent cations and 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, bone defect repairing and other effects.

[0018] 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

[0019] 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.

[0020] Figure 2 Appearance of CoWPP with different compositions, the photo is taken after the sample is pressed once by a punch and lifted.

[0021] 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).

[0022] 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.

[0023] Figure 5 Viscoelasticity of CoWPP sample showing loss modulus and storage modulus.

[0024] Figure 6 Viscoelasticity of commercial bone wax.

[0025] Figure 7Viscosity-shear rate for CaMg samples after 4 days of preparation.

[0026] Figure 8 Appearance of CaSr samples after 4 days of preparation showed that the samples solidified.

[0027] Figure 9 Cumulative ion concentration of Ca, Mg and P ions released from CaMg2.5 samples in PBS solution.

[0028] 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.

[0029] Figure 11 Representative SEM morphology of CoWPP powder after drying at 120 °C for 72 h.

[0030] 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.

[0031] 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.

[0032] 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) in KEGG metabolic pathway enrichment.

[0033] 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.

[0034] 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

[0035] The application provides a preparation method of a pure inorganic waxy active material, comprising 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+ .

[0036] 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.

[0037] In the present application, the divalent cation solution is a solution including any one or several of Ca 2+ , Mg 2+ and Sr 2+ , the solution including Ca 2+ is a calcium chloride or calcium nitrate solution, the solution including Mg 2+ is a magnesium chloride or magnesium nitrate solution, and the solution including Sr 2+ is a strontium chloride or strontium nitrate solution, 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 includes 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 includes 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 includes 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 includes 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 including Ca 2+ is a calcium chloride or calcium nitrate solution, the solution including Mg 2+ is a magnesium chloride or magnesium nitrate solution, and the solution including Sr 2+ is a strontium chloride or strontium nitrate solution.

[0038] In the present application, after mixing the sodium polyphosphate solution with the divalent cation solution, chelation is performed, the chelation preferably being accompanied by stirring, the rotation speed of the stirring being preferably 100-500 rpm; the time of the chelation is preferably 0.15-2 h, and the temperature of the chelation 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.

[0039] 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%.

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

[0041] The application further provides application of the wax-like co-doped polyphosphate CoPPW in preparation of a material for repairing bone defects.

[0042] 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 osteogenic genes, inducing new bone formation or inducing extracellular matrix formation.

[0043] 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.

[0044] Examples

[0045] The average polymerization degree of the commercial sodium polyphosphate (NaPP) used in the examples is 20 (Thermo Fisher Scientific Inc., USA). The NaPP powder is dissolved in deionized water to prepare a NaPP solution with a concentration of 0.5 mol / L. 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, magnesium chloride and 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 A M B 3,

[0046] M A M B 2.5 and M A M B 1.5.

[0047] Table 1 Sample composition and water content of waxy co-doped polyphosphate CoPPW

[0048]

[0049]

[0050] Material characterization:

[0051] Chemical composition and water content of CoPPW:

[0052] The CoPPW obtained from the above preparation was washed with deionized water and dried in an oven at 120 °C for 72 h. The dried powder was analyzed by x-ray diffractometer (XRD, D8 advance, Bruker, USA) in the range of 10 ° to 80 ° with a scan step of 0.2 s / step and a step size of 0.02 °. Its microstructure and elemental distribution were analyzed by scanning electron microscope (SEM, Merlin, Zeiss, Germany) equipped with energy dispersive x-ray spectroscopy (EDS). The water content of CoPPW was calculated using the formula: Water content (%) = (W 湿 -W 干 ) / W 湿 x 100%, where W 湿 and W 干 are the weights of wet and dry CoPPW, respectively.

[0053] The results are shown in Figure 2 , and the images of the newly synthesized CoPPW are shown in Figure 2 and Figure 3 A. It can be seen that the CaSr sample presents a white opaque appearance, while the CaMg and MgSr samples present a whitish appearance. The MgSr sample is more translucent than the other samples. The elemental composition was analyzed using SEM-EDS, as shown in Figure 3 D, and the EDS elemental map of dried MgSr2.5 shows a uniform distribution of Mg, Sr, and P elements. The molar ratio of cations to phosphorus (Mii / P) obtained from the experiment is lower than the theoretical value, indicating that some cations were not chelated to NaPP during the preparation process but were retained in the supernatant. As shown in Table 1, the percentage of water in CoPPW is between 30 wt% and 43 wt%.

[0054] The concentration of divalent cations plays an important role in determining the chelation ability. In divalent cation-phosphate systems, the occupation of metal ions depends on the concentration of divalent cations; at low concentrations, cations are trapped inside the polyphosphate cage, while at higher concentrations, the cage is saturated, and thus cations are chelated to the external polyphosphate chains15,16. Therefore, the precipitation of the condensation layer depends on van der Waals attraction and electrostatic repulsion. The critical concentration (M*) that leads to precipitation is determined by the concentration and the average degree of polymerization of NaPP.

[0055] Rheological properties of CoPPW:

[0056] 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 The viscoelastic properties of the coacervates were determined by the storage modulus (G') and loss modulus (G") in the frequency range of 0.1-100 Hz. To study the effect of temperature, rheological measurements were performed at 25° and 37°, respectively. Rheological tests were performed one hour after coacervation. All samples were measured after 1 day and 4 days to determine the change in viscosity over time.

[0057] The effect of divalent cation chelation on the rheological properties of CoPPW was studied in dynamic frequency sweep. All CoPPW exhibited shear thinning behavior, with the viscosity decreasing as the shear rate increased (Fig. B in Figure 3 The viscosities of CaSr samples were higher than those of other samples, except for the MgSr3 sample. The viscoelastic behavior of CoPPW varied with the type and amount of cation chelation. 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 the 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. C in Figure 9 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. D in Figure 10 Specifically, the viscosity of the MgSr2.5 sample compared to that of bone wax is shown in Fig. C in Figure 3 After 1 day, the MgSr2.5 was still in a liquid-like gel structure, with a lower viscosity than that of the wax; after 4 days, the MgSr2.5 became solidified and could be shaped.

[0058] CoPPW had very high viscosity, which was consistent with its wax-like appearance. This could be due to the multiple effects of double cation chelation. In polyelectrolyte solutions, the addition of cations has opposite effects on viscosity. At low concentrations, cation chelation decreases viscosity due to chain breakage caused by charge shielding effects, while at high concentrations, cation chelation increases viscosity due to increased interactions 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 viscosities of MgSr and CaSr were also higher than that of CaMg, which could be due to the higher charge density of Sr2+ More effective in cross-linking polyphosphate chains.

[0059] In vitro degradation behavior and ion release:

[0060] The in vitro degradation behavior of CoPPW was evaluated by immersing the samples in PBS solution at a temperature of 37°C. The ratio of PBS volume (mL) to sample weight (g) was 10mL:1g. On days 1, 7, 14, 21, 28, 35 and 46, the PBS solution was removed and the weight loss was measured. The percentage of weight loss was determined by the ratio of (original weight - residual weight) to the original weight. The release of ions was also studied. At each time interval, 1mL of supernatant was taken and replaced with fresh PBS solution. The collected supernatant was analyzed for Sr by inductively coupled optical emission spectrometry (PerkinElmer, Inc., USA). 2+ Mg 2+ and P 4+ Cumulative concentration of ion release. Solutions were collected on days 1, 2, 4, 6, 8, 14, and 21.

[0061] The degradation of MgSr2.5 and CaMg2.5 was studied by immersing the samples in PBS solution. The weight loss percentage of MgSr2.5 over time is shown in Figure 3 As shown in Figure E. During the first week, the MgSr2.5 sample absorbed water and increased in weight by 10%, followed by a gradual weight loss 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%. In addition, the Mg released from MgSr2.5 was analyzed using ICP-OES. 2+ 、Sr 2+ and P 5+ ion concentration. Consistent with the weight loss results, the ions released from MgSr2.5 were released slowly within the first week without sudden release. 5+ The release of ions was higher than that of divalent cations, which indicated that there was a higher 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 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 a 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.

[0062] Hemostatic ability evaluation:

[0063] To compare the hemostatic ability of CoPPW with commercial bone wax (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 is composed 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.

[0064] 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

[0065] Biocompatibility evaluation of CoPPW

[0066] 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.

[0067] Human bone marrow mesenchymal stem cells (h-BMSC; ScienCell, #7500, USA) were cultured in h-MSC basal medium (Cyagen, HUXMA-90,011, China), which contained 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 with 95% air and 5% CO2 at 37°C for 24 hours. The supernatant was collected and filtered three times with a 0.22 μm filter membrane (MerckMillipore, USA) to obtain a 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.

[0068] The effects of the biomaterial MgSr2.5-CoPPW on h-MSC proliferation and cytotoxicity were evaluated by CCK-8 (CCK-8, purchased from Dojindo, Japan). Briefly, h-MSCs were cultured at 2×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured in h-MSC culture medium and MgSr2.5-CoPPW extract 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 of each well was measured at 450 nm. h-MSCs were cultured at a density of 1.5×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates. A blank control group was cultured with normal cell culture medium, while the control and experimental groups were cultured with high- and low-concentration extracts. On day 7 of culture, EdU (5-ethynyl-2'-deoxyuridine) staining was performed using an EDU kit (Biyuntian, China) according to the manufacturer's protocol, and photographs were taken.

[0069] h-MSCs were cultured at a rate of 2 × 10 5 Cells were seeded at a density of 1 / well in 12-well plates and stimulated with cell culture medium and biomaterial extracts for 14 days. Total RNA from BMSCs was extracted using Trizol reagent (Invitrogen, California, USA). The one-step SYBR PrimeScript qPCR kit (TaKaRa Bio, Otsu, Japan) was used to synthesize cDNA according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed using SYBR Premix Ex Taq (TaKaRa) in a Bio-Rad CFX96 real-time PCR system using thermal cycling conditions.

[0070] The cDNA fragments were amplified by real-time PCR using the following specific primers.

[0071] Table 2 Specific primer sequences

[0072]

[0073]

[0074] The results are as follows Figure 12 As shown in Figure A, after culturing h-BMScs for 1, 2, 3, 5, and 7 days, the optical density (OD) values ​​of bone wax and CoPPW-MSP gradually increased with the culture time, indicating an increase in the number of viable cells. After the 5th day, the cell viability of the low-concentration and high-concentration CoPPW-MSP groups was higher than that of the bone wax group and the blank group. To further confirm the CCK-8 results, on the 7th day of h-BMSC culture, the different concentration treatment groups were subjected to edu (5-ethynyl-2'-deoxyuridine) staining ( Figure 12 In the low-concentration CoPPW-MSP group, the number of mesenchymal stem cells that replicated and divided was significantly higher than that in the other groups. The cell proliferation rate in the low-concentration CoPPW-MSP group was 29.3% ( Figure 12 A), which was higher than that of the blank group (20.8%) and the low-concentration bone wax group (21.7%).

[0075] Alizarin red staining and alkaline phosphatase staining

[0076] h-BMSCs were cultured at a rate of 2 × 10 5 Cells / well were seeded in 6-well plates and treated with cell culture medium containing or without biomaterial extracts. After 14 days of culture, calcium deposition in h-BMSCs was detected using Alizarin Red staining (Cyagen, HUXMA-90,021, China). Mineralized particles were observed using an inverted microscope (Nikon, Japan). After 14 days of culture in MSCs treated with cell culture medium containing or without biomaterial extracts, all groups were stained for alkaline phosphatase (Beyotime, China) and photographed under a microscope (Nikon, Japan).

[0077] Ions released from CoPPW-MSP materials can stimulate cell proliferation and replication at specific concentrations, and there was no significant difference between CoPPW-MSP and bone wax in the first 3 days ( Figure 12 A), which is attributed to Mg 2+ and Sr 2+ Slow release of ions (eg Figure 3 (E in Figure 5). After 5 days, the Mg released by CoPPW-MSP 2+ and Sr 2+ Ions increase, promoting cell proliferation and division.

[0078] In vitro osteogenic properties of CoPPW-MSP

[0079] After h-BMScs were stimulated with the extracted medium for 14 days, the osteogenic properties induced by CoPPW-MSP and bone wax were further investigated by alkaline phosphatase (ALP) and Alizarin Red S staining. Figure 13 As shown in Figure A, there were more ALP-positive cells in the high- and low-concentration CoPPW-MSP groups compared with the bone wax group and the blank group. In addition, the ALP-positive rate in the high-concentration CoPPW-MSP group was higher than that in the low-concentration CoPPW-MSP group. Figure 13 As shown in Figure B, calcium deposition, an indicator of late osteogenesis, was assessed by staining the extracellular matrix of h-MSCs with alizarin red. Compared with the blank group and the bone wax group, the calcium nodule staining in the CoPPW-MSP group was more intense and obvious. 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 osteogenesis mechanism of CoPPW-MSP at the gene expression level. Figure 13 As shown in Figure C, the gene expressions of osteoglycin (OGN), osteopontin (OPN), type I collagen α1 (col1a1), and osteocalcin (BGLAP) in the CoPPW-MSP group were significantly higher than those in the control group and the bone wax group. In addition, high concentrations of 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 osteogenesis ability. The typical bone formation process includes cell proliferation, extracellular maturation, and mineralization. ALP activity and the formation of mineralized nodules were used to evaluate the early stages of osteogenesis. In the present invention, CoPPW may promote osteogenic differentiation and mineralization because it showed a better expression effect on osteogenesis-related genes ( Figure 13 C in). This may be due to Sr 2+ and Mg 2+ Ion release. Strontium-doped calcium polyphosphate bioceramics (Sr-CaPP) have been studied for their osteogenic and angiogenic abilities; however, their hardness does not meet the clinical requirements of waxy and viscous properties. In the present invention, CoPPW-MSP exhibits excellent osteogenic ability while maintaining excellent waxy properties, suggesting promising application prospects in bone repair and regeneration.

[0080] Potential mechanisms of osteogenesis and coagulation in CoPPW-MSP

[0081] Transcriptome sequencing and data analysis

[0082] The samples were divided into three groups: a blank group (undifferentiated h-BMSCs), a bone wax group, and a CoPPW-MSP group. The h-BMSCs in the three groups were cultured in a 6-well Trans well plate for 14 days to allow the stimulated differentiation of h-BMSCs. There were three replicates in each group. The cells were detached using trypsin, suspended to remove the supernatant, and stored in liquid nitrogen. RNA sequencing analysis was performed at the Beijing Institute of Genomics. In addition, network and pathway analysis was performed using the analysis software IPA.

[0083] h-BMSCs were further subjected to transcriptome sequencing to explore the potential osteogenesis mechanism induced by CoPPW-MSP. The differentially expressed genes of h-BMSCs were revealed in the heat map ( Figure 14 (A), where up-regulated genes are marked in red and down-regulated 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 up-regulated genes and 1345 down-regulated genes (e.g. Figure 14 GO enrichment was applied to analyze these differentially expressed genes, most of which were involved in osteogenesis and extracellular matrix, including extracellular matrix and structural organization, collagen metabolism, cell adhesion molecule binding, growth factor binding, and collagen binding ( Figure 14 Based on these significantly differentially expressed genes, the KEGG metabolic pathway was used to analyze key osteogenic signaling pathways, such as Figure 14 As shown in D. KEGG signaling pathway analysis showed that the PI3K / Akt signaling pathway and the ECM receptor interaction signaling pathway were highly enriched and played an important role in promoting the osteogenic differentiation of h-BMSCs. The PI3K / Akt signaling pathway is an important lipid kinase activation pathway that significantly regulates the osteogenic differentiation, proliferation and apoptosis of MSCs. AKt phosphorylation can upregulate the Runx2 gene and induce the expression of bone repair and reconstruction related genes, such as BGLAP, COL1, OPN and ALP26. In the present invention, gene sequencing ( Figure 14 Figure E) shows that CoPPW-MSP effectively activates the PI3K / Akt signaling pathway, which further affects the mTOR signaling pathway through REDD1 overexpression. The activated AKT-mTOR signaling pathway promotes MSC differentiation into osteoblasts and increases the mRNA translation rate, thereby increasing cellular protein synthesis. In addition, overexpression of the downstream target protein p53, activated by AKT, can promote cell proliferation and cell cycle progression ( Figure 14 E). Mg release from CoPPW-MSP 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 osteogenesis. At the same time, CoPPW-MSP also activates the ECM-receptor interaction signaling pathway (Figure 14 D in ). This signaling pathway affects collagen metabolism and synthesis in bone and plays an important role in regulating cell adhesion, thereby promoting the formation of extracellular matrix. The upregulation of the ECM-receptor interaction signaling pathway is consistent with the significant increase in type I collagen expression in CoPPW-MSP by QT-PCR ( Figure 13 On the other hand, CoPPW-MSP activates complement and coagulation cascade signaling pathways ( Figure 14 E), which plays an important role in subsequent cellular responses that influence anti-inflammatory and tissue repair. In the coagulation cascade of CoPPW-MSP, expression of PAR1, PAR3, and PAR4 is upregulated due to activated coagulation factors VIII and X, leading to the release of anti-inflammatory mediators and increased endothelial permeability. Polyps have been shown to accelerate blood coagulation, with increased thrombin generation in the presence of polyps activating factor XIII. Furthermore, polyps slow fibrinolysis by modulating the fibrin clot structure, making it more resistant to fibrinolysis.

[0084] In vivo study: rabbit femoral defect model surgical procedure

[0085] Based on the method reported in reference (Sun, Y., Helmholz, H. & R.Surgical Classification for Preclinical Rat Femoral Bone Defect Model: Standardization Based on Systematic Review, Anatomical Analysis and Virtual Surgery. Bioengineering 9, (2022).) A modified femoral defect model was constructed to evaluate rabbit bone repair. First, rabbits were anesthetized with an intraauricular injection of 1% pentobarbital (50 mg / kg). After the surgical area was shaved and disinfected, the animal was 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 and deep fascia were dissected, the protuberance was identified as the drilling target. Kirschner wires with a diameter of 5 mm were drilled continuously into the target area, forming a cavity defect with a diameter of 5 mm and a depth of 10 mm from the outside to the inside in a direction perpendicular to the longitudinal axis of the femoral shaft. Critical defect size was created in 12 rabbits. The left diaphysis received bone wax for the cavity defect, while CoPPW-MSPs were implanted for the cavity defect in the right diaphysis. All procedures were performed in strict adherence to aseptic principles, and each subject received an intramuscular injection of antibiotics on the first postoperative day. Rabbits were allowed to move freely after surgery without immobilization. At 4 and 8 weeks after surgery, the rabbits were sacrificed, and bilateral femurs were harvested for evaluation.

[0086] Micro-CT evaluation

[0087] To evaluate the bone volume and density at a specific Hu value, the lateral region of the femur was scanned using micro-CT (Siemens, Germany). The resolution of the micro-CT scan was 10.3 microns, the voltage was 70 kilovolts, and the current was 400 milliamps. The resulting images were used to construct a 3D model of the femur using the Dslicer software (Inveon Research Workplace software). The bone volume was calculated by identifying the region of interest with a Hu signal ranging from 700 to 2100. The bone volume / total bone volume (BV / TV) ratio was then calculated using the bone volume of the selected region. Histological analysis decalcified the femur samples and cut them into 4-micron-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's trichrome (G1340, Solarbio, China) and examined under a microscope (Ci-S, Nikon, Japan) to detect bone formation.

[0088] Figure 15 Fig. 1 shows the μ-CT images of bone wax and CoPPW-MSP at 4 and 8 weeks after implantation. At 4 weeks, newly formed bone was evident in the CoPPW-MSP group, while the femoral defect was still hollow in the bone wax group. Over time, the femoral defect gradually healed, with newly formed tissue partially filling the defect site. By 8 weeks, 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 edge of the defect. Quantitative micro-CT analysis of bone volume is shown in Fig. 1B. Figure 15 Fig. 1 shows the μ-CT images of bone wax and CoPPW-MSP at 4 and 8 weeks after implantation. At 4 weeks, newly formed bone was evident in the CoPPW-MSP group, while the femoral defect was still hollow in the bone wax group. Over time, the femoral defect gradually healed, with newly formed tissue partially filling the defect site. By 8 weeks, 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 edge of the defect. Quantitative micro-CT analysis of bone volume is shown in Fig. 1B. Figure 15 Fig. 1 shows the μ-CT images of bone wax and CoPPW-MSP at 4 and 8 weeks after implantation. At 4 weeks, newly formed bone was evident in the CoPPW-MSP group, while the femoral defect was still hollow in the bone wax group. Over time, the femoral defect gradually healed, with newly formed tissue partially filling the defect site. By 8 weeks, 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 edge of the defect. Quantitative micro-CT analysis of bone volume is shown in Fig. 1B.

[0089] To observe the blood occlusion, the femur of New Zealand white rabbits was drilled, resulting in a large amount of blood flowing out of the defect. CoPPW-MSP and bone wax were used to stop bleeding, respectively. Both groups showed good sealing effect (Figure 16 ) In orthopedic surgery, hemostasis at bone defects is challenging. It is often necessary to fill the defect with a bioadhesive waxy material such as beeswax to physically seal 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.

[0090] 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 in 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.

[0091] 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 within the scope of protection of the present application.

Claims

1. A method for preparing a pure inorganic waxy active material, characterized in that: The following steps are involved: Sodium polyphosphate solution was mixed with divalent cation solution to obtain waxy co-doped polyphosphate CoPPW through chelation; The divalent cation solution includes Ca 2+ Mg 2+ and Sr 2+ Any one or more solutions; The concentration of the sodium polyphosphate solution is 0.1-1.5 mol / L, and the average degree of polymerization of the sodium polyphosphate is 10-100; The total divalent cation concentration in the divalent cation solution is 0.1 to 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 to 1:5; The chelation process is accompanied by stirring, the chelation time is 0.15-2 hours, and the chelation temperature is 10-60°C; The water content of the waxy co-doped polyphosphate CoPPW is 25% to 50%.

2. The preparation method according to claim 1, characterized in that Including Ca 2+ The solution is calcium chloride or calcium nitrate solution, including Mg 2+ The solution is magnesium chloride or magnesium nitrate solution, including Sr 2+ The solution is strontium chloride or strontium nitrate solution.

3. The waxy co-doped polyphosphate CoPPW prepared by the preparation method according to claim 1 or 2, characterized in that: The invention comprises sodium polyphosphate, divalent cations and water; the molar ratio of the divalent cations to the sodium polyphosphate is 5:1 to 1:1; and the water content of the waxy co-doped polyphosphate CoPPW is 25% to 50%.

4. Use of the waxy co-doped polyphosphate CoPPW according to claim 3 in the preparation of hemostatic materials.

5. Use of the waxy co-doped polyphosphate CoPPW according to claim 3 in preparing a material for repairing bone defects.

6. Use of the waxy co-doped polyphosphate CoPPW according to claim 3 in the preparation of an agent for promoting cell proliferation, promoting the expression of osteogenic genes, inducing new bone formation, or inducing extracellular matrix production.

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