Easily-injectable and anti-collapsing calcium phosphate-based bone cement as well as preparation method and application thereof

Abstract

The invention discloses easy-to-inject anti-collapse calcium phosphate-based bone cement as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The calcium phosphate-based bone cement disclosed by the invention is prepared from solid-phase powder and curing liquid, the solid powder comprises alpha-tricalcium phosphate and tungsten sulfide; the curing liquid at least comprises an aqueous solution of gellan gum. The bone cement prepared by the invention is easy to inject, can be self-cured, has excellent collapsibility resistance and high mechanical strength, can induce deposition of bone-like hydroxyapatite, and has good biocompatibility and bone induction capability. Therefore, the bone cement is suitable for the field of dental and orthopedic filling repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an easily injectable, anti-collapse calcium phosphate-based bone cement and a preparation method and application thereof. Background Art

[0002] Bone defects and bone injuries caused by trauma, tumors, etc. are common clinical problems. At present, the bone defect repair materials used in clinical practice mainly include autologous bone / allogeneic bone and artificial bone repair materials. Autologous bone is an ideal bone transplant material, but its source is limited, and the transplantation process may cause secondary damage. Although allogeneic bone can solve the source problem, the risks of immune rejection, infection and inflammation are inevitable. Therefore, the development of non-destructive and widely available artificial bone repair materials has become an important direction of biomedical materials research.

[0003] Calcium phosphate-based bone cement is a bone repair material with excellent performance, among which α-tricalcium phosphate bone cement has attracted widespread attention due to its excellent biocompatibility, plasticity and self-curing properties. However, α-tricalcium phosphate bone cement also has several limitations, such as long setting time, poor injectability and weak anti-collapse ability. When α-tricalcium phosphate is mixed with water, its preparation process is more complicated, injection is difficult, and it is easy to collapse in the water environment, which may cause adverse reactions such as inflammation during bone repair. In addition, the long setting time not only affects the efficiency of surgery, but also may affect the performance development of the material. In response to the key challenges of calcium phosphate-based bone cement, researchers have promoted hydration reactions and accelerated the setting process by doping with inorganic salts (such as α-calcium sulfate hemihydrate, calcium carbonate and calcium chloride). Although these additives can shorten the setting time, the anti-collapse and injectability of calcium phosphate bone cement are still limited. The addition of inorganic salts often leads to a decrease in the fluidity of the material while promoting the hydration reaction, or produces a more fragile gel structure, which makes the bone cement unable to be injected.

[0004] In order to overcome the problem of poor anti-disintegration, researchers have improved its anti-disintegration ability by adding gel polymers (such as sodium alginate, modified starch and chitosan, etc.). For example, CN202210052612.X discloses an anti-disintegration calcium phosphate bone cement containing sodium polyacrylate and a preparation method thereof. By introducing acrylic acid monomers and performing radiation treatment, the anti-disintegration and injectability of calcium phosphate bone cement are significantly improved; radiation treatment can change the molecular structure of the polymer, so that it plays a better stabilizing role in the cement system. However, there is an obvious problem with this method: the setting time is still relatively long, which may not be suitable for clinical applications that urgently need rapid solidification. On the other hand, although the use of organic gelling agents can form a protective film on the surface of the particles, increase adhesion and improve resistance to water shock, the introduction of these organic substances may also inhibit the hydration reaction, thereby affecting the curing time of the material. Existing anti-disintegration agents are difficult to meet the synergistic improvement of anti-disintegration, injectability and curing characteristics. Therefore, there is an urgent application demand for the development of a calcium phosphate-based bone cement filling material that is easy to inject, anti-disintegration, and has good self-curing properties.

[0005] Tungsten sulfide is a semiconductor metal dichalcogenide compound with a two-dimensional structure. It is widely used in bioimaging, sensors, drug delivery, photothermal therapy and other fields because of its certain antibacterial activity, good biocompatibility and photothermal properties. Gellan gum has excellent cohesion and rheological properties due to its excellent gel-forming ability and rheological properties, and has good biocompatibility and non-toxicity. Therefore, it is widely used in biomedical fields such as bone tissue engineering, drug delivery and wound healing.

[0006] At present, there is no report on the potential application of tungsten sulfide and gellan gum composite in calcium phosphate bone cement. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide an easily injectable, anti-collapse calcium phosphate-based bone cement and its preparation method and application in view of the deficiencies of the prior art. The preparation method of the present invention can obtain a calcium phosphate-based bone cement material with high osteogenic activity, which can solve the problems of insufficient injection performance and poor anti-collapse of existing calcium phosphate-based materials.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The invention provides an easily injectable, anti-collapse calcium phosphate-based bone cement, which is prepared by mixing solid phase powder and solidifying liquid; the solid phase powder comprises α-tricalcium phosphate and tungsten sulfide; and the solidifying liquid comprises at least an aqueous solution of gellan gum.

[0010] The present invention uses tungsten sulfide and gellan gum to form an organic-inorganic composite material for the first time. Tungsten sulfide is used to increase the solidification ability of calcium phosphate-based materials, shorten the solidification time, and make the material have good biocompatibility and bone induction ability. At the same time, gellan gum is used to enhance the cohesion of bone cement particles, thereby improving the operability and anti-collapse property of bone cement.

[0011] Preferably, the bone cement is prepared by blending a solidifying liquid and a solid phase powder at a ratio of 0.3 to 0.6 g / g.

[0012] Preferably, in the solid phase powder, the weight percentage of α-tricalcium phosphate is 96% to 99%, the weight percentage of tungsten sulfide is 1% to 4%, and the sum of the weight percentages of the two is 100%.

[0013] Preferably, the solidifying liquid is an aqueous solution containing gellan gum.

[0014] Furthermore, in the solidifying liquid, the weight percentage of gellan gum is 1% to 3%.

[0015] The present invention also provides a method for preparing the above-mentioned easily injectable, anti-collapse calcium phosphate-based bone cement, comprising the following steps:

[0016] (1) uniformly mixing α-tricalcium phosphate powder and tungsten sulfide powder to obtain a solid phase powder;

[0017] (2) adding gellan gum into water and fully dissolving it to obtain a uniform liquid phase to obtain a solidified liquid;

[0018] (3) The solid phase powder and the solidifying liquid are uniformly blended in proportion to obtain the injectable and anti-collapse calcium phosphate-based bone cement.

[0019] The present invention also provides the application of the above-mentioned easy-to-inject and anti-collapse calcium phosphate-based bone cement in the field of dental and orthopedic filling and repair. The easy-to-inject and anti-collapse calcium phosphate-based bone cement provided by the present invention can be applied to biomedical fields such as bone injuries.

[0020] Beneficial effects:

[0021] 1. The present invention combines tungsten sulfide and gellan gum for the first time to form an organic-inorganic composite material. Tungsten sulfide increases the solidification ability of calcium phosphate-based materials, shortens the solidification time, and makes the materials have good biocompatibility and bone induction ability. At the same time, gellan gum enhances the cohesion of bone cement particles, thereby improving the operability and anti-collapse property of bone cement.

[0022] 2. The calcium phosphate-based bone cement of the present invention has excellent apatite mineralization ability, can promote apatite crystal deposition, has excellent biological activity, and can enhance the mineralization ability of osteoblasts.

[0023] 3. The present invention can prepare an injectable bone cement material with good flowability by adjusting the ratio of solid powder to solid liquid to meet clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The static anti-collapse performance test diagram of the calcium phosphate bone cement prepared in Examples 1-3 of the present invention and Comparative Examples 1 and 2;

[0025] Figure 2 The dynamic anti-collapse performance test diagram of the calcium phosphate bone cement prepared in Examples 1-3 of the present invention and Comparative Example 2;

[0026] Figure 3 This is a test diagram of the dynamic anti-collapse performance of the calcium phosphate bone cement prepared in Examples 4 and 5 of the present invention;

[0027] Figure 4 It is a graph of the curing time of the calcium phosphate bone cement prepared in Examples 1-3 of the present invention and Comparative Example 2;

[0028] Figure 5 It is a graph of the injection rate of calcium phosphate bone cement prepared in Examples 1-3 of the present invention and Comparative Example 2;

[0029] Figure 6 The compressive strength diagram of the calcium phosphate bone cement prepared in Example 1 and Comparative Example 2 of the present invention;

[0030] Figure 7 The cell activity diagram of the calcium phosphate bone cement prepared in Example 1, Example 3 and Comparative Example 2 of the present invention;

[0031] Figure 8 It is a quantitative analysis diagram of Alizarin Red (ARS) staining of calcium phosphate bone cement prepared in Example 1, Example 2 and Comparative Example 2 of the present invention;

[0032] Fig. 9 This is a SEM image of apatite deposition on the surface of calcium phosphate bone cement prepared in Example 2 of the present invention;

[0033] Fig.10 The XRD spectra of the hydration products of calcium phosphate bone cement prepared in Example 2 of the present invention and Comparative Example 2 are shown. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0035] The gellan gum in the examples and comparative examples of the present invention was purchased from MacLean Reagent Company.

[0036] Example 1 Preparation of easily injectable, anti-collapse calcium phosphate-based bone cement

[0037] (1) 99% α-tricalcium phosphate powder and 1% tungsten sulfide powder are uniformly mixed according to weight percentage to obtain a solid phase powder.

[0038] (2) Gellan gum is added into deionized water and fully dissolved to obtain a uniform liquid phase, thereby obtaining a solidified liquid having a gellan gum weight percentage of 2%.

[0039] (3) The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.3 g / g to obtain a bone cement material.

[0040] Example 2 Preparation of easily injectable, anti-collapse calcium phosphate-based bone cement

[0041] (1) 98% α-tricalcium phosphate powder and 2% tungsten sulfide powder are uniformly mixed according to weight percentage to obtain a solid phase powder.

[0042] (2) Gellan gum is added into deionized water and fully dissolved to obtain a uniform liquid phase, thereby obtaining a solidified liquid having a gellan gum weight percentage of 2%.

[0043] (3) The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.3 g / g to obtain a bone cement material.

[0044] Example 3 Preparation of easily injectable, anti-collapse calcium phosphate-based bone cement

[0045] (1) 96% α-tricalcium phosphate powder and 4% tungsten sulfide powder are uniformly mixed according to weight percentage to obtain a solid phase powder.

[0046] (2) Gellan gum is added into deionized water and fully dissolved to obtain a uniform liquid phase, thereby obtaining a solidified liquid having a gellan gum weight percentage of 2%.

[0047] (3) The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.3 g / g to obtain a bone cement material.

[0048] Example 4 Preparation of easily injectable, anti-collapse calcium phosphate-based bone cement

[0049] (1) 99% α-tricalcium phosphate powder and 1% tungsten sulfide powder are uniformly mixed according to weight percentage to obtain a solid phase powder.

[0050] (2) Gellan gum was added into deionized water and fully dissolved to obtain a uniform liquid phase, thereby obtaining a solidified liquid having a gellan gum weight percentage of 1%.

[0051] (3) The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.4 g / g to obtain a bone cement material.

[0052] Example 5 Preparation of easily injectable, anti-collapse calcium phosphate-based bone cement

[0053] (1) 96% α-tricalcium phosphate powder and 4% tungsten sulfide powder are uniformly mixed according to weight percentage to obtain a solid phase powder.

[0054] (2) Gellan gum was added into deionized water and fully dissolved to obtain a uniform liquid phase, thereby obtaining a solidified liquid having a gellan gum weight percentage of 3%.

[0055] (3) The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.6 g / g to obtain a bone cement material.

[0056] Comparative Example 1

[0057] A single α-tricalcium phosphate powder is used as a solid phase powder, and deionized water is used as a solidifying liquid. The solidifying liquid and the solid phase powder are uniformly mixed at a ratio of 0.3 g / g to obtain a bone cement material.

[0058] Comparative Example 2

[0059] A single α-tricalcium phosphate powder is used as the solid phase powder, gellan gum is added into deionized water and fully dissolved to obtain a uniform liquid phase, and a solidified liquid with a gellan gum weight percentage of 2% is obtained. The solidified liquid and the solid phase powder are uniformly mixed at a ratio of 0.3 g / g to obtain a bone cement material.

[0060] The following are performance characteristics of each embodiment and comparative example:

[0061] Anti-collapse performance test: This performance test is divided into static anti-collapse performance test and dynamic anti-collapse performance test.

[0062] 1. Static anti-collapse performance test: Fill the prepared bone cement into a syringe, squeeze it into water and place it for 10 minutes. Observe the degree of collapse of the slurry with the naked eye to see whether it can maintain its initial shape and whether particles are free.

[0063] 2. Dynamic anti-collapse performance test: The prepared bone cement was squeezed into a glass dish filled with deionized water, placed in a vibrator and oscillated at a frequency of 100 r / min for 10 minutes, and then the bone cement collapse phenomenon was observed.

[0064] The bone cement materials of Examples 1-5 and Comparative Examples 1-2 were used to measure the static anti-collapse performance. Some of the test results are shown in the figure. Figure 1 As shown, the bone cement in Comparative Example 1 has poor anti-collapse performance and collapses immediately, while the bone cement prepared in Comparative Example 2 and Examples 1-3 can still maintain the initial shape after being soaked in water for 10 minutes, and no particle liberation occurs, and the anti-collapse performance is significantly enhanced, and the results of Examples 1-5 are similar, all maintaining the initial shape. This result shows that the addition of gellan gum greatly enhances the anti-collapse performance of bone cement, proving that gellan gum can improve the anti-collapse property of bone cement.

[0065] In order to further investigate the anti-collapse effect of tungsten sulfide in bone cement, the dynamic anti-collapse performance test was carried out on Comparative Example 2 and Examples 1-5. The test results of Comparative Example 2 and Examples 1-3 are as follows: Figure 2 As shown. It can be seen from the figure that: when bone cement containing different proportions of tungsten sulfide is placed in water, it can maintain its original shape and has good anti-collapse performance. After being placed in a vibrator and shaken for 10 minutes, the bone cement in Comparative Example 2 has disintegrated particles and is free, while the bone cement in Examples 1-3 basically maintains its original shape. In Example 1, some particles of the bone cement at the end of the bone cement collapse, and in Example 2, the bone cement has slight particle collapse, while in Example 3, the bone cement does not collapse, maintains its original shape, and has excellent anti-collapse properties. This result shows that the introduction of tungsten sulfide also enhances the anti-collapse performance of bone cement. The dynamic anti-collapse performance test results of the calcium phosphate bone cement prepared in Examples 4 and 5 are shown in Figure 2. Figure 3 As shown, only a small amount of particles are free and overflowed in Example 4 and Example 5, and both maintain excellent anti-collapse performance.

[0066] Curing time test: Fill the prepared bone cement material (paste) into a plastic mold (2 mm in height and 8 mm in diameter), and place it in a 37.5°C water bath for curing. Take out the sample quickly at regular intervals and use a Vicat instrument to check whether the sample is cured. If it is not cured, continue to place it in the water bath for curing until it is cured.

[0067] Comparative Example 2 and Examples 1-3 were tested and the results were as follows. Figure 4 As shown, the curing time of the bone cement tested in Comparative Example 2 is about 32 minutes, while the curing time tested in Examples 1-3 is significantly less than the curing time of Comparative Example 2, and the curing time is 19 minutes, 16 minutes, and 13 minutes, respectively. This shows that tungsten sulfide significantly shortens the curing time of bone cement paste.

[0068] Injection performance test: The prepared bone cement material (paste) was filled into a 2.5 ml syringe and loaded and injected using a universal testing machine. The pressure head was extruded at a constant speed of 15 mm / min, and the maximum load was 100 N. The injection rate was determined by the ratio of the weight of the injected paste to the weight of the original paste.

[0069] Injectability test was carried out using Examples 1-5 and Comparative Example 2. The test results of Comparative Example 2 and Examples 1-3 are as follows: Figure 5 As shown in Table 1, the injection rate in Comparative Example 2 is about 88%, while the injection rates of Examples 1-3 decrease as the tungsten sulfide content increases, and the injection rates are 73.2%, 56.5%, and 42.9%, respectively. The injectability test results of Examples 4 and 5 are shown in Table 1. The bone cement in these examples maintains excellent injectability, indicating that increasing the content of the solidifying liquid is beneficial to improving the injectability of the bone cement.

[0070] Table 1 Injection rate results of calcium phosphate bone cement

[0071]

[0072] Compression strength measurement: Inject bone cement material (paste) into a mold (12 mm in height and 6 mm in diameter), place it in a 37.5°C water bath for 1 day, then demould it, continue to cure for 6 days, rinse it with deionized water, place it in anhydrous ethanol (EtOH) for 1 day to terminate the curing. The compressive strength of the sample was tested using a universal testing machine at a loading rate of 1 mm / min.

[0073] Figure 6 The compressive strength results of Example 1 and Comparative Example 2 of the present invention show that the addition of tungsten sulfide improves the compressive strength of bone cement.

[0074] Cell activity assay: Grind the solidified bone cement into powder, then add the powder into α-MEM cell culture medium for extraction, where the content of bone cement powder in the culture medium is 0.5 mg / mL, collect the supernatant after centrifugation to obtain bone cement extract. BMSCs were used as experimental cells, and the cell activity of different bone cement materials was evaluated by CCK-8 cell technology kit. BMSCs were added to the bone cement extract for culture, and then CCK-8 reagent was added, incubated at 37°C in the dark, and then the cell activity was measured by microplate reader.

[0075] Example 1, Example 3 and Comparative Example 2 were tested, and the cell activity results after 3 days of culture were as follows Figure 7 As shown, the cell activity of Example 1 is higher than that of the cells cultured in the culture medium, and the cell activity of Example 3 is similar to the result of the culture medium, indicating that the bone cement has good cell activity and the introduction of tungsten sulfide does not show cytotoxicity.

[0076] Alizarin red (ARS) staining quantitative determination: First, BMSCs were cultured in α-MEM medium or bone cement extract for 14 days, then fixed with 4% formaldehyde and washed with PBS. The fixed cells were stained with ARS solution, and then 10% cetylpyridinium chloride was added for elution for 15 minutes for quantitative analysis. The absorbance was measured at 562 nm using an ELISA reader (Agilent BioTek), and the absorbance value obtained could be used to compare the size of the mineralized calcium nodule content.

[0077] ARS staining was performed on Example 1, Example 2 and Comparative Example 2 and the results were as follows: Figure 8 As shown in the figure, with the increase of tungsten sulfide content, the mineralization ability of BMSCs cells gradually increased, indicating that tungsten sulfide makes bone cement have good osteoinduction ability.

[0078] Apatite mineralization performance determination: The bone cement material (paste) was placed in a 37.5°C water bath for one day to obtain a solidified disc. The disc sample was immersed in simulated body fluid (SBF) for 7 days, and the liquid sample was replaced every three days. The ratio of the immersed surface area to the SBF volume was set to 0.1 cm 2 After the immersion time was over, the samples were collected, the sample surface was washed with deionized water, and then placed in a 60°C oven to fully dry, and then SEM was used to observe the apatite formation on the sample surface.

[0079] Fig. 9 This is a graph of apatite deposition on the surface of the bone cement of Example 2 of the present invention. As can be seen from the figure, after being soaked in SBF, a large amount of apatite can be deposited on the surface of the paste, indicating that the bone cement paste has excellent apatite deposition ability.

[0080] Determination of hydration products: Fill the prepared bone cement material (paste) into a mold (12 mm in height and 6 mm in diameter), demold after curing in a water bath at 37.5°C for 1 day, continue curing for 6 days, rinse with deionized water, place in EtOH for 1 day to terminate curing, and then fully dry in an oven at 60°C, grind into powder, and finally use XRD to perform phase analysis on the cured sample.

[0081] The hydration products of the bone cement materials prepared in Example 2 and Comparative Example 2 were analyzed. Fig.10 As shown, the results indicate that the addition of tungsten sulfide promotes the rate of hydration reaction.

[0082] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An easily injectable, anti-collapse calcium phosphate-based bone cement, characterized in that: The bone cement is prepared by mixing solid phase powder and solidifying liquid; the solid phase powder contains α-tricalcium phosphate and tungsten sulfide; and the solidifying liquid contains at least an aqueous solution of gellan gum.

2. The easily injectable, anti-collapse calcium phosphate-based bone cement according to claim 1, characterized in that: The bone cement is prepared by blending a solidifying liquid and a solid phase powder at a ratio of 0.3 to 0.6 g / g.

3. The easily injectable, anti-collapse calcium phosphate-based bone cement according to claim 1, characterized in that: In the solid phase powder, the weight percentage of α-tricalcium phosphate is 96% to 99%, the weight percentage of tungsten sulfide is 1% to 4%, and the sum of the weight percentages of the two is 100%.

4. The easily injectable, anti-collapse calcium phosphate-based bone cement according to claim 1, characterized in that: The solidifying liquid is an aqueous solution containing gellan gum.

5. The easily injectable, anti-collapse calcium phosphate-based bone cement according to claim 4, characterized in that: In the solidified liquid, the weight percentage of gellan gum is 1% to 3%.

6. The method for preparing the easily injectable, anti-collapse calcium phosphate-based bone cement according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) uniformly mixing α-tricalcium phosphate powder and tungsten sulfide powder to obtain a solid phase powder; (2) adding gellan gum into water and fully dissolving it to obtain a uniform liquid phase to obtain a solidified liquid; (3) The solid phase powder and the solidifying liquid are uniformly blended in proportion to obtain the easily injectable and anti-collapse calcium phosphate-based bone cement.

7. Use of the easily injectable, anti-collapse calcium phosphate-based bone cement according to any one of claims 1 to 5 in the field of dental and orthopedic filling and repair.

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