阅读说明：本技术 一种空心磷酸钙微球/丙三醇改性pmma骨水泥及其制备方法 (Hollow calcium phosphate microsphere/glycerol modified PMMA bone cement and preparation method thereof ) 是由 肖东琴 罗栩伟 赵桥 冯刚 刘康 张成栋 李艳 于 2021-10-26 设计创作，主要内容包括：本发明提供了一种空心磷酸钙微球/丙三醇改性PMMA骨水泥及其制备方法,包括以下步骤：将钙盐溶液与磷酸盐溶液混合形成悬浊液,滴加植酸至悬浊液重新清亮,向其中加入尿素调节溶液pH值,然后升温反应,再收集沉淀,洗净后冻干,得到磷酸钙微球；将磷酸钙微球加入骨水泥粉剂中,混合均匀,得粉剂混合物；将丙三醇加入骨水泥液剂中,混合均匀,得液剂混合物；将粉剂混合物与液剂混合物混合均匀,制得磷酸钙微球改性PMMA骨水泥。该骨水泥可有效改善临床骨水泥生物活性差及PMMA弹性模量高易导致邻近锥体骨折发生的问题。(The invention provides a hollow calcium phosphate microsphere/glycerol modified PMMA bone cement and a preparation method thereof, and the preparation method comprises the following steps: mixing a calcium salt solution and a phosphate solution to form a suspension, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution, heating for reaction, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate microspheres; adding the calcium phosphate microspheres into the bone cement powder, and uniformly mixing to obtain a powder mixture; adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture; and uniformly mixing the powder mixture and the liquid mixture to prepare the calcium phosphate microsphere modified PMMA bone cement. The bone cement can effectively solve the problems that the clinical bone cement has poor biological activity and PMMA has high elastic modulus and is easy to cause fracture of an adjacent cone.)

1. A preparation method of hollow calcium phosphate microsphere/glycerol modified PMMA bone cement is characterized by comprising the following steps:

(1) mixing a calcium salt solution and a phosphate solution to form a suspension, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution, heating for reaction, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate microspheres;

(2) adding the calcium phosphate microspheres obtained in the step (1) into bone cement powder, and uniformly mixing to obtain a powder mixture;

(3) adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture;

(4) and (3) uniformly mixing the powder mixture in the step (2) and the liquid mixture in the step (3) to prepare the calcium phosphate microsphere modified PMMA bone cement.

2. The method for preparing hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, characterized in that the calcium salt solution in the step (1) has a molar concentration of 0.05-0.2mol/L and the molar ratio of calcium salt to calcium and phosphorus in phosphate is 1.5-2: 1.

3. The method for preparing a hollow calcium phosphate microsphere/glycerol-modified PMMA bone cement according to claim 1, wherein the phosphate is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium pyrophosphate and disodium hydrogen phosphate; the calcium salt is anhydrous calcium chloride or calcium nitrate.

4. The method for preparing hollow calcium phosphate microsphere/glycerol-modified PMMA bone cement according to claim 1, wherein the pH is adjusted to 9-10 in the step (1).

5. The method for preparing the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, wherein the reaction is carried out for 1-3h at the temperature of 130-170 ℃ in the step (1).

6. The method for preparing hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, characterized in that in step (1), the reaction is carried out for 2 hours at 150 ℃.

7. The method for preparing hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, wherein in step (2) the calcium phosphate microspheres account for 8-12% of the weight of the powder mixture.

8. The method for preparing hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, characterized in that the glycerol accounts for 15-25% of the liquid mixture volume in the step (3).

9. The method for preparing the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement as claimed in claim 1, characterized in that in the step (4), the powder mixture and the liquid mixture are uniformly mixed according to the mass-volume ratio of 1.5-2.5: 1.

10. A hollow calcium phosphate microsphere/glycerol modified PMMA bone cement, characterized in that it is obtained by the method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the field of bone cement, and particularly relates to hollow calcium phosphate microsphere/glycerol modified PMMA bone cement and a preparation method thereof.

Background

Along with the rapid development of medical technology and economy in China, arousal of population, aging and health consciousness and obvious increase of the diagnosis rate of various degenerative diseases, wherein more and more patients with osteoporosis vertebral compression fracture gradually become social problems which puzzle the health of the old people. For the patients, due to the age, the general condition is poor, and the factors of bone mass reduction, bone strength reduction, bone fragility increase, poor bone healing capability and the like caused by the old age make the patients difficult to achieve the requirements of restoring the spinal structure and functional integrity by self-repairing function.

At present, the clinical common methods for treating the osteoporotic vertebral compression fracture are as follows: (1) conservative treatment: comprises bed rest, drug analgesia, brace external fixation and the like. But conservative treatment can not correct spinal deformity, and other related complications are easily caused because long-term bed rest is needed. (2) Open surgery: trauma is great and, because patients are generally ill, often without surgical conditions, internal fixation devices often fail to loosen due to severe osteoporosis. (3) Minimally invasive surgery: most of the methods adopt PKP or PVP, and PMMA bone cement is injected into a vertebral body with compression fracture to re-expand the vertebral body, recover the spine structure and stress, quickly relieve pain and recover normal activities at an early stage.

The PMMA bone cement used clinically at present has the following problems: (1) high heat is released during solid-liquid polymerization, cells around bone cement are killed, and the bone regeneration and repair process is inhibited; (2) the material has poor biocompatibility and certain cytotoxicity, and inhibits the proliferation of peripheral osteoblasts; (3) in the aspect of mechanical strength, the elastic modulus of the vertebral body is higher, and the adjacent vertebral body is easy to fracture again due to the difference of the elastic modulus of the vertebral body and the modulus of the human vertebral body; (4) when PMMA is modified, the elastic modulus is reduced and the compressive strength is also greatly reduced.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement and the preparation method thereof, and the bone cement can effectively solve the problems that the clinical bone cement has poor biological activity and PMMA elastic modulus is high, which is easy to cause the adjacent cone fracture.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of hollow calcium phosphate microsphere/glycerol modified PMMA bone cement comprises the following steps:

(1) mixing a calcium salt solution and a phosphate solution to form a suspension, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution, heating for reaction, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate microspheres;

(2) adding the calcium phosphate microspheres obtained in the step (1) into bone cement powder, and uniformly mixing to obtain a powder mixture;

(3) adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture;

(4) and (3) uniformly mixing the powder mixture in the step (2) and the liquid mixture in the step (3) to prepare the calcium phosphate microsphere modified PMMA bone cement.

Further, in the step (1), the molar concentration of the calcium salt solution is 0.05-0.2mol/L, and the molar ratio of calcium salt to calcium and phosphorus in phosphate is 1.5-2: 1.

Further, the phosphate is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium pyrophosphate and disodium hydrogen phosphate; the calcium salt is anhydrous calcium chloride or calcium nitrate.

Further, the pH is adjusted to 9-10 in step (1).

Further, the reaction is carried out for 1-3h at the temperature of 130-170 ℃ in the step (1).

Further, in the step (1), the reaction is carried out for 2 hours at the temperature of 150 ℃.

Further, in the step (2), the calcium phosphate microspheres account for 8-12% of the weight of the powder mixture.

Further, in the step (3), the glycerol accounts for 15-25% of the volume of the liquid mixture.

Further, in the step (4), the powder mixture and the liquid mixture are uniformly mixed according to the mass-volume ratio of 1.5-2.5: 1.

The beneficial effects produced by the invention are as follows:

in the application, the calcium phosphate hollow microspheres with good biocompatibility and excellent osteogenic performance are dispersed into PMMA, on one hand, due to the hollow calcium phosphate microsphere structure, the elastic modulus of PMMA can be reduced, so that the performance of the bone cement is close to that of a normal bone of a human body; on the other hand, the calcium phosphate material has excellent bone inductivity, can firmly connect the bone cement with surrounding tissues, and avoids the loosening and sinking in vivo. The glycerol is added into the bone cement, so that the elastic modulus of the traditional PMMA bone cement is reduced, and the mechanical strength of PMMA is kept.

The calcium phosphate microsphere/glycerol modified PMMA bone cement has good cell proliferation and osteoinductivity, maintains the mechanical strength and simultaneously reduces the elastic modulus of the bone cement.

Drawings

FIG. 1 is an electron microscope scan of bone cements in comparative examples 1-3 and example 3, a being the bone cement in comparative example 1, b being the bone cement in comparative example 2, c being the bone cement in comparative example 3, d being the bone cement in example 3;

FIG. 2 is a statistical chart of elastic modulus measurements of bone cements in comparative examples 1 to 3 and example 3, wherein 1 is the bone cement in comparative example 1, 2 is the bone cement in comparative example 2, 3 is the bone cement in comparative example 3, and 4 is the bone cement in example 3;

FIG. 3 is a staining pattern of living and dead cells of co-culture of bone cement and mesenchymal stem cells in comparative examples 1-3 and example 3, wherein the staining pattern includes firstly the bone cement in comparative example 1, secondly the bone cement in comparative example 2, thirdly the bone cement in comparative example 3, and fourthly the bone cement in example 3;

FIG. 4 is a statistical view of cell proliferation performed by the bone cements in comparative examples 1 to 3 and example 3, wherein the bone cement in comparative example 1, the bone cement in comparative example 2, the bone cement in comparative example 3, and the bone cement in example 3 are shown;

FIG. 5 is a photograph showing the alizarin red staining after cell culture of the bone cements in comparative examples 1-3 and example 3, wherein the bone cement in comparative example 1, the bone cement in comparative example 2, the bone cement in comparative example 3 and the bone cement in example 3 are shown.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings.

Example 1

A preparation method of the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement comprises the following steps:

(1) mixing an anhydrous calcium chloride solution with the molar concentration of 0.05mol/L and a disodium hydrogen phosphate solution to form a suspension, wherein the molar ratio of calcium to phosphorus of the two is 1.5:1, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution to be 9, heating to 130 ℃, reacting for 1h, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate powder;

(2) adding the calcium phosphate powder obtained in the step (1) into bone cement powder, and uniformly mixing to obtain a powder mixture, wherein the calcium phosphate microspheres account for 8% of the weight of the powder mixture;

(3) adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture, wherein the glycerol accounts for 15% of the volume of the liquid mixture;

(4) and (3) uniformly mixing the powder mixture in the step (2) and the liquid mixture in the step (3) to prepare the modified bone cement.

Example 2

A preparation method of the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement comprises the following steps:

(1) mixing a calcium nitrate solution with the molar concentration of 0.2mol/L and an ammonium dihydrogen phosphate solution to form a suspension, wherein the molar ratio of calcium to phosphorus is 2:1, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution to be 10, heating to 170 ℃, reacting for 2 hours, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate powder;

(2) adding the calcium phosphate powder obtained in the step (1) into the bone cement powder, and uniformly mixing to obtain a powder mixture, wherein the calcium phosphate powder accounts for 12% of the weight of the powder mixture;

(3) adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture, wherein the glycerol accounts for 25% of the volume of the liquid mixture;

(4) and (3) uniformly mixing the powder mixture in the step (2) and the liquid mixture in the step (3) to prepare the modified bone cement.

Example 3

A preparation method of the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement comprises the following steps:

(1) mixing an anhydrous calcium chloride solution with the molar concentration of 0.1mol/L and a disodium hydrogen phosphate solution to form a suspension, wherein the molar ratio of calcium to phosphorus of the anhydrous calcium chloride solution to the disodium hydrogen phosphate solution is 1.67:1, dripping phytic acid into the suspension until the suspension is clear again, adding urea into the suspension to adjust the pH value of the solution to be 9, heating the solution to 150 ℃, reacting for 2 hours, collecting precipitates, washing and freeze-drying to obtain calcium phosphate powder;

(2) adding the calcium phosphate powder obtained in the step (1) into the bone cement powder, and uniformly mixing to obtain a powder mixture, wherein the calcium phosphate powder accounts for 10% of the weight of the powder mixture;

(3) adding glycerol into the bone cement liquid, and uniformly mixing to obtain a liquid mixture, wherein the glycerol accounts for 20% of the volume of the liquid mixture;

(4) and (3) uniformly mixing the powder mixture in the step (2) and the liquid mixture in the step (3) to prepare the modified bone cement.

Comparative example 1

The preparation method of the bone cement comprises the following steps: and directly and uniformly mixing the bone cement solid phase and the bone cement liquid phase according to the mass volume percentage ratio of 200% to prepare the bone cement.

Comparative example 2

The preparation method of the modified PMMA bone cement comprises the following steps:

(1) adding glycerol into the bone cement liquid phase, and uniformly mixing to obtain a liquid mixture, wherein the glycerol accounts for 20% of the volume of the liquid mixture;

(2) and (2) uniformly mixing the liquid mixture in the step (1) with a bone cement solid phase to obtain the bone cement.

Comparative example 3

A preparation method of the hollow calcium phosphate microsphere/glycerol modified PMMA bone cement comprises the following steps:

(1) mixing an anhydrous calcium chloride solution with the molar concentration of 0.1mol/L and a disodium hydrogen phosphate solution to form a suspension, wherein the molar ratio of calcium to phosphorus of the anhydrous calcium chloride solution to the disodium hydrogen phosphate solution is 1.67:1, dripping phytic acid until the suspension is clear again, adding urea to adjust the pH value of the solution, heating to 150 ℃, reacting for 2 hours, collecting precipitates, cleaning, and freeze-drying to obtain calcium phosphate powder;

(2) adding the calcium phosphate powder in the step (1) into a bone cement solid phase, and uniformly mixing to obtain a solid phase mixture, wherein the calcium phosphate powder accounts for 10% of the weight of the solid phase mixture;

(3) and (3) uniformly mixing the powder mixture obtained in the step (2) with the bone cement liquid to obtain the bone cement.

Test examples

The obtained bone cements in example 3 and comparative examples 1 to 3 were subjected to physical characterization, elastic modulus measurement, live and dead cell measurement, cell proliferation, alizarin staining, respectively.

1. The physical characterization experiment comprises the following specific operation processes: the bone cement in comparative examples 1 to 3 and example 3 was ground, round pieces of 10mm in diameter and 2mm in thickness, respectively designated as four groups a, b, c and d, were observed under a scanning electron microscope, and the results are shown in fig. 1.

As can be seen from fig. 1, comparative examples 1 and 2 are similar in surface and have no obvious difference; comparative example 3 it can be seen that calcium phosphate microspheres are dispersed inside the cement; example 3 it can be seen that the calcium phosphate powder is actually hollow calcium phosphate microspheres embedded inside the cement.

2. The specific operation process of the elastic modulus detection experiment is as follows: the bone cements in comparative examples 1 to 3 and example 3 were ground into cylinders with a diameter of 5mm and a height of 10mm, which were respectively labeled as four groups a, b, c, and d, and the elastic modulus and the compressive modulus were measured using a universal mechanical tester, and the specific test results are shown in fig. 2.

As can be seen from fig. 2, the elastic modulus of comparative example 1 is the largest, and the other three examples have no statistical difference. While example 3 had the least compressive strength, the remaining three cases were not statistically different.

3. The specific process of the live and dead cell staining experiment is as follows: inoculating rBMSCs of P3 generation into 6-hole plate, dividing into five groups, each group has 3 parallel holes of 1 × 10⁵And adding the PMMA bone cement leaching liquor obtained in comparative examples 1-3 and example 3 into the components I to II, culturing for 3 days, and dyeing by using a live and dead cell dyeing reagent, wherein the specific result is shown in figure 3.

As can be seen from FIG. 3, in the case of group (r), i.e., group (3), the material leaching solution has the strongest ability to maintain the cell activity, and the strong to weak groups are group (c), and group (r) in sequence.

4. The specific operation process of the cell proliferation test experiment is as follows: rBMSCs of generation P3 (inoculated in a 96-well plate, divided into five groups, each group is provided with 12 parallel holes, each hole is about 1 multiplied by 10³Adding the PMMA bone cement leaching liquor obtained in the comparative examples 1-3 and the example 3 into the components I to II, respectively, culturing for 1, 3, 5 and 7 days, detecting the proliferation condition of the cells by adopting a CCK-8 method, namely removing the culture medium, adding 90 mu l of fresh culture medium and 10 mu l of CCK-8 cell proliferation detection solution into each hole, incubating for 2 hours, and detecting the absorbance value at the wavelength of 450nm by using an enzyme-labeling instrument, wherein the specific result is shown in figure 4.

As can be seen from FIG. 4, the group (r), that is, the group (3) of the example, has the strongest proliferation promoting property of the material leaching solution, and the group (c), the group (g) and the group (r) are sequentially from strong to weak.

5. In the alizarin red staining experiment, the process is as follows: inoculating rBMSCs of P3 generation into 6-hole plate, dividing into blank group, first group, second group, third group and fourth group, each group is provided with 3 parallel holes, each hole is about 1 × 10⁵And adding the PMMA bone cement leaching liquor obtained in comparative examples 1-3 and example 3 into the components I to II, culturing for 14 days, and dyeing by using an alizarin red dyeing kit, wherein the specific result is shown in figure 5.

As can be seen from FIG. 5, group (r), i.e., group (3), the mineralization promoting ability of the material leaching solution is strongest, and the group (c), and the group (r) are sequentially from strong to weak.