Method for evaluating safety of bone cement by using release amount of methyl methacrylate in curing process

Abstract

The invention belongs to the technical field of bone cement, and discloses a method for evaluating the safety of bone cement by using the release amount of methyl methacrylate in a curing process. The method comprises the following steps: 1) respectively injecting to-be-detected bone cement and known bone cement into an artificial joint simulation material, recording the injection amounts of the to-be-detected bone cement and the known bone cement, and soaking the artificial joint simulation material in water or an ethanol water solution; (2) testing the content of methyl methacrylate in the simulated leaching solution and the limit leaching solution by adopting a liquid chromatography; 3) respectively calculating the release amount of methyl methacrylate in the bone cement to be detected and the known bone cement; and 4) when the simulation extraction result is not significant, the bone cement to be tested is safe in the aspect of methyl methacrylate release, then limit extraction is used as supplementary verification safety test, and significance analysis is performed on the limit extraction result. The method is simple and easy to implement, and fills the blank of safety evaluation of the bone cement in the aspect of monomer release.

Description

Technical Field

[0001] The invention belongs to the technical field of bone cement, and particularly relates to a method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process. Background Art

[0002] Bone cement is a radiopaque, fast-setting polymethylmethacrylate bone cement used to fill and stabilize vertebral bodies. It is prepared by mixing a powdered component with a liquid component. After mixing, a low-viscosity paste is formed, which is injected into the body as a stabilizing substance and then solidifies.

[0003] After using bone cement technology, the long-term loosening rate is significantly reduced, but there are also disadvantages. The main harmful substance released into the human body from bone cement is methyl methacrylate (MMA). Long-term exposure to methyl methacrylate can lead to chronic poisoning, mainly manifested by comprehensive symptoms of nervous system damage, and even toxic encephalopathy, which has certain carcinogenicity.

[0004] How to evaluate the safety of bone cement in the human body is one of the problems that people need to solve. Currently, there is no literature involving the evaluation of the safety of bone cement. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method for evaluating the safety of bone cement by using the amount of methyl methacrylate released during the curing process. The method of the present invention is simple, reliable, and easy to implement, and supplements the deficiencies of the safety evaluation of bone cement.

[0006] The purpose of the present invention is achieved by the following method:

[0007] A method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process comprises the following steps:

[0008] 1) Simulated leaching: Mix the components of the bone cement to be tested and the components of the known bone cement, then inject the bone cement to be tested and the known bone cement into the artificial joint simulation material respectively, and record the injection amount of the bone cement to be tested and the injection amount of the known bone cement, and the artificial joint simulation material is immersed in water;

[0009] 2) Extreme extraction: Mix the components of the bone cement to be tested and the components of the known bone cement, then inject the bone cement to be tested and the known bone cement into the artificial joint simulation material respectively, and record the injection amount of the bone cement to be tested and the injection amount of the known bone cement, and the artificial joint simulation material is immersed in an ethanol aqueous solution with a density of 0.9373-0.9378 g / mL;

[0010] 3) For simulated extraction, the same amount of simulated extract was taken out at different extraction times; for extreme extraction, the same amount of extreme extract was taken out at different extraction times, and the contents of methyl methacrylate in the simulated extract and the extreme extract were tested by liquid chromatography respectively;

[0011] 4) Calculate the release amount of methyl methacrylate in the test bone cement and known bone cement in simulated extraction and extreme extraction respectively;

[0012] 5) The results of simulated extraction of the bone cement to be tested and the known bone cement are subjected to significance analysis. When it is not significant, the bone cement to be tested is safe in terms of methyl methacrylate release; then, extreme extraction is used as a supplementary safety verification test, and the results of extreme extraction of the bone cement to be tested and the known bone cement are subjected to significance analysis.

[0013] The extreme extraction is to extract methyl methacrylate to the maximum extent, and is used as another method to verify the sample leaching results. The simulated extraction was not significant, and the extreme extraction was supplemented for verification analysis. The difference in the results was small and not significant, indicating that the bone cement to be tested is safe.

[0014] The known bone cement is a commercially available safe product, and bone cements from Kellytech and / or Heraeus can be selected.

[0015] The soaking temperature in step 1) is 36-38°C. The soaking temperature in step 2) is 36-38°C.

[0016] In the simulated extraction in step 3), the different times are 1 min, 3 min, 5 min, 7 min, 10 min, 15 min, 30 min, 1 h, 24 h, 48 h, and 72 h.

[0017] In the extreme extraction, the different times are 24h and 72h.

[0018] The detection conditions of the liquid chromatography in step 3) are:

[0019] Chromatographic column: Shimadzu WondaSil C18-WR (4.6×250mm, 5μm)

[0020] Mobile phase A: water

[0021] Mobile phase B: acetonitrile

[0022] Isocratic elution: A:B = 30:70

[0023] Column temperature: 30°C

[0024] Flow rate: 1mL / min

[0025] Detection wavelength: 210nm

[0026] Injection volume: 20 μL.

[0027] The extract in step 3) is filtered using a 0.22 μm filter membrane before liquid chromatography detection.

[0028] The release amount of methyl methacrylate in step 4) is calculated by the following method:

[0029] MMA weight (mg) / bone cement volume (mL) = [MMA concentration in the extract measured by liquid chromatography (mg / mL)*extract volume+MMA weight taken out at the previous extraction time points (mg)] / bone cement volume;

[0030] The volume of the extract refers to the volume of the extract at the extraction time point and includes the volume of the extract for liquid chromatography test. The volume of the extract refers to the volume of water or the volume of ethanol-water solution; the volume of water in simulated extraction and the volume of ethanol-water solution in extreme extraction;

[0031] The weight of MMA taken out at the first few extraction time points (mg) = the sum of the MMA concentrations measured by liquid chromatography at the first few extraction time points * the volume of the extracted extract.

[0032] Specific formula:

[0033] Release amount W = [C tn *V tn +(C t1 +C t2 +......+C t(n-1) )*V] / V 骨水泥 ; where C tn is the MMA concentration in the extract when the extraction time is tn, V tn is the volume of the extract when the extraction time is tn, V is the volume of the extract taken out, that is, the volume of the extract used for liquid chromatography, C t(n-1) is the MMA concentration in the extract when the extraction time is t(n-1); t1, t2, ..., t(n-1), tn represent the extraction time in order; V tn =V t(n-1) -V.

[0034] The artificial joint simulation materials in step 1) and step 2) are respectively sawbones foam and hard polyurethane foam, and the density of the material is 7-8 pcf.

[0035] The volume ratio of the injected amount of the bone cement to the artificial joint simulation material is 1:(15-25).

[0036] The volume ratio of the artificial joint simulation material to the soaking liquid is 1:2 to 6. The soaking liquid in step 1) is water, and the soaking liquid in step 2) is ethanol water solution.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] The invention simulates the environment of bone cement release in human bones and human blood, tests the MMA monomer release during the bone cement curing process, and evaluates the safety performance of the product through significance analysis. The method of the invention is simple, easy to implement, and fills the gap in the safety evaluation of bone cement in terms of monomer release. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a bar graph of the amount of MMA released during the curing process of bone cement when simulating leaching; sample A to be tested: A20220412001. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto.

[0041] Example

[0042] (1) Sample processing

[0043] (1-1) The sample to be tested (sample A) and the known sample (Kellytech, Heraeus) were mixed to prepare the bone cement sample to be tested and the known bone cement sample, respectively. 3.60 mL of the bone cement sample was injected into sawbone foam, respectively. The sawbone foam containing bone cement (volume 4.1 cm*4.0 cm*4.0 cm, model 1522-507, 7.5 PCF, 0.12 g / mL, volume fraction 0.11) was placed in a 300 mL deionized water preheated in a 37°C water bath. The device was sealed and extracted in a 37°C water bath for 72 hours; 2 mL of deionized water samples were taken out at 1 minute, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 24 hours, 48 ​​hours, and 72 hours and placed in a sealable 20 mL glass bottle, and the samples were filtered with a 0.22 μm filter membrane and then the MMA monomer concentration was detected by liquid chromatography; two identical samples were taken out of each bone cement sample at each extraction time point, 1# and 2# represent two identical samples whose immersion liquid is water, and the specific sample labels are shown in Table 1;

[0044] (1-2) The sample to be tested (sample A to be tested (A20220412001)) and the known sample (Kellytech, Heraeus) were mixed to prepare the bone cement sample to be tested and the known bone cement sample, respectively. 3.60 mL of the mixture was added to sawbone foam. The sawbone foam containing bone cement was placed in a device containing 300 mL of deionized water containing 30% ethanol at 37°C, sealed, and extracted in a 37°C water bath for 72 h. After 24 and 72 hours, 2 mL of deionized water containing 30% ethanol was taken out and placed in a sealable 20 mL glass bottle. The sample was filtered with a 0.22 μm filter membrane and then the MMA monomer concentration was detected by liquid chromatography. Two identical samples were taken out from each bone cement sample at each extraction time point. Ethanol 1# and ethanol 2# represent two identical samples whose immersion solution is ethanol-water solution. The specific sample labels are shown in Table 2.

[0045] (2) Liquid chromatography detection conditions

[0046] Chromatographic column: Shimadzu WondaSil C18-WR 4.6×250mm, 5μm

[0047] Mobile phase A: water

[0048] Mobile phase B: acetonitrile

[0049] Isocratic elution: A:B = 30:70

[0050] Column temperature: 30°C

[0051] Flow rate: 1mL / min

[0052] Detection wavelength: 210nm

[0053] Injection volume: 20 μL;

[0054] When the extracting solution is water, the concentration of MMA in the extracting solution is specifically shown in Table 3.

[0055] When the extracting solution is an ethanol aqueous solution, the concentration of MMA in the extracting solution is specifically shown in Table 5.

[0056] (3) Calculate the release amount of MMA monomer in bone cement based on the MMA monomer concentration measured by liquid chromatography:

[0057] MMA weight (mg) / bone cement volume (mL) = [MMA concentration of the extract obtained by HPLC (mg / mL)*volume of the extract (mL) + weight of MMA removed at the previous extraction time points (mg)] / 3.6mL bone cement;

[0058] The weight of MMA removed at the first few elution time points (mg) = the sum of the MMA concentrations (mg / mL) in the extract measured by HPLC at the first few extraction time points * 2mL.

[0059] When the extracting liquid is water, the release amount of MMA in the bone cement measured in this embodiment is shown in Table 4. Figure 1 The bar graph of MMA release during the bone cement solidification process during simulated leaching is shown in Figure 1 shown.

[0060] When the extracting solution is ethanol aqueous solution, the release amount of MMA in the bone cement measured in this example is shown in Table 6.

[0061] (4) Results analysis

[0062] Analyze and compare the extraction results of the sample to be tested and the known bone cement:

[0063] From the results of the simulated leaching test, it can be seen that the monomer release trend of the test sample A (A20220412001) bone cement is similar to that of the Kelly and Heraeus bone cements after injection. The amount of monomer released by the two products continued to increase until it remained relatively stable from 10 minutes to 1 hour after injection.

[0064] From the extreme extraction results, it can be seen that the monomer release trend of the test sample A (A20220412001) bone cement is comparable to that of Kellytech and Heraeus bone cements after injection.

[0065] (5) Significance analysis

[0066] The release amount of the test results was analyzed by software, and the results are shown in Tables 7 and 8, indicating that there is no significant difference between the three groups of data.

[0067] Table 1 Markings of samples taken at different times during simulated leaching of different bone cements

[0068] Extraction time Sample A Kelly Tai Heraeus 1 minute T1#1,T1#2 K1#1,K1#2 H1#1,H1#2 3 minutes T3#1,T3#2 K3#1,K3#2 H3#1,H3#2 5 minutes T5#1,T5#2 K5#1,K5#2 H5#1,H5#2 7 minutes T7#1,T7#2 K7#1,K7#2 H7#1,H7#2 10 minutes T10#1,T10#2 K10#1,K10#2 H10#1,H10#2 15 minutes T15#1,T15#2 K15#1,K15#2 H15#1,H15#2 30 minutes T30#1,T30#2 K30#1,K30#2 H30#1,H30#2 1 hour T60#1,T60#2 K60#1,K60#2 H60#1,H60#2 24 hours T24#1,T24#2 K24#1,K24#2 H24#1,H24#2 48 hours T48#1,T48#2 K48#1,K48#2 H48#1,H48#2 72 hours T72#1,T72#2 K72#1,K72#2 H72#1,H72#2

[0069] Table 2 Markings of samples taken at different times during extreme extraction of different bone cements

[0070] Extraction time Sample A Kelly Tai Heraeus 24 hours T24 Ethanol #1, T24 Ethanol #2 K24 Ethanol #1, K24 Ethanol #2 H24 ethanol #1, H24 ethanol #2 72 hours T72 Ethanol #1, T72 Ethanol #2 K72 Ethanol #1, K72 Ethanol #2 H72 ethanol #1, H72 ethanol #2

[0071] Table 3 MMA concentration of different bone cements at different extraction times during simulated extraction

[0072]

[0073] Table 4 Cumulative amount of MMA released per mL of bone cement during simulated extraction (mg)

[0074]

[0075] Table 5 MMA concentration of different bone cements at different extraction times during extreme extraction

[0076]

[0077] Table 6 Cumulative amount of MMA released per mL of bone cement during extreme extraction (mg)

[0078]

[0079] Table 7 Significance analysis of simulated leaching test results

[0080]

[0081] Note: 1 indicates the test result of sample A bone cement; 2 indicates the test result of Kellytech bone cement; 3 indicates the test result of Heraeus bone cement.

[0082] Table 8 Significance analysis of limit immersion test results

[0083]

[0084] Note: 1 indicates the test result of the bone cement of the tested sample; 2 indicates the test result of Kellytech bone cement; 3 indicates the test result of Heraeus bone cement.

[0085] in conclusion:

[0086] In water, within 1 minute to 30 minutes, the MMA monomer (9.33 mg / mL) released by the test sample A (A20220412001) bone cement was lower than the MMA monomer released by Kellytech bone cement (14.42 mg / mL), and slightly lower than the MMA monomer released by Heraeus bone cement (10.00 mg / mL). Within 1 hour to 72 hours, the MMA monomer (8.47 mg / mL) released by the test sample bone cement was lower than the MMA monomer released by Kellytech bone cement (13.53 mg / mL), and higher than the MMA monomer released by Heraeus bone cement (9.29 mg / mL). A significance analysis of the test results showed that there was no significant difference in the levels of MMA monomer released by the three products, so the product is safe and can be used in terms of monomer release.

[0087] In an ethanol aqueous solution with a density of 0.9373-0.9378 g / mL, the monomer release value of the bone cement of the test sample (8.86 mg / mL) at 24 hours was lower than that of Kellyte bone cement (12.52 mg / mL), and higher than the MMA monomer released by Heraeus bone cement (6.33 mg / mL). Within 72 hours, the monomer release value of the bone cement of the test sample (1.75 mg / mL) was slightly lower than that of Kellyte bone cement (3.36 mg / mL), and higher than the MMA monomer released by Heraeus bone cement (1.19 mg / mL). The test results were analyzed by software for significance, and there was no significant difference in the levels of MMA monomer released by the three products. Therefore, the product is safe and can be used in terms of monomer release.

Claims

1. A method for evaluating the safety of bone cement using the amount of methyl methacrylate released during the curing process, characterized in that: The following steps are involved: 1) Simulated leaching: Mix the components of the bone cement to be tested and the components of the known bone cement, then inject the bone cement to be tested and the known bone cement into the artificial joint simulation material respectively, and record the injection amount of the bone cement to be tested and the injection amount of the known bone cement, and the artificial joint simulation material is immersed in water; 2) Extreme extraction: Mix the components of the bone cement to be tested and the components of the known bone cement, then inject the bone cement to be tested and the known bone cement into the artificial joint simulation material respectively, and record the injection amount of the bone cement to be tested and the injection amount of the known bone cement, and the artificial joint simulation material is immersed in an ethanol aqueous solution with a density of 0.9373-0.9378 g / mL; 3) For simulated extraction, the same amount of simulated extract was taken out at different extraction times; for extreme extraction, the same amount of extreme extract was taken out at different extraction times; and the content of methyl methacrylate in the simulated extract and the extreme extract was tested by liquid chromatography; 4) Calculate the release amount of methyl methacrylate in the test bone cement and known bone cement in simulated extraction and extreme extraction respectively; 5) The results of simulated extraction of the bone cement to be tested and the known bone cement are subjected to significance analysis. When it is not significant, the bone cement to be tested is safe in terms of methyl methacrylate release; then, extreme extraction is used as a supplementary safety verification test, and the results of extreme extraction of the bone cement to be tested and the known bone cement are subjected to significance analysis.

2. The method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process according to claim 1, characterized in that: The detection conditions of the liquid chromatography in step 3) are: Chromatographic column: Shimadzu WondaSil C18-WR, 4.6×250mm, 5μm; Mobile phase A: water Mobile phase B: acetonitrile Isocratic elution: A:B = 30:70 Column temperature: 30°C Flow rate: 1mL / min Detection wavelength: 210nm Injection volume: 20 μL.

3. The method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process according to claim 1, characterized in that: The release amount of methyl methacrylate in step 4) is calculated by the following method: Release amount W = [C tn *V tn +(C t1 +C t2 +......+C t(n-1) )*V] / V 骨水泥 ; where C tn is the MMA concentration in the extract when the extraction time is tn, V tn is the volume of the extract when the extraction time is tn, V is the volume of the extract taken out, that is, the volume of the extract used for liquid chromatography, C t(n-1) is the MMA concentration in the extract when the extraction time is t(n-1); t1, t2, ..., t(n-1), tn represent the extraction time in order; V tn =V t(n-1) -V; The volume of the extract refers to the volume of water or the volume of ethanol-water solution; in simulated extraction, it is the volume of water, and in extreme extraction, it is the volume of ethanol-water solution.

4. The method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process according to claim 1, characterized in that: The artificial joint simulation materials in step 1) and step 2) are respectively sawbones foam and hard polyurethane foam, and the density of the material is 7-8 pcf; The volume ratio of the injected amount of bone cement to the artificial joint simulation material is 1:(15-25); The volume ratio of the artificial joint simulation material to the soaking liquid is 1:2-6 respectively; the soaking liquid in step 1) is water, and the soaking liquid in step 2) is ethanol aqueous solution.

5. The method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process according to claim 1, characterized in that: The soaking temperature in step 1) is 36-38°C; the soaking temperature in step 2) is 36-38°C; In the simulated extraction in step 3), the different extraction times are 1 min, 3 min, 5 min, 7 min, 10 min, 15 min, 30 min, 1 h, 24 h, 48 h, and 72 h; In the extreme extraction, the different extraction times are 24h and 72h; The extract in step 3) is filtered using a 0.22 μm filter membrane before liquid chromatography detection.

6. The method for evaluating the safety of bone cement by utilizing the amount of methyl methacrylate released during the curing process according to claim 1, characterized in that: The known bone cement is a commercially available safe bone cement.

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