Mutagenicity test of magnetic nanocomposite for interface fixation

Abstract

The aim of the study is to evaluate the biocompatibility and safety of the Nano-HA/PLLA/Fe₂O₃ magnetic composites material from toxicological aspects for the clinical use of the interface fixation, produce the Nano-HA/PLLA/Fe₂O₃ magnetic composites material and its diffusion, and examine the diffusion through Ames test. The observation markers of the test are the mutagenic rates (MRs) of murine typhoid salmonella. The results of MR are less than 2 in the experimental groups of all dosage levels. The Nano-HA/PLLA/Fe₂O₃ magnetic composites material will not cause the increase of the back mutation in the MTS. It indicates that this artificial bone will not cause gene mutation.

Introduction

Interface Screw is a commonly used tendon fixation material in the arthroscopic anterior cruciate ligament (ACL) reconstruction. However, this material is associated with the defects of poor mechanical strength, foreign body reaction, inconsistency between the degradation rate and the rate of bone tunnel healing, and the absence of osteoinductive activity. The poor union of tendon–bone or biomaterial–bone interface is likely to occur as a result of these defects, resulting in postoperative bone tunnel enlargement and relaxation of ligament. ¹ In this study, low temperature-rapid prototyping machine was used to prepare for the scaffold of the Nano-HA/PLLA/Fe₂O₃ magnetic composites material with the raw materials of Nano-HA, PLLA, and Fe₂O₃. ^2,3 By the standards about the evaluation of biomaterials, ^3,4 the biocompatibility of the Nano-HA/PLLA/Fe₂O₃ scaffold for interface fixation was evaluated by Ames test from the perspective of genetic toxicology. The results provide biosecurity data for the clinical application of the Nano-HA/PLLA/Fe₂O₃ magnetic composites material in the fixation of tendon-to-bone interface in ACL reconstruction.

Materials and methods

Material preparation

The Nano-HA/PLLA/Fe₂O₃ magnetic composites material for interface fixation was developed by State Key Laboratory of Tissue Engineering of Shenzhen Second People’s Hospital in conjunction with Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The magnetic composite was cubic, as shown in Figure 1. The Nano-HA/PLLA/Fe₂O₃ magnetic composites material was first irradiated by ultraviolet ray for half an hour before being soaked and rinsed with PBS. Then the material was disinfected with 75% ethanol and dried for later use.

Figure 1.

Nano-HA/PLLA/Fe₂O₃ composite.

Experimental methods

The Nano-HA/PLLA/Fe₂O₃ magnetic composites material was mixed with 2.5% (weight ratio) carboxymethyl cellulose sodium to prepare suspensions with the concentrations of 5, 0.5, and 0.01 mg/ml, respectively. ⁵ The histidine-defective strains of Salmonella typhimurium (TA-97, TA-98, TA-100, and TA-102) were provided by Ames Laboratory (USA) and reproduced and preserved by Hunan Center for Disease Prevention and Control. The strains were used for the experiment after being confirmed as qualified by trait identification. The Nano-HA/PLLA/Fe₂O₃ magnetic composites material for interface fixation is implanted into the human body, and the human hepatic microsomal enzyme (S9) may have a mutagenic action on HA. However, the cells cultured in vitro do not contain such enzyme. To make the experimental conditions resemble with the human in vivo environment, we divide the culture plates into activation group (with the induction of S9 solution) and non-activation group (without the induction of S9 solution). Rat hepatic microsomal enzyme (S9) was induced by polychlorinated biphenyl and prepared into liver homogenate to be stored in the fridge at −80°C. The activity determination was carried out using 2-aminofluorene. Daunomyc in (25 ug/0.1 ml) was used as the direct indicator of mutagenic activity in bacterial culture (without the addition of S9). 2-Aminofluorene (50 ug/0.1 ml) was used as the indirect indicator of mutagenic activity (with the addition of S9). Sodium azide (5 ug/0.1 ml) was used as the positive control and physiological saline of equal volume as the negative control. Three replicates were set up for each strain and each dosage, respectively.

Plate infiltration method was employed for the mixed culture of suspension with different concentrations and strain on the plate with the lowest nutritional level. After culture at 37°C for 48 h, the colonies showing reverse mutation were counted. The average number of colonies showing reverse mutation was calculated for three replicates of each group. The mutagenic substances were detected in nano-HA material according to mutagenicity ratio calculated by the following formula:

$\text{Mutagenicity ratio} \text{(MR})\text{=Number of reversely mutated colonies in the experimental group }\left(\text{Rt}\right)\text{/number of reversely mutated colonies in the negative control }\left(\text{Rc}\right).$

Results

Under different test concentrations with and without the addition of S9, the Rt/Rc ratios of all four strains were lower than 2.0. However, the Rt/Rc ratio in the positive control was larger than 2 (see Table 1). It could be inferred that Nano-HA/PLLA/Fe₂O₃ magnetic composites material for interface fixation did not have mutagenicity in Ames test.

Table 1.

Results of Ames test of nano-HA artificial bone.

	TA97				TA98				TA100				TA102
	+S9		−S9		+S9		−S9		+S9		−S9		+S9		−S9
Strain	R	MR	R	MR	R	MR	R	MR	R	MR	R	MR	R	MR	R	MR
5 mg/ml group	123	1.12	123	1.02	39	0.72	42	0.88	93	0.81	84	0.72	85	0.76	97	0.75
0.5 mg/ml group	134	1.15	111	0.98	79	1.75	45	0.87	146	1.37	95	0.80	109	0.88	114	0.97
0.01 mg/ml group	117	0.93	129	1.06	81	1.59	39	0.83	138	1.33	94	0.80	122	0.92	92	0.84
Negative control	116		125		52		43		105		118		125		129
Positive control	1679	17	1689	13	1405	28	1412	31	1886	19	1899	19	2218	16	2225	20

R: number of colonies showing reverse mutation; MR: mutagenicity ratio.

Discussion

Biocompatibility refers to the sum of complex biological, physical, and chemical reactions induced by the interaction between the biomaterial and the human body as well as the human tolerance to these reactions. According to standard ISO10993, we will perform biological tests on biomedical materials that are in long-term close contact with or implanted into the human body for potential genetic toxicology. ⁶ Ames test was established by Ames (USA) in 1975 for reverse mutation test of S. typhimurium. It is a widely adopted method for mutagenicity detection. ⁷ The detection principle is as follows: for the histidine-defective strains of S. typhimurium (TAhis-) cultured on the histidine-defective medium, only a few colonies undergoing spontaneous reverse mutation can grow. If the test medium contains the mutagenic substances, the reverse mutation will occur with a greater probability. It follows that more colonies will be growing on the histidine-defective medium. This method can detect the short-term mutagenicity of medical materials.

In the present experiment, the Nano-HA/PLLA/Fe₂O₃ magnetic composites material was mixed with different standard bacterial liquids on the plate with the lowest nutritional level. The presence of mutagenic substances in the artificial bone was detected according to the number of reversely mutated colonies and MR. After the biomaterial is implanted into the human body, the real human in vivo experiment was simulated by the induction or non-induction of the human hepatic microsomal enzyme (S9). In other words, the experiment consisted of the S9 (+) group and S9 (−) group to detect the possibility of mutagenicity activated by S9. We observed whether the colonies undergoing reverse mutation are increased after the induction of S9. Moreover, we performed the strict aseptic technique to prevent the contamination by other microorganisms. In this way, the reliability of the experimental results was enhanced.

The results show that the average number of reversely mutated colonies of each strain under each concentration did not exceed the average number in the negative control by 2 times (MR < 2). It indicates that the biomaterial did not have mutagenic activity with or without the addition of S9. The average number of reversely mutated colonies of each strain in the positive control all exceeded that of the negative control by over 2 times (MR > 2). It reflects that the histidine-defective strains of S. typhimurium are effective in detecting the mutagenicity of the relevant biomaterial. We come to the conclusion that the Nano-HA/PLLA/Fe₂O₃ magnetic composites material has no genetic toxicology and can be safely implanted into the human body. Our research may provide reference for future animal experiments and clinical application.