| 1 ObjectivesDiscogenic low back and leg pain is a common orthopedic disease. Case number of low back and leg pain has been growing with the development of modern society and change of working habits and lifestyle. Traditional surgeries like resection of nucleus pulposus and interbody fusion are effective, while they may lead to lumbar instability and lumbar load change, and quicken the deterioration of adjacent lumber segments. With the fast development of dynamic lumbar stabilization and reconstruction concepts and wide application of various dynamic stabilization devices and technologies in recent 10 years, certain clinical efficacy has been obtained, but many problems still remains. To solve these problems, people are still exploring and perfecting non-fusion technologies Prosthetic disc nucleus replacement has shown promising application future. It can retain the integrity of anatomic intervertebral structure and alleviate degeneration of adjacent segments, and will lead to small trauma and fast recovery, so it is more suitable to early and metaphase intervertebral disc degeneration. However, prosthetic disc nucleus replacement still has many disadvantages like prosthesis extrusion, so it is not widely clinically applied. Therefore, we invented a new injectable prosthetic disc nucleus, which is made of two parts: the external saccule is made of polyurethane and the internal nucleus is made of aggregated silicon rubber. Polyurethane and silicon rubber have been widely used in medical industry, while as a kind of long-term implant, to evaluate its bio-safety, a series of tests need to be carried out to study genotoxicity of prosthetic disc nucleus in accordance with the biocompatibility standards of medial implants. 2 Methods1. In accordance with the biological standards of medical devices, accurately weigh 25 g polyurethane and silicon rubber samples, cut them into grains of 2~3mm, prepare the extraction solution by adding samples into 125 ml extraction medium as per 0.2 g/ml proportion. Choose healthy male adult SD rats, intraperitoneally inject 500 mg/kg ArOclorl254, mutate the animals, sacrifice them in 5 days, harvest their livers, make the liver into even liver homogenate in a tissue homogenizer, centrifuge at low temperature and high speed, the supernatant aspired is S9 component, and prepare S9 component into S9 mixed liquid.2. Salmonella Typhimurium Reverse Mutation Assay (Ames test): it adopts plate permeating method. The samples are divided into polyurethane group and silicon rubber group, and also negative control group, solvent control group and positive control group. Choose histidine-deficient salmonella typhimurium TA97, TA98, TA100, TA102 and TA1535 test strains. Dilute polyurethane and silicon rubber with leaching liquor, prepare them into 5 doses: 4ul/dish, 10ul/dish, 40ul/dish, 100ul/dish and 200ul/dish. With leaching medium as solvent control and not any treatment to negative control, positive controls are as follows: when metabolic activation system (S9) is not added, use atabrine (500 ug/dish) for TA97, use nitroquinoline (200 ug/dish) for TA98, use methyl methanesulphonate (1ul/dish) for TA100 and TA 102, use NaN3 (3ug/dish) for TA1535; when metabolic activation system (S9) is added, use 2-FA (10 ug/dish) for TA97, TA 98 and TA100, use 2,8-dihydroxyanthraquinone (50 ug/dish) for TA 102, use 2-aminoanthracene (2ug/dish) for TA1535. Accurately tranfer 0.1 ml enrichment broth of test strains, 0.2 ml test sample solution and 0.5 ml S9 mixed solution (if metabolism activation is needed), fully mixed them on the plate, put the plate in an incubator at 7℃, incubate for 48 h, record the number of reverse colony in each dish. Each test sample will have three parallel dishes whether S9 mixed solution is added or not.3. Chromosomal aberration test: divide samples into polyurethane group and silicon rubber group, use DMEM cell culture medium as negative control, use mitomycin C (0.25ug/ml) as positive control when S9 is not added and use cyclophosphamide as positive control (20ug/ml) as positive control when S9 is added. Prepare the samples into the following concentrates: 5000ug/ml for high concentrate group, 2500ug/ml for middle concentrate group and 1250 ug/ml for low concentrate group. During the day prior to the experiment, choose well-developed CHO cells, prepare them into suspension of 2×105 cells/ml, accurate transfer 5ml suspension, inoculate it in a culture flask, put the flask in a CO2 incubator, culture for 24h. Take the culture solution, divide it into different groups, add 4.5 ml test sample solution or negative/positive control and 0.5 ml S9 mixed solution (make compensation with relevant solutions if S9 mixed solution is not added), put them in an incubator, culture for 4h, take culture media out, wash cells with Hanks solution for 3 times, add 5ml 10% fetal calf serum culture media, put it back into the incubator, culture for 4h, add colchicine (1μg/ml) 4h before the harvest. After digestion, low permeating, sterilization, silde preparation and staining with normal procedures, observe the number of mitotic cells of each group. Choose 200 well-developed metaphase cells, observe the number and types of chromosome aberrations, and calculate the percentage of cells with chromosome aberrations.4. Micronucleus test: The samples are divided into polyurethane group and silicon rubber group, with physical saline as negative control and cyclophosphamide (40mg/kg) as positive control. Select 32 one-month-old, 20～32g-weight Kunming rice, divide them into 2 groups, with 8 in each group and half-and-half male and female. Using oral lavage method, infuse extraction solution (25ml/kg) into rats in test group, infuse physical saline (25ml/kg) into rats in negative control group, and infuse cyclophosphamide physical solution (1.6 mg/kg) into rats in positive control group. The rats have twice administrations during 30 hours, with 24h interval between two administrations, and harvest material from rats 6h after the second administration. Sacrifice the rats through cervical dislocation, separate and resect femur at one side, flush bone marrow cells into a centrifuge tube with 1ml calf serum, fully mix and centrifuge the cells, discard the supernatant, fully mixed the cells left, transfer one drop of cells onto a slide, dry the slide and have Gimsa staining. Count 1000 bone marrow polychromatic erythrocytes of each mouse, record the number of micronuclei and micronucleated cells, and calculate MCNR.3 Results1. Ames test. Whether adding S9 or not, when metabolism activation system is added to 5 strains in different doses of test samples, number of reverse colonies is less than twice the number of negative control group, therefore, Ames test is negative.2. Chromosomal aberration test. When polyurethane is 5.0mg/ml, 2.5mg/ml and 1.25mg/ml, mitotic indices of CHO cells are 2.72%, 2.60% and 2.72% respectively; while silicon rubber is of the same concentration with polyurethane, mitotic indices of CHO cells are 2.54%, 2.74% and 2.68% respectively, no significant difference when compared with 2.78% of negative control group (P>0.05). Except some test groups (1.25 polyurethane with S9, 1.25 polyurethane without S9, 2.5 mg/ml silicone rubber without S9) have 0.5% chromosome aberration rate, other test groups have 0% chromosome aberration rate. Chromosome aberration rates of all test groups do not have significant difference when compared to that of negative control group (0.5% when S9 is added, 0% when S9 is not added), but significantly less than that of positive control group (29.5% when S9 is added, 26.5% when S9 is not added) (P<0.01).3. Micronucleus test. When 5g/kg polyurethane takes effect, MNCR of mouse bone marrow is 2.38‰±1.06‰, and when 5g/kg silicon rubber takes effect, MNCR of mouse bone marrow is 2.88‰±2.17‰, without significant difference when compared with that of negative control group (1.63±1.19‰) and significantly less than that of positive control group (36.25‰±8.54‰) (P<0.01).4 ConclusionAmes test, chromosome aberration test and micronucleus test obtained negative results in terms of DNA, chromosome and gene in this study. This shows that polyurethane and silicone rubber do not have impact on the inheritance of organisms and also provides biological evidence for further clinical study. |
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