- Email: [email protected]
Biological evaluation of a novel copper-containing composite for contraception
Biological evaluation of a novel copper-containing composite for contraception Lu Sun, M.D.,a Xun-bin Huang, M.D.,a Jin-ping Suo, Ph.D.,b Bo-lin Fan, M.D.,c Zong-lin Chen, M.D.,a Wen-xiang Yang, M.D.,c and Juan Li, Ph.D.b a Family Planning Research Institute, Tongji Medical College, and b State Key Laboratory of Mould Technology, College of Materials Science and Engineering, Huazhong University of Science and Technology; and c Hubei Provincial Academy of Preventive Medicine, Wuhan, People’s Republic of China
Objective: To investigate the biocompatibility of a novel copper-containing composite to provide preclinical data for clinical application of intrauterine device (IUD) or intra-vas device (IVD). Design: Prospective experimental study. Setting: Good laboratory practices laboratories. Animals: Twenty healthy adult mice (SPF grade Kunming white mice, animal code SCXK 2003-0005). Intervention(s): Cytotoxicity tests in vitro were conducted to evaluate the influence of the materials on the morphology, growth, and proliferation of cultured L929 mouse fibroblasts. Acute systemic toxicity tests were conducted to investigate the acute systemic toxic reaction with mice, and then the materials were implanted into the spinal muscle of rabbits (n ¼ 15). The rabbits were sacrificed for pathologic examination at 1, 4, and 12 weeks after surgery. Main Outcome Measure(s): Evaluation of cytotoxicity by MTT assay, cytotoxicity test by direct contact assay, acute systemic toxicity test, and material implantation test. Result(s): The cytotoxicity grade of the copper-containing composite was 0–1, suggesting that the material was free of cytotoxicity; no acute systemic toxicity was found in any mice; mild inflammatory reaction was observed in the surrounding tissues of the implanted material in the early implantation stage, which was similar to that of the sham-operated sides. Twelve weeks after implantation, the inflammatory reaction was completely disappeared in the implanted tissue, similarly to the sham-operated sides. The fibrosis membrane surrounding the material became stable gradually over time. Conclusion: The copper-containing composite has excellent biocompatibility, which is feasible and safe for the clinical application as a novel contraceptive material. (Fertil Steril 2011;95:1416–20. 2011 by American Society for Reproductive Medicine.) Key Words: Copper, contraception, composite, intrauterine device, intra-vas device
A novel cross-linked composite based on polyvinyl alcohol (PVA) and nanoparticles of silica complex containing cupric ions instead of metallic copper was devised as an intra-vas device (IVD) or intrauterine device (IUD) material by our research team. Previous research revealed that burst release of cupric ions could be avoided and that the effective utility of cupric ions could be improved in this composite (1–2). Although Suzuki et al. (3) reported that the PVA derivative hydrogel was safe for clinical use owing to its biocompatibility, the biocompatibility of this complex composite needed to be tested before clinical usage. The aim of the present study was to evaluate the biocompatibility of this novel composite, such as cell cytotoxicity in vitro, acute systemic toxicity, and muscle implantation in vivo. This may help us to examine the feasibility and safety of applying it as contraceptive material and to determine whether the material meets the biocompatibility standards for imReceived January 6, 2010; revised April 15, 2010; accepted April 16, 2010; published online June 8, 2010. L.S. has nothing to disclose. X.-b.H. has nothing to disclose. J.-p.S. has nothing to disclose. B.-l.F. has nothing to disclose. Z.-l.C. has nothing to disclose. W.-x.Y. has nothing to disclose. J.L. has nothing to disclose. Supported by the 11th Five-Year National Technology R&D Program of China (grant no. 2006BAI03B03). Reprint requests: Xun-bin Huang, M.D., Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13, Hang Kong Road, Wuhan 430030, People’s Republic of China (E-mail: [email protected]).
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plantable medical products. All these tests are mandatory in preclinical trials and are required by licensing authorities to demonstrate clinical phase 1 safety (5–12).
MATERIALS AND METHODS RPMI 1640 culture medium and fetal bovine serum (FBS) were from Gibco (Grand Island, NY), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was from Sigma (Ronkonkoma, NY), and parenzyme was
TABLE 1 Cytotoxicity assay determined as the relative growth rate (RGR) and cytotoxicity grade (CTG) of the L929 mouse fibroblasts. 2d
4d
7d
Group
RGR
CTG
RGR
CTG
RGR
CTG
100% extracts 50% extracts 25% extracts Negative control Positive control
94.3 97.9 104.5 100 19.3
1 1 0 0 4
90.4 98.9 101.4 100 9.9
1 1 0 0 4
90.9 92.8 95.1 100 8.5
1 1 1 0 4
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.04.041
FIGURE 1 Morphologies of the cultured L929 mouse fibroblasts for 7 days and direct-contact assay. (A) sample; (B) negative control; and (C) positive control (200). The morphologies in the sample group were indistinguishable from those in the negative control group. Fibroblasts in the sample and negative control groups were polygonal, and cell division could be observed. In the positive control group, dying and dead fibroblasts were significantly smaller. (D–G) Morphologies of the L929 cells which were cultured by direct contact with material after 24 h (D), 48 h (E), and 72 h (F), and negative control (G). Both the cells on the materials (left) and the cells which did not contact the materials (right) were shuttle-shaped and spread homogeneously on the surface of the material.
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
from Amresco (Solon, OH). All other chemicals and reagents used were of analytical grade from Shanghai Chemical Reagent Company (Shanghai, China). L929 mouse fibroblasts were from the China Center for Type Culture Collection, Wuhan University, Wuhan, China.
Cytotoxicity Test: MTT Assay According to the International Organization for Standardization (ISO) standard ISO 10993-5 (12), an MTT assay was used to evaluate the cytotoxicity of the composite. By measuring the optical density (OD) value of the formazan, the percentage of viable cells could be determined.
Preparation of the Extracts The composite sample was first disinfected with ethylene oxide and then put into phosphate-buffered saline solution (PBS) or RPMI-1640 culture medium supplemented with 10% FBS, and penicillin-streptomycin solution (100 U penicillin and 100 mg of streptomycin per mL) at the ratio of 1.0 g sample in 5 mL solution. After the sample was soaked for 24 hours in a 37 C incubator, the solution was disinfected and stored ar 4 C (4).
Fertility and Sterility
Cytotoxicity Test: Direct Contact Assay Third-generation fibroblasts were suspended at a concentration of 5 104 cells/mL after detachment with 0.25% parenzyme. Composite material was cut into pieces (18 18 1 mm) and disinfected with ethylene oxide. The square-shaped composite discs were placed at the bottom of each well
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Statistical Evaluation
TABLE 2 Indicators of hematology between experimental group and control group (mean ± SD; n [ 9).
Indicator
Experimental group
Control group
28.1 5.8
33.3 8.5
117 15.4
124 45.9
64.5 2.3 39.2 1.4 9.3 1.5 7.3 0.3
63.9 2.8 39.6 2.3 9.6 1.5 7.1 2.0
55.0 2.4
50.7 5.0
Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Total protein (g/L) Albumin (g/L) Glucose (mmol/L) Blood urea nitrogen (mmol/L) Creatinine (mmol/L)
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
of a 12-well plate, and 1 mL of cell suspension was added to each well. After 72 hours of incubation, the shape of the fibroblasts in the experimental group and control group was examined with an inverted microscope (Olympus, Germany).
Acute Systemic Toxicity Test Animal experimentation was performed in compliance with the local Ethics Committee and was approved by the Institutional Review Board at Huazhong University of Science and Technology. Twenty healthy adult mice (SPFgrade Kunming white mice, animal code SCXK 2003-0005) of 20 g body weight were equally divided between male and female and then divided into two groups randomly: experimental group and negative control group. The material extracts were prepared in a ratio of 0.2 g solid materials with 1 mL PBS soaked for 24 hours at 37 C. Mice in the experimental group were injected with the material extracts by intraperitoneal injection. The dosage was 50 mL/kg body weight, whereas mice in the negative control group were injected with PBS at the same volume. After 24 hours, 48 hours, and 72 hours, the general behavior (aggressiveness, anorexia, or mating behaviors), toxicity manifestation (diarrhea, vomiting, spasm), and death number were observed. Their venous blood was drawn to detect indicators of biochemistry (alanine aminotransferase, aspartate aminotransferase, total protein, albumin, glucose, blood urea nitrogen, and creatinine) after the experiment.
Material Implantation Test Implantation experiments were carried out according to ISO 10993-6 (13). Fifteen healthy adult New Zealand white rabbits (Wuhan University Laboratory Animal Center, China), body weight 3.0–4.0 kg, were the subjects of the implantation experiment. The animals were divided into three groups of different implantation periods (1, 4, and 12 wk), with five animals tested at each time period. All animal experimental procedures were carried out in accordance with the Guidelines and Regulations for the Use and Care of Animals of the Review Board of Hubei Medical Laboratory Animal Center. Each animal was anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). The material was then implanted into the spinal muscle of the rabbits at four points, at 2-cm intervals, and the material inserted into the muscle at a 1–2 cm in depth. The contralateral side was sham-operated. Rabbits were sacrificed by CO2 asphyxiation sequentially at 1, 4, and 12 weeks after the implantation. Both sites of the spinal muscles adjacent to the materials were carefully excised from the surrounding tissue and immersed in 10 vol.% buffered formalin solution for 48 hours. The specimens were paraffin embedded. The paraffin blocks were parallel to the long axis of the tubes. Six-micrometer serial sections were obtained and stained with hematoxylin and eosin. The stained sections were assessed with light microscopy (Olympus, Germany).
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All of the data was processed with one-way analysis of variance and Student t test. A probability value < .05 was considered to be significant.
RESULTS Evaluation of Cytotoxicity (MTT Assay) No significant growth inhibition effect for different concentrations of the extracts of this novel composite (25%, 50%, and 100%, v/v) was found (Table 1). The cytotoxicity grade (CTG) of the composite is obtained by the relationship between relative growth rate and CTG, according to standard GB/T 16886-1997 (7). The morphologies of the cultured fibroblasts for 7 days also showed the excellent cytocompatibility of the studied material, as shown in Figures 1A–1C.
Evaluation of the Direct Contact Assay The L929 cells cultured on the surface of the materials for 1, 2 and 3 days are shown in Figures 1D–1G. The L929 cells adhered and grew well on the surface of the material.
Acute Systemic Toxicity The mice administrated with the extracts from the material by intraperitoneal injection (50 mL/kg body wt.) showed no symptoms of lethargy, anorexia, or diarrhea in the period of observation (3 days). No death of the mice was seen, and no macroscopic abnormalities were noted in all of the mice during the 3 days of observation. The body weight gains of the mice in the sample control groups were not significantly different (P>.05). The indicators of biochemistry between the experimental and control groups are presented in Table 2. Data of all the indicators showed no significant differences between the experimental and control groups (P>.05).
Material Implanting Test For all of the rabbits, the insertion hole could still be found on the muscle fascia for all implants after 1, 4, and 12 weeks. After 1 week of implantation, the implants were located in a muscular pocket that was clearly separated from the surrounding tissue. At 4 weeks, fibrous tissue formation started resulting in encapsulation of the implanted material. At 12 weeks, the implants adhered tightly to the surrounding tissue, and the area of the implantation resembled the surrounding original tissue. Light micrographs of histologic sections after 1, 4, and 12 weeks of implantation are shown in Figure 2.
DISCUSSION The ISO-10993-5 guidelines for ‘‘Biological Evaluation of Medical Devices’’ allow for the use of MTT assay and direct contact assay in cytotoxicity determinations (5). L929 cells incubated in direct contact with biomaterial samples is a valid method for rapidly estimating the cytotoxicity of new materials. Direct contact testing of novel biomaterials with in vitro cell cultures may provide an early indicator of potential problems in vivo. Systemic toxicity studies evaluate potential adverse effects of medical products on other tissues and organs other than those caused at the initial contact. These tests evaluate the effects that any substance released by the implantable product may have on the host general health or organs, such as liver, heart, kidneys, or brain. The ISO 10993-11 (8) standard, which is applicable to study designs to investigate such effects, has established systematic international testing guidelines that have increased the safety of biomedical products. Study protocols should be based on classic toxicologic
Evaluation of novel composite contraceptive
Vol. 95, No. 4, March 15, 2011
FIGURE 2 Light micrographs of the novel composite (B, D, F) which was implanted into the spinal muscle of adult rabbits compared with sham-operated group (A, C, E) for 1 (A, B), 4 (C, D), and 12 weeks (E, F). All sections were stained with hematoxylin and eosin. At 1 week, acute inflammatory reaction was the main characteristic. A lot of heterophil granulocyte and a small amount of lymph-cell infiltration were seen within and around the implant, but without formation of complete capsule. Evidently, a phagocytotic and inflammatory reaction occurred (B). Similar inflammatory reaction was observed on the sham-operated side (A). After 4 weeks of implantation, the number of inflammatory cells decreased sharply, heterophil granulocyte disappeared, lymph cells were reduced, and compact fibrous capsule formed on the material surface of the tissue was clearly observed (D). A very small number of lymphocytes were observed on the sham-operated side, which was closed to normal tissue (C). At 12 weeks, the inflammatory reaction of the two groups had almost disappeared (E, F). The implant was completely penetrated with fibrous tissue. Furthermore, it was noticed that the surrounding fibrous capsule had become more stable (F).
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
evaluations of drugs and other chemical substances. However, these protocols should be modified to evaluate solid components on medical products (6). The product tested in the present study did not show any systemic reaction, confirming its safety, an essential requirement for its application as a contraceptive material. The experimental materials were regarded to be biocompatible if the intensity of connective reaction decreased over time. Consequently, to be considered biocompatible, at 90 days the connective tissue surrounding the implant must show a thin fibrous capsule forFertility and Sterility
mation surrounding the tube as well as an absence of inflammatory reaction and/or macrophages/giant cells. On the other hand, the material is considered to be nonbiocompatible when a persistent inflammatory reaction occurrs related to macrophages and giant cells with a thick fibrous capsule development even at 90 days after implantation (8). Cupric ions rather than copper were added into the PVA matrix. PVA, as a chelating resin, has recently drawn considerable attention for its capability to chelate hydroxyl groups to retain metal ions
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(10). In the composite, the release of cupric ions depended on the chelated equilibrium, which is quite different from the corrosion of metallic copper. By avoiding the formation of cupric oxide, the effectiveness of copper may be improved. Metallic copper in bulk generates soluble ions together with Cu2O films on its surface during corrosion, and the deposition could interact with the endometrium, resulting in side effects and incompatibility of the copper IUD (Cu-IUD). In places with higher pressure, such as the exposure of the surface of Cu-IUD, copper wires or sleeves might be broken or fragmented after longterm corrosion, resulting in the failure of the Cu-IUD (11). The corrosion products and long-term release behavior of the novel composite after soaking in FBS for different time spans were studied previously (1–2). It was shown that there were no other new elements, such as P, Cl, and Ca, appearing on the surface of the composite and no Cu2O formed after immersing in FBS for 1 year, indicating that the release channels would not be obstructed by the deposition of these composites and that the effectiveness of copper could be improved significantly. Also, because of its characteristics of flexibility and ease of controlling micropore formation, we recently tried to produce a filtering-type IVD with this composite (12).
The toxicity of nanoparticles of silica was paid attention to, because it is widely used in food and biomaterials nowdays. Chang et al. (13) investigated the response of several normal fibroblast and tumor cells to varying doses of amorphous silica or composite nanoparticles of silica and chitosan. A cell proliferation assay indicates that silica nanoparticles are nontoxic at low dosages but that cell viability decreases at high dosages. A lactate dehydrogenase assay indicates that high dosages of silica induce cell membrane damage (13). Endothelial cells exposure to silica nanoparticles causes cytotoxic damage and a decrease in cell survival in the EAHY926 cell line in a dose-related manner (14). The silica nanoparticles that we used were cross-linked with PVA, the dosage was low, and the gravitational force between molecules also may limit their effluence from the complex, possible explaining why no significant toxicity was found in this composite. In conclusion, results from all of the tests demonstrated that this novel composite exhibited steady-state release of copper ions and longer-term stability/material integrity than found in current Cu-IUDs, suggesting that it might be a safe contraceptive material for future IUD or IVD.
REFERENCES 1. Li J, Suo J, Huang X, Ye C, Wu X. Release behavior of copper ion in a novel contraceptive composite. Contraception 2007;76:233–7. 2. Li J, Suo J, Huang X, Jia L. Study on a novel coppercontaining composite for contraception. Contraception 2009;79:439–44. 3. Suzuki Y, Tanihara M, Nishimura Y, Suzali K, Kakimaru Y, Shimizu Y. A new drug delivery system with controlled release of antibiotic only in the presence of infection. J Biomed Mater Res 1998;2:112–6. 4. International Organization for Standardization. ISO 10993–12:2002. Biological evaluation of medical devices—part 12: sample preparation and reference materials. 5. International Organization for Standardization. ISO 10993–10995:1999. Biological evaluation of medical devices—part 5: tests or in vitro cytotoxicity.
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6. International Organization for Standardization. ISO 10993–10996:2007. Biological evaluation of medical devices—part 6: tests for local effects after implantation. 7. State Standards of the People’s Republic of China. GBT 16886–1997. Biological evaluation of medical devices. 8. International Organization for Standardization. ISO 10993–11:2006. Biological evaluation of medical devices—part 11: tests for systemic toxicity. 9. Bhatia SK, and A.B. Yetter. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements. Cell Biol Toxicol 2008;24:315–9. 10. Lin H, Watanabe Y, Kimura M, Hanabusa K, Shirai H. Preparation of magnetic poly (vinyl alcohol) (PVA) materials by in situ synthesis of magnetite in a PVA matrix. J Appl Polym Sci 2003;87:1239–47.
Evaluation of novel composite contraceptive
11. Xia XP, Cai SZ, Hu JH, Xie CS. Water absorption characteristics of novel/LDPE nanocomposite for use in intrauterine devices. J Biomed Mater Res B 2006;79B:345–52. 12. Chen ZL, Huang XB, Suo JP, Li J, Sun L. The contraceptive effect of a novel filtering-type nanocopper complex/polymer composite intra-vas device on male animals. Int J Androl 2010: Mar 7. [Epub ahead of print]. 13. Chang JS, Chang KL, Hwang DF, Kong ZL. In vitro cytotoxicitiy of silica nanoparticles at high concentrations strongly depends on the metabolic activity type of the cell line. Environ Sci Technol 2007;41:2064–8. 14. Napierska D, Thomassen LC, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, et al. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 2009;5:846–53.
Vol. 95, No. 4, March 15, 2011
Objective: To investigate the biocompatibility of a novel copper-containing composite to provide preclinical data for clinical application of intrauterine device (IUD) or intra-vas device (IVD). Design: Prospective experimental study. Setting: Good laboratory practices laboratories. Animals: Twenty healthy adult mice (SPF grade Kunming white mice, animal code SCXK 2003-0005). Intervention(s): Cytotoxicity tests in vitro were conducted to evaluate the influence of the materials on the morphology, growth, and proliferation of cultured L929 mouse fibroblasts. Acute systemic toxicity tests were conducted to investigate the acute systemic toxic reaction with mice, and then the materials were implanted into the spinal muscle of rabbits (n ¼ 15). The rabbits were sacrificed for pathologic examination at 1, 4, and 12 weeks after surgery. Main Outcome Measure(s): Evaluation of cytotoxicity by MTT assay, cytotoxicity test by direct contact assay, acute systemic toxicity test, and material implantation test. Result(s): The cytotoxicity grade of the copper-containing composite was 0–1, suggesting that the material was free of cytotoxicity; no acute systemic toxicity was found in any mice; mild inflammatory reaction was observed in the surrounding tissues of the implanted material in the early implantation stage, which was similar to that of the sham-operated sides. Twelve weeks after implantation, the inflammatory reaction was completely disappeared in the implanted tissue, similarly to the sham-operated sides. The fibrosis membrane surrounding the material became stable gradually over time. Conclusion: The copper-containing composite has excellent biocompatibility, which is feasible and safe for the clinical application as a novel contraceptive material. (Fertil Steril 2011;95:1416–20. 2011 by American Society for Reproductive Medicine.) Key Words: Copper, contraception, composite, intrauterine device, intra-vas device
A novel cross-linked composite based on polyvinyl alcohol (PVA) and nanoparticles of silica complex containing cupric ions instead of metallic copper was devised as an intra-vas device (IVD) or intrauterine device (IUD) material by our research team. Previous research revealed that burst release of cupric ions could be avoided and that the effective utility of cupric ions could be improved in this composite (1–2). Although Suzuki et al. (3) reported that the PVA derivative hydrogel was safe for clinical use owing to its biocompatibility, the biocompatibility of this complex composite needed to be tested before clinical usage. The aim of the present study was to evaluate the biocompatibility of this novel composite, such as cell cytotoxicity in vitro, acute systemic toxicity, and muscle implantation in vivo. This may help us to examine the feasibility and safety of applying it as contraceptive material and to determine whether the material meets the biocompatibility standards for imReceived January 6, 2010; revised April 15, 2010; accepted April 16, 2010; published online June 8, 2010. L.S. has nothing to disclose. X.-b.H. has nothing to disclose. J.-p.S. has nothing to disclose. B.-l.F. has nothing to disclose. Z.-l.C. has nothing to disclose. W.-x.Y. has nothing to disclose. J.L. has nothing to disclose. Supported by the 11th Five-Year National Technology R&D Program of China (grant no. 2006BAI03B03). Reprint requests: Xun-bin Huang, M.D., Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, 13, Hang Kong Road, Wuhan 430030, People’s Republic of China (E-mail: [email protected]).
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plantable medical products. All these tests are mandatory in preclinical trials and are required by licensing authorities to demonstrate clinical phase 1 safety (5–12).
MATERIALS AND METHODS RPMI 1640 culture medium and fetal bovine serum (FBS) were from Gibco (Grand Island, NY), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was from Sigma (Ronkonkoma, NY), and parenzyme was
TABLE 1 Cytotoxicity assay determined as the relative growth rate (RGR) and cytotoxicity grade (CTG) of the L929 mouse fibroblasts. 2d
4d
7d
Group
RGR
CTG
RGR
CTG
RGR
CTG
100% extracts 50% extracts 25% extracts Negative control Positive control
94.3 97.9 104.5 100 19.3
1 1 0 0 4
90.4 98.9 101.4 100 9.9
1 1 0 0 4
90.9 92.8 95.1 100 8.5
1 1 1 0 4
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.04.041
FIGURE 1 Morphologies of the cultured L929 mouse fibroblasts for 7 days and direct-contact assay. (A) sample; (B) negative control; and (C) positive control (200). The morphologies in the sample group were indistinguishable from those in the negative control group. Fibroblasts in the sample and negative control groups were polygonal, and cell division could be observed. In the positive control group, dying and dead fibroblasts were significantly smaller. (D–G) Morphologies of the L929 cells which were cultured by direct contact with material after 24 h (D), 48 h (E), and 72 h (F), and negative control (G). Both the cells on the materials (left) and the cells which did not contact the materials (right) were shuttle-shaped and spread homogeneously on the surface of the material.
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
from Amresco (Solon, OH). All other chemicals and reagents used were of analytical grade from Shanghai Chemical Reagent Company (Shanghai, China). L929 mouse fibroblasts were from the China Center for Type Culture Collection, Wuhan University, Wuhan, China.
Cytotoxicity Test: MTT Assay According to the International Organization for Standardization (ISO) standard ISO 10993-5 (12), an MTT assay was used to evaluate the cytotoxicity of the composite. By measuring the optical density (OD) value of the formazan, the percentage of viable cells could be determined.
Preparation of the Extracts The composite sample was first disinfected with ethylene oxide and then put into phosphate-buffered saline solution (PBS) or RPMI-1640 culture medium supplemented with 10% FBS, and penicillin-streptomycin solution (100 U penicillin and 100 mg of streptomycin per mL) at the ratio of 1.0 g sample in 5 mL solution. After the sample was soaked for 24 hours in a 37 C incubator, the solution was disinfected and stored ar 4 C (4).
Fertility and Sterility
Cytotoxicity Test: Direct Contact Assay Third-generation fibroblasts were suspended at a concentration of 5 104 cells/mL after detachment with 0.25% parenzyme. Composite material was cut into pieces (18 18 1 mm) and disinfected with ethylene oxide. The square-shaped composite discs were placed at the bottom of each well
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Statistical Evaluation
TABLE 2 Indicators of hematology between experimental group and control group (mean ± SD; n [ 9).
Indicator
Experimental group
Control group
28.1 5.8
33.3 8.5
117 15.4
124 45.9
64.5 2.3 39.2 1.4 9.3 1.5 7.3 0.3
63.9 2.8 39.6 2.3 9.6 1.5 7.1 2.0
55.0 2.4
50.7 5.0
Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Total protein (g/L) Albumin (g/L) Glucose (mmol/L) Blood urea nitrogen (mmol/L) Creatinine (mmol/L)
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
of a 12-well plate, and 1 mL of cell suspension was added to each well. After 72 hours of incubation, the shape of the fibroblasts in the experimental group and control group was examined with an inverted microscope (Olympus, Germany).
Acute Systemic Toxicity Test Animal experimentation was performed in compliance with the local Ethics Committee and was approved by the Institutional Review Board at Huazhong University of Science and Technology. Twenty healthy adult mice (SPFgrade Kunming white mice, animal code SCXK 2003-0005) of 20 g body weight were equally divided between male and female and then divided into two groups randomly: experimental group and negative control group. The material extracts were prepared in a ratio of 0.2 g solid materials with 1 mL PBS soaked for 24 hours at 37 C. Mice in the experimental group were injected with the material extracts by intraperitoneal injection. The dosage was 50 mL/kg body weight, whereas mice in the negative control group were injected with PBS at the same volume. After 24 hours, 48 hours, and 72 hours, the general behavior (aggressiveness, anorexia, or mating behaviors), toxicity manifestation (diarrhea, vomiting, spasm), and death number were observed. Their venous blood was drawn to detect indicators of biochemistry (alanine aminotransferase, aspartate aminotransferase, total protein, albumin, glucose, blood urea nitrogen, and creatinine) after the experiment.
Material Implantation Test Implantation experiments were carried out according to ISO 10993-6 (13). Fifteen healthy adult New Zealand white rabbits (Wuhan University Laboratory Animal Center, China), body weight 3.0–4.0 kg, were the subjects of the implantation experiment. The animals were divided into three groups of different implantation periods (1, 4, and 12 wk), with five animals tested at each time period. All animal experimental procedures were carried out in accordance with the Guidelines and Regulations for the Use and Care of Animals of the Review Board of Hubei Medical Laboratory Animal Center. Each animal was anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). The material was then implanted into the spinal muscle of the rabbits at four points, at 2-cm intervals, and the material inserted into the muscle at a 1–2 cm in depth. The contralateral side was sham-operated. Rabbits were sacrificed by CO2 asphyxiation sequentially at 1, 4, and 12 weeks after the implantation. Both sites of the spinal muscles adjacent to the materials were carefully excised from the surrounding tissue and immersed in 10 vol.% buffered formalin solution for 48 hours. The specimens were paraffin embedded. The paraffin blocks were parallel to the long axis of the tubes. Six-micrometer serial sections were obtained and stained with hematoxylin and eosin. The stained sections were assessed with light microscopy (Olympus, Germany).
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All of the data was processed with one-way analysis of variance and Student t test. A probability value < .05 was considered to be significant.
RESULTS Evaluation of Cytotoxicity (MTT Assay) No significant growth inhibition effect for different concentrations of the extracts of this novel composite (25%, 50%, and 100%, v/v) was found (Table 1). The cytotoxicity grade (CTG) of the composite is obtained by the relationship between relative growth rate and CTG, according to standard GB/T 16886-1997 (7). The morphologies of the cultured fibroblasts for 7 days also showed the excellent cytocompatibility of the studied material, as shown in Figures 1A–1C.
Evaluation of the Direct Contact Assay The L929 cells cultured on the surface of the materials for 1, 2 and 3 days are shown in Figures 1D–1G. The L929 cells adhered and grew well on the surface of the material.
Acute Systemic Toxicity The mice administrated with the extracts from the material by intraperitoneal injection (50 mL/kg body wt.) showed no symptoms of lethargy, anorexia, or diarrhea in the period of observation (3 days). No death of the mice was seen, and no macroscopic abnormalities were noted in all of the mice during the 3 days of observation. The body weight gains of the mice in the sample control groups were not significantly different (P>.05). The indicators of biochemistry between the experimental and control groups are presented in Table 2. Data of all the indicators showed no significant differences between the experimental and control groups (P>.05).
Material Implanting Test For all of the rabbits, the insertion hole could still be found on the muscle fascia for all implants after 1, 4, and 12 weeks. After 1 week of implantation, the implants were located in a muscular pocket that was clearly separated from the surrounding tissue. At 4 weeks, fibrous tissue formation started resulting in encapsulation of the implanted material. At 12 weeks, the implants adhered tightly to the surrounding tissue, and the area of the implantation resembled the surrounding original tissue. Light micrographs of histologic sections after 1, 4, and 12 weeks of implantation are shown in Figure 2.
DISCUSSION The ISO-10993-5 guidelines for ‘‘Biological Evaluation of Medical Devices’’ allow for the use of MTT assay and direct contact assay in cytotoxicity determinations (5). L929 cells incubated in direct contact with biomaterial samples is a valid method for rapidly estimating the cytotoxicity of new materials. Direct contact testing of novel biomaterials with in vitro cell cultures may provide an early indicator of potential problems in vivo. Systemic toxicity studies evaluate potential adverse effects of medical products on other tissues and organs other than those caused at the initial contact. These tests evaluate the effects that any substance released by the implantable product may have on the host general health or organs, such as liver, heart, kidneys, or brain. The ISO 10993-11 (8) standard, which is applicable to study designs to investigate such effects, has established systematic international testing guidelines that have increased the safety of biomedical products. Study protocols should be based on classic toxicologic
Evaluation of novel composite contraceptive
Vol. 95, No. 4, March 15, 2011
FIGURE 2 Light micrographs of the novel composite (B, D, F) which was implanted into the spinal muscle of adult rabbits compared with sham-operated group (A, C, E) for 1 (A, B), 4 (C, D), and 12 weeks (E, F). All sections were stained with hematoxylin and eosin. At 1 week, acute inflammatory reaction was the main characteristic. A lot of heterophil granulocyte and a small amount of lymph-cell infiltration were seen within and around the implant, but without formation of complete capsule. Evidently, a phagocytotic and inflammatory reaction occurred (B). Similar inflammatory reaction was observed on the sham-operated side (A). After 4 weeks of implantation, the number of inflammatory cells decreased sharply, heterophil granulocyte disappeared, lymph cells were reduced, and compact fibrous capsule formed on the material surface of the tissue was clearly observed (D). A very small number of lymphocytes were observed on the sham-operated side, which was closed to normal tissue (C). At 12 weeks, the inflammatory reaction of the two groups had almost disappeared (E, F). The implant was completely penetrated with fibrous tissue. Furthermore, it was noticed that the surrounding fibrous capsule had become more stable (F).
Sun. Evaluation of novel composite contraceptive. Fertil Steril 2011.
evaluations of drugs and other chemical substances. However, these protocols should be modified to evaluate solid components on medical products (6). The product tested in the present study did not show any systemic reaction, confirming its safety, an essential requirement for its application as a contraceptive material. The experimental materials were regarded to be biocompatible if the intensity of connective reaction decreased over time. Consequently, to be considered biocompatible, at 90 days the connective tissue surrounding the implant must show a thin fibrous capsule forFertility and Sterility
mation surrounding the tube as well as an absence of inflammatory reaction and/or macrophages/giant cells. On the other hand, the material is considered to be nonbiocompatible when a persistent inflammatory reaction occurrs related to macrophages and giant cells with a thick fibrous capsule development even at 90 days after implantation (8). Cupric ions rather than copper were added into the PVA matrix. PVA, as a chelating resin, has recently drawn considerable attention for its capability to chelate hydroxyl groups to retain metal ions
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(10). In the composite, the release of cupric ions depended on the chelated equilibrium, which is quite different from the corrosion of metallic copper. By avoiding the formation of cupric oxide, the effectiveness of copper may be improved. Metallic copper in bulk generates soluble ions together with Cu2O films on its surface during corrosion, and the deposition could interact with the endometrium, resulting in side effects and incompatibility of the copper IUD (Cu-IUD). In places with higher pressure, such as the exposure of the surface of Cu-IUD, copper wires or sleeves might be broken or fragmented after longterm corrosion, resulting in the failure of the Cu-IUD (11). The corrosion products and long-term release behavior of the novel composite after soaking in FBS for different time spans were studied previously (1–2). It was shown that there were no other new elements, such as P, Cl, and Ca, appearing on the surface of the composite and no Cu2O formed after immersing in FBS for 1 year, indicating that the release channels would not be obstructed by the deposition of these composites and that the effectiveness of copper could be improved significantly. Also, because of its characteristics of flexibility and ease of controlling micropore formation, we recently tried to produce a filtering-type IVD with this composite (12).
The toxicity of nanoparticles of silica was paid attention to, because it is widely used in food and biomaterials nowdays. Chang et al. (13) investigated the response of several normal fibroblast and tumor cells to varying doses of amorphous silica or composite nanoparticles of silica and chitosan. A cell proliferation assay indicates that silica nanoparticles are nontoxic at low dosages but that cell viability decreases at high dosages. A lactate dehydrogenase assay indicates that high dosages of silica induce cell membrane damage (13). Endothelial cells exposure to silica nanoparticles causes cytotoxic damage and a decrease in cell survival in the EAHY926 cell line in a dose-related manner (14). The silica nanoparticles that we used were cross-linked with PVA, the dosage was low, and the gravitational force between molecules also may limit their effluence from the complex, possible explaining why no significant toxicity was found in this composite. In conclusion, results from all of the tests demonstrated that this novel composite exhibited steady-state release of copper ions and longer-term stability/material integrity than found in current Cu-IUDs, suggesting that it might be a safe contraceptive material for future IUD or IVD.
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6. International Organization for Standardization. ISO 10993–10996:2007. Biological evaluation of medical devices—part 6: tests for local effects after implantation. 7. State Standards of the People’s Republic of China. GBT 16886–1997. Biological evaluation of medical devices. 8. International Organization for Standardization. ISO 10993–11:2006. Biological evaluation of medical devices—part 11: tests for systemic toxicity. 9. Bhatia SK, and A.B. Yetter. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements. Cell Biol Toxicol 2008;24:315–9. 10. Lin H, Watanabe Y, Kimura M, Hanabusa K, Shirai H. Preparation of magnetic poly (vinyl alcohol) (PVA) materials by in situ synthesis of magnetite in a PVA matrix. J Appl Polym Sci 2003;87:1239–47.
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11. Xia XP, Cai SZ, Hu JH, Xie CS. Water absorption characteristics of novel/LDPE nanocomposite for use in intrauterine devices. J Biomed Mater Res B 2006;79B:345–52. 12. Chen ZL, Huang XB, Suo JP, Li J, Sun L. The contraceptive effect of a novel filtering-type nanocopper complex/polymer composite intra-vas device on male animals. Int J Androl 2010: Mar 7. [Epub ahead of print]. 13. Chang JS, Chang KL, Hwang DF, Kong ZL. In vitro cytotoxicitiy of silica nanoparticles at high concentrations strongly depends on the metabolic activity type of the cell line. Environ Sci Technol 2007;41:2064–8. 14. Napierska D, Thomassen LC, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, et al. Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 2009;5:846–53.
Vol. 95, No. 4, March 15, 2011