Background: This biomechanical study evaluates the effect of N-acetylcysteine alone and in combination with the most commonly used antibiotic-loaded bone cement mixtures. Methods: We mixed eight bone cement mixture groups including combinations of N-acetylcysteine, gentamicin, teicoplanin, and vancomycin and applied a four-point bending test individually to each sample on days 1 and 15 using an MTS Acumen test device. Results: The result was less than 50 MPa—the limit declared by the ISO (International Standards Organization)—in only the “gentamicin + bone cement + N-acetylcysteine” group. Mechanical fatigue resistance of the bone cement decreased significantly with the addition of N-acetylcysteine both on day 1 and day 15 (p < 0.001). With the addition of N-acetylcysteine into the “gentamicin + bone cement” and “vancomycin + bone cement” mixtures, a significant decrease in mechanical fatigue resistance was observed both on day 1 and day 15 (p < 0.001). In contrast, with the addition of N-acetylcysteine into the “teicoplanin + bone cement” mixture, no significant difference in mechanical fatigue resistance was observed on days 1 and 15 (p =0.093, p =0.356). Conclusion: Preliminary results indicate that adding N-acetylcysteine to teicoplanin-loaded bone cement does not significantly affect the cement’s mechanical resistance, potentially leading to a new avenue for preventing and treating peri-prosthetic joint infection. N-acetylcysteine may, therefore, be considered as an alternative agent to be added to antibiotic-loaded bone cement mixtures used in the prevention of peri-prosthetic joint infection. Keywords: N-acetylcysteine, Antibiotic-loaded, Bone cement, Peri-prosthetic joint infection Background surgical site to inhibit biofilm formation and thereby Peri-prosthetic joint infection (PJI) is one of the most avoiding the occurrence of systemic side effects caused feared and devastating complications following total by an over-dosage of antibiotics [1, 3]. For the prophy- joint arthroplasty (TJA). PJI treatment through antibi- lactic use of ALBC, the antibiotic should not be used at otics is complex and challenging on account of biofilm high doses because increasing quantities of antibiotic formation, which tends to protect pathogens from the powder may reduce the compressive and tensile effects of systemic antibiotics as well as host immune strengths of bone cement . As a result, the amount of system . antibiotic that can be added to bone cement is limited, Antibiotic-loaded bone cement (ALBC) prophylaxis thereby limiting their effectiveness against certain promises to be an effective strategy towards reducing micro-organisms. Moreover, adding antibiotics to ALBC the risk of infection following TJA  and is commonly may theoretically contribute to an increased resistance used for high-dose local delivery of antibiotics to the towards antibiotics; as a consequence, the probability of obtaining a negative culture result in subsequent aspira- tions also increases [3, 4]. * Correspondence: email@example.com N-acetylcysteine (NAC) is a non-antibiotic drug and Department of Orthopedics and Traumatology, Sakarya University Research antioxidant amino acid that is generally safe and and Training Hospital, 54050 Sakarya, Turkey Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sukur et al. Journal of Orthopaedic Surgery and Research (2018) 13:132 Page 2 of 6 well-tolerated, even at high doses; further, it has a highly the molds were pressed between two metal plates through favorable risk/benefit ratio and a low rate of adverse use of a clamp and maintained between the plates for events [5, 6]. Studies have shown that NAC reduces bio- 15 min to facilitate cement-mixture hardening. Burrs, cre- film formation, inhibits bacterial adherence, and ated during removal of mixture samples from the molds decreases the production of extracellular polysaccharide using a special remover, were subsequently cleaned. The matrix and cell viability [7–10]. Since NAC exhibits a samples were then macroscopically examined for manu- synergistic antibacterial and anti-biofilm activity, the facturing defects (as depicted in Fig. 1c, d). Defected sam- proposed study has been designed to evaluate its effects ples, wherein more than 10% of the section surface on the mechanical properties of bone cement when used exhibited signs of defects, were discarded from the study. exclusively as well as in combination with commonly Twenty samples were prepared from each group, 10 of used antibiotic mixtures, such as gentamicin, teicopla- which were evaluated on day 1 while the remaining were nin, and vancomycin. It has been hypothesized that its evaluated on day 15. Samples scheduled for evaluation on addition would cast a curtailing effect on the biomech- day 15 were set apart for 15 days to be maintained in a anical properties of bone cement, and its combination 37 °C normal saline bath, wherein antibiotics were with antibiotics causes further aggravation of this effect. released from the surface of the sample. In order to pre- vent saturation of the environment due to antibiotic Methods release and facilitate maximum antibiotic secretion, the Cement mixture preparation saline solution containing the samples was changed on a Eight bone cement mixture groups were prepared con- daily basis. taining various combinations of bone cement, NAC along with varying concentrations of gentamicin, teico- Biomechanical testing planin, and vancomycin, as described in Table 1. Each Acumen (MTS Systems Corporation, MN, USA) electro- group was prepared by mixing the ingredients together dynamic testing device was used for biomechanical for 45 s using commercially available mechanical mixing evaluation of the prepared samples. A four-point bend- bowls under a constant vacuum pressure of approxi- ing test was independently performed on 10 samples mately 200 mbar in a controlled environment with each on day 1 and day 15. During the test, a certain temperature and relative humidity values corresponding bending force was exerted upon the samples at a speed to 22 ± 1 °C and 40–60%, respectively . of 3 mm/min, and corresponding uniaxial bending stress values were calculated in MPa (N/mm2). The value of Sample preparation the bending stress recorded at the instant at which the Special rectangular-prism-shaped molds measuring 3.3 × sample failed was considered as the mechanical fatigue 10 × 75 mm, as shown in Fig. 1a, b), were prepared in ac- resistance limit (Fig. 2a, b). cordance with the size recommended by the International Standards Organization (ISO 5833). Upon attainment of Statistical analysis dough-like viscosity values, the prepared mixtures were Statistical evaluation in the proposed study was performed made to fill the prism-shaped molds by pouring them into using the SPSS 22.0 application for Windows (SPSS Inc., the molds simultaneously by hand and applying pressure Chicago, IL, USA). Descriptive statistics were provided to to completely occupy all molds and gaps. Subsequently, represent the mean and standard deviation for numeric variables. Student’s t test was performed when numeric Table 1 Cement mixture groups variables were normally distributed, and multiple groups Group Cement mixture ingredients were compared using the one-way ANOVA test. Add- 1 Bone cement itionally, the Mann–Whitney U test and the Kruskal– Wallis test were performed when numeric variables did 2 Bone cement + 600 mg NAC not exhibit normal distribution. For a parametric 3 Bone cement + 0.5 g gentamicin sub-group analysis comprising more than two groups, the 4 Bone cement + 0.5 gentamicin + 600 mg NAC Tukey test was performed. For non-parametric analyses, 5 Bone cement + 1 g vancomycin on the other hand, the Mann–Whitney U test with Bon- 6 Bone cement + 1 g vancomycin + 600 mg NAC ferroni correction was preferred. The significance level for 7 Bone cement + 400 mg teicoplanin each of the above tests was set at 0.05. 8 Bone cement + 400 mg teicoplanin + 600 mg NAC Results Versabond cement (40 g polymer powder + 20 mL monomer liquid) Acetyl-L-cysteine (Sigma Aldrich, A7250) Results of the biomechanical evaluation described Cemex Genta d above are summarized and presented in Table 2.As Edicin 1 g (Sandoz, Serbia) Targocid, Sanofi Aventis, Italy seen in Table 2, only samples belonging to group 4 Sukur et al. Journal of Orthopaedic Surgery and Research (2018) 13:132 Page 3 of 6 Fig. 1 a–d Rectangular-prism-shaped molds demonstrated a value of the failure bending stress less Discussion than 50 MPa (the limiting value declared by ISO). Although some in vitro studies have evaluated the anti- The mechanical fatigue resistance (MFR) of bone biofilm and antimicrobial effects of NAC in bone cement samples was found to have significantly cement, to our knowledge, this is the first study to decreased upon addition of NAC. This result was evaluate the biomechanical features of NAC and ALBC consistent in the biomechanical testing of samples applied together. We observed that adding NAC to performed on day 1 as well as day 15 (p < 0.001). As teicoplanin-loaded bone cement did not affect its MFR depicted in Fig. 3a, NAC addition to the group 3 on day 1 or day 15. On the other hand, adding NAC into mixture causes a significant decrease in MFR (p gentamicin- and vancomycin-loaded bone cement de- < 0.001), as observed on day 1 as well as day 15. In creased biomechanical features of the bone cement. the case of NAC addition to the group 5 mixture, a Biofilm formation is a bacterial behavior that impedes significant difference was observed between MFR the effectiveness of PJI therapy , and inadequate values recorded on day 1 and day 15 (p < 0.001), as ALBC application may lead to the proliferation of resist- depicted in Fig. 3b. NAC addition to the group 7 ant bacteria strains and cannot obviate biofilm formation mixture, however, demonstrated no significant differ- . In our study, we have chosen to evaluate a combin- ence between MFR values recorded on day 1 and day ation of ALBC and NAC, because NAC is a new 15 (p = 0.093 and p = 0.356, respectively), as depicted pharmacological approach to inhibiting biofilm forma- in Fig. 3c. tion, eradicating mature biofilms and increasing the Fig. 2 a, b Demonstration of four-point bending test Sukur et al. Journal of Orthopaedic Surgery and Research (2018) 13:132 Page 4 of 6 Table 2 Results of the biomechanical tests permeability of antibiotics to overcome antibiotic resist- ance . We expected that adding NAC into ALBC may Peak load N (day 1) Peak load N (day 15) worsen its mechanical properties by increasing the total Mean ± SD Mean ± SD p number of particles added to the cement. Although Group 1 107.7 ± 3.5 100.4 ± 4.1 0.001 compression tests are commonly performed to evaluate Group 2 93.3 ± 3.4 87.3 ± 4.6 0.017 the biomechanical resistance of bone cement, we per- p < 0.001 < 0.001 formed four-point bending tests as described by ISO, Group 3 91.2 ± 3.5 86.8 ± 5.1 0.005 because they are more sensitive to surface defects and Group 4 77.3 ± 6.3 49.2 ± 4.2 < 0.001 internal porosity [13, 14]. ALBC is used to avoid systemic toxicity from antibi- p < 0.001 < 0.001 otics and maximize their local concentration, inhibiting Group 5 93.2 ± 6.1 87.6 ± 3.2 0.077 biofilm formation at the infection site in the treatment Group 6 76.3 ± 7.6 62.4 ± 3.3 0.002 and prophylaxis of PJI [15–17]. Currently, antibiotic p < 0.001 < 0.001 doses in ALBC vary depending on the application. When Group 7 93.2 ± 4.3 83.9 ± 4.0 < 0.001 ALBC is used to treat an existing infection, appropriate Group 8 88.5 ± 2.8 81.9 ± 4.4 0.001 therapeutic levels can be achieved by loading more than 2 g of antibiotics into 40 g of bone cement . However, p 0.093 0.356 the addition of antibiotics into bone cement decreases its compressive and tensile strength. Furthermore, this decrease is augmented by increasing the antibiotic dose . Hence, a maximum of 1–2 g of antibiotics can be loaded into 40 g of bone cement for infection prophy- laxis [1, 18, 19]. In the current study, the cement mixtures were formed by adding prophylactic doses of three types of antibiotics (0.5 g gentamicin, 1 g vancomycin, and 400 mg teicoplanin) and 600 mg NAC into bone cement to mimic prophylactic application. Consistent with the literature, we observed a significant decrease in mechan- ical properties with the addition of antibiotics alone into the bone cement in a four-point bending test [1, 18]. Similarly, adding only NAC negatively affected the bone cement’s mechanical properties. However, we found no available literature on the effect of NAC addition into bone cement on its biomechanical properties. Thus, it was not possible to compare our results. We postulate that adding NAC to bone cement decreased its mechan- ical properties by increasing the number of particles in the cement. A homogenous mixing and pressurization during implantation are crucial for better mechanical properties of the bone cement. The use of vacuum-mixing of the cement causes less porosity and less reduction of the tensile fatigue strength of ALBC than hand-mixing . In a current study, it was concluded that the standard method using a plastic pressurizer with cement gun after vacuum mixing appears to be adequate for achievement of optimum pressurization during femoral cementing without increased risk of embolization . In our study, we used the vacuum mixing method to optimize the cement porosity. Our results showed that 600 mg of NAC may be safely added to bone cement while maintaining sufficient bio- Fig. 3 a–c The comparison of the results mechanical resistance against bending and shearing Sukur et al. Journal of Orthopaedic Surgery and Research (2018) 13:132 Page 5 of 6 forces. However, in this study, adding NAC to Ethics approval and consent to participate Not applicable gentamicin-loaded bone cement significantly decreased the mechanical strength of the mixture, consistent with our hy- Competing interests pothesis. Our four-point bending test result was less than The authors declare that they have no competing interests. the minimum ISO requirement of 50 MPa in the 0.5 genta- micin+600mgNAC+bonecement group. Similarly, Lautenschlager et al. observed decreased mechanical resist- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in ance in gentamicin-loaded bone cement . In our study, published maps and institutional affiliations. adding NAC to vancomycin-loaded bone cement signifi- cantly decreased its biomechanical resistance; however, this Author details Department of Orthopedics and Traumatology, Sakarya University Research mixture achieved bending test results of at least 50 MPa on and Training Hospital, 54050 Sakarya, Turkey. Department of Orthopedics both days 1 and 15. By contrast, adding NAC to 400 mg and Traumatology, Kocaeli Medical Park Hospital, Kocaeli, Turkey. teicoplanin-loaded bone cement did not adversely affect its Received: 10 March 2018 Accepted: 24 May 2018 mechanical properties. Gogus et al. investigated the effects of increasing doses of teicoplanin in bone cement on the cement’s biomechanical properties and found that doses References greater than 2000 mg added into bone cement (40 g poly- 1. Hinarejos P, Guirro P, Puig-Verdie L, Torres-Claramunt R, Leal-Blanquet J, mer powder + 20 mL monomer liquid) decreased its mech- Sanchez-Soler J, Monllau JC. Use of antibiotic-loaded cement in total knee arthroplasty. World J Orthop. 2015;6(11):877–85. anical resistance past the critical lower limit. In addition to 2. Randelli P, Evola FR, Cabitza P, Polli L, Denti M, Vaienti L. Prophylactic use of the concentration and type of antibiotics, the brand of the antibiotic-loaded bone cement in primary total knee replacement. Knee antibiotic and the bone cement also affects biomechanical Surg Sports Traumatol Arthrosc. 2010;18(2):181–6. 3. Chen AF, Parvizi J. Antibiotic-loaded bone cement and periprosthetic joint properties [20–22], which might cause the variability of the infection. J Long-Term Eff Med Implants. 2014;24(2–3):89–97. results in the available literature. 4. Walker LC, Baker P, Holleyman R, Deehan D. Microbial resistance related to The main limitation of our study was that the antibi- antibiotic-loaded bone cement: a historical review. Knee Surg Sports Traumatol Arthrosc. 2017;25(12):3808–17. otics were added to the bone cement only in prophylac- 5. Dinicola S, De Grazia S, Carlomagno G, Pintucci JP. N-acetylcysteine as tic doses, and the effect of only a single dose of NAC powerful molecule to destroy bacterial biofilms: a systematic review. Eur was evaluated. Determining the correct dose of NAC for Rev Med Pharmacol Sci. 2014;18:2942–8. 6. Millea PJ. N-acetylcysteine: multiple clinical applications. Am Fam Physician. use in combination with optimized antibiotic doses in 2009;80(3):265–9. bone cement requires further study. Additionally, only a 7. Leite B, Gomes F, Teixeria P, Souza C, Pizzolitto E, Oliveria R. Combined four-point bending test was performed; compressive effect of linezolid and N-acetylcysteine against Staphylococcus epidermidis biofilms. Enferm Infecc Microbiol Clin. 2013;31(10):655–9. resistance limits were not evaluated in our study. 8. Drago L, De Vecchi E, Mattina R, Romano CL. Activity of N-acetyl-L-cysteine against biofilm of Staphylococcus aureus and Pseudomonas aeruginosa on orthopedic prosthetic materials. Int J Artif Organs. 2013;36(1):39–46. Conclusions 9. Gomes F, Leite B, Teixeria P, Azeredo J, Oliveria R. Farnesol in combination This study examines the mechanical properties of with N-acetylcysteine against Staphylococcus epidermidis planktonic and antibiotic-loaded bone cement after the addition of N-acet- biofilm cells. Braz J Microbiol. 2012;43(1):235–42. 10. Olofsson AC, Hermansson M, Elwing H. N-acetyl-L-cysteine affects growth, ylcysteine. We have shown preliminary results indicating extracellular polysaccharide production, and bacterial biofilm formation on that adding N-acetylcysteine to teicoplanin-loaded bone ce- solid surfaces. Appl Environ Microbiol. 2003;69(8):4814–22. ment does not significantly affect the cement’s mechanical 11. Pithankuakul K, Samranvedhya W, Visutipol B, Rojviroj S. The effects of different mixing speeds on the elution and strength of high-dose antibiotic- resistance, potentially leading to a new avenue for prevent- loaded bone cement created with the hand-mixed technique. J Arthroplast. ing and treating PJI. However, this effect varies with the 2015;30(5):858–63. type and amount of antibioticusedinthe mixtures.Bone 12. Jiranek WA, Hanssen AD, Greenwald AS. Antibiotic-loaded bone cement for infection prophylaxis in total joint replacement. J Bone Joint Surg Am. 2006; cement mixtures containing a combination of NAC and an- 88(11):2487–500. tibiotics may be manufactured and used clinically. 13. American Society for Testing and Materials. Standard specification for acrylic bone cement F 451-99a. In: Annual Book of ASTM Standards; Abbreviations 1999. p. 56–62. ALBC: Antibiotic-loaded bone cement; ANOVA: Analysis of variance; 14. Kühn KD. Bone cements: up-to-date comparison of physical and chemical ISO: International Standards Organization; MFR: Mechanical fatigue resistance; properties of commercial materials. Berlin, Heidelberg: Springer Verlag; 2000. NAC: N-acetylcysteine; PJI: Peri-prosthetic joint infection; TJA: Total joint p. 126–30. arthroplasty 15. Parvizi J, Adeli B, Zmistowski B, Restrepo C, Greenwald AS. Management of periprosthetic joint infection: the current knowledge: AAOS exhibit Authors’ contributions selection. J Bone Joint Surg Am. 2012;94(14):e104. ES participated in the design of the study and the manuscript preparation 16. Cui Q, Mihalko WM, Shields JS, Ries M, Saleh KJ. Antibiotic-impregnated and made the data collection and literature search. AA participated in the cement spacers for the treatment of infection associated with total hip or data collection and helped to draft the manuscript. HNT and OC participated knee arthroplasty. J Bone Joint Surg Am. 2007;89(4):871–82. in the data interpretation. AK participated in the statistical analysis. MT 17. Jiranek W. Antibiotic-loaded cement in total hip replacement: current participated in the study design and funds collection. All authors read and indications, efficacy, and complications. Orthopedics. 2005;28(8 Suppl): approved the final manuscript. s873–7. Sukur et al. Journal of Orthopaedic Surgery and Research (2018) 13:132 Page 6 of 6 18. Lautenschlager EP, Jacobs JJ, Marshall GW, Meyer PR. Mechanical properties of bone cements containing large doses of antibiotic powders. J Biomed Mater Res. 1976;10(6):929–38. 19. Kapoor B, Datir SP, Davis B, Wynn-Jones CH, Maffulli N. Femoral cement pressurization in hip arthroplasty: a laboratory comparison of three techniques. Acta Orthop Scand. 2004;75(6):708–12. 20. Lee SH, Tai CL, Chen SY, Chang CH, Chang YH, Hsieh PH. Elution and mechanical strength of vancomycin-loaded bone cement: in vitro study of the influence of brand combination. PLoS One. 2016;11(11):e0166545. 21. Göğüş A, Akman S, Göksan SB, Bozdağ E. Mechanical strength of antibiotic- impregnated bone cement on day 0 and day 15: a biomechanical study with surgical simplex P and teicoplanin. Acta Orthop Traumatol Turc. 2002; 36(1):63–71. 22. Gölge UH, Oztemur Z, Parlak M, Tezeren G, Oztürk H, Bulut O. Investigation of mechanical strength of teicoplanin and ciprofloxacin impregnated bone cement on day 1 and day 15. Acta Orthop Traumatol Turc. 2014;48(3):333–8.
Journal of Orthopaedic Surgery and Research – Springer Journals
Published: May 31, 2018
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