- Email: [email protected]
A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants
Journal of Clinical Neuroscience 31 (2016) 219–223
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory studies
A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants Shalin S. Patel a, Wilson Aruni b, Serkan Inceoglu a, Yusuf T. Akpolat a, Gary D. Botimer a, Wayne K. Cheng a, Olumide A. Danisa a,⇑ a b
Department of Orthopedic Surgery, Loma Linda University Medical Center, 11406 Loma Linda Drive, Suite 218, Loma Linda, CA 92354, USA Division of Microbiology and Molecular Genetics, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA
a r t i c l e
i n f o
Article history: Received 5 March 2016 Accepted 11 March 2016
Keywords: Biofilm Cobalt chrome Spinal implant Staphylococcus aureus Titanium alloy
a b s t r a c t The use of cobalt chrome (CoCr) implants in spinal surgery has become increasingly popular. However, there have been no studies specifically comparing biofilm formation on CoCr with that of titaniumalloy spinal implants. The objective of this study was to compare the difference in propensity for biofilm formation between these two materials, as it specifically relates to spinal rods. Staphylococcus aureus subsp. Aureus (ATCC 6538) were incubated with two different types of spinal rods composed of either CoCr or titanium-alloy. The spinal rods were then subject to a trypsin wash to allow for isolation of the colonized organism and associated biofilms. The associated optical density values (OD) from the bacterial isolates were obtained and the bacterial solutions were plated on brain-heart infusion agar plates and the resultant colony-forming units (CFU) were counted. The OD values for the titanium-alloy rods were 1.105 ± 0.096 nm (mean ± SD) and 1.040 ± 0.026 nm at 48 hours and 96 hours, respectively. In contrast, the OD values for the CoCr rods were 1.332 ± 0.161 nm and 1.115 ± 0.207 nm at 48 and 96 hours, respectively (p < 0.05). The CFU values were 1481 ± 417/100 mm2 and 745 ± 159/100 mm2 at 48 and 96 hours, respectively for the titanium-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721 ± 605/100 mm2 and 928 ± 88/100 mm2 (p < 0.001) at both 48 and 96 hours respectively. Our findings, evaluating both the OD and CFU values, indicate that implants composed of CoCr had a higher proclivity towards biofilm formation compared to titanium-alloy implants. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Biofilm formation after implantation of spinal hardware is a serious complication associated with spine surgery. The initiation of biofilm formation begins with bacterial adherence onto the surface of implanted hardware. If favorable environmental conditions exist, bacteria have the ability to bind to implanted hardware and multiply, thus forming a resistant coating [1–3]. Given the resistance of this biofilm layer to host immune defenses, it is often necessary to undertake explantation of hardware to fully eliminate the infectious process [4]. The intrinsic characteristics of implant material are implicated in the formation of biofilms [5]. Staphylococcus aureus is one of the most heavily implicated organisms in post-surgical hardware infections [6]. As a relatively new material, cobalt chrome (CoCr) is imaging friendly (MRI), has greater fatigue life [7], and has ⇑ Corresponding author. Tel.: +1 909 558 6444; fax: +1 909 558 6118. E-mail address: [email protected] (O.A. Danisa). http://dx.doi.org/10.1016/j.jocn.2016.03.013 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.
higher correction forces then titanium-alloys (Ti-alloy) [8], yet literature on biofilm formation on CoCr implants is limited. To our knowledge there are no published studies comparing biofilm formation on CoCr and Ti-alloy spinal implants. We hypothesized that there was no difference in biofilm formation properties between CoCr and Ti-alloy implants. This study involves a direct comparison of the degree of biofilm formation between CoCr and Ti-alloy spinal rods by examining both the difference in optical density (OD) values of bacterial isolates and the resultant number of colonyforming units (CFU); thus enabling us to investigate which material had a higher tendency towards biofilm formation. 2. Materials and methods 2.1. Preparation of bacteria Lyophilized pellets of Staphylococcus aureus subsp. Aureus (ATCC 6538) were obtained and incubated in brain-heart infusion medium for a period of 24 hours to allow for sufficient rehydration.
220
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
Twelve well plates were prepared (Fig. 1) in which 12 Ti-alloy (10 mm 5.5 mm) (Depuy Synthes, Raynham, MA, USA) and 12 CoCr rods (10 mm 5.5 mm) (Depuy Synthes) were placed.
and a bacterial pellet was isolated. This pellet was then dissolved in 200 ug of phosphate buffer and plated on brain-heart infusion agar plates and incubated for 24 hours (Fig. 2). The resulting number of CFU for each plate was evaluated, using a CFU counter.
2.2. Spinal implant preparation 3. Results Each rod was machine cut to allow for uniform diameter and length, and thus allow for identical surface area. With regard to rod surface treatment during the post-production process, each rod underwent identical treatment first involving glass bead blasting to allow for a matte finish. This was performed to remove any tool marks that were incurred during the production process. Furthermore, during the post-production process, both rods underwent an identical passivation process allowing for removal of any free ions and residual oil. Both rods were autoclaved at 121°C (249°F). Each rod was then immersed in 5 ml of brain-heart infusion medium (Fig. 1). To each of these well plates, 1 ml of overnight culture of Staphylococcus aureus subsp. Aureus (ATCC 6538) was added. The implants were then divided into two groups and incubated at 37°C for a period of either 48 (n = 4) or 96 hours (n = 8). 2.3. Isolation of biofilm from spinal implant & evaluation of OD After the respectively designated incubation periods had elapsed the implants were washed with a 0.25% trypsin solution to extract the biofilms from the rods. This bacterial solution was then collected and the overall turbidity of the solution was evaluated by determining the OD measurement via a spectrophotometer. 2.4. Evaluation of CFU After the OD values were obtained the bacterial solution was centrifuged and the residual trypsin solution was decanted off
Two-way ANOVA analysis showed both OD and CFU were significantly influenced by duration of incubation and implant material. The CoCr rods yielded both a higher OD and CFU values compared to Ti-alloy (p < 0.05). Higher OD and CFU values were encountered at both 4-day incubation and 2-day incubation (p < 0.05). The mean (± SD) OD values for the bacterial solution obtained from the Ti-alloy implants were 1.105 ± 0.096 nm and 1.040 ± 0.026 nm at 48 hours and 96 hours, respectively. In contrast, the OD values for the bacterial solution obtained from the CoCr rods were 1.332 ± 0.161 nm and 1.115 ± 0.207 nm at 48 and 96 hours, respectively, which were significantly higher than the Ti-alloy implants (p = 0.025) (Fig. 3). Similarly, the CFU values were 1481 ± 417/100 mm2 and 745 ± 159/100 mm2 at 48 and 96 hours, respectively for the Ti-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721 ± 605/100 mm2 and 928 ± 88/100 mm2 (p < 0.001) at both 48 and 96 hours respectively (Fig. 4). 4. Discussion Implant associated infections are a major source of morbidity and mortality after spine surgery. The characteristic features of different metal implants are a contributory factor in the development of biofilm on spinal implants. The formation of biofilms makes implant associated infections difficult to treat and often requires
Fig. 1. Autoclaved titanium-alloy and cobalt chrome rods in 5 cc brain-heart infusion medium prior to incubation with Staphylococcus aureus subsp. Aureus (ATCC 6538).
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
221
Fig. 2. Bacterial solution isolated from titanium-alloy (left) and cobalt chrome (CoCr) (right) rods after 24 hours incubation on brain-heart infusion agar plates. Evaluation of resultant colony forming unit values show higher values associated with CoCr rods.
Fig. 3. Optical Density (OD) values obtained after 48 and 96 hours incubation of spinal rods in Staphylococcus aureus subsp. Aureus (ATCC 6538) solution. OD values are significantly higher on cobalt chrome (CoCr) rods (p = 0.025). Ti-alloy = titanium-alloy.
explantation of hardware [6]. The process of biofilm formation is complex and is the result of an interaction between the implant surface and the adherent microorganism. The formation of biofilm
involves the development of a community of microorganisms adhering to the biomaterial embedded in a matrix of extracellular polymeric substance that they produce [9]. This large aggregate,
222
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
Fig. 4. Colony forming unit values obtained for bacterial solution derived from 48 and 96 hours incubation of spinal rods in Staphylococcus aureus subsp. Aureus (ATCC 6538) solution. CFU values significantly were higher on cobalt chrome (CoCr) rods (p < 0.001). Ti-alloy = titanium-alloy.
enables bacteria to escape host defenses and antibiotic infiltration [10]. In our experimentation, we evaluate the adherence and biofilm formation of Staphylococcus aureus subsp. Aureus (ATCC 6538) on CoCr and Ti-alloy implants. Each of the metal implants were machine cut to precisely 10 mm length with a diameter of 5.5 mm to allow for uniform surface areas. Additionally, both metal rods were cultured in the Staphylococcal aureus bacterial solution for a period of 48 and 96 hours. Evaluation of the resulting CFU from the various implants showed that CoCr implants had a greater proclivity toward biofilm formation compared to Ti-alloy implants at both 48 and 96 hours of incubation. This finding serves to support the notion that the intrinsic physical characteristics of CoCr are implicated in promoting biofilm formation. To our knowledge there have been no head to head comparisons specifically evaluating biofilm formation between CoCr and Ti-alloy spinal implants. Not only do Ti-alloy implants most closely mimic the characteristic of cortical bone in terms of its biomechanical properties [11] but—as demonstrated by our study here—Tialloy implants have a lower proclivity for biofilm formation. It has been reported in in vitro studies that Staphylococcal epidermidis has greater adherence to stainless steel implants than Ti-alloy [12]. Additionally, it has been observed in animal studies, specifically involving rabbits, that Ti-alloy implants have a lower rate of infection compared to stainless steel [13]. The lower adherence of bacteria to Ti-alloy implants in vivo is believed to be the result of Ti-alloy’s higher biocompatibility. It has been postulated that Ti-alloy implants allow for host cells to rapidly grow over the implant and encapsulate it in a fibrous tissue coating, thus preventing bacterial adherence onto the surface of the implant [14]. This phenomenon was described by Gristina et al., as ‘‘a race for the surface” between the host connective tissue and the bacteria for ownership of the implant material [5]. Our study shows that at both 48 and 96 hours CoCr implants had a higher degree of biofilm formation and an associated higher OD value for the resultant bacterial solution that was obtained
after trypsin wash in comparison to Ti-alloy implants. The use of OD values serves to directly evaluate the turbidity of the bacterial solution obtained from each rod. Application of a 0.25% trypsin solution to remove biofilm from the metallic implants has been previously described by Ryoo et al. [6]. In their study, electron microscopy was employed before and after 0.25% trypsin wash, to demonstrate effective biofilm extraction off the titanium and stainless steel implants. Additionally, the use of OD values to evaluate the degree of biofilm formation has been previously described in the literature by Kalvenes et al. [15] with regards to evaluation of Pseudomonas aeruginosa biofilm formation. This method was found to be effective in the quantification of biofilm formation given that light absorption by the bacterial solution was found to correlate with the degree of biofilm cell mass. The decision to use CoCr versus Ti-alloy implants in patients undergoing spinal instrumentation is largely dependent on the overall clinical context in which operative intervention is being performed. The use of CoCr rods provides a number of advantages in terms of its biomechanical properties. In particular CoCr implants have a greater rigidity and stiffness in comparison to other biomaterials [16]. It has been shown in biomechanical studies that CoCr implants have the ability to perform the highest corrective forces in comparison to Ti-alloy and stainless steel rods [8]. Given these intrinsic qualities CoCr implants are particularly efficacious in major spinal deformity correction surgery. Our study shows that CoCr implants have a significantly higher incidence of biofilm formation compared to Ti-alloy implants. It is important to note that we specifically confirmed with the rod manufacturers that both metals have different surface property due to atomic structures, but not due to difference in surface treatment. Given this finding further investigation will be needed to understand why this phenomenon occurs on a cellular level. Additionally, these finding can certainly help guide surgical planning. Perhaps additional steps with regard to infection prevention should be employed in patients undergoing spinal instrumentation with CoCr implants. Steps such as restricting operating room traffic during
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
surgery, copious wound irrigation with antibacterial solution, and even the use of vancomycin powder prior to wound closure [17], may all help to minimize the risk of post-operative wound infection and eventual biofilm formation. In the material science literature, there are several studies that have extensively examined the surface properties of orthopedic implants [18–21]. According to their results, surface roughness, surface hydrophobicity and surface free radicals are the most important intrinsic properties of the materials studied. It is possible that these factors could have contributed to the variability seen in terms of the biofilm formation between Ti-alloy and CoCr implants. Given the differences observed in this study, we feel it would be worthwhile to study the aforementioned characteristics and its impact on biofilm production on CoCr and Ti-alloy. One of the limitations of our study is that this was an in-vitro study; certainly conditions maybe differ in the in-vivo setting. Additionally, given the importance of Staphylococcus epidermidis in orthopedic infections, it would certainly be worthwhile to repeat this study with use of Staphylococcus epidermidis. 5. Conclusion The degree of biofilm formation of Staphylococcus aureus on spinal implants composed of CoCr and Ti-alloy were directly compared using OD values and CFU values of the trypsinized biofilm from incubated rods. Spinal implants composed of CoCr showed a significantly higher rate of biofilm formation after both 48 and 96 hours of incubation. Further investigation will be required to understand why CoCr implants have a higher affinity for bacterial adherence and biofilm formation on a cellular level. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. References [1] Wang X, Qiu S, Yao X, et al. Berberine inhibits Staphylococcus epidermidis adhesion and biofilm formation on the surface of titanium alloy. J Orthop Res 2009;27:1487–92.
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[2] Wadstrom T. Molecular aspects of bacterial adhesion, colonization, and development of infections associated with biomaterials. J Invest Surg 1989;2:353–60. [3] Veerachamy S, Yarlagadda T, Manivasagam G, et al. Bacterial adherence and biofilm formation on medical implants: a review. Proc Inst Mech Eng H 2014;228:1083–99. [4] Gracia E, Fernandez A, Conchello P, et al. Adherence of Staphylococcus aureus slime-producing strain variants to biomaterials used in orthopaedic surgery. Int Orthop 1997;21:46–51. [5] Gristina AG, Costerton JW. Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis. J Bone Joint Surg Am 1985;67:264–73. [6] Ha KY, Chung YG, Ryoo SJ. Adherence and biofilm formation of Staphylococcus epidermidis and Mycobacterium tuberculosis on various spinal implants. Spine (Phila Pa 1976) 2005;30:38–43. [7] Nguyen TQ, Buckley JM, Ames C, et al. The fatigue life of contoured cobalt chrome posterior spinal fusion rods. Proc Inst Mech Eng H 2011;225:194–8. [8] Serhan H, Mhatre D, Newton P, et al. Would CoCr rods provide better correctional forces than stainless steel or titanium for rigid scoliosis curves? J Spinal Disord Tech 2013;26:E70–4. [9] Braem A, Van Mellaert L, Mattheys T, et al. Staphylococcal biofilm growth on smooth and porous titanium coatings for biomedical applications. J Biomed Mater Res Part A 2014;102:215–24. [10] Montanaro L, Speziale P, Campoccia D, et al. Scenery of Staphylococcus implant infections in orthopedics. Future Microbiol 2011;6:1329–49. [11] Xu J, Weng XJ, Wang X, et al. Potential use of porous titanium-niobium alloy in orthopedic implants: preparation and experimental study of its biocompatibility in vitro. PLoS One 2013;8:e79289. [12] Chang CC, Merritt K. Infection at the site of implanted materials with and without preadhered bacteria. J Orthop Res 1994;12:526–31. [13] Arens S, Schlegel U, Printzen G, et al. Influence of materials for fixation implants on local infection. An experimental study of steel versus titanium DCP in rabbits. J Bone Joint Surg Br 1996;78:647–51. [14] Gristina AG, Naylor PT, Webb LX. Molecular mechanisms in musculoskeletal sepsis: the race for the surface. Instr Course Lect 1990;39:471–82. [15] Bakke R, Kommedal R, Kalvenes S. Quantification of biofilm accumulation by an optical approach. J Microbiol Methods 2001;44:13–26. [16] Ahmad FU, Sidani C, Fourzali R, et al. Postoperative magnetic resonance imaging artifact with cobalt-chromium versus titanium spinal instrumentation: presented at the 2013 Joint Spine Section Meeting. Clinical article. J Neurosurg Spine 2013;19:629–36. [17] Khan NR, Thompson CJ, DeCuypere M, et al. A meta-analysis of spinal surgical site infection and vancomycin powder. J Neurosurg Spine 2014;21:974–83. [18] Shida T, Koseki H, Yoda I, et al. Adherence ability of Staphylococcus epidermidis on prosthetic biomaterials: an in vitro study. Int J Nanomed 2013;8:3955–61. [19] Hu X, Neoh KG, Zhang J, et al. Bacterial and osteoblast behavior on titanium, cobalt-chromium alloy and stainless steel treated with alkali and heat: a comparative study for potential orthopedic applications. J Colloid Interface Sci 2014;417:410–9. [20] Koseki H, Yonekura A, Shida T, et al. Early staphylococcal biofilm formation on solid orthopaedic implant materials: in vitro study. PLoS One 2014;9:e107588. [21] Castellanos J, Gonzalez-Cuevas A, Sierra JM, et al. Adherence of S. epidermidis on different metals. A comparative in vitro study. J Appl Biomater Funct Mater 2014;12:141–4.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory studies
A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants Shalin S. Patel a, Wilson Aruni b, Serkan Inceoglu a, Yusuf T. Akpolat a, Gary D. Botimer a, Wayne K. Cheng a, Olumide A. Danisa a,⇑ a b
Department of Orthopedic Surgery, Loma Linda University Medical Center, 11406 Loma Linda Drive, Suite 218, Loma Linda, CA 92354, USA Division of Microbiology and Molecular Genetics, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA
a r t i c l e
i n f o
Article history: Received 5 March 2016 Accepted 11 March 2016
Keywords: Biofilm Cobalt chrome Spinal implant Staphylococcus aureus Titanium alloy
a b s t r a c t The use of cobalt chrome (CoCr) implants in spinal surgery has become increasingly popular. However, there have been no studies specifically comparing biofilm formation on CoCr with that of titaniumalloy spinal implants. The objective of this study was to compare the difference in propensity for biofilm formation between these two materials, as it specifically relates to spinal rods. Staphylococcus aureus subsp. Aureus (ATCC 6538) were incubated with two different types of spinal rods composed of either CoCr or titanium-alloy. The spinal rods were then subject to a trypsin wash to allow for isolation of the colonized organism and associated biofilms. The associated optical density values (OD) from the bacterial isolates were obtained and the bacterial solutions were plated on brain-heart infusion agar plates and the resultant colony-forming units (CFU) were counted. The OD values for the titanium-alloy rods were 1.105 ± 0.096 nm (mean ± SD) and 1.040 ± 0.026 nm at 48 hours and 96 hours, respectively. In contrast, the OD values for the CoCr rods were 1.332 ± 0.161 nm and 1.115 ± 0.207 nm at 48 and 96 hours, respectively (p < 0.05). The CFU values were 1481 ± 417/100 mm2 and 745 ± 159/100 mm2 at 48 and 96 hours, respectively for the titanium-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721 ± 605/100 mm2 and 928 ± 88/100 mm2 (p < 0.001) at both 48 and 96 hours respectively. Our findings, evaluating both the OD and CFU values, indicate that implants composed of CoCr had a higher proclivity towards biofilm formation compared to titanium-alloy implants. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Biofilm formation after implantation of spinal hardware is a serious complication associated with spine surgery. The initiation of biofilm formation begins with bacterial adherence onto the surface of implanted hardware. If favorable environmental conditions exist, bacteria have the ability to bind to implanted hardware and multiply, thus forming a resistant coating [1–3]. Given the resistance of this biofilm layer to host immune defenses, it is often necessary to undertake explantation of hardware to fully eliminate the infectious process [4]. The intrinsic characteristics of implant material are implicated in the formation of biofilms [5]. Staphylococcus aureus is one of the most heavily implicated organisms in post-surgical hardware infections [6]. As a relatively new material, cobalt chrome (CoCr) is imaging friendly (MRI), has greater fatigue life [7], and has ⇑ Corresponding author. Tel.: +1 909 558 6444; fax: +1 909 558 6118. E-mail address: [email protected] (O.A. Danisa). http://dx.doi.org/10.1016/j.jocn.2016.03.013 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.
higher correction forces then titanium-alloys (Ti-alloy) [8], yet literature on biofilm formation on CoCr implants is limited. To our knowledge there are no published studies comparing biofilm formation on CoCr and Ti-alloy spinal implants. We hypothesized that there was no difference in biofilm formation properties between CoCr and Ti-alloy implants. This study involves a direct comparison of the degree of biofilm formation between CoCr and Ti-alloy spinal rods by examining both the difference in optical density (OD) values of bacterial isolates and the resultant number of colonyforming units (CFU); thus enabling us to investigate which material had a higher tendency towards biofilm formation. 2. Materials and methods 2.1. Preparation of bacteria Lyophilized pellets of Staphylococcus aureus subsp. Aureus (ATCC 6538) were obtained and incubated in brain-heart infusion medium for a period of 24 hours to allow for sufficient rehydration.
220
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
Twelve well plates were prepared (Fig. 1) in which 12 Ti-alloy (10 mm 5.5 mm) (Depuy Synthes, Raynham, MA, USA) and 12 CoCr rods (10 mm 5.5 mm) (Depuy Synthes) were placed.
and a bacterial pellet was isolated. This pellet was then dissolved in 200 ug of phosphate buffer and plated on brain-heart infusion agar plates and incubated for 24 hours (Fig. 2). The resulting number of CFU for each plate was evaluated, using a CFU counter.
2.2. Spinal implant preparation 3. Results Each rod was machine cut to allow for uniform diameter and length, and thus allow for identical surface area. With regard to rod surface treatment during the post-production process, each rod underwent identical treatment first involving glass bead blasting to allow for a matte finish. This was performed to remove any tool marks that were incurred during the production process. Furthermore, during the post-production process, both rods underwent an identical passivation process allowing for removal of any free ions and residual oil. Both rods were autoclaved at 121°C (249°F). Each rod was then immersed in 5 ml of brain-heart infusion medium (Fig. 1). To each of these well plates, 1 ml of overnight culture of Staphylococcus aureus subsp. Aureus (ATCC 6538) was added. The implants were then divided into two groups and incubated at 37°C for a period of either 48 (n = 4) or 96 hours (n = 8). 2.3. Isolation of biofilm from spinal implant & evaluation of OD After the respectively designated incubation periods had elapsed the implants were washed with a 0.25% trypsin solution to extract the biofilms from the rods. This bacterial solution was then collected and the overall turbidity of the solution was evaluated by determining the OD measurement via a spectrophotometer. 2.4. Evaluation of CFU After the OD values were obtained the bacterial solution was centrifuged and the residual trypsin solution was decanted off
Two-way ANOVA analysis showed both OD and CFU were significantly influenced by duration of incubation and implant material. The CoCr rods yielded both a higher OD and CFU values compared to Ti-alloy (p < 0.05). Higher OD and CFU values were encountered at both 4-day incubation and 2-day incubation (p < 0.05). The mean (± SD) OD values for the bacterial solution obtained from the Ti-alloy implants were 1.105 ± 0.096 nm and 1.040 ± 0.026 nm at 48 hours and 96 hours, respectively. In contrast, the OD values for the bacterial solution obtained from the CoCr rods were 1.332 ± 0.161 nm and 1.115 ± 0.207 nm at 48 and 96 hours, respectively, which were significantly higher than the Ti-alloy implants (p = 0.025) (Fig. 3). Similarly, the CFU values were 1481 ± 417/100 mm2 and 745 ± 159/100 mm2 at 48 and 96 hours, respectively for the Ti-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721 ± 605/100 mm2 and 928 ± 88/100 mm2 (p < 0.001) at both 48 and 96 hours respectively (Fig. 4). 4. Discussion Implant associated infections are a major source of morbidity and mortality after spine surgery. The characteristic features of different metal implants are a contributory factor in the development of biofilm on spinal implants. The formation of biofilms makes implant associated infections difficult to treat and often requires
Fig. 1. Autoclaved titanium-alloy and cobalt chrome rods in 5 cc brain-heart infusion medium prior to incubation with Staphylococcus aureus subsp. Aureus (ATCC 6538).
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
221
Fig. 2. Bacterial solution isolated from titanium-alloy (left) and cobalt chrome (CoCr) (right) rods after 24 hours incubation on brain-heart infusion agar plates. Evaluation of resultant colony forming unit values show higher values associated with CoCr rods.
Fig. 3. Optical Density (OD) values obtained after 48 and 96 hours incubation of spinal rods in Staphylococcus aureus subsp. Aureus (ATCC 6538) solution. OD values are significantly higher on cobalt chrome (CoCr) rods (p = 0.025). Ti-alloy = titanium-alloy.
explantation of hardware [6]. The process of biofilm formation is complex and is the result of an interaction between the implant surface and the adherent microorganism. The formation of biofilm
involves the development of a community of microorganisms adhering to the biomaterial embedded in a matrix of extracellular polymeric substance that they produce [9]. This large aggregate,
222
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
Fig. 4. Colony forming unit values obtained for bacterial solution derived from 48 and 96 hours incubation of spinal rods in Staphylococcus aureus subsp. Aureus (ATCC 6538) solution. CFU values significantly were higher on cobalt chrome (CoCr) rods (p < 0.001). Ti-alloy = titanium-alloy.
enables bacteria to escape host defenses and antibiotic infiltration [10]. In our experimentation, we evaluate the adherence and biofilm formation of Staphylococcus aureus subsp. Aureus (ATCC 6538) on CoCr and Ti-alloy implants. Each of the metal implants were machine cut to precisely 10 mm length with a diameter of 5.5 mm to allow for uniform surface areas. Additionally, both metal rods were cultured in the Staphylococcal aureus bacterial solution for a period of 48 and 96 hours. Evaluation of the resulting CFU from the various implants showed that CoCr implants had a greater proclivity toward biofilm formation compared to Ti-alloy implants at both 48 and 96 hours of incubation. This finding serves to support the notion that the intrinsic physical characteristics of CoCr are implicated in promoting biofilm formation. To our knowledge there have been no head to head comparisons specifically evaluating biofilm formation between CoCr and Ti-alloy spinal implants. Not only do Ti-alloy implants most closely mimic the characteristic of cortical bone in terms of its biomechanical properties [11] but—as demonstrated by our study here—Tialloy implants have a lower proclivity for biofilm formation. It has been reported in in vitro studies that Staphylococcal epidermidis has greater adherence to stainless steel implants than Ti-alloy [12]. Additionally, it has been observed in animal studies, specifically involving rabbits, that Ti-alloy implants have a lower rate of infection compared to stainless steel [13]. The lower adherence of bacteria to Ti-alloy implants in vivo is believed to be the result of Ti-alloy’s higher biocompatibility. It has been postulated that Ti-alloy implants allow for host cells to rapidly grow over the implant and encapsulate it in a fibrous tissue coating, thus preventing bacterial adherence onto the surface of the implant [14]. This phenomenon was described by Gristina et al., as ‘‘a race for the surface” between the host connective tissue and the bacteria for ownership of the implant material [5]. Our study shows that at both 48 and 96 hours CoCr implants had a higher degree of biofilm formation and an associated higher OD value for the resultant bacterial solution that was obtained
after trypsin wash in comparison to Ti-alloy implants. The use of OD values serves to directly evaluate the turbidity of the bacterial solution obtained from each rod. Application of a 0.25% trypsin solution to remove biofilm from the metallic implants has been previously described by Ryoo et al. [6]. In their study, electron microscopy was employed before and after 0.25% trypsin wash, to demonstrate effective biofilm extraction off the titanium and stainless steel implants. Additionally, the use of OD values to evaluate the degree of biofilm formation has been previously described in the literature by Kalvenes et al. [15] with regards to evaluation of Pseudomonas aeruginosa biofilm formation. This method was found to be effective in the quantification of biofilm formation given that light absorption by the bacterial solution was found to correlate with the degree of biofilm cell mass. The decision to use CoCr versus Ti-alloy implants in patients undergoing spinal instrumentation is largely dependent on the overall clinical context in which operative intervention is being performed. The use of CoCr rods provides a number of advantages in terms of its biomechanical properties. In particular CoCr implants have a greater rigidity and stiffness in comparison to other biomaterials [16]. It has been shown in biomechanical studies that CoCr implants have the ability to perform the highest corrective forces in comparison to Ti-alloy and stainless steel rods [8]. Given these intrinsic qualities CoCr implants are particularly efficacious in major spinal deformity correction surgery. Our study shows that CoCr implants have a significantly higher incidence of biofilm formation compared to Ti-alloy implants. It is important to note that we specifically confirmed with the rod manufacturers that both metals have different surface property due to atomic structures, but not due to difference in surface treatment. Given this finding further investigation will be needed to understand why this phenomenon occurs on a cellular level. Additionally, these finding can certainly help guide surgical planning. Perhaps additional steps with regard to infection prevention should be employed in patients undergoing spinal instrumentation with CoCr implants. Steps such as restricting operating room traffic during
S.S. Patel et al. / Journal of Clinical Neuroscience 31 (2016) 219–223
surgery, copious wound irrigation with antibacterial solution, and even the use of vancomycin powder prior to wound closure [17], may all help to minimize the risk of post-operative wound infection and eventual biofilm formation. In the material science literature, there are several studies that have extensively examined the surface properties of orthopedic implants [18–21]. According to their results, surface roughness, surface hydrophobicity and surface free radicals are the most important intrinsic properties of the materials studied. It is possible that these factors could have contributed to the variability seen in terms of the biofilm formation between Ti-alloy and CoCr implants. Given the differences observed in this study, we feel it would be worthwhile to study the aforementioned characteristics and its impact on biofilm production on CoCr and Ti-alloy. One of the limitations of our study is that this was an in-vitro study; certainly conditions maybe differ in the in-vivo setting. Additionally, given the importance of Staphylococcus epidermidis in orthopedic infections, it would certainly be worthwhile to repeat this study with use of Staphylococcus epidermidis. 5. Conclusion The degree of biofilm formation of Staphylococcus aureus on spinal implants composed of CoCr and Ti-alloy were directly compared using OD values and CFU values of the trypsinized biofilm from incubated rods. Spinal implants composed of CoCr showed a significantly higher rate of biofilm formation after both 48 and 96 hours of incubation. Further investigation will be required to understand why CoCr implants have a higher affinity for bacterial adherence and biofilm formation on a cellular level. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. References [1] Wang X, Qiu S, Yao X, et al. Berberine inhibits Staphylococcus epidermidis adhesion and biofilm formation on the surface of titanium alloy. J Orthop Res 2009;27:1487–92.
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