Clinical Orthopaedics and Related Research®
Clin Orthop Relat Res DOI 10.1007/s11999-015-4280-3
A Publication of The Association of Bone and Joint Surgeons®
SYMPOSIUM: RESEARCH ADVANCES AFTER A DECADE OF WAR
Cathodic Electrical Stimulation Combined With Vancomycin Enhances Treatment of Methicillin-resistant Staphylococcus aureus Implant-associated Infections Scott Nodzo MD, Menachem Tobias MS, Lisa Hansen MS, Nicole R. Luke-Marshall PhD, Ross Cole BS, Linda Wild MD, Anthony A. Campagnari PhD, Mark T. Ehrensberger PhD
Ó The Association of Bone and Joint Surgeons1 2015
Abstract Background Effective treatments for implant-associated infections are often lacking. Cathodic voltage-controlled electrical stimulation has shown potential as a treatment of implant-associated infections of methicillin-resistant Staphylococcus aureus (MRSA). Questions/purposes The primary purpose of this study was to (1) determine if cathodic voltage-controlled electrical stimulation combined with vancomycin therapy is more effective at reducing the MRSA bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. We also
The institution of one or more of the authors (MTE) has received, during the study period, funding from Congressionally Directed Medical Research Program, Peer Reviewed Orthopedic Research Program, and the Bruce Holm Memorial Catalyst Fund. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research1 editors and board members are on file with the publication and can be viewed on request. Clinical Orthopaedics and Related Research1 neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDAapproval status, of any drug or device prior to clinical use. Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research. S. Nodzo, M. Tobias, R. Cole, M. T. Ehrensberger Department of Orthopedics, State University of New York at Buffalo, Buffalo, NY, USA L. Hansen, N. R. Luke-Marshall, A. A. Campagnari Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, NY, USA
sought to (2) evaluate the histologic effects of the various treatments on the surrounding bone; and to (3) determine if the cathodic voltage-controlled electrical stimulation treatment had an effect on the mechanical properties of the titanium implant as a result of possible hydrogen embrittlement. Methods Thirty-two adult male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) with surgically placed shoulder titanium implants were infected with a clinical strain of MRSA (NRS70). One week after infection, eight animals received a treatment of cathodic voltage-controlled electrical stimulation at 1.8 V versus Ag/AgCl for 1 hour (STIM), eight received vancomycin twice daily for 1 week (VANCO), eight received the cathodic voltage-controlled electrical stimulation and vancomycin therapy combined (STIM + VANCO), and eight served as controls with no treatment (CONT). Two weeks after initial infection, the implant, bone, and synovial fluid were collected for colony-forming unit (CFU) enumeration, qualitative histological analysis by a pathologist blinded to the treatments each animal received, and implant three-point bend testing. Results The implant-associated CFU enumerated from the STIM + VANCO (mean, 3.7 9 103; SD, 6.3 9 103) group were less than those from the CONT (mean, M. T. Ehrensberger (&) Department of Biomedical Engineering, State University of New York at Buffalo, 162 Farber Hall, 3435 Main Street, Buffalo, NY 14214, USA e-mail: [email protected]
L. Wild Department of Pathology and Anatomical Sciences, State University of New York at Buffalo, Buffalo, NY, USA
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1.3 9 106; SD, 2.8 9 106; 95% confidence interval [CI] of difference, 4.3 9 105 to 9.9 9 103; p \ 0.001), STIM (mean, 1.4 9 106; SD, 2.0 9 106; 95% CI of difference, 2.1 9 106 to 1.8 9 103; p = 0.002), and VANCO (mean, 5.8 9 104; SD, 5.7 9 104; 95% CI of difference, 6.4 9 104 to 1.7 9 104; p \ 0.001) group. The boneassociated CFU enumerated from the STIM + VANCO group (6.3 9 101; SD, 1.1 9 102) were less than those from the CONT (mean, 2.8 9 105; SD, 4.8 9 105; 95% CI of difference, 9.4 9 104 to 5.0 9 103; p \ 0.001) and STIM (mean, 2.6 9 104; SD, 2.5 9 104; 95% CI of difference, 4.1 9 104 to 1.6 9 103; p \ 0.001) groups. The VANCO group (4.3 9 105; SD, 6.3 9 102) also had lower bone-associated CFU as compared with the CONT (mean 95% CI of difference, 9.3 9 104 to 4.5 9 103; p \ 0.001) and STIM (95% CI of difference, 4.0 9 104 to 1.5 9 103; p \ 0.001) groups. In comparison to the synovial fluid CFU enumerated from the CONT group (mean, 3.3 9 104; SD, 6.0 9 104), lower synovial CFU were reported for both the STIM + VANCO group (mean, 4.6 9 101; SD, 1.2 9 102; 95% CI of difference, 4.9 9 103 to 3.0 9 102; p \ 0.001) and the VANCO group (mean, 6.8 9 101; SD, 9.2 9 101; 95% CI of difference, 4.9 9 103 to 2.8 9 102; p = 0.007). The histological analysis showed no discernable deleterious effects on the surrounding tissue as a result of the treatments. No brittle fracture occurred during mechanical testing and with the numbers available, no differences in implant flexural yield strength were detected between the groups. Conclusions In this rodent model, cathodic voltage-controlled electrical stimulation combined with vancomycin is an effective treatment for titanium implant-associated infections showing greater than 99.8% reduction in bacterial burden on the implant, surrounding bone, and synovial fluid as compared with the controls and the stimulation alone groups. Clinical Relevance Cathodic voltage-controlled electrical stimulation combined with vancomycin may enable successful treatment of titanium orthopaedic implantassociated infections with implant retention. Future studies will focus on optimization of the stimulation parameters for complete eradication of infection and the ability to promote beneficial host tissue responses.
Introduction Extremity trauma has been a frequent combat-related injury in the conflicts in Iraq and Afghanistan. The US Military Amputee Database indicates over 1450 US service members have undergone traumatic limb amputation [3]. A promising approach to amputee care is the development of titanium
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(Ti) percutaneous osseointegrated prosthetic limbs. Despite many advantages [34], the use of osseointegrated prostheses has been limited as a result of concerns about infection. A previous clinical study of these prostheses reported a deep infection rate of 18% (seven of 39 patients) and a superficial infection rate of approximately 28% (11 of 39 patients) [29]. The most common bacteria isolated were Staphylococcus aureus and coagulase-negative staphylococcus. Other investigators have reported 18% of osseointegrated prostheses had to be removed as a result of infection [28]. A recent prospective study reported superficial infection, at a rate of 55%, as the most frequent complication and identified four patients with deep infection resulting in removal of one implant [4]. Although overall this prospective study showed a 2-year survival rate of 92% with enhanced prosthetic use and mobility, the anticipated service life of these devices will require durability far beyond that, and so infection risk remains a clinical concern. Periprosthetic joint infection also represents an important source of morbidity in both civilian and military patient populations. The frequency of infection after primary hip and knee arthroplasty has been reduced to 0.3% to 2% with modern aseptic techniques, but this has reached 20% in some revision procedures [17, 20]. Currently, the preferred treatment for periprosthetic joint infection is a two-staged revision arthroplasty with antibiotic spacer placement resulting from bacterial biofilm formation on the retained components [15, 27, 30]. Although effective, this method requires multiple surgical procedures, increases healthcare costs, limits mobility, and increases soft tissue and skeletal damage often resulting in additional reconstructive procedures at the time of replantation of permanent components [15, 27]. Previous work has provided proof of principle that cathodic voltage-controlled electrical stimulation of orthopaedic implants has potential for treating implant-associated infections of methicillin-resistant S aureus (MRSA). Specifically, using a rodent model of implantassociated infections, cathodic voltage-controlled electrical stimulation of 1.8 V versus Ag/AgCl applied to a Ti implant for 1 hour reduced MRSA bacterial colony-forming units (CFU) recovered immediately after treatment from the implant and surrounding bone by one to two orders of magnitude [13]. Also of importance, the cathodic voltagecontrolled electrical stimulation treatment had no discernible deleterious effects on the bone as confirmed with histological analysis. Although this treatment was effective in reducing the CFU, clinically the goal is complete eradication of the bacteria. Because it is envisioned that the cathodic voltage-controlled electrical stimulation would be used as an adjunctive therapy, there is a need to evaluate this treatment approach combined with antibiotic therapy. The primary purpose of this study was to (1) determine if cathodic voltage-controlled electrical stimulation combined
CVCES + Vancomycin as Infection Treatment
with vancomycin therapy is more effective at reducing the MRSA bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. We also sought to (2) evaluate the histologic effects of the various treatments on the surrounding bone; and to (3) determine if the cathodic voltage-controlled electrical stimulation treatment had an effect on the mechanical properties of the Ti implant as a result of possible hydrogen embrittlement.
Materials and Methods Study Design All of the experimental protocols used in this study were reviewed and approved by the University at Buffalo Institutional Animal Care and Use Committee. Using a previously developed rodent septic joint model of MRSA implant-associated infections [13], this study sought to evaluate the antimicrobial, histological, and mechanical effects of treatment with cathodic voltage-controlled electrical stimulation alone (STIM), vancomycin alone (VANCO), or cathodic voltage-controlled electrical stimulation combined with vancomycin (STIM + VANCO). In addition to these three treatment groups, an additional group that received no treatment served as a control group (CONT). A power analysis, conducted in R statistical software (R Foundation for Statistical Computing, Vienna, Austria) using data from previous work with this stimulation animal model showing an implant CFU population SD of 3.3 9 104, determined eight rodents should be used in each experimental group to detect a 90% reduction in CFU at a power of 80% and p value of 0.05.
(Indianapolis, IN, USA). The ventral periaxillary area of the anesthetized (using a ketamine, xylazine, and acepromazine mix prepared by the university veterinary services) rat was shaved, sterilized with chlorhexidine and alcohol, and injected with local anesthetic (bupivacaine and lidocaine mix) and analgesic (buprenorphine) subcutaneously. As described previously [13], an oblique 1-cm skin incision was made over the shoulder, and sharp dissection through the subcutaneous fat and superficial muscle layers was carried down to expose the anterior portion of the rotator cuff. The anterior one-third of the rotator cuff was sharply incised with the shoulder in abduction and external rotation to expose the humeral head. A 1.7-mm drill bit was used to drill through the center of the humeral head and out through the lateral cortex. The canal created was inoculated with approximately 105 CFU (0.1 cc) of NRS70. After inoculation, a custom 1.6 mm 9 16-mm, sterilized Ti rod (Titanium Industries, Inc, Rockaway, NJ, USA) was implanted in the canal so that it sat flush with the articular surface of the humeral head (so to not impede with normal joint mechanics postoperatively) and extended slightly beyond the lateral cortex of the humerus (Fig. 1). After implantation, the deep tissue and skin incisions were closed using 4-0 Webcryl (Patterson Veterinary, Devens, MA, USA), and carprofen was administered for postoperative pain management. Rats were maintained 6 days postoperatively for establishment of a septic shoulder [13].
Bacteria All experiments were conducted with MRSA strain NRS70, a recent respiratory isolate [19]. The minimal inhibitory concentration for vancomycin against NRS70 was determined by the clinical microbiology laboratory at the Buffalo VA Hospital to be 0.5 lg/mL. Bacteria were suspended in tryptic soy broth supplemented with 0.25% glucose to an optical density at 600 nm (OD600) of 0.1 (corresponding to approximately 107 CFU per mL) and diluted 1:10 to generate the inoculum.
Surgical Implantation and Infection A total of 32 adult Long-Evans rats (males, 175–200 g/ 8–9 weeks) were acquired from Harlan Laboratories
Fig. 1 Radiograph showing placement of the Ti implant in the rat humerus.
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Treatment Protocol A total of eight animals were randomly assigned to each of the four experimental groups: STIM, VANCO, STIM + VANCO, or CONT. On postoperative day (POD) 6, the rats in the VANCO and STIM + VANCO groups started receiving subcutaneous injections of vancomycin at 150 mg/ kg (V8138; Sigma-Aldrich, St Louis, MO, USA) twice a day. This dosing was derived from other experimental models [1, 6, 18, 26, 33] and pilot studies. On POD 7, all animals were anesthetized and the skin over the lateral cortex of the humerus was incised to expose the end of the Ti rod. Subsequently, an Ag/AgCl pellet reference electrode (E242ML; In Vivo Metric, Healdsburg, CA, USA) and a dual platinum wire counter electrode (CHI115; CH Instruments, Austin, TX, USA) were placed subcutaneously at an incision site immediately adjacent to the incision site providing access to the Ti rod (Fig. 2). In the STIM and STIM + VANCO groups the electrodes were connected to a potentiostat (Ref 600; Gamry Instruments, Warminster, PA, USA) that provided cathodic voltagecontrolled electrical stimulation of 1.8 V to the Ti rod (working electrode) for 1 hour. Animals in the VANCO and CONT groups had the electrodes placed, but no stimulation was delivered during the sham procedure. After the stimulation/sham procedure, the reference electrode,
Clinical Orthopaedics and Related Research1
counter electrode, and the contact to the implant were removed, all incisions sutured, and the animals were returned to their cages. Animals in the VANCO and STIM + VANCO groups continued to receive twice-daily subcutaneous injections of vancomycin. On POD 14, all animals were anesthetized for harvesting of specimens. The Ti implant was extracted for enumeration of CFU and subsequent mechanical testing. The humeral head and diaphysis were harvested for enumeration of CFU and histological analysis. Synovial fluid was aspirated from the joint immediately after joint capsule penetration for enumeration of CFU. Additionally, peripheral venous blood samples were collected by venipuncture to assess the presence of systemic bacteremia. Subsequently, the animals were euthanized by exsanguination under isoflurane anesthesia.
Enumeration of Colony-forming Units The Ti rod and the proximal segment of the humeral head collected at the end of the studies were individually rinsed with sterile phosphate-buffered saline (PBS), immersed in PBS-0.1% saponin, and subjected to a sonicating water bath for 10 minutes. Viable CFU were enumerated from the Ti, humeral head bone specimens, synovial fluid, and peripheral blood samples by plating 100 lL of serial 10-fold dilutions onto Mueller Hinton agar plates and incubating at 37 °C in 5% CO2 for 18 hours.
Histological Evaluation Portions of the humeral head and diaphysis were immediately fixed in 10% buffered formalin phosphate (Fisher Scientific, Waltham, MA, USA) after harvest. Briefly, samples were decalcified for approximately 24 hours, paraffin wax-embedded, sliced to 5-lm thick sections (perpendicular to the axis of the implant canal), set to glass slides, and stained using hematoxylin and eosin. The serial sections from two animals in all experimental groups were evaluated by a pathologist (LW) under blinded review for qualitative histological analysis of the bone and surrounding tissue as a result of the treatment protocol.
Fig. 2 Schematic showing the in vivo stimulation using a threeelectrode system. The Ti implant (working electrode) was accessed through an internal cortex incision while Ag/AgCl (reference) and platinum (counter) electrodes were placed subcutaneously adjacent to the Ti implant. Reprinted from Biomaterials, 41, Cathodic voltagecontrolled electrical stimulation of titanium implants as treatment for methicillin-resistant Staphylococcus aureus periprosthetic infection, 97–105, 2015, with permission from Elsevier.
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Implant Mechanical Testing After sonication to release bacteria, implants from the STIM and CONT groups were subjected to a three-point bending test to evaluate the potential influence of hydrogen generation and implant embrittlement during the cathodic polarization [16, 25]. The implants were placed in a three-
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point bending test fixture mounted within a load frame system (ElectroForce1 3200; Bose, Eden Prairie, MN, USA). Implants were loaded at the midpoint of the 14-mm span length at a rate of 2.5 mm per second for a total displacement of 5 mm. The primary outcome of this testing was to assess if brittle fracture or plastic yielding occurred when implants were loaded. The intention was to report either flexural yield strength or fracture strength depending on the test outcomes. However, no samples displayed brittle fracture during testing and therefore only flexural yield strength, defined as the load where the load-displacement curve deviates from the initial linear region, was reported as a characterization of the implant mechanical properties.
Statistical Analysis A Kruskal-Wallis nonparametric test followed by serial Mann-Whitney post hoc tests with a Bonferroni correction were used to compare the log-transformed CFU data across all experimental groups. As a result of the Bonferroni correction, a p value \ 0.0125 was considered statistically
significant for the Mann-Whitney tests. A Mann-Whitney test was used to compare the flexural yield strength between the STIM and CONT groups. A p value \ 0.05 was considered statistically significant for the strength statistical test. All statistical tests were performed with SPSS statistical software, Version 12 (Armonk, NY, USA).
Results The STIM + VANCO treatment decreased the CFU on the implant, bone, and synovial fluid by 99.8% when compared with the CONT and STIM groups. As compared with VANCO, STIM + VANCO decreased implant CFU by 94% but showed no differences in terms of bone and synovial fluid CFU. The implant-associated CFU (Fig. 3A) enumerated from the STIM + VANCO (mean, 3.7 9 103; SD, 6.3 9 103) group were less than those from the CONT (mean, 1.3 9 106; SD, 2.8 9 106; 95% confidence interval [CI] of difference, 4.3 9 105 to 9.9 9 103; p \ 0.001), STIM (mean, 1.4 9 106; SD, 2.0 9 106; 95% CI of difference, 2.1 9 106 to 1.8 9 103; p = 0.002), and 4 VANCO (mean, 5.8 9 10 ; SD, 5.7 9 104; 95% CI of
Fig. 3A–C Plots showing the CFU enumerated from the Ti implant (A), bone (B), and synovial fluid (C) for the control (CONT), stimulation alone (STIM), vancomycin alone (VANCO), and combined stimulation and vancomycin treatment (STIM + VANCO) groups. The data sets are plotted as the mean values ± SD and presented on a log scale. *Statistically significant differences where p \ 0.0125.
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difference, 6.4 9 104 to 1.7 9 104; p \ 0.001) groups. There was no difference in implant-associated CFU found among the VANCO, CONT, and STIM groups. In addition, the bone-associated CFU (Fig. 3B) enumerated from the STIM + VANCO group (6.3 9 101; SD, 1.1 9 102) were less than those from the CONT (mean, 2.8 9 105; SD, 4.8 9 105; 95% CI of difference, 9.4 9 104 to 3 5.0 9 10 ; p \ 0.001) and STIM (mean, 2.6 9 104; SD, 2.5 9 104; 95% CI of difference, 4.1 9 104 to 3 1.6 9 10 ; p \ 0.001) groups. The VANCO group (4.3 9 105; SD, 6.3 9 102) also had lower bone-associated CFU as compared with the CONT (mean 95% CI of difference, 9.3 9 104 to 4.5 9 103; p \ 0.001) and STIM (95% CI of difference, 4.0 9 104 to 1.5 9 103; p \ 0.001) groups. In comparison to the synovial fluid CFU (Fig. 3C) enumerated from the CONT group (mean, 3.3 9 104; SD, 6.0 9 104), lower synovial CFU were reported for both the STIM + VANCO group (mean, 4.6 9 101; SD, 1.2 9 102; 95% CI of difference, 4.9 9 103 to 3.0 9 102; p \ 0.001) and the VANCO group (mean, 6.8 9 101; SD, 9.2 9 101; 95% CI of difference, 4.9 9 103 to 2.8 9 102; p = 0.007). The synovial fluid CFU enumerated from the STIM group (mean, 4.5 9 104; SD, 1.2 9 105) was not different from any of the other groups. Peripheral blood cultures were negative in all animals suggesting the infection remained localized and did not become systemic. On review of histopathology specimens by a reviewer (LW) blinded to the treatments used, we were able to discern
Fig. 4A–D Representative histological sections of the rat humerus after Ti implant removal from the control (A, Stain: hematoxylin and eosin, original magnification: 9 40), stimulation alone (B, Stain: hematoxylin and eosin, original magnification: 9 40), vancomycin alone (C, Stain: hematoxylin and eosin, original magnification: 9 40), and combined stimulation and vancomycin treatment (D, Stain: hematoxylin and eosin, original magnification: 9 40) groups.
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no deleterious histological effects of treatment. Microscopic sections of decalcified bone from specimens in all experimental groups of rats showed open epiphyseal growth plates with endochondral ossification and circular defects from the implant that extend from the subchondral region beneath the articular surface to the proximal diaphysis. Abundant new bone formation surrounded the implant defects and in the subperiosteum of cortical bone in all specimens. Surrounding skeletal muscle fibers and connective tissue appeared viable and unremarkable. Varying amounts of inflammation and repair tissue were noted between the groups in the histological sections of the implant circular defects. The CONT group showed the most abundant layers of edematous and hemorrhagic granulation tissue containing inflammatory exudate with numerous neutrophils and macrophages lining the implant defect and extending into adjacent cancellous bone (Fig. 4A). The STIM group showed a moderate layer of granulation tissue with occasional neutrophils and lymphocytes coating the defect edges (Fig. 4B). The VANCO group showed the least amount of granulation tissue (Fig. 4C), whereas the STIM + VANCO group showed a moderate layer of granulation tissue around the implant defect (Fig. 4D). Recent hemorrhage was observed in several sections; however, there was no apparent or consistent relationship between evidence of hemorrhage and experimental groups. There was no evidence of bone necrosis in any of the experimental groups. All implants exhibited plastic yielding without catastrophic brittle fracture during the mechanical testing. The
CVCES + Vancomycin as Infection Treatment
flexural yield strengths of stimulated (mean, 122.9 N; SD, 5.5 N) and nonstimulated (mean, 127.4 N; SD, 4.9 N) implants were not different based on the available numbers (p = 0.28).
Discussion Infection subsequent to placement of orthopaedic implants is a devastating outcome associated with increased patient morbidity, longer hospital stays, and increased costs to the healthcare system. Furthermore, effective treatment options are often limited for implant-associated orthopaedic infections. However, it has recently been shown that cathodic voltage-controlled electrical stimulation of Ti implants at 1.8 V versus Ag/AgCl for 1 hour significantly reduces the MRSA CFU immediately recovered from both the Ti implant and the surrounding bone tissue by one to two orders of magnitude [13]. Because it is envisioned that the cathodic voltage-controlled electrical stimulation would be used as an adjunctive therapy, the primary purpose of this study was to determine if cathodic voltagecontrolled electrical stimulation combined with vancomycin therapy is more effective at reducing the bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. This study found that the combined stimulation and vancomycin treatment was highly effective and reduced the bacterial burden of the implant, bone, and synovial fluid by 99.8% as compared with the control and stimulation alone groups. In addition, the combined treatment produced a 94% reduction of bacteria on implant as compared with the vancomycin alone group. Although this study reports encouraging antimicrobial results when cathodic voltage-controlled electrical stimulation is combined with vancomycin, there are some limitations to consider. First, although these are in vivo results, they are from a rodent model, which may not completely represent the clinical picture in a human patient. Also, the animal model used is well aligned with studying bone-embedded implant-associated infections but does not have a percutaneous site and, therefore, does not completely model all of the infection risks of osseointegrated prosthetic limbs. Future studies will be conducted to address the effects of cathodic voltage-controlled electrical stimulation at the percutaneous site. We also note a 1-week course of antibiotics is subtherapeutic for treatment for implant-associated infections. A more prolonged antibiotic therapy protocol or local, high-dose antibiotic delivery may have further lowered the CFU counts in the VANCO and STIM + VANCO groups. Another limitation was that the histological analysis in this study consisted of qualitative descriptions rather than a quantitative scoring system to
allow for statistical analysis of histological outcomes. In addition, we used an indirect method for assessment of hydrogen embrittlement and rigorous chemical composition and fatigue studies should be conducted to rule out the presence of hydrogen embrittlement. Finally, we did not evaluate varying durations or magnitudes of cathodic voltage-controlled electrical stimulation. We chose stimulation at 1.8 V for 1 hour based on previous in vitro and in vivo animal work [13]; however, future studies are needed to optimize these parameters. Despite these limitations, our data show that this cathodic voltage-controlled electrical stimulation treatment modality holds promise as an adjunctive treatment for orthopaedic implant-associated infections and warrants further investigation. The outcomes of the CONT group showed the rodent model used is a localized and sustained implant-associated infection. Although previous work showed respective bacterial reductions of 98% and 87% for implants and bone tissue harvested immediately after cathodic voltage-controlled electrical stimulation of 1.8 V for 1 hour [13], the present study has shown that the bacterial burden for the STIM group was not different from the CONT at 1 week poststimulation/sham. This indicates that the antimicrobial effects of cathodic voltage-controlled electrical stimulation treatment are immediate but not sustained. This could be attributable to the biofilm bacteria on the implant being released on electrical stimulation but then subsequently adhering back to the implant. Compared with the CONT group, the VANCO group showed CFU reductions in the bone and synovial fluid, but not for the implant. This may indicate that the bacteria are adherent to the implant in a biofilm, which is known to enhance resistance to antimicrobials [7, 8]. The combined treatment was most effective and compared with the controls showed CFU reductions of 99.8% for the implant, bone, and synovial fluid. Although the antimicrobial effect on the implant CFU in the STIM + VANCO group was robust, it may have been blunted by bacteria in the surrounding tissues reattaching to the implant after the effects of stimulation subsided. We theorize that the implant CFU count after cathodic voltagecontrolled electrical stimulation may become even lower if the synovial fluid and bone bacterial burden are minimized before the cathodic voltage-controlled electrical stimulation treatment, and future research will be focused on optimal antibiotic and stimulation protocols. The cathodic voltage-controlled electrical stimulation had no discernible deleterious effect on the surrounding bone based on qualitative histologic evaluation. Histological evaluation showed inflammation in the bone tissue, which is expected with implant-associated infections. It may seem paradoxical that although the STIM + VANCO group had the best reductions in all CFU categories, there appeared to be more granulation tissue surrounding the
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implant defect in this group as compared with the VANCO group. Because the STIM + VANCO group had significantly less implant-associated CFU as compared with the VANCO, we postulate that the stimulation resulted in a large bacterial load being released from the biofilm on the implant into the surrounding environment in a planktonic form, which are more susceptible to killing by antibiotics and the host response. Therefore, the increased granulation tissue observed in STIM + VANCO as compared with VANCO may actually be indicative of this increased killing of bacteria and the associated increased neutrophil respiratory burst and release of proinflammatory cytokines. Importantly, there were no regions of bone necrosis observed, and because the cathodic voltage-controlled electrical stimulation had been applied 7 days before harvesting, there was enough time for bone necrosis to occur. These histologic results are similar to previous work [13] using this model showing no signs of bone damage in histologic sections evaluated immediately after cathodic voltage-controlled electrical stimulation suggesting that 1.8 V for 1 hour appears to have no negative short-term or long-term impact on the surrounding bone tissues. The three-point bending tests of the CONT and STIM implants revealed no differences in the flexural yield strength based on the numbers available and also displayed no brittle fracture. Although Ti is known to be susceptible to hydrogen embrittlement [5, 16, 25], the present outcomes suggest the interfacial water and oxygen reduction electrochemical processes during cathodic voltage-controlled electrical stimulation [13] did not result in hydrogen embrittlement of the Ti. However, this single loading mechanical evaluation was performed after one cathodic voltage-controlled electrical stimulation treatment; further research is needed to assess effects on fatigue strength and also the effects of multiple cathodic voltage-controlled electrical stimulation treatments on implant mechanical properties. Although there have been numerous in vitro reports on the antimicrobial effects of direct electrical simulation with or without combination with antibiotics [2, 9, 11, 12, 21– 23, 32], there are only a few in vivo reports. Using a rabbit model of osteomyelitis with Staphylococcal epidermidis, it was shown that a constant current of 200 lA delivered to an intramedullary stainless steel electrode for 21 days produced a 1.5 order of magnitude reduction in bacteria when compared with the use of doxycycline alone [10]. It has also been shown that delivery of a constant current of 100 lA for 21 days to stainless steel external fixator pins in a goat model successfully prevented pin site infection of S epidermidis in 89% of sites evaluated [31]. The present study is different from these previous in vivo studies in a few important ways. As described before [13], two-electrode techniques used to deliver constant current are
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fundamentally different from the three-electrode cathodic voltage-controlled electrical stimulation method used in the present study to deliver constant voltage. The ability to enforce a constant voltage of the Ti implant allows for precise modulation of the voltage-dependent interfacial electrochemical processes at the surface oxide film that are thought to be involved in the antimicrobial effects of cathodic voltage-controlled electrical stimulation [13, 14]. In addition, the present study showed that antibiotics combined with cathodic voltage-controlled electrical stimulation of only 1 hour produced robust antimicrobial effects as compared with the longer 21-day treatments using in previous in vivo constant current studies. Furthermore, bone discoloration was noted in the study that delivered 200 lA for the 21 days, but there was no histological assessment of the tissues [10]. The present study includes histological outcomes showing no discernible deleterious effects on the tissue surround the implant that received cathodic voltage-controlled electrical stimulation for 1 hour. It is also important to point out that although the present study was conducted with Ti, most of the metals used for orthopaedic implants are also passivated with surface oxide films that have voltage-dependent electrochemical properties [24]. Therefore, the application of cathodic voltagecontrolled electrical stimulation to metallic orthopaedic devices may represent a new and widely applicable treatment modality. In conclusion, in this rat model of implant-associated infection, cathodic voltage-controlled electrical stimulation combined with vancomycin has been shown to reduce the bacterial burden on the implant, surrounding bone, and synovial fluid by 99.8% as compared with the controls. Although these large bacterial reductions are promising and may provide a solution for effective treatment with implant retention, further optimization of the treatment parameters may be needed to reach the ultimate goal of complete bacterial eradication. Future directions will include evaluations of stimulation magnitude, pattern, and duration as well as optimal timing for adjunctive stimulation during the time course of antibiotic therapy. In addition to optimizing the antimicrobial outcomes, subsequent studies will also seek to evaluate the ability of cathodic voltage-controlled electrical stimulation to promote beneficial host tissue responses such as enhanced bone and skin integration for osseointegrated and/or percutaneous orthopaedic implants. Finally, we are also focused on developing a translational protocol that would facilitate clinical utilization. Application of this stimulation to percutaneous implants is envisioned to be noninvasive, and on further optimization, a minimally invasive application of this stimulation could be potentially used to treat infections of fully implanted joint prostheses.
CVCES + Vancomycin as Infection Treatment
References 1. Aronoff GR, Sloan RS, Dinwiddie CB, Jr., Glant MD, Fineberg NS, Luft FC. Effects of vancomycin on renal function in rats. Antimicrob Agents Chemother. 1981;19:306–308. 2. Blenkinsopp SA, Khoury AE, Costerton JW. Electrical enhancement of biocide efficacy against Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 1992;58:3770–3773. 3. Bosse MJ, Ficke JR, Andersen RC. Extremity war injuries: current management and research priorities. J Am Acad Orthop Surg. 2012;20(Suppl):viii–x. 4. Branemark R, Berlin O, Hagberg K, Bergh P, Gunterberg B, Rydevik B. A novel osseointegrated percutaneous prosthetic system for the treatment of patients with transfemoral amputation: a prospective study of 51 patients. Bone Joint J. 2014;96:106–113. 5. Briant CL, Wang ZF, Chollocoop N. Hydrogen embrittlement of commercial purity titanium. Corrosion Science. 2002;44:1875– 1888. 6. Chilukuri DM, Shah JC. Local delivery of vancomycin for the prophylaxis of prosthetic device-related infections. Pharm Res. 2005;22:563–572. 7. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. Bacterial biofilms in nature and disease. Annu Rev Microbiol. 1987;41:435–464. 8. Costerton JW. Biofilm theory can guide the treatment of devicerelated orthopaedic infections. Clin Orthop Relat Res. 2005;437:7–11. 9. Costerton JW, Ellis B, Lam K, Johnson F, Khoury AE. Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria. Antimicrob Agents Chemother. 1994;38:2803–2809. 10. Del Pozo JL, Rouse MS, Euba G, Kang CI, Mandrekar JN, Steckelberg JM, Patel R. The electricidal effect is active in an experimental model of Staphylococcus epidermidis chronic foreign body osteomyelitis. Antimicrob Agents Chemother. 2009;53:4064–4068. 11. del Pozo JL, Rouse MS, Mandrekar JN, Sampedro MF, Steckelberg JM, Patel R. Effect of electrical current on the activities of antimicrobial agents against Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother. 2009;53:35–40. 12. del Pozo JL, Rouse MS, Mandrekar JN, Steckelberg JM, Patel R. The electricidal effect: reduction of staphylococcus and pseudomonas biofilms by prolonged exposure to low-intensity electrical current. Antimicrob Agents Chemother. 2009;53:41–45. 13. Ehrensberger M, Tobias M, Nodzo S, Hansen L, Luke-Marshall N, Cole R, Wild L, Campagnari A. Cathodic Voltage-controlled electrical stimulation of titanium implants as treatment for methicillin-resistant Staphylococcus aureus periprosthetic infections. Biomaterials. 2015;41:97–105. 14. Ehrensberger MT, Gilbert JL. The effect of static applied potential on the 24 hour impedance of commercially pure titanium in simulated biological conditions. J Biomed Mater Res B. 2010;93:106–112. 15. Gardner J, Gioe TJ, Tatman P. Can this prosthesis be saved? Implant salvage attempts in infected primary TKA. Clin Orthop Relat Res. 2011;469:970–976. 16. Jones D. Principles and Prevention of Corrosion. Upper Saddle River, NJ, USA: Prentice-Hall, Inc; 1996. 17. Joseph TN, Chen AL, Di Cesare PE. Use of antibiotic-impregnated cement in total joint arthroplasty. J Am Acad Orthop Surg. 2003;11:38–47. 18. Karau MJ, Schmidt-Malan SM, Greenwood-Quaintance KE, Mandrekar JN, Cai J, Pierce WM, Merten K, Patel R. Treatment
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Clin Orthop Relat Res DOI 10.1007/s11999-015-4280-3
A Publication of The Association of Bone and Joint Surgeons®
SYMPOSIUM: RESEARCH ADVANCES AFTER A DECADE OF WAR
Cathodic Electrical Stimulation Combined With Vancomycin Enhances Treatment of Methicillin-resistant Staphylococcus aureus Implant-associated Infections Scott Nodzo MD, Menachem Tobias MS, Lisa Hansen MS, Nicole R. Luke-Marshall PhD, Ross Cole BS, Linda Wild MD, Anthony A. Campagnari PhD, Mark T. Ehrensberger PhD
Ó The Association of Bone and Joint Surgeons1 2015
Abstract Background Effective treatments for implant-associated infections are often lacking. Cathodic voltage-controlled electrical stimulation has shown potential as a treatment of implant-associated infections of methicillin-resistant Staphylococcus aureus (MRSA). Questions/purposes The primary purpose of this study was to (1) determine if cathodic voltage-controlled electrical stimulation combined with vancomycin therapy is more effective at reducing the MRSA bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. We also
The institution of one or more of the authors (MTE) has received, during the study period, funding from Congressionally Directed Medical Research Program, Peer Reviewed Orthopedic Research Program, and the Bruce Holm Memorial Catalyst Fund. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research1 editors and board members are on file with the publication and can be viewed on request. Clinical Orthopaedics and Related Research1 neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDAapproval status, of any drug or device prior to clinical use. Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research. S. Nodzo, M. Tobias, R. Cole, M. T. Ehrensberger Department of Orthopedics, State University of New York at Buffalo, Buffalo, NY, USA L. Hansen, N. R. Luke-Marshall, A. A. Campagnari Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, NY, USA
sought to (2) evaluate the histologic effects of the various treatments on the surrounding bone; and to (3) determine if the cathodic voltage-controlled electrical stimulation treatment had an effect on the mechanical properties of the titanium implant as a result of possible hydrogen embrittlement. Methods Thirty-two adult male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) with surgically placed shoulder titanium implants were infected with a clinical strain of MRSA (NRS70). One week after infection, eight animals received a treatment of cathodic voltage-controlled electrical stimulation at 1.8 V versus Ag/AgCl for 1 hour (STIM), eight received vancomycin twice daily for 1 week (VANCO), eight received the cathodic voltage-controlled electrical stimulation and vancomycin therapy combined (STIM + VANCO), and eight served as controls with no treatment (CONT). Two weeks after initial infection, the implant, bone, and synovial fluid were collected for colony-forming unit (CFU) enumeration, qualitative histological analysis by a pathologist blinded to the treatments each animal received, and implant three-point bend testing. Results The implant-associated CFU enumerated from the STIM + VANCO (mean, 3.7 9 103; SD, 6.3 9 103) group were less than those from the CONT (mean, M. T. Ehrensberger (&) Department of Biomedical Engineering, State University of New York at Buffalo, 162 Farber Hall, 3435 Main Street, Buffalo, NY 14214, USA e-mail: [email protected]
L. Wild Department of Pathology and Anatomical Sciences, State University of New York at Buffalo, Buffalo, NY, USA
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1.3 9 106; SD, 2.8 9 106; 95% confidence interval [CI] of difference, 4.3 9 105 to 9.9 9 103; p \ 0.001), STIM (mean, 1.4 9 106; SD, 2.0 9 106; 95% CI of difference, 2.1 9 106 to 1.8 9 103; p = 0.002), and VANCO (mean, 5.8 9 104; SD, 5.7 9 104; 95% CI of difference, 6.4 9 104 to 1.7 9 104; p \ 0.001) group. The boneassociated CFU enumerated from the STIM + VANCO group (6.3 9 101; SD, 1.1 9 102) were less than those from the CONT (mean, 2.8 9 105; SD, 4.8 9 105; 95% CI of difference, 9.4 9 104 to 5.0 9 103; p \ 0.001) and STIM (mean, 2.6 9 104; SD, 2.5 9 104; 95% CI of difference, 4.1 9 104 to 1.6 9 103; p \ 0.001) groups. The VANCO group (4.3 9 105; SD, 6.3 9 102) also had lower bone-associated CFU as compared with the CONT (mean 95% CI of difference, 9.3 9 104 to 4.5 9 103; p \ 0.001) and STIM (95% CI of difference, 4.0 9 104 to 1.5 9 103; p \ 0.001) groups. In comparison to the synovial fluid CFU enumerated from the CONT group (mean, 3.3 9 104; SD, 6.0 9 104), lower synovial CFU were reported for both the STIM + VANCO group (mean, 4.6 9 101; SD, 1.2 9 102; 95% CI of difference, 4.9 9 103 to 3.0 9 102; p \ 0.001) and the VANCO group (mean, 6.8 9 101; SD, 9.2 9 101; 95% CI of difference, 4.9 9 103 to 2.8 9 102; p = 0.007). The histological analysis showed no discernable deleterious effects on the surrounding tissue as a result of the treatments. No brittle fracture occurred during mechanical testing and with the numbers available, no differences in implant flexural yield strength were detected between the groups. Conclusions In this rodent model, cathodic voltage-controlled electrical stimulation combined with vancomycin is an effective treatment for titanium implant-associated infections showing greater than 99.8% reduction in bacterial burden on the implant, surrounding bone, and synovial fluid as compared with the controls and the stimulation alone groups. Clinical Relevance Cathodic voltage-controlled electrical stimulation combined with vancomycin may enable successful treatment of titanium orthopaedic implantassociated infections with implant retention. Future studies will focus on optimization of the stimulation parameters for complete eradication of infection and the ability to promote beneficial host tissue responses.
Introduction Extremity trauma has been a frequent combat-related injury in the conflicts in Iraq and Afghanistan. The US Military Amputee Database indicates over 1450 US service members have undergone traumatic limb amputation [3]. A promising approach to amputee care is the development of titanium
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(Ti) percutaneous osseointegrated prosthetic limbs. Despite many advantages [34], the use of osseointegrated prostheses has been limited as a result of concerns about infection. A previous clinical study of these prostheses reported a deep infection rate of 18% (seven of 39 patients) and a superficial infection rate of approximately 28% (11 of 39 patients) [29]. The most common bacteria isolated were Staphylococcus aureus and coagulase-negative staphylococcus. Other investigators have reported 18% of osseointegrated prostheses had to be removed as a result of infection [28]. A recent prospective study reported superficial infection, at a rate of 55%, as the most frequent complication and identified four patients with deep infection resulting in removal of one implant [4]. Although overall this prospective study showed a 2-year survival rate of 92% with enhanced prosthetic use and mobility, the anticipated service life of these devices will require durability far beyond that, and so infection risk remains a clinical concern. Periprosthetic joint infection also represents an important source of morbidity in both civilian and military patient populations. The frequency of infection after primary hip and knee arthroplasty has been reduced to 0.3% to 2% with modern aseptic techniques, but this has reached 20% in some revision procedures [17, 20]. Currently, the preferred treatment for periprosthetic joint infection is a two-staged revision arthroplasty with antibiotic spacer placement resulting from bacterial biofilm formation on the retained components [15, 27, 30]. Although effective, this method requires multiple surgical procedures, increases healthcare costs, limits mobility, and increases soft tissue and skeletal damage often resulting in additional reconstructive procedures at the time of replantation of permanent components [15, 27]. Previous work has provided proof of principle that cathodic voltage-controlled electrical stimulation of orthopaedic implants has potential for treating implant-associated infections of methicillin-resistant S aureus (MRSA). Specifically, using a rodent model of implantassociated infections, cathodic voltage-controlled electrical stimulation of 1.8 V versus Ag/AgCl applied to a Ti implant for 1 hour reduced MRSA bacterial colony-forming units (CFU) recovered immediately after treatment from the implant and surrounding bone by one to two orders of magnitude [13]. Also of importance, the cathodic voltagecontrolled electrical stimulation treatment had no discernible deleterious effects on the bone as confirmed with histological analysis. Although this treatment was effective in reducing the CFU, clinically the goal is complete eradication of the bacteria. Because it is envisioned that the cathodic voltage-controlled electrical stimulation would be used as an adjunctive therapy, there is a need to evaluate this treatment approach combined with antibiotic therapy. The primary purpose of this study was to (1) determine if cathodic voltage-controlled electrical stimulation combined
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with vancomycin therapy is more effective at reducing the MRSA bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. We also sought to (2) evaluate the histologic effects of the various treatments on the surrounding bone; and to (3) determine if the cathodic voltage-controlled electrical stimulation treatment had an effect on the mechanical properties of the Ti implant as a result of possible hydrogen embrittlement.
Materials and Methods Study Design All of the experimental protocols used in this study were reviewed and approved by the University at Buffalo Institutional Animal Care and Use Committee. Using a previously developed rodent septic joint model of MRSA implant-associated infections [13], this study sought to evaluate the antimicrobial, histological, and mechanical effects of treatment with cathodic voltage-controlled electrical stimulation alone (STIM), vancomycin alone (VANCO), or cathodic voltage-controlled electrical stimulation combined with vancomycin (STIM + VANCO). In addition to these three treatment groups, an additional group that received no treatment served as a control group (CONT). A power analysis, conducted in R statistical software (R Foundation for Statistical Computing, Vienna, Austria) using data from previous work with this stimulation animal model showing an implant CFU population SD of 3.3 9 104, determined eight rodents should be used in each experimental group to detect a 90% reduction in CFU at a power of 80% and p value of 0.05.
(Indianapolis, IN, USA). The ventral periaxillary area of the anesthetized (using a ketamine, xylazine, and acepromazine mix prepared by the university veterinary services) rat was shaved, sterilized with chlorhexidine and alcohol, and injected with local anesthetic (bupivacaine and lidocaine mix) and analgesic (buprenorphine) subcutaneously. As described previously [13], an oblique 1-cm skin incision was made over the shoulder, and sharp dissection through the subcutaneous fat and superficial muscle layers was carried down to expose the anterior portion of the rotator cuff. The anterior one-third of the rotator cuff was sharply incised with the shoulder in abduction and external rotation to expose the humeral head. A 1.7-mm drill bit was used to drill through the center of the humeral head and out through the lateral cortex. The canal created was inoculated with approximately 105 CFU (0.1 cc) of NRS70. After inoculation, a custom 1.6 mm 9 16-mm, sterilized Ti rod (Titanium Industries, Inc, Rockaway, NJ, USA) was implanted in the canal so that it sat flush with the articular surface of the humeral head (so to not impede with normal joint mechanics postoperatively) and extended slightly beyond the lateral cortex of the humerus (Fig. 1). After implantation, the deep tissue and skin incisions were closed using 4-0 Webcryl (Patterson Veterinary, Devens, MA, USA), and carprofen was administered for postoperative pain management. Rats were maintained 6 days postoperatively for establishment of a septic shoulder [13].
Bacteria All experiments were conducted with MRSA strain NRS70, a recent respiratory isolate [19]. The minimal inhibitory concentration for vancomycin against NRS70 was determined by the clinical microbiology laboratory at the Buffalo VA Hospital to be 0.5 lg/mL. Bacteria were suspended in tryptic soy broth supplemented with 0.25% glucose to an optical density at 600 nm (OD600) of 0.1 (corresponding to approximately 107 CFU per mL) and diluted 1:10 to generate the inoculum.
Surgical Implantation and Infection A total of 32 adult Long-Evans rats (males, 175–200 g/ 8–9 weeks) were acquired from Harlan Laboratories
Fig. 1 Radiograph showing placement of the Ti implant in the rat humerus.
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Treatment Protocol A total of eight animals were randomly assigned to each of the four experimental groups: STIM, VANCO, STIM + VANCO, or CONT. On postoperative day (POD) 6, the rats in the VANCO and STIM + VANCO groups started receiving subcutaneous injections of vancomycin at 150 mg/ kg (V8138; Sigma-Aldrich, St Louis, MO, USA) twice a day. This dosing was derived from other experimental models [1, 6, 18, 26, 33] and pilot studies. On POD 7, all animals were anesthetized and the skin over the lateral cortex of the humerus was incised to expose the end of the Ti rod. Subsequently, an Ag/AgCl pellet reference electrode (E242ML; In Vivo Metric, Healdsburg, CA, USA) and a dual platinum wire counter electrode (CHI115; CH Instruments, Austin, TX, USA) were placed subcutaneously at an incision site immediately adjacent to the incision site providing access to the Ti rod (Fig. 2). In the STIM and STIM + VANCO groups the electrodes were connected to a potentiostat (Ref 600; Gamry Instruments, Warminster, PA, USA) that provided cathodic voltagecontrolled electrical stimulation of 1.8 V to the Ti rod (working electrode) for 1 hour. Animals in the VANCO and CONT groups had the electrodes placed, but no stimulation was delivered during the sham procedure. After the stimulation/sham procedure, the reference electrode,
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counter electrode, and the contact to the implant were removed, all incisions sutured, and the animals were returned to their cages. Animals in the VANCO and STIM + VANCO groups continued to receive twice-daily subcutaneous injections of vancomycin. On POD 14, all animals were anesthetized for harvesting of specimens. The Ti implant was extracted for enumeration of CFU and subsequent mechanical testing. The humeral head and diaphysis were harvested for enumeration of CFU and histological analysis. Synovial fluid was aspirated from the joint immediately after joint capsule penetration for enumeration of CFU. Additionally, peripheral venous blood samples were collected by venipuncture to assess the presence of systemic bacteremia. Subsequently, the animals were euthanized by exsanguination under isoflurane anesthesia.
Enumeration of Colony-forming Units The Ti rod and the proximal segment of the humeral head collected at the end of the studies were individually rinsed with sterile phosphate-buffered saline (PBS), immersed in PBS-0.1% saponin, and subjected to a sonicating water bath for 10 minutes. Viable CFU were enumerated from the Ti, humeral head bone specimens, synovial fluid, and peripheral blood samples by plating 100 lL of serial 10-fold dilutions onto Mueller Hinton agar plates and incubating at 37 °C in 5% CO2 for 18 hours.
Histological Evaluation Portions of the humeral head and diaphysis were immediately fixed in 10% buffered formalin phosphate (Fisher Scientific, Waltham, MA, USA) after harvest. Briefly, samples were decalcified for approximately 24 hours, paraffin wax-embedded, sliced to 5-lm thick sections (perpendicular to the axis of the implant canal), set to glass slides, and stained using hematoxylin and eosin. The serial sections from two animals in all experimental groups were evaluated by a pathologist (LW) under blinded review for qualitative histological analysis of the bone and surrounding tissue as a result of the treatment protocol.
Fig. 2 Schematic showing the in vivo stimulation using a threeelectrode system. The Ti implant (working electrode) was accessed through an internal cortex incision while Ag/AgCl (reference) and platinum (counter) electrodes were placed subcutaneously adjacent to the Ti implant. Reprinted from Biomaterials, 41, Cathodic voltagecontrolled electrical stimulation of titanium implants as treatment for methicillin-resistant Staphylococcus aureus periprosthetic infection, 97–105, 2015, with permission from Elsevier.
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Implant Mechanical Testing After sonication to release bacteria, implants from the STIM and CONT groups were subjected to a three-point bending test to evaluate the potential influence of hydrogen generation and implant embrittlement during the cathodic polarization [16, 25]. The implants were placed in a three-
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point bending test fixture mounted within a load frame system (ElectroForce1 3200; Bose, Eden Prairie, MN, USA). Implants were loaded at the midpoint of the 14-mm span length at a rate of 2.5 mm per second for a total displacement of 5 mm. The primary outcome of this testing was to assess if brittle fracture or plastic yielding occurred when implants were loaded. The intention was to report either flexural yield strength or fracture strength depending on the test outcomes. However, no samples displayed brittle fracture during testing and therefore only flexural yield strength, defined as the load where the load-displacement curve deviates from the initial linear region, was reported as a characterization of the implant mechanical properties.
Statistical Analysis A Kruskal-Wallis nonparametric test followed by serial Mann-Whitney post hoc tests with a Bonferroni correction were used to compare the log-transformed CFU data across all experimental groups. As a result of the Bonferroni correction, a p value \ 0.0125 was considered statistically
significant for the Mann-Whitney tests. A Mann-Whitney test was used to compare the flexural yield strength between the STIM and CONT groups. A p value \ 0.05 was considered statistically significant for the strength statistical test. All statistical tests were performed with SPSS statistical software, Version 12 (Armonk, NY, USA).
Results The STIM + VANCO treatment decreased the CFU on the implant, bone, and synovial fluid by 99.8% when compared with the CONT and STIM groups. As compared with VANCO, STIM + VANCO decreased implant CFU by 94% but showed no differences in terms of bone and synovial fluid CFU. The implant-associated CFU (Fig. 3A) enumerated from the STIM + VANCO (mean, 3.7 9 103; SD, 6.3 9 103) group were less than those from the CONT (mean, 1.3 9 106; SD, 2.8 9 106; 95% confidence interval [CI] of difference, 4.3 9 105 to 9.9 9 103; p \ 0.001), STIM (mean, 1.4 9 106; SD, 2.0 9 106; 95% CI of difference, 2.1 9 106 to 1.8 9 103; p = 0.002), and 4 VANCO (mean, 5.8 9 10 ; SD, 5.7 9 104; 95% CI of
Fig. 3A–C Plots showing the CFU enumerated from the Ti implant (A), bone (B), and synovial fluid (C) for the control (CONT), stimulation alone (STIM), vancomycin alone (VANCO), and combined stimulation and vancomycin treatment (STIM + VANCO) groups. The data sets are plotted as the mean values ± SD and presented on a log scale. *Statistically significant differences where p \ 0.0125.
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difference, 6.4 9 104 to 1.7 9 104; p \ 0.001) groups. There was no difference in implant-associated CFU found among the VANCO, CONT, and STIM groups. In addition, the bone-associated CFU (Fig. 3B) enumerated from the STIM + VANCO group (6.3 9 101; SD, 1.1 9 102) were less than those from the CONT (mean, 2.8 9 105; SD, 4.8 9 105; 95% CI of difference, 9.4 9 104 to 3 5.0 9 10 ; p \ 0.001) and STIM (mean, 2.6 9 104; SD, 2.5 9 104; 95% CI of difference, 4.1 9 104 to 3 1.6 9 10 ; p \ 0.001) groups. The VANCO group (4.3 9 105; SD, 6.3 9 102) also had lower bone-associated CFU as compared with the CONT (mean 95% CI of difference, 9.3 9 104 to 4.5 9 103; p \ 0.001) and STIM (95% CI of difference, 4.0 9 104 to 1.5 9 103; p \ 0.001) groups. In comparison to the synovial fluid CFU (Fig. 3C) enumerated from the CONT group (mean, 3.3 9 104; SD, 6.0 9 104), lower synovial CFU were reported for both the STIM + VANCO group (mean, 4.6 9 101; SD, 1.2 9 102; 95% CI of difference, 4.9 9 103 to 3.0 9 102; p \ 0.001) and the VANCO group (mean, 6.8 9 101; SD, 9.2 9 101; 95% CI of difference, 4.9 9 103 to 2.8 9 102; p = 0.007). The synovial fluid CFU enumerated from the STIM group (mean, 4.5 9 104; SD, 1.2 9 105) was not different from any of the other groups. Peripheral blood cultures were negative in all animals suggesting the infection remained localized and did not become systemic. On review of histopathology specimens by a reviewer (LW) blinded to the treatments used, we were able to discern
Fig. 4A–D Representative histological sections of the rat humerus after Ti implant removal from the control (A, Stain: hematoxylin and eosin, original magnification: 9 40), stimulation alone (B, Stain: hematoxylin and eosin, original magnification: 9 40), vancomycin alone (C, Stain: hematoxylin and eosin, original magnification: 9 40), and combined stimulation and vancomycin treatment (D, Stain: hematoxylin and eosin, original magnification: 9 40) groups.
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no deleterious histological effects of treatment. Microscopic sections of decalcified bone from specimens in all experimental groups of rats showed open epiphyseal growth plates with endochondral ossification and circular defects from the implant that extend from the subchondral region beneath the articular surface to the proximal diaphysis. Abundant new bone formation surrounded the implant defects and in the subperiosteum of cortical bone in all specimens. Surrounding skeletal muscle fibers and connective tissue appeared viable and unremarkable. Varying amounts of inflammation and repair tissue were noted between the groups in the histological sections of the implant circular defects. The CONT group showed the most abundant layers of edematous and hemorrhagic granulation tissue containing inflammatory exudate with numerous neutrophils and macrophages lining the implant defect and extending into adjacent cancellous bone (Fig. 4A). The STIM group showed a moderate layer of granulation tissue with occasional neutrophils and lymphocytes coating the defect edges (Fig. 4B). The VANCO group showed the least amount of granulation tissue (Fig. 4C), whereas the STIM + VANCO group showed a moderate layer of granulation tissue around the implant defect (Fig. 4D). Recent hemorrhage was observed in several sections; however, there was no apparent or consistent relationship between evidence of hemorrhage and experimental groups. There was no evidence of bone necrosis in any of the experimental groups. All implants exhibited plastic yielding without catastrophic brittle fracture during the mechanical testing. The
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flexural yield strengths of stimulated (mean, 122.9 N; SD, 5.5 N) and nonstimulated (mean, 127.4 N; SD, 4.9 N) implants were not different based on the available numbers (p = 0.28).
Discussion Infection subsequent to placement of orthopaedic implants is a devastating outcome associated with increased patient morbidity, longer hospital stays, and increased costs to the healthcare system. Furthermore, effective treatment options are often limited for implant-associated orthopaedic infections. However, it has recently been shown that cathodic voltage-controlled electrical stimulation of Ti implants at 1.8 V versus Ag/AgCl for 1 hour significantly reduces the MRSA CFU immediately recovered from both the Ti implant and the surrounding bone tissue by one to two orders of magnitude [13]. Because it is envisioned that the cathodic voltage-controlled electrical stimulation would be used as an adjunctive therapy, the primary purpose of this study was to determine if cathodic voltagecontrolled electrical stimulation combined with vancomycin therapy is more effective at reducing the bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. This study found that the combined stimulation and vancomycin treatment was highly effective and reduced the bacterial burden of the implant, bone, and synovial fluid by 99.8% as compared with the control and stimulation alone groups. In addition, the combined treatment produced a 94% reduction of bacteria on implant as compared with the vancomycin alone group. Although this study reports encouraging antimicrobial results when cathodic voltage-controlled electrical stimulation is combined with vancomycin, there are some limitations to consider. First, although these are in vivo results, they are from a rodent model, which may not completely represent the clinical picture in a human patient. Also, the animal model used is well aligned with studying bone-embedded implant-associated infections but does not have a percutaneous site and, therefore, does not completely model all of the infection risks of osseointegrated prosthetic limbs. Future studies will be conducted to address the effects of cathodic voltage-controlled electrical stimulation at the percutaneous site. We also note a 1-week course of antibiotics is subtherapeutic for treatment for implant-associated infections. A more prolonged antibiotic therapy protocol or local, high-dose antibiotic delivery may have further lowered the CFU counts in the VANCO and STIM + VANCO groups. Another limitation was that the histological analysis in this study consisted of qualitative descriptions rather than a quantitative scoring system to
allow for statistical analysis of histological outcomes. In addition, we used an indirect method for assessment of hydrogen embrittlement and rigorous chemical composition and fatigue studies should be conducted to rule out the presence of hydrogen embrittlement. Finally, we did not evaluate varying durations or magnitudes of cathodic voltage-controlled electrical stimulation. We chose stimulation at 1.8 V for 1 hour based on previous in vitro and in vivo animal work [13]; however, future studies are needed to optimize these parameters. Despite these limitations, our data show that this cathodic voltage-controlled electrical stimulation treatment modality holds promise as an adjunctive treatment for orthopaedic implant-associated infections and warrants further investigation. The outcomes of the CONT group showed the rodent model used is a localized and sustained implant-associated infection. Although previous work showed respective bacterial reductions of 98% and 87% for implants and bone tissue harvested immediately after cathodic voltage-controlled electrical stimulation of 1.8 V for 1 hour [13], the present study has shown that the bacterial burden for the STIM group was not different from the CONT at 1 week poststimulation/sham. This indicates that the antimicrobial effects of cathodic voltage-controlled electrical stimulation treatment are immediate but not sustained. This could be attributable to the biofilm bacteria on the implant being released on electrical stimulation but then subsequently adhering back to the implant. Compared with the CONT group, the VANCO group showed CFU reductions in the bone and synovial fluid, but not for the implant. This may indicate that the bacteria are adherent to the implant in a biofilm, which is known to enhance resistance to antimicrobials [7, 8]. The combined treatment was most effective and compared with the controls showed CFU reductions of 99.8% for the implant, bone, and synovial fluid. Although the antimicrobial effect on the implant CFU in the STIM + VANCO group was robust, it may have been blunted by bacteria in the surrounding tissues reattaching to the implant after the effects of stimulation subsided. We theorize that the implant CFU count after cathodic voltagecontrolled electrical stimulation may become even lower if the synovial fluid and bone bacterial burden are minimized before the cathodic voltage-controlled electrical stimulation treatment, and future research will be focused on optimal antibiotic and stimulation protocols. The cathodic voltage-controlled electrical stimulation had no discernible deleterious effect on the surrounding bone based on qualitative histologic evaluation. Histological evaluation showed inflammation in the bone tissue, which is expected with implant-associated infections. It may seem paradoxical that although the STIM + VANCO group had the best reductions in all CFU categories, there appeared to be more granulation tissue surrounding the
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implant defect in this group as compared with the VANCO group. Because the STIM + VANCO group had significantly less implant-associated CFU as compared with the VANCO, we postulate that the stimulation resulted in a large bacterial load being released from the biofilm on the implant into the surrounding environment in a planktonic form, which are more susceptible to killing by antibiotics and the host response. Therefore, the increased granulation tissue observed in STIM + VANCO as compared with VANCO may actually be indicative of this increased killing of bacteria and the associated increased neutrophil respiratory burst and release of proinflammatory cytokines. Importantly, there were no regions of bone necrosis observed, and because the cathodic voltage-controlled electrical stimulation had been applied 7 days before harvesting, there was enough time for bone necrosis to occur. These histologic results are similar to previous work [13] using this model showing no signs of bone damage in histologic sections evaluated immediately after cathodic voltage-controlled electrical stimulation suggesting that 1.8 V for 1 hour appears to have no negative short-term or long-term impact on the surrounding bone tissues. The three-point bending tests of the CONT and STIM implants revealed no differences in the flexural yield strength based on the numbers available and also displayed no brittle fracture. Although Ti is known to be susceptible to hydrogen embrittlement [5, 16, 25], the present outcomes suggest the interfacial water and oxygen reduction electrochemical processes during cathodic voltage-controlled electrical stimulation [13] did not result in hydrogen embrittlement of the Ti. However, this single loading mechanical evaluation was performed after one cathodic voltage-controlled electrical stimulation treatment; further research is needed to assess effects on fatigue strength and also the effects of multiple cathodic voltage-controlled electrical stimulation treatments on implant mechanical properties. Although there have been numerous in vitro reports on the antimicrobial effects of direct electrical simulation with or without combination with antibiotics [2, 9, 11, 12, 21– 23, 32], there are only a few in vivo reports. Using a rabbit model of osteomyelitis with Staphylococcal epidermidis, it was shown that a constant current of 200 lA delivered to an intramedullary stainless steel electrode for 21 days produced a 1.5 order of magnitude reduction in bacteria when compared with the use of doxycycline alone [10]. It has also been shown that delivery of a constant current of 100 lA for 21 days to stainless steel external fixator pins in a goat model successfully prevented pin site infection of S epidermidis in 89% of sites evaluated [31]. The present study is different from these previous in vivo studies in a few important ways. As described before [13], two-electrode techniques used to deliver constant current are
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fundamentally different from the three-electrode cathodic voltage-controlled electrical stimulation method used in the present study to deliver constant voltage. The ability to enforce a constant voltage of the Ti implant allows for precise modulation of the voltage-dependent interfacial electrochemical processes at the surface oxide film that are thought to be involved in the antimicrobial effects of cathodic voltage-controlled electrical stimulation [13, 14]. In addition, the present study showed that antibiotics combined with cathodic voltage-controlled electrical stimulation of only 1 hour produced robust antimicrobial effects as compared with the longer 21-day treatments using in previous in vivo constant current studies. Furthermore, bone discoloration was noted in the study that delivered 200 lA for the 21 days, but there was no histological assessment of the tissues [10]. The present study includes histological outcomes showing no discernible deleterious effects on the tissue surround the implant that received cathodic voltage-controlled electrical stimulation for 1 hour. It is also important to point out that although the present study was conducted with Ti, most of the metals used for orthopaedic implants are also passivated with surface oxide films that have voltage-dependent electrochemical properties [24]. Therefore, the application of cathodic voltagecontrolled electrical stimulation to metallic orthopaedic devices may represent a new and widely applicable treatment modality. In conclusion, in this rat model of implant-associated infection, cathodic voltage-controlled electrical stimulation combined with vancomycin has been shown to reduce the bacterial burden on the implant, surrounding bone, and synovial fluid by 99.8% as compared with the controls. Although these large bacterial reductions are promising and may provide a solution for effective treatment with implant retention, further optimization of the treatment parameters may be needed to reach the ultimate goal of complete bacterial eradication. Future directions will include evaluations of stimulation magnitude, pattern, and duration as well as optimal timing for adjunctive stimulation during the time course of antibiotic therapy. In addition to optimizing the antimicrobial outcomes, subsequent studies will also seek to evaluate the ability of cathodic voltage-controlled electrical stimulation to promote beneficial host tissue responses such as enhanced bone and skin integration for osseointegrated and/or percutaneous orthopaedic implants. Finally, we are also focused on developing a translational protocol that would facilitate clinical utilization. Application of this stimulation to percutaneous implants is envisioned to be noninvasive, and on further optimization, a minimally invasive application of this stimulation could be potentially used to treat infections of fully implanted joint prostheses.
CVCES + Vancomycin as Infection Treatment
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