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Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study

Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by... Purpose: Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. Methods: Tibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone. Results: Except two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group. Conclusions: In a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis. Keywords: NBD peptide, Osteomyelitis, Methicillin-resistant Staphylococcus aureus, Vancomycin, Rabbit * Correspondence: gd2hxcp@163.com; gd2hqy@163.com Department of Orthopaedics, Guangdong Second Provincial General Hospital, NO.466 Xingang Road, Haizhu District, Guangzhou 510317, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 2 of 12 Introduction complex is a crucial step in pathways leading to NF-κBacti- Chronic osteomyelitis is a bone infection with hallmarks of vation. The IKK complex contains two catalytic subunits progressive bone necrosis and sequestrum formation [1]. (IKKα and IKKβ) and a regulatory component named NF- Due to the recurrence and chronicity of osteomyelitis, it is κB essential modulator (NEMO) or IKKγ [13]. A short cell- often necessary that the infected bone be debrided, tissues permeable peptide spanning the NEMO-binding domain reconstructed, and long-term antibiotic therapy adminis- (NBD) disrupts the association of NEMO with IKKβ,blocks tered [2]. The Gram-positive organism Staphylococcus TNF-α-induced NF-κB activation in vitro, and effectively aureus (S. aureus) is the most common causative agent of ameliorates responses in distinct animal models of inflam- osteomyelitis, accounting for approximately 80% of all hu- mation [14]. Recent identification and characterization of man cases [3]. The growing incidence of antibiotic-resistant NBD peptide have provided an opportunity to selectively S. aureus strains can explain the recurrent attacks of osteo- abrogate the inflammation-induced activation of NF-κBby myelitis in patients undergoing therapy [4]. To encounter targeting the NBD-NEMO interaction [15]. A previous the multidrug-resistance challenge, developed from the sys- study [11] in our laboratory had shown that application of temic antibiotic usage, local antibiotic delivery including NBD peptide could ameliorate the osteoblast differentiation polymethylmethacrylate (PMMA) cement and biodegrad- inhibition caused by TNF-α-induced NF-κB activation. able materials has been introduced, which increases the Taken together, we hypothesized that NBD peptide could local antibiotic concentration and simultaneously mini- be a promising tool for osteomyelitis treatment, owing to mizes their systemic toxicity [5]. However, the cement may its usefulness in controlling inflammatory bone resorption act as a foreign body when antibiotics are no longer being and accelerating bone regeneration. The present study released, requiring additional surgery for its removal. In aimed to investigate the efficacy of NBD peptide, based on addition, finding new antibiotic substances for antibiotic NF-κB blockade, in inflammation suppression and bone re- resistance is expensive and often involves compromised generation using a rabbit model of osteomyelits. efficacy within short time periods due to the enormous potential of rapid adaptation in microorganisms. Recently, Materials and methods “cellular hysteresis” strategy, using the currently available Animals antibiotics, was harnessed to optimize the antibiotic ther- Approval was obtained from Institutional Animal Care apy, in order to achieve both enhanced elimination of bac- and Use Committee prior to performing this study. All teria and reduced evolution of resistance [6]. animals were treated according to the guidelines for la- S. aureus is a capable bone pathogen with adhesion boratory animal treatment and care, and all protocols molecules that facilitate its binding to the bone matrix were approved by the local animal welfare committee. and toxin secretion, thereby stimulating bone resorption Fifty healthy, pathogen-free adult New Zealand white [7]. Proinflammatory cytokines such as interleukin 1 (IL- rabbits (weighing 2.42–2.76 kg) were chosen for the ex- 1), interleukin 6 (IL-6), or tumor necrosis factor alpha periment; they were individually caged, fed a standard (TNF-α), are produced in S. aureus-induced osteomye- balanced chow, and provided with water ad libitum. litis [8]. S. aureus may also activate the nuclear factor kappa B (NF-κB) pathway and ensure that NF-κB activa- NBD peptide synthesis tion is required for the phagocytosis of S. aureus by A 23-amino acid cell-permeable NBD peptide (YGRK macrophages [9]. TNF-α acts on marrow-derived macro- KRRQRRR-G-TTLDWSWLQME) was synthesized, and phages to promote the induction of differentiation to purified by high performance liquid chromatography, ac- osteoclast cells, and the receptor activator of nuclear fac- cording to our previous study [11]. tor kappa B ligand (RANKL) acts on mature osteoclast cells to induce bone resorption activity [10]. Our group Osteomyelitis model induction and Yamazaki et al. [11, 12] had previously shown that The rabbit model of chronic osteomyelitis was induced as TNF-α inhibits osteoblast differentiation through the ac- described in Nijhof et al. [16]. Rabbit weights were re- tivation of NF-κB, which directly leads to the abrogation corded, and ketamine (35 mg/kg) and xylazine (5 mg/kg), of Smad1 signaling to hamper bone formation activity. for anesthesia, were injected subcutaneously under strictly Taken together, NF-κB seems to be a critical molecular aseptic conditions. The operation area of proximal right switch for several downstream events that affect host re- tibia was shaved, disinfected with povidone-iodine, and sponses to bone infection in osteomyelitis. covered by sterile drapes, before injecting lidocaine (5 mg/ Drugs that selectively target inflammation-induced kg) for local anesthesia. After making a 2.0-cm skin inci- NF-κB activity while sparing the protective functions of sion on the anterolateral surface of the right proximal basal NF-κB levels would be of greater therapeutic value tibia, the cortex of metaphysis was partially exposed. A and would likely display fewer undesired side-effects. hole was drilled in the cortex using a 2.0-mm Kirschner Phosphorylation of IκB proteins by the IκB kinase (IKK) wire, saline irrigated, and bone marrow extracted by Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 3 of 12 means of 18-gauge needle, which was inserted into the Radiological, microbiological, and histological medullary canal. Sequentially, 0.1 ml of 5% sodium morrh- examination uate, 0.1 ml of bacterial suspension of MRSA, ATCC After the debridement procedure, standard anteropos- 43300 (1 × 10 CFU/ml), and 0.1 ml of saline (0.9%) were terior- and lateral-view radiographs were taken on day 1, injected into the medullary cavity. After sealing the holes and 3 and 6 weeks post-operation, for all animals. Ac- with sterile bone wax to prevent leakage of the injection, cording to the severity scoring system for osteomyelitis, the fascia, subcutaneous layer, and skin were irrigated and described by Norden et al., all the radiographs were closed with 4/0 vicryl. General conditions, including food assessed in a blinded fashion by an orthopedic surgeon. and water intake, activity, and presence of localized and At 6 weeks after debridement, the animals were eutha- systemic infections, were monitored daily for 3 weeks after nized by an intravenous injection of excess pentobarbital initial operation. There was no death of rabbits during the sodium. In the same way as the first operation, the tibiae follow-up period, and standard anteroposterior- and were excised and freed from soft tissues under aseptic lateral-view radiographs were taken for all animals, at 3 conditions. With sterile curettes, and swabbing the initial weeks post-operation, to determine the severity of osteo- bone window, samples were obtained and sent for a cul- myelitis according to the parameters for osteomyelitis de- ture study to analyze the infection rates. scribed by Norden et al. [17]. Meanwhile, five parameters, Tibia samples were used for micro-computed tomog- including periosteal new bone, bone destruction, seques- raphy. Morphology of the reconstructed tibial cortex was tra, soft tissue calcification, and soft tissue swelling, were assessed using an animal micro-CT scanner (eXplore determined for each tibia, and animals with three points Locus, GE Healthcare Biosciences, London, UK). Briefly, or more were diagnosed with osteomyelitis. the specimens were scanned with a 55 kVp energy setting, having intensity of 145 mA with 200 ms acquisition time, and no frame average in high-resolution mode, which pro- Treatment groups vided a voxel resolution of 12 μm. After micro-CT scan, Forty-five animals were finally diagnosed with osteomye- the defect region was identified by a contour as a traced litis and randomly divided into four groups: Control, region of interest (ROI), and the relative measurements Van, NBD, and NBD + Van groups. Animals in the Con- were calculated, including bone mineral densities (BMDs) trol group (n = 9) were left untreated. Animals in the and bone volume/total volume (BV/TV). Van group (n = 12) were treated with debridement and Next, conventional pathological sections stained with intravenous injection of vancomycin (30 mg/kg, twice hematoxylin and eosin were prepared for bone samples, daily) for 4 weeks. Animals in the NBD group (n = 12) and decalcified cross sections (5-μm) were assessed by a were treated with debridement and local percutaneous pathologist in a blinded fashion according to the scoring injection of NBD peptides (5 mg/kg BW) into the me- system of Smeltzer et al. [18]. Animals were diagnosed dullary cavity (immediately after debridement, 2 and 4 with osteomyelitis when they possessed four or more weeks post-operation). Animals in the NBD + Van group points. (n = 12) received the same treatment as the Van group, plus local injection with NBD peptides as the NBD Statistical analysis group. The post-operative follow-up after debridement The Statistical Package for the Social Sciences (SPSS) lasted for 6 weeks, during which the animals were (SPSS Inc., Chicago, IL) version 11.0 for Windows was housed in individual cages and fed a normal diet. used to analyze the results. Mann-Whitney tests for For the debridement procedure, an 8 mm × 20 mm independent samples were performed to compare the la- cortical bone window in the anteromedial surface of boratory values and radiological and histological scores right proximal tibia was generated to clean out all infec- across the groups. Newly formed bone volume was com- tious and necrotic tissues, according to the varying pared by analysis of variance. Fisher’s exact test was used radiological severity of osteomyelitis. Before wound clos- to compare infection rates across the groups. Unless ure, the NBD peptides were injected into the medullary stated otherwise, results are presented as mean ± stand- cavity of animals in the NBD + Van and NBD groups. Fi- ard deviation, and performed in triplicate; p-values of nally, the removed samples were sent for a culture study. 0.05 or less were considered to be significant. Laboratory monitoring Results After debridement procedure, all animals in the four General conditions groups were followed closely for any clinical sign of sep- The clinical signs of infection varied from mild to severe, tic spread. Blood samples were collected weekly for the following induction, and in the one-week follow-up after measurement of leucocyte count, erythrocyte sedimenta- debridement, two rabbits in the Control group and one in tion rate (ESR), and C-reactive protein (CRP) levels. the NBD group died from severe infection, as confirmed Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 4 of 12 by autopsy. The remaining 42 animals recovered well, was not significant (P = 0.101). NBD + Van group showed with no obvious sign of systemic complication in the 6- the highest infection clearance rate, although there was no week follow-up, and were included in statistical analysis. significant difference with the Van (P = 0.667) or NBD Furthermore, laboratory analyses did not reveal any sig- group (P = 0.214). Infection clearance rate of the NBD group nificant change of leucocyte count, and ESR and CRP was a little lower than that of the Van group, although the levels (Fig. 1), although there were fluctuations throughout difference was not significant (P = 0.684). Altogether, there the follow-up period after debridement. was no significant differenceamong thetreatment groups (Van, NBD + Van, and NBD) with regard to infection clear- Microbiology ance rate, although the best therapeutic efficacy was con- While performing debridement, the culture of curetted firmed by the optimal infection elimination in the NBD samples showed all 42 rabbits to have MRSA. At sacri- peptide + Van group. fice, 7 of 7, 5 of 12, 3 of 12, and 6 of 11 animals in the Control, Van, NBD + Van, and NBD groups, respectively, Radiography were tested positive for MRSA (Fig. 2). Infection clear- At 3 weeks after infection introduction, typical signs of ance rate of the Control group was significantly lower bone infection, including periosteal new bone, bone de- than that of the Van (P = 0.017) and NBD + Van groups struction, and sequestra, could be seen in the radio- (P = 0.003). Infection clearance rate of the Control group graphs of tibiae (Fig. 3a). After treatment for 6 weeks, was also lower than that of the NBD group, although it elimination of bone infection and bone window healing Fig. 1 Leucocyte count (a), and ESR (b) and CRP (c) levels in the Control, Van, NBD + Van, and NBD groups during the 6 weeks follow-up period after debridement Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 5 of 12 Fig. 2 Infection clearance rates of MRSA in the Control, Van, NBD + Van, and NBD groups at 6 weeks after debridement. The Control group showed the lowest infection clearance rate, while the highest infection clearance rate was detected in the NBD + Van group. Fisher’s exact test determined statistical significance where *p < 0.05 Fig. 3 Lateral radiographs of tibiae in different groups, taken before and 1 day, 3 and 6 weeks after debridement. a 3 weeks after infection induction, the tibia showed a typical character of chronic osteomyelitis, including periosteal new bone formation (white arrow), bone destruction (black arrow), and sequestra (black arrows). The tibiae in the NBD + Van group have shown a gradually healing of osteomyelitis after treatment (b, c, and d), whereas the tibial osteomyelitis in other groups did not ameliorate obviously (e, f, and g) Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 6 of 12 were observed in the NBD + Van group (Fig. 3b–d), with The BV/TV results exhibited a similar trend as the BMD only 3 of 12 animals having a score of three or above. data. Mean BV/TV percentage in the NBD + Van group Different degrees of infection were observed in the Con- was significantly higher than that in the Van and NBD trol, Van, and NBD groups (Fig. 3e–g), with 7 of 7, 5 of groups (P = 0.001); there was also a significant difference 12, and 6 of 11 animals having a score of three or above, in BV/TV between the Van and NBD groups (P = 0.001). respectively. Total radiological score of the Control group was significantly higher than that of the Van (P = Histology 0.031), NBD + Van (P = 0.001), and NBD groups (P = The varying severity of osteomyelitis in the four groups was 0.03). The NBD + Van group showed a significantly further confirmed by histological results (Figs. 7 and 8). lower score than the Van (P = 0.03) and NBD groups Typical signs of osteomyelitis, including acute and chronic (P = 0.007). No statistically significant difference was intraosseous inflammation, periosteal inflammation, and found between the Van and NBD groups (P = 0.767) bone necrosis could be seen in some animals, whereas par- (Fig. 4). tial signs were noted in others. A severity score of four or As shown in Fig. 5, morphology of the newly formed more was found in 7 of 7, 6 of 12, 3 of 12, and 7 of 11 ani- bone was reconstructed using micro-CT in the Van, mals in the Control, Van, NBD + Van, and NBD groups, re- NBD + Van, and NBD groups (Fig. 5a, c, e), and the re- spectively. Histological score of the Control group was sults were almost consistent with the x-ray images. significantly higher than that of the Van (P = 0.001), NBD + Micro-CT cross sections of the defect area showed more Van (P = 0.001), and NBD groups (P = 0.038), respectively. obvious new bone formation in the NBD + Van group The NBD + Van group showed a significantly lower score (Fig. 5d). Further, there were several cavities scattered than the Van (P = 0.039) and NBD groups (P =0.01). The over the area in the Van and NBD groups (Fig. 5b, f). Van group also showed a significantly lower score than the Compared to the NBD group, Van group exhibited NBD group (P = 0.047). greater new bone formation, although the cortex was in- complete and there were voids beneath the cortex. Com- pared to the Van group, NBD + Van group showed Discussion improved new cortex formation. In all groups, sporadic The present in-vivo study investigated the effects of trabecular bone was observed in the marrow canal. NBD peptide on infected bone; it played a key role in The amount of newly formed bone in the defect sites inhibiting inflammation and promoting bone remodeling was calculated by morphometric analysis (Fig. 6). The towards osteogenesis, and the effects were better than NBD group exhibited the lowest levels of both BMDs those produced by antibiotics alone. Despite the failure and BV/TV at the defect site when compared to other in demonstrating antibacterial effectiveness of NBD pep- groups (P = 0.001). BMDs at the defect site of NBD + tide in vivo, antibiotics combined with NBD peptide Van group were significantly higher than that of the Van seemed to be a preferred therapeutic option in chronic and NBD groups (P = 0.001 and P = 0.001, respectively). osteomyelitis. Fig. 4 Radiological scores in the Control, Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Mann-Whitney test determined statistical significance where * P < 0.05 Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 7 of 12 Fig. 5 Representative 3D micro-CT reconstruction and Micro-CT cross sections analysis of the proximal tibia in the Van (a, b), NBD + Van (c, d), and NBD (e, f) group 6 weeks after debridement. The bone window in the NBD + Van group was covered by newly formed bone, while no sufficient bone formed in other two groups In general, a number of research groups have reported regulates the inflammatory response leading to the develop- the successful treatment of synovial inflammation [19], ment of bone infection, and selective blocking of NF-κB inflammatory osteolysis [20, 21], inflammatory arthritis could have therapeutic implication in treating the disease. [22], cartilage degradation [23], and muscular dystrophy In this study, our results indicated that specific and selective [24] by the use of NBD peptide. Existing rationale for inhibition of NF-κB by the NBD peptide is an effective treating chronic inflammatory diseases involving bone approach to treat chronic osteomyelitis. The anti- resorption by NBD peptide includes the documented re- inflammatory efficacy of NBD peptide in infected bone was duction of both TNF-α levels and bone destruction assessed by radiologic and histologic means. Extensive based on NF-κB blockade [20]. Furthermore, proinflam- chronic osteomyelitis was achieved 3 weeks after infection matory cytokines, such as TNF-α, are known to be pro- induction, followed by debridement in all groups, except duced in S. aureus-induced osteomyelitis [8], which in the Control group. Six weeks after debridement, combin- turn, results in progressive inflammatory destruction of ation treatment with NBD peptide and vancomycin exhib- the bone. We aimed to address whether NBD peptide ited the most curative antimicrobial efficacy, with the could be made accessible to subjects with osteomyelitis, lowest infection rate, as well as radiological and histological or whether it could be a therapeutic option in chronic scores. All these values were also lowered in the bone infections. vancomycin-only group. Meanwhile, local NBD peptide in- Previous in-vitro studies had shown that a number of jection alone exhibited infection clearance efficacy in vivo, bacteria, including Streptococcus pneumoniae [25, 26], although it was not comparable to that by vancomycin. Streptococcus pyogenes [27], and S. aureus [28], could acti- Based on these observations, our data may be considered to vate NF-κB in response to infection. S. aureus is the major be in favor of NBD peptide application to chronic osteo- pathogen among staphylococci and the most common myelitis; it exhibited infection elimination efficacy, perhaps cause of bone infections, which may also activate the NF- owing to the NBD peptide providing an opportunity to se- κB pathway and ensure that NF-κB activation is required lectively abrogate the inflammation-induced activation of for the phagocytosis of S. aureus by macrophages [9]. A re- NF-κB by targeting the NBD-NEMO interaction. cent investigation [29]demonstratedthatNF-κBis acti- At present, vancomycin has been introduced in most vated during the development of bone infection. Consistent cases of chronic osteomyelitis treatment [3]. However, it with this finding, we had shown that selective inhibition of has been losing potency against S. aureus, and various NF-κB, using the NBD peptide, blocks RANKL-induced forms of resistance to vancomycin have emerged in osteoblast differentiation in vitro [11]. Thus, NF-κB MRSA. Vancomycin could not cause adequate microbial Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 8 of 12 Fig. 6 Morphometric evaluation of the local bone mineral densities (BMDs) (a) in the new bone formation area and bone volume/total volume (BV/TV) (b) in the Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Analysis of variance test determined statistical significance where * P < 0.05 clearance for treating chronic osteomyelitis induced by microbiological examination was increased after NBD MRSA, in this study, and microbiological investigations peptide therapy compared to that in the untreated con- demonstrated persistence of infection with positive tis- trol group. Based on these observations, our data are in sue cultures even at study endpoint. Considering the favor of anti-inflammatory NBD application for treating complexity of chronic infections, with antibiotic resist- chronically infected bone, consistent with the previously ance of MRSA, biofilm formation, low drug penetration, published in-vitro data [29]. Despite the failure to defin- drug activity, and much more, anti-MRSA combinations itely demonstrate anti-MRSA effectiveness of NBD pep- might be preferable for treating MRSA-osteomyelitis. tide in vivo, there is a possible U-shaped response of Compared to vancomycin therapy alone, vancomycin in bacterial growth to pro-inflammatory cytokines [30], and conjunction with NBD peptide therapy was not expected we speculate that NBD peptide would ameliorate inflam- to elicit a more pronounced antibacterial effect. How- matory responses and may be expected to have a posi- ever, infection clearance rate was enhanced with the tive influence on host biological response against MRSA combination therapy compared to that in vancomycin infections. Moreover, based on the cellular hysteresis alone group. Additionally, infection clearance rate of strategy [6], there may be a synergistic effect of Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 9 of 12 that NBD peptide could promote bone regeneration ef- fectively only when infection was controlled. We had previously demonstrated that NF-κB activation inhibits osteoblast differentiation induced by TNF-α, and that application of NBD peptide ameliorates this inhibitory effect [11]. Our in-vivo results in the present study showed that NBD peptide is able to effectively amelior- ate the inhibition of osteoblast differentiation by chronic inflammation and promote bone regeneration. In con- clusion, the NBD peptide not only seemed to prevent bone destruction in chronic osteomyelitis but also accel- erated bone regeneration, obliterating the dead space left by debridement. In the current study, we have utilized an NBD peptide that binds NEMO and compromises the formation of active IKK complex. This sequestration disables IKK functions, primarily the immediate activation of NF-κB. However, physiologically, NF-κB has a crucial anti- apoptotic role in survival, and its pharmacological inhib- ition may be of limited benefit, owing to the likelihood of generalized apoptosis [20]. In support of this notion, a study reporting the effects of conditional deletion of IKK suggested complete blockage of NF-κBto possibly lead to enhanced apoptosis and damage [33]. Nevertheless, the ef- fects of highly selective pharmacological inhibition of only proinflammatory IKK activity has not yet been fully inves- tigated and reported. This is supported by a number of previous reports [20, 22, 34], showing little or no adverse Fig. 7 Photomicrographs of the control group (a and b) shows effect, owing to the blockade of NF-κB, on therapeutic destruction of cortical bone (black arrow), sequestrum formation purposes in chronic inflammatory diseases in vivo. In pre- (white arrow), intramedullary abscess (black arrow-head), fibrosis vious collagen-induced arthritis experiments, to prevent (white arrow-head), and proliferated foamy histotcytes (red inflammatory bone destruction [20], NBD peptide was arrow-head). The van group (c)and NBDgroup (d)showed injected into mice, daily for over 4 weeks, without observ- relatively moderate osteomyelitis characteristics, with some new bone formation (red arrow). Photomicrographs of the NBD + Van ing any detrimental side effect, or liver or kidney toxicity; group (e and f) were shown mild destruction of cortical bone injection of the peptide for only the first 5 days was and intramedullary new bone formation, without signs of severe enough to maintain the therapeutic effects for nearly 3 inflammation (haematoxylin and eosin) weeks after administration. In our current experiments, we locally injected the rabbits with the NBD peptide, vancomycin in conjunction with NBD peptide therapy, thrice for over 6 weeks, without observing any detrimental which was harnessed to optimize the antibiotic therapy, side effect or bacterial spread. Local NBD peptide delivery subsequently achieving enhanced bacterial elimination. in our experiments minimized the probability of their sys- To establish these events in vivo, future studies should temic toxicity caused by systemic usage. However, exten- be conducted to explore inducible physiological effects sive pharmacological evaluation of this approach would be of NBD peptide, which may, subsequently help to find required to fully determine the effects of long-term use of direct antibacterial effects of NBD peptide for improving the NBD peptide to treat chronic osteomyelitis induced by antibiotic therapy. MRSA. Although temporary inflammation may promote bone There are several well-established animal models with healing, persistent and serious infection-associated in- some kind of implantation that support the establish- flammation could result in suboptimal and impaired ment of infection and allow the study of chronic osteo- bone regeneration [31, 32]. The newly formed bone was myelitis in vivo. However, the implantation of foreign quantified by micro-CT, and results showed the volume bodies usually does not allow the infection to heal unless of newly formed bone in the NBD + vancomycin group the implant is removed. In the present study, we used to be significantly higher than that in the Van or NBD the model described by Nijhof et al. [16], which aimed group; the bone window eventually healed, indicating to induce infection via the injection of MRSA bacterial Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 10 of 12 Fig. 8 Histological scores in the Control, Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Mann-Whitney U-test determined statistical significance where * P < 0.05 suspension without implant infections. The establish- inhibition caused by NF-κB activation in vitro. These ment of active infection at 3 weeks, in all animals, could studies may be performed in other laboratories and rep- clearly be confirmed by local clinical signs and radio- licated further using a larger sample size, thereby helping graphs. Our data are consistent with the previous finding to re-confirm the reliability of our conclusion. Finally, [35], and demonstrate that a high rate of chronic osteo- further studies regarding the efficacy of NBD peptides myelitis could be achieved within 3 weeks of infection against other types of pathogenic microorganisms, as induction. The use of sodium morrhuate in the present well as studies considering the combined use of NBD osteomyelitis model, with the inherent variability of in- peptides and antibiotics, should be performed in vivo. fection intensity, has certain limitations in assessing the effects of NBD peptide in chronic bone infections; how- Conclusion ever, the model used herein was appropriate to study We conclude that NBD peptide induced neither bacterial treatment safety. Clinical assessment and laboratory spread nor worsening of infection. Together with the values were used to study the systemic side effects and previous reports of successful and safe treatment of in- bacterial spread after debridement. Neither clinical nor flammatory bone destruction using NBD peptide, results laboratory signs of bacterial spread was observed. We, of the present study are in favor of the applicability of therefore, concluded that NBD peptide neither worsened antibiotics combined with NBD peptide to treat osteo- bone infections, nor induced bacterial sepsis. myelitis. Since bone defect and residual infection are A limitation of the current study was the absence of a common problems after debridement, in the treatment group of rabbits with osteomyelitis that received de- of osteomyelitis, despite the failure to definitely demon- bridement without antibiotics or NBD peptide adminis- strate antibacterial effectiveness of NBD peptide in vivo, tration. Therefore, we cannot figure out the specific role antibiotics in conjunction with NBD peptide are sug- of debridement in eliminating infection and stimulating gested to possibly have promising therapeutic potential bone regeneration. Another limitation was that a reason- in osteomyelitis. able sample size was not calculated due to lack of a pilot study before the experiment. Our sample size refers to Abbreviations BMDs: Bone mineral densities; BV/TV: Bone volume/total volume; IKK: IκB the previous experiments [20, 35], which is more than kinase; IL-1: Interleukin 1; IL-6: Interleukin 6; MRSA: Methicillin-resistant that in the above reports. Meanwhile, our results have Staphylococcus aureus; NBD: NEMO-binding domain; NEMO: NF-κB essential effectively evaluated the anti-inflammatory effect of modulator; NF-κB: Nuclear factor kappa B; PMMA: Polymethylmethacrylate; RANKL: Nuclear factor kappa B ligand; ROI: Region of interest; vancomycin in conjunction with NBD peptide and its ef- S.aureus: Staphylococcus aureus; TNF-α: Tumour necrosis factor alpha fect on promoting bone remodeling in MRSA-induced chronic osteomyelitis. The current results are consistent Acknowledgments with our previous investigation, showing that NBD pep- The authors would like to thank Editage (www.editage.com) for English tide could ameliorate the osteoblast differentiation language editing. In addition, Chang-Peng Xu thanks the inimitable care and Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 11 of 12 support of Xiao-Jie Zheng over the years. I love you. Will you spend the rest demonstrate potent bone resorbing activity. J Bone Miner Res. 1995; of your life with me? 10(5):726–34. 8. Yoshii T, Magara S, Miyai D, Nishimura H, Kuroki E, Furudoi S, Komori T, Ohbayashi C. Local levels of interleukin-1β, −4, −6, and tumor necrosis Authors’ contributions factor α in an experimental model of murine osteomyelitis due to CP Xu and Y Qi designed the study. Y Chen contributed in the animal staphylococcus aureus. Cytokine. 2002;19(2):59–65. feeding. HT Sun contributed in the osteomyelitis model induction. QP Yang contributed in peptide synthesis. Z Cui contributed in the experimental 9. Zhu F, Yue W, Wang Y. The nuclear factor kappa B (NF-κB) activation is studies. Bin Yu and L Huang contributed in radiological and microbiological required for phagocytosis of staphylococcus aureus by RAW 264.7 cells. Exp examination. YJ Yang contributed in histological examination. FZ Wang Cell Res. 2014;327(2):256–63. performed the analysis of the data. CP Xu contributed in drafting the work. 10. Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, All authors read and approved the final manuscript. Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Morinaga T, Higashio K, Martin TJ, Suda T. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL- Funding RANK interaction. J Exp Med. 2000;191(2):275–86. This work was supported by the National Natural Science Fundation of China 11. Li W, Yu B, Li M, Sun D, Hu Y, Zhao M, Cui CB, Hou S. NEMO-binding (No.81972083, 81501902), Guangdong Province Natural Science Foundation domain peptide promotes osteoblast differentiation impaired by tumor (No.2014A030310451, 2017A030313736), Science and Technology Planning necrosis factor alpha. Biochem Biophys Res Commun. 2010;391(2):1228–33. Project of Guangzhou (No. 201804010226), Science Foundation of 12. Yamazaki M, Fukushima H, Shin M, Katagiri T, Doi T, Takahashi T, Jimi E. Guangdong Second Provincial General Hospital (No. YQ2016–003), Science Tumor necrosis factor alpha represses bone morphogenetic protein (BMP) and Technology Assistance Project of Xinjiang Province (No. 2018E02056), signaling by interfering with the DNA binding of Smads through the Natural Science Foundation of Xinjiang Province (No. 2018D01C014). activation of NF-kappaB. J Biol Chem. 2009;284(51):35987–95. 13. Karin M, Delhase M. The IκB kinase (IKK) and NF-κB: key elements of Availability of data and materials proinflammatory signaling. Semin Immunol. 2000;12(1):85–98. The data can be accessible to the interested researchers by the 14. May MJ, D'Acquisto F, Madge LA, Glöckner J, Pober JS, Ghosh S. Selective corresponding authors on reasonable request. inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science. 2000;55(15):1550–4. Ethics approval and consent to participate 15. Strickland I, Ghosh S. Use of cell permeable NBD peptides for suppression This study was approved by the animal ethics committee of Nanfang of inflammation. Ann Rheum Dis. 2006;65(Suppl 3):75–82. Hospital, Southern Medical University (No: NFYY-2015-61). 16. Nijhof MW, Stallmann HP, Vogely HC, Fleer A, Schouls LM, Dhert WJ, Verbout AJ. Prevention of infection with tobramycin-containing bone Consent for publication cement or systemic cefazolin in an animal model. 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Inhibition of IKK activation, Received: 2 June 2019 Accepted: 16 October 2019 through sequestering NEMO, blocks PMMA-induced osteoclastogenesis and calvarial inflammatory osteolysis. J Orthop Res. 2006;24(7):1358–65. 22. Dai S, Hirayama T, Abbas S, Abu-Amer Y. The IκB kinase (IKK) inhibitor, References NEMO-binding domain peptide, blocks Osteoclastogenesis and bone 1. Lazzarini L, Mader JT, Calhoun JH. Osteomyelitis in long bones. J Bone Joint Erosion in inflammatory arthritis. J Biol Chem. 2004;279(36):37219–22. Surg Am Vol. 2004;86(10):2305–18. 23. Liu YD, Yang H, Liao LF, Jiao K, Zhang HY, Lu L, Zhang M, Zhang J, He JJ, 2. Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Wu YP, Chen D, Wang MQ. Systemic administration of strontium or NBD Elliott G, Kelley MJ, Sarosi I, Wang L, Xia XZ, Elliott R, Chiu L, Black T, Scully S, peptide ameliorates early stage cartilage degradation of mouse mandibular Capparelli C, Morony S, Shimamoto G, Bass MB, Boyle WJ. 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Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study

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Biomedicine; Medical Microbiology; Drug Resistance; Infectious Diseases
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10.1186/s13756-019-0627-y
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Abstract

Purpose: Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. Methods: Tibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone. Results: Except two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group. Conclusions: In a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis. Keywords: NBD peptide, Osteomyelitis, Methicillin-resistant Staphylococcus aureus, Vancomycin, Rabbit * Correspondence: gd2hxcp@163.com; gd2hqy@163.com Department of Orthopaedics, Guangdong Second Provincial General Hospital, NO.466 Xingang Road, Haizhu District, Guangzhou 510317, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 2 of 12 Introduction complex is a crucial step in pathways leading to NF-κBacti- Chronic osteomyelitis is a bone infection with hallmarks of vation. The IKK complex contains two catalytic subunits progressive bone necrosis and sequestrum formation [1]. (IKKα and IKKβ) and a regulatory component named NF- Due to the recurrence and chronicity of osteomyelitis, it is κB essential modulator (NEMO) or IKKγ [13]. A short cell- often necessary that the infected bone be debrided, tissues permeable peptide spanning the NEMO-binding domain reconstructed, and long-term antibiotic therapy adminis- (NBD) disrupts the association of NEMO with IKKβ,blocks tered [2]. The Gram-positive organism Staphylococcus TNF-α-induced NF-κB activation in vitro, and effectively aureus (S. aureus) is the most common causative agent of ameliorates responses in distinct animal models of inflam- osteomyelitis, accounting for approximately 80% of all hu- mation [14]. Recent identification and characterization of man cases [3]. The growing incidence of antibiotic-resistant NBD peptide have provided an opportunity to selectively S. aureus strains can explain the recurrent attacks of osteo- abrogate the inflammation-induced activation of NF-κBby myelitis in patients undergoing therapy [4]. To encounter targeting the NBD-NEMO interaction [15]. A previous the multidrug-resistance challenge, developed from the sys- study [11] in our laboratory had shown that application of temic antibiotic usage, local antibiotic delivery including NBD peptide could ameliorate the osteoblast differentiation polymethylmethacrylate (PMMA) cement and biodegrad- inhibition caused by TNF-α-induced NF-κB activation. able materials has been introduced, which increases the Taken together, we hypothesized that NBD peptide could local antibiotic concentration and simultaneously mini- be a promising tool for osteomyelitis treatment, owing to mizes their systemic toxicity [5]. However, the cement may its usefulness in controlling inflammatory bone resorption act as a foreign body when antibiotics are no longer being and accelerating bone regeneration. The present study released, requiring additional surgery for its removal. In aimed to investigate the efficacy of NBD peptide, based on addition, finding new antibiotic substances for antibiotic NF-κB blockade, in inflammation suppression and bone re- resistance is expensive and often involves compromised generation using a rabbit model of osteomyelits. efficacy within short time periods due to the enormous potential of rapid adaptation in microorganisms. Recently, Materials and methods “cellular hysteresis” strategy, using the currently available Animals antibiotics, was harnessed to optimize the antibiotic ther- Approval was obtained from Institutional Animal Care apy, in order to achieve both enhanced elimination of bac- and Use Committee prior to performing this study. All teria and reduced evolution of resistance [6]. animals were treated according to the guidelines for la- S. aureus is a capable bone pathogen with adhesion boratory animal treatment and care, and all protocols molecules that facilitate its binding to the bone matrix were approved by the local animal welfare committee. and toxin secretion, thereby stimulating bone resorption Fifty healthy, pathogen-free adult New Zealand white [7]. Proinflammatory cytokines such as interleukin 1 (IL- rabbits (weighing 2.42–2.76 kg) were chosen for the ex- 1), interleukin 6 (IL-6), or tumor necrosis factor alpha periment; they were individually caged, fed a standard (TNF-α), are produced in S. aureus-induced osteomye- balanced chow, and provided with water ad libitum. litis [8]. S. aureus may also activate the nuclear factor kappa B (NF-κB) pathway and ensure that NF-κB activa- NBD peptide synthesis tion is required for the phagocytosis of S. aureus by A 23-amino acid cell-permeable NBD peptide (YGRK macrophages [9]. TNF-α acts on marrow-derived macro- KRRQRRR-G-TTLDWSWLQME) was synthesized, and phages to promote the induction of differentiation to purified by high performance liquid chromatography, ac- osteoclast cells, and the receptor activator of nuclear fac- cording to our previous study [11]. tor kappa B ligand (RANKL) acts on mature osteoclast cells to induce bone resorption activity [10]. Our group Osteomyelitis model induction and Yamazaki et al. [11, 12] had previously shown that The rabbit model of chronic osteomyelitis was induced as TNF-α inhibits osteoblast differentiation through the ac- described in Nijhof et al. [16]. Rabbit weights were re- tivation of NF-κB, which directly leads to the abrogation corded, and ketamine (35 mg/kg) and xylazine (5 mg/kg), of Smad1 signaling to hamper bone formation activity. for anesthesia, were injected subcutaneously under strictly Taken together, NF-κB seems to be a critical molecular aseptic conditions. The operation area of proximal right switch for several downstream events that affect host re- tibia was shaved, disinfected with povidone-iodine, and sponses to bone infection in osteomyelitis. covered by sterile drapes, before injecting lidocaine (5 mg/ Drugs that selectively target inflammation-induced kg) for local anesthesia. After making a 2.0-cm skin inci- NF-κB activity while sparing the protective functions of sion on the anterolateral surface of the right proximal basal NF-κB levels would be of greater therapeutic value tibia, the cortex of metaphysis was partially exposed. A and would likely display fewer undesired side-effects. hole was drilled in the cortex using a 2.0-mm Kirschner Phosphorylation of IκB proteins by the IκB kinase (IKK) wire, saline irrigated, and bone marrow extracted by Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 3 of 12 means of 18-gauge needle, which was inserted into the Radiological, microbiological, and histological medullary canal. Sequentially, 0.1 ml of 5% sodium morrh- examination uate, 0.1 ml of bacterial suspension of MRSA, ATCC After the debridement procedure, standard anteropos- 43300 (1 × 10 CFU/ml), and 0.1 ml of saline (0.9%) were terior- and lateral-view radiographs were taken on day 1, injected into the medullary cavity. After sealing the holes and 3 and 6 weeks post-operation, for all animals. Ac- with sterile bone wax to prevent leakage of the injection, cording to the severity scoring system for osteomyelitis, the fascia, subcutaneous layer, and skin were irrigated and described by Norden et al., all the radiographs were closed with 4/0 vicryl. General conditions, including food assessed in a blinded fashion by an orthopedic surgeon. and water intake, activity, and presence of localized and At 6 weeks after debridement, the animals were eutha- systemic infections, were monitored daily for 3 weeks after nized by an intravenous injection of excess pentobarbital initial operation. There was no death of rabbits during the sodium. In the same way as the first operation, the tibiae follow-up period, and standard anteroposterior- and were excised and freed from soft tissues under aseptic lateral-view radiographs were taken for all animals, at 3 conditions. With sterile curettes, and swabbing the initial weeks post-operation, to determine the severity of osteo- bone window, samples were obtained and sent for a cul- myelitis according to the parameters for osteomyelitis de- ture study to analyze the infection rates. scribed by Norden et al. [17]. Meanwhile, five parameters, Tibia samples were used for micro-computed tomog- including periosteal new bone, bone destruction, seques- raphy. Morphology of the reconstructed tibial cortex was tra, soft tissue calcification, and soft tissue swelling, were assessed using an animal micro-CT scanner (eXplore determined for each tibia, and animals with three points Locus, GE Healthcare Biosciences, London, UK). Briefly, or more were diagnosed with osteomyelitis. the specimens were scanned with a 55 kVp energy setting, having intensity of 145 mA with 200 ms acquisition time, and no frame average in high-resolution mode, which pro- Treatment groups vided a voxel resolution of 12 μm. After micro-CT scan, Forty-five animals were finally diagnosed with osteomye- the defect region was identified by a contour as a traced litis and randomly divided into four groups: Control, region of interest (ROI), and the relative measurements Van, NBD, and NBD + Van groups. Animals in the Con- were calculated, including bone mineral densities (BMDs) trol group (n = 9) were left untreated. Animals in the and bone volume/total volume (BV/TV). Van group (n = 12) were treated with debridement and Next, conventional pathological sections stained with intravenous injection of vancomycin (30 mg/kg, twice hematoxylin and eosin were prepared for bone samples, daily) for 4 weeks. Animals in the NBD group (n = 12) and decalcified cross sections (5-μm) were assessed by a were treated with debridement and local percutaneous pathologist in a blinded fashion according to the scoring injection of NBD peptides (5 mg/kg BW) into the me- system of Smeltzer et al. [18]. Animals were diagnosed dullary cavity (immediately after debridement, 2 and 4 with osteomyelitis when they possessed four or more weeks post-operation). Animals in the NBD + Van group points. (n = 12) received the same treatment as the Van group, plus local injection with NBD peptides as the NBD Statistical analysis group. The post-operative follow-up after debridement The Statistical Package for the Social Sciences (SPSS) lasted for 6 weeks, during which the animals were (SPSS Inc., Chicago, IL) version 11.0 for Windows was housed in individual cages and fed a normal diet. used to analyze the results. Mann-Whitney tests for For the debridement procedure, an 8 mm × 20 mm independent samples were performed to compare the la- cortical bone window in the anteromedial surface of boratory values and radiological and histological scores right proximal tibia was generated to clean out all infec- across the groups. Newly formed bone volume was com- tious and necrotic tissues, according to the varying pared by analysis of variance. Fisher’s exact test was used radiological severity of osteomyelitis. Before wound clos- to compare infection rates across the groups. Unless ure, the NBD peptides were injected into the medullary stated otherwise, results are presented as mean ± stand- cavity of animals in the NBD + Van and NBD groups. Fi- ard deviation, and performed in triplicate; p-values of nally, the removed samples were sent for a culture study. 0.05 or less were considered to be significant. Laboratory monitoring Results After debridement procedure, all animals in the four General conditions groups were followed closely for any clinical sign of sep- The clinical signs of infection varied from mild to severe, tic spread. Blood samples were collected weekly for the following induction, and in the one-week follow-up after measurement of leucocyte count, erythrocyte sedimenta- debridement, two rabbits in the Control group and one in tion rate (ESR), and C-reactive protein (CRP) levels. the NBD group died from severe infection, as confirmed Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 4 of 12 by autopsy. The remaining 42 animals recovered well, was not significant (P = 0.101). NBD + Van group showed with no obvious sign of systemic complication in the 6- the highest infection clearance rate, although there was no week follow-up, and were included in statistical analysis. significant difference with the Van (P = 0.667) or NBD Furthermore, laboratory analyses did not reveal any sig- group (P = 0.214). Infection clearance rate of the NBD group nificant change of leucocyte count, and ESR and CRP was a little lower than that of the Van group, although the levels (Fig. 1), although there were fluctuations throughout difference was not significant (P = 0.684). Altogether, there the follow-up period after debridement. was no significant differenceamong thetreatment groups (Van, NBD + Van, and NBD) with regard to infection clear- Microbiology ance rate, although the best therapeutic efficacy was con- While performing debridement, the culture of curetted firmed by the optimal infection elimination in the NBD samples showed all 42 rabbits to have MRSA. At sacri- peptide + Van group. fice, 7 of 7, 5 of 12, 3 of 12, and 6 of 11 animals in the Control, Van, NBD + Van, and NBD groups, respectively, Radiography were tested positive for MRSA (Fig. 2). Infection clear- At 3 weeks after infection introduction, typical signs of ance rate of the Control group was significantly lower bone infection, including periosteal new bone, bone de- than that of the Van (P = 0.017) and NBD + Van groups struction, and sequestra, could be seen in the radio- (P = 0.003). Infection clearance rate of the Control group graphs of tibiae (Fig. 3a). After treatment for 6 weeks, was also lower than that of the NBD group, although it elimination of bone infection and bone window healing Fig. 1 Leucocyte count (a), and ESR (b) and CRP (c) levels in the Control, Van, NBD + Van, and NBD groups during the 6 weeks follow-up period after debridement Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 5 of 12 Fig. 2 Infection clearance rates of MRSA in the Control, Van, NBD + Van, and NBD groups at 6 weeks after debridement. The Control group showed the lowest infection clearance rate, while the highest infection clearance rate was detected in the NBD + Van group. Fisher’s exact test determined statistical significance where *p < 0.05 Fig. 3 Lateral radiographs of tibiae in different groups, taken before and 1 day, 3 and 6 weeks after debridement. a 3 weeks after infection induction, the tibia showed a typical character of chronic osteomyelitis, including periosteal new bone formation (white arrow), bone destruction (black arrow), and sequestra (black arrows). The tibiae in the NBD + Van group have shown a gradually healing of osteomyelitis after treatment (b, c, and d), whereas the tibial osteomyelitis in other groups did not ameliorate obviously (e, f, and g) Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 6 of 12 were observed in the NBD + Van group (Fig. 3b–d), with The BV/TV results exhibited a similar trend as the BMD only 3 of 12 animals having a score of three or above. data. Mean BV/TV percentage in the NBD + Van group Different degrees of infection were observed in the Con- was significantly higher than that in the Van and NBD trol, Van, and NBD groups (Fig. 3e–g), with 7 of 7, 5 of groups (P = 0.001); there was also a significant difference 12, and 6 of 11 animals having a score of three or above, in BV/TV between the Van and NBD groups (P = 0.001). respectively. Total radiological score of the Control group was significantly higher than that of the Van (P = Histology 0.031), NBD + Van (P = 0.001), and NBD groups (P = The varying severity of osteomyelitis in the four groups was 0.03). The NBD + Van group showed a significantly further confirmed by histological results (Figs. 7 and 8). lower score than the Van (P = 0.03) and NBD groups Typical signs of osteomyelitis, including acute and chronic (P = 0.007). No statistically significant difference was intraosseous inflammation, periosteal inflammation, and found between the Van and NBD groups (P = 0.767) bone necrosis could be seen in some animals, whereas par- (Fig. 4). tial signs were noted in others. A severity score of four or As shown in Fig. 5, morphology of the newly formed more was found in 7 of 7, 6 of 12, 3 of 12, and 7 of 11 ani- bone was reconstructed using micro-CT in the Van, mals in the Control, Van, NBD + Van, and NBD groups, re- NBD + Van, and NBD groups (Fig. 5a, c, e), and the re- spectively. Histological score of the Control group was sults were almost consistent with the x-ray images. significantly higher than that of the Van (P = 0.001), NBD + Micro-CT cross sections of the defect area showed more Van (P = 0.001), and NBD groups (P = 0.038), respectively. obvious new bone formation in the NBD + Van group The NBD + Van group showed a significantly lower score (Fig. 5d). Further, there were several cavities scattered than the Van (P = 0.039) and NBD groups (P =0.01). The over the area in the Van and NBD groups (Fig. 5b, f). Van group also showed a significantly lower score than the Compared to the NBD group, Van group exhibited NBD group (P = 0.047). greater new bone formation, although the cortex was in- complete and there were voids beneath the cortex. Com- pared to the Van group, NBD + Van group showed Discussion improved new cortex formation. In all groups, sporadic The present in-vivo study investigated the effects of trabecular bone was observed in the marrow canal. NBD peptide on infected bone; it played a key role in The amount of newly formed bone in the defect sites inhibiting inflammation and promoting bone remodeling was calculated by morphometric analysis (Fig. 6). The towards osteogenesis, and the effects were better than NBD group exhibited the lowest levels of both BMDs those produced by antibiotics alone. Despite the failure and BV/TV at the defect site when compared to other in demonstrating antibacterial effectiveness of NBD pep- groups (P = 0.001). BMDs at the defect site of NBD + tide in vivo, antibiotics combined with NBD peptide Van group were significantly higher than that of the Van seemed to be a preferred therapeutic option in chronic and NBD groups (P = 0.001 and P = 0.001, respectively). osteomyelitis. Fig. 4 Radiological scores in the Control, Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Mann-Whitney test determined statistical significance where * P < 0.05 Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 7 of 12 Fig. 5 Representative 3D micro-CT reconstruction and Micro-CT cross sections analysis of the proximal tibia in the Van (a, b), NBD + Van (c, d), and NBD (e, f) group 6 weeks after debridement. The bone window in the NBD + Van group was covered by newly formed bone, while no sufficient bone formed in other two groups In general, a number of research groups have reported regulates the inflammatory response leading to the develop- the successful treatment of synovial inflammation [19], ment of bone infection, and selective blocking of NF-κB inflammatory osteolysis [20, 21], inflammatory arthritis could have therapeutic implication in treating the disease. [22], cartilage degradation [23], and muscular dystrophy In this study, our results indicated that specific and selective [24] by the use of NBD peptide. Existing rationale for inhibition of NF-κB by the NBD peptide is an effective treating chronic inflammatory diseases involving bone approach to treat chronic osteomyelitis. The anti- resorption by NBD peptide includes the documented re- inflammatory efficacy of NBD peptide in infected bone was duction of both TNF-α levels and bone destruction assessed by radiologic and histologic means. Extensive based on NF-κB blockade [20]. Furthermore, proinflam- chronic osteomyelitis was achieved 3 weeks after infection matory cytokines, such as TNF-α, are known to be pro- induction, followed by debridement in all groups, except duced in S. aureus-induced osteomyelitis [8], which in the Control group. Six weeks after debridement, combin- turn, results in progressive inflammatory destruction of ation treatment with NBD peptide and vancomycin exhib- the bone. We aimed to address whether NBD peptide ited the most curative antimicrobial efficacy, with the could be made accessible to subjects with osteomyelitis, lowest infection rate, as well as radiological and histological or whether it could be a therapeutic option in chronic scores. All these values were also lowered in the bone infections. vancomycin-only group. Meanwhile, local NBD peptide in- Previous in-vitro studies had shown that a number of jection alone exhibited infection clearance efficacy in vivo, bacteria, including Streptococcus pneumoniae [25, 26], although it was not comparable to that by vancomycin. Streptococcus pyogenes [27], and S. aureus [28], could acti- Based on these observations, our data may be considered to vate NF-κB in response to infection. S. aureus is the major be in favor of NBD peptide application to chronic osteo- pathogen among staphylococci and the most common myelitis; it exhibited infection elimination efficacy, perhaps cause of bone infections, which may also activate the NF- owing to the NBD peptide providing an opportunity to se- κB pathway and ensure that NF-κB activation is required lectively abrogate the inflammation-induced activation of for the phagocytosis of S. aureus by macrophages [9]. A re- NF-κB by targeting the NBD-NEMO interaction. cent investigation [29]demonstratedthatNF-κBis acti- At present, vancomycin has been introduced in most vated during the development of bone infection. Consistent cases of chronic osteomyelitis treatment [3]. However, it with this finding, we had shown that selective inhibition of has been losing potency against S. aureus, and various NF-κB, using the NBD peptide, blocks RANKL-induced forms of resistance to vancomycin have emerged in osteoblast differentiation in vitro [11]. Thus, NF-κB MRSA. Vancomycin could not cause adequate microbial Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 8 of 12 Fig. 6 Morphometric evaluation of the local bone mineral densities (BMDs) (a) in the new bone formation area and bone volume/total volume (BV/TV) (b) in the Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Analysis of variance test determined statistical significance where * P < 0.05 clearance for treating chronic osteomyelitis induced by microbiological examination was increased after NBD MRSA, in this study, and microbiological investigations peptide therapy compared to that in the untreated con- demonstrated persistence of infection with positive tis- trol group. Based on these observations, our data are in sue cultures even at study endpoint. Considering the favor of anti-inflammatory NBD application for treating complexity of chronic infections, with antibiotic resist- chronically infected bone, consistent with the previously ance of MRSA, biofilm formation, low drug penetration, published in-vitro data [29]. Despite the failure to defin- drug activity, and much more, anti-MRSA combinations itely demonstrate anti-MRSA effectiveness of NBD pep- might be preferable for treating MRSA-osteomyelitis. tide in vivo, there is a possible U-shaped response of Compared to vancomycin therapy alone, vancomycin in bacterial growth to pro-inflammatory cytokines [30], and conjunction with NBD peptide therapy was not expected we speculate that NBD peptide would ameliorate inflam- to elicit a more pronounced antibacterial effect. How- matory responses and may be expected to have a posi- ever, infection clearance rate was enhanced with the tive influence on host biological response against MRSA combination therapy compared to that in vancomycin infections. Moreover, based on the cellular hysteresis alone group. Additionally, infection clearance rate of strategy [6], there may be a synergistic effect of Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 9 of 12 that NBD peptide could promote bone regeneration ef- fectively only when infection was controlled. We had previously demonstrated that NF-κB activation inhibits osteoblast differentiation induced by TNF-α, and that application of NBD peptide ameliorates this inhibitory effect [11]. Our in-vivo results in the present study showed that NBD peptide is able to effectively amelior- ate the inhibition of osteoblast differentiation by chronic inflammation and promote bone regeneration. In con- clusion, the NBD peptide not only seemed to prevent bone destruction in chronic osteomyelitis but also accel- erated bone regeneration, obliterating the dead space left by debridement. In the current study, we have utilized an NBD peptide that binds NEMO and compromises the formation of active IKK complex. This sequestration disables IKK functions, primarily the immediate activation of NF-κB. However, physiologically, NF-κB has a crucial anti- apoptotic role in survival, and its pharmacological inhib- ition may be of limited benefit, owing to the likelihood of generalized apoptosis [20]. In support of this notion, a study reporting the effects of conditional deletion of IKK suggested complete blockage of NF-κBto possibly lead to enhanced apoptosis and damage [33]. Nevertheless, the ef- fects of highly selective pharmacological inhibition of only proinflammatory IKK activity has not yet been fully inves- tigated and reported. This is supported by a number of previous reports [20, 22, 34], showing little or no adverse Fig. 7 Photomicrographs of the control group (a and b) shows effect, owing to the blockade of NF-κB, on therapeutic destruction of cortical bone (black arrow), sequestrum formation purposes in chronic inflammatory diseases in vivo. In pre- (white arrow), intramedullary abscess (black arrow-head), fibrosis vious collagen-induced arthritis experiments, to prevent (white arrow-head), and proliferated foamy histotcytes (red inflammatory bone destruction [20], NBD peptide was arrow-head). The van group (c)and NBDgroup (d)showed injected into mice, daily for over 4 weeks, without observ- relatively moderate osteomyelitis characteristics, with some new bone formation (red arrow). Photomicrographs of the NBD + Van ing any detrimental side effect, or liver or kidney toxicity; group (e and f) were shown mild destruction of cortical bone injection of the peptide for only the first 5 days was and intramedullary new bone formation, without signs of severe enough to maintain the therapeutic effects for nearly 3 inflammation (haematoxylin and eosin) weeks after administration. In our current experiments, we locally injected the rabbits with the NBD peptide, vancomycin in conjunction with NBD peptide therapy, thrice for over 6 weeks, without observing any detrimental which was harnessed to optimize the antibiotic therapy, side effect or bacterial spread. Local NBD peptide delivery subsequently achieving enhanced bacterial elimination. in our experiments minimized the probability of their sys- To establish these events in vivo, future studies should temic toxicity caused by systemic usage. However, exten- be conducted to explore inducible physiological effects sive pharmacological evaluation of this approach would be of NBD peptide, which may, subsequently help to find required to fully determine the effects of long-term use of direct antibacterial effects of NBD peptide for improving the NBD peptide to treat chronic osteomyelitis induced by antibiotic therapy. MRSA. Although temporary inflammation may promote bone There are several well-established animal models with healing, persistent and serious infection-associated in- some kind of implantation that support the establish- flammation could result in suboptimal and impaired ment of infection and allow the study of chronic osteo- bone regeneration [31, 32]. The newly formed bone was myelitis in vivo. However, the implantation of foreign quantified by micro-CT, and results showed the volume bodies usually does not allow the infection to heal unless of newly formed bone in the NBD + vancomycin group the implant is removed. In the present study, we used to be significantly higher than that in the Van or NBD the model described by Nijhof et al. [16], which aimed group; the bone window eventually healed, indicating to induce infection via the injection of MRSA bacterial Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 10 of 12 Fig. 8 Histological scores in the Control, Van, NBD + Van, and NBD groups 6 weeks after debridement. Results were shown as mean ± standard deviation. Mann-Whitney U-test determined statistical significance where * P < 0.05 suspension without implant infections. The establish- inhibition caused by NF-κB activation in vitro. These ment of active infection at 3 weeks, in all animals, could studies may be performed in other laboratories and rep- clearly be confirmed by local clinical signs and radio- licated further using a larger sample size, thereby helping graphs. Our data are consistent with the previous finding to re-confirm the reliability of our conclusion. Finally, [35], and demonstrate that a high rate of chronic osteo- further studies regarding the efficacy of NBD peptides myelitis could be achieved within 3 weeks of infection against other types of pathogenic microorganisms, as induction. The use of sodium morrhuate in the present well as studies considering the combined use of NBD osteomyelitis model, with the inherent variability of in- peptides and antibiotics, should be performed in vivo. fection intensity, has certain limitations in assessing the effects of NBD peptide in chronic bone infections; how- Conclusion ever, the model used herein was appropriate to study We conclude that NBD peptide induced neither bacterial treatment safety. Clinical assessment and laboratory spread nor worsening of infection. Together with the values were used to study the systemic side effects and previous reports of successful and safe treatment of in- bacterial spread after debridement. Neither clinical nor flammatory bone destruction using NBD peptide, results laboratory signs of bacterial spread was observed. We, of the present study are in favor of the applicability of therefore, concluded that NBD peptide neither worsened antibiotics combined with NBD peptide to treat osteo- bone infections, nor induced bacterial sepsis. myelitis. Since bone defect and residual infection are A limitation of the current study was the absence of a common problems after debridement, in the treatment group of rabbits with osteomyelitis that received de- of osteomyelitis, despite the failure to definitely demon- bridement without antibiotics or NBD peptide adminis- strate antibacterial effectiveness of NBD peptide in vivo, tration. Therefore, we cannot figure out the specific role antibiotics in conjunction with NBD peptide are sug- of debridement in eliminating infection and stimulating gested to possibly have promising therapeutic potential bone regeneration. Another limitation was that a reason- in osteomyelitis. able sample size was not calculated due to lack of a pilot study before the experiment. Our sample size refers to Abbreviations BMDs: Bone mineral densities; BV/TV: Bone volume/total volume; IKK: IκB the previous experiments [20, 35], which is more than kinase; IL-1: Interleukin 1; IL-6: Interleukin 6; MRSA: Methicillin-resistant that in the above reports. Meanwhile, our results have Staphylococcus aureus; NBD: NEMO-binding domain; NEMO: NF-κB essential effectively evaluated the anti-inflammatory effect of modulator; NF-κB: Nuclear factor kappa B; PMMA: Polymethylmethacrylate; RANKL: Nuclear factor kappa B ligand; ROI: Region of interest; vancomycin in conjunction with NBD peptide and its ef- S.aureus: Staphylococcus aureus; TNF-α: Tumour necrosis factor alpha fect on promoting bone remodeling in MRSA-induced chronic osteomyelitis. The current results are consistent Acknowledgments with our previous investigation, showing that NBD pep- The authors would like to thank Editage (www.editage.com) for English tide could ameliorate the osteoblast differentiation language editing. In addition, Chang-Peng Xu thanks the inimitable care and Xu et al. Antimicrobial Resistance and Infection Control (2019) 8:182 Page 11 of 12 support of Xiao-Jie Zheng over the years. I love you. Will you spend the rest demonstrate potent bone resorbing activity. J Bone Miner Res. 1995; of your life with me? 10(5):726–34. 8. Yoshii T, Magara S, Miyai D, Nishimura H, Kuroki E, Furudoi S, Komori T, Ohbayashi C. Local levels of interleukin-1β, −4, −6, and tumor necrosis Authors’ contributions factor α in an experimental model of murine osteomyelitis due to CP Xu and Y Qi designed the study. Y Chen contributed in the animal staphylococcus aureus. Cytokine. 2002;19(2):59–65. feeding. HT Sun contributed in the osteomyelitis model induction. 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