Longitudinal intravital imaging of the bone marrow for analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Evidence of bisphosphonate-conjugated sitafloxacin eradication of established methicillin-resistant S. aureus infection with osseointegration in murine models of implant-associated osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Epidemiological updates of post-traumatic related limb osteomyelitis in china: a 10 years multicentre cohort study.

BACKGROUND: Post-traumatic related limb osteomyelitis (PTRLO) is a complex bone infection. Currently, there are no available microbial data on a national scale that can guide appropriate antibiotic selection, and explore the dynamic changes in dominant pathogens over time. This study aimed to conduct a comprehensive epidemiological analysis of PTRLO in China. METHODS: The study was approved by the Institutional Research Board (IRB), and 3526 PTRLO patients were identified from 212 394 traumatic limb fracture patients at 21 hospitals between 1 January 2008 and 31 December 2017. A retrospective analysis was conducted to investigate the epidemiology of PTRLO, including changes in infection rate (IR), pathogens, infection risk factors and antibiotic resistance and sensitivity. RESULTS: The IR of PTRLO increased gradually from 0.93 to 2.16% (Z=14.392, P <0.001). Monomicrobial infection (82.6%) was significantly higher than polymicrobial infection (17.4%) ( P <0.001). The IR of Gram-positive (GP) and Gram-negative (GN) pathogens showed a significant increase from the lowest 0.41% to the highest 1.15% (GP) or 1.62% (GN), respectively. However, the longitudinal trend of GP vs. GN's composition did not show any significance (Z=±1.1918, P >0.05). The most prevalent GP strains were Methicillin-sensitive Staphylococcus aureus (MSSA) (17.03%), Methicillin-resistant Staphylococcus aureus (MRSA) (10.46%), E. faecalis (5.19%) and S. epidermidis (4.87%). In contrast, the dominant strains GN strains were Pseudomonas Aeruginosa (10.92%), E. cloacae (10.34%), E. coli (9.47%), Acinetobacter Baumannii (7.92%) and Klebsiella Pneumoniae (3.33%). In general, the high-risk factors for polymicrobial infection include opened-fracture (odds ratio, 2.223), hypoproteinemia (odds ratio, 2.328), and multiple fractures (odds ratio, 1.465). It is important to note that the antibiotics resistance and sensitivity analysis of the pathogens may be influenced by complications or comorbidities. CONCLUSIONS: This study provides the latest data of PTRLO in China and offers trustworthy guidelines for clinical practice. (China Clinical Trials.gov number, ChiCTR1800017597).

Evidence of Bisphosphonate-Conjugated Sitafloxacin Eradication of Established Methicillin-Resistant S. aureus Infection with Osseointegration in Murine Models of Implant-Associated Osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (bisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy, while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Reduced angiogenesis and delayed endochondral ossification in CD163-/- mice highlights a role of M2 macrophages during bone fracture repair.

While recent studies showed that macrophages are critical for bone fracture healing, and lack of M2 macrophages have been implicated in models of delayed union, functional roles for specific M2 receptors have yet to be defined. Moreover, the M2 scavenger receptor CD163 has been identified as a target to inhibit sepsis following implant-associated osteomyelitis, but potential adverse effects on bone healing during blockage therapy have yet to be explored. Thus, we investigated fracture healing in C57BL/6 versus CD163-/- mice using a well-established closed, stabilized, mid-diaphyseal femur fracture model. While gross fracture healing in CD163-/- mice was similar to that of C57BL/6, plain radiographs revealed persistent fracture gaps in the mutant mice on Day 14, which resolved by Day 21. Consistently, 3D vascular micro-CT demonstrated delayed union on Day 21, with reduced bone volume (74%, 61%, and 49%) and vasculature (40%, 40%, and 18%) compared to C57BL/6 on Days 10, 14, and 21 postfracture, respectively (p < 0.01). Histology confirmed large amounts of persistent cartilage in CD163-/- versus C57BL/6 fracture callus on Days 7 and 10 that resolves over time, and immunohistochemistry demonstrated deficiencies in CD206+ M2 macrophages. Torsion testing of the fractures confirmed the delayed early union in CD163-/- femurs, which display decreased yield torque on Day 21, and a decreased rigidity with a commensurate increase in rotation at yield on Day 28 (p < 0.01). Collectively, these results demonstrate that CD163 is required for normal angiogenesis, callus formation, and bone remodeling during fracture healing, and raise potential concerns about CD163 blockade therapy.

Relapsed boyhood tibia polymicrobial osteomyelitis linked to dermatophytosis: a case report.

BACKGROUND: Relapsed childhood polymicrobial osteomyelitis associated with dermatophytosis has not been reported in the literature. CASE PRESENTATION: Here we report on a case of a 45-year-old man who had left tibial osteomyelitis for 29 years, accompanied by skin fungal infection of the ipsilateral heel for 20 years, and underwent a second operation due to recurrence of polymicrobial infection 6 years ago. The patient had a history of injury from a rusty object, which penetrated the anterior skin of the left tibia middle segment causing subsequent bone infection, but was asymptomatic after receiving treatments in 1983. The patient was physically normal until dermatophytosis occurred on the ipsilateral heel skin in 1998. The patient complained that the dermatophytosis was gradually getting worse, and the tibial wound site became itchy, red, and swollen. The left tibial infection resurged in May 2012, leading to the patient receiving debridement and antibiotic treatment. H&E and Gram-stained histology was performed on biopsy specimens of sequestrum and surrounding inflammatory tissue. Tissue culture and microbiology examination confirmed polymicrobial infection with Staphylococcus aureus (S. aureus) and Corynebacterium and a fungus. Additionally, the patient also received potassium permanganate for dermatophytosis when he was admitted into the hospital. CONCLUSIONS: Together with longitudinal follow-up of medical history, surgical findings, histopathological and microbiology culture evidence, we conclude that boyhood tibia polymicrobial osteomyelitis with S. aureus and Corynebacterium occurred in this patient, and the fungal activation of dermatophytosis may have led to osteomyelitis relapse.

Therapeutic Effectiveness of Percutaneous Endoscopic Spinal Surgery for Intraspinal Cement Leakage Following Percutaneous Vertebroplasty: An Early Clinical Study of 12 Cases.

BACKGROUND: Intraspinal cement leakage is a catastrophic complication of percutaneous vertebroplasty (PVP). Percutaneous endoscopic spinal surgery (PESS) for intraspinal cement leakage has rarely been reported. OBJECTIVES: To evaluate the therapeutic effectiveness of PESS for intraspinal cement leakage following PVP. STUDY DESIGN: This was a retrospective study approved by the ethics committee of our institution. SETTING: Department of Orthopedics from an affiliated hospital. METHODS: Twelve patients with neurologic impairments resulting from intraspinal cement leakage after PVP were treated with PESS for spinal decompression from May 2014 to June 2018. Computed tomography and 3-dimensional reconstruction were used to confirm the vertebral level of cement leakage. The surgical index, neurologic function, and clinical results were recorded in this study. RESULTS: The leaked cement of all patients was successfully removed under PESS, and no severe intraoperative complications were reported in our study. The operation time ranged from 43 to 119 minutes (mean, 65.5 minutes). The amount of intraoperative blood loss was 64.25 ± 9.62 mL. The lengths of postoperative hospital stays were 5.25 ± 2.53 days. The follow-up rate was 83.3% (10/12). The follow-up time ranged from 14 to 30 months (mean, 22 months). The Visual Analog Scale scores of foraminal leaks improved from 6.50 ± 0.93 preoperatively to 1.75 ± 0.71 at the last follow-up (P < 0.05). Neurologic function was evaluated by Japanese Orthopaedic Association 29 scores, which improved from 18.75 ± 1.06 to 22.70 ± 1.64 (P < 0.0001). The good and excellent rates were 80% according to the modified Macnab criteria. LIMITATIONS: This study is limited by the volume of patients and the deep learning curve needed for PESS. CONCLUSIONS: PESS, as a minimally invasive technique, can achieve targeted spinal cord decompression and may be a safe and effective alternative approach to conventional procedures for cement leakage after PVP. KEY WORDS: Endoscopes, cement leakage, minimally invasive surgery, percutaneous vertebroplasty.

Outcomes of Discectomy by Using Full-Endoscopic Visualization Technique via the Transcorporeal and Transdiscal Approaches in the Treatment of Cervical Intervertebral Disc Herniation: A Comparative Study.

OBJECTIVE: To compare the difference in clinical and radiographic outcomes between anterior transcorporeal and transdiscal percutaneous endoscopic cervical discectomy (ATc-PECD/ATd-PECD) approaches for treating patients with cervical intervertebral disc herniation (CIVDH). METHOD: We selected 77 patients with single-segment CIVDH and received ATc-PECD or ATd-PECD in the Second Affiliated Hospital of Chongqing Medical University between March 1, 2010, and July 1, 2015. 35 patients suffered from ATc-PECD, and there were 42 patients in the ATd-PECD group. Obtaining the data of 1, 3, 6, 12, and 24 months postoperatively, the VAS for neck and arm pain and the modified MacNab criteria were used to evaluate the clinical outcomes, comparing radiographic outcomes and complications of these two groups. RESULTS: We found that the mean operative time was significantly longer in the ATc-PECD group (P < 0.05). At the 2-year follow-up, the mean VAS score for neck and arm pain was significantly decreased in both two groups. There was no significant difference in the VAS score for arm pain and neck pain between the two groups at the 2-year follow-up (P=0.783 and P=0.785, respectively). For the ATc-PECD group, the difference in the height of IVS or vertebral body was significant between the preoperative and postoperative groups (P < 0.05, respectively). For the ATd-PECD group, there was only a significant decrease in the height of the IVS (P < 0.05); the decrease in the surgical vertebral body was not significant between the preoperative and postoperative groups (P > 0.05). CONCLUSION: In the 2-year follow-up, there is no significant difference in the clinical outcomes between the 2 approaches. While the longer time was consumed in the ATc-PECD group, the lower rate of disc collapse and recurrence is notable. Additionally, when the center diameter of tunnel was limited to 6 mm, the bony defect can be healed without the occurrence of the collapse of the superior endplate, and ATc-PECD may be preferable in the endoscopic treatment of CIVDH.

The combinations of multiple factors to improve the diagnostic sensitivity and specificity after artificial joint infection.

OBJECTIVE: To discuss the sensitivity and specificity of the combinations of multiple factors that work on bone infection after artificial joint, and provide evidence-based medical basis for the early diagnosis of infection after artificial joint. METHODS: A retrospective review was conducted on 35 patients diagnosed with periprosthetic joint infections (PJI) or aseptic loosening (AL) who both received revision operation from January 2011 to January 2015. Analyzing and comparing their epidemiology indexes and expounded a series of auxiliary examinations corresponding positive diagnosis ratio. RESULTS: Thirty-five patients were divided into two groups. One is called group PJI which includes 16 patients, and the other is called group AL which contains 19 patients. There was no statistical difference between in age (p = 0.536), gender ratio (p = 0.094), and the time of catching infection or getting loose (p = 0.055). Swelling was statistical significant (p = 0.0435 < 0.05). AUC of CRP = 0.947, ESR = 0.893, IL-6 = 0.893, PCT = 0.781, WBC = 0.839, and PMN = 0.755, respectively, CRP has a high diagnostic value to PJI, ESR, IL-6, PCT, WBC, and PMN% possess a moderate diagnostic value. There were 3 cases of PJI whose pathological paraffin section showed infectious inflammatory cells (100%). three PJI patients and one AL patient whose 99mTc-MDP examination presented 100% infection or looseness rate. CONCLUSION: CRP has a high diagnostic value to PJI. Histopathology HE staining, Gram staining, and 99mTc-MDP provide a highly accurate diagnosis for PJI. Therefore, the results suggest combining the unique clinical symptoms of PJI patients with relevant laboratory indexes, histopathologic characteristics, and imageological examinations that can improve diagnostic sensitivity and specificity of PJI in its early stage.

Schnurri-3 regulates BMP9-induced osteogenic differentiation and angiogenesis of human amniotic mesenchymal stem cells through Runx2 and VEGF.

Human amniotic mesenchymal stem cells (hAMSCs) are multiple potent progenitor cells (MPCs) that can differentiate into different lineages (osteogenic, chondrogenic, and adipogenic cells) and have a favorable capacity for angiogenesis. Schnurri-3 (Shn3) is a large zinc finger protein related to Drosophila Shn, which is a critical mediator of postnatal bone formation. Bone morphogenetic protein 9 (BMP9), one of the most potent osteogenic BMPs, can strongly upregulate various osteogenesis- and angiogenesis-related factors in MSCs. It remains unclear how Shn3 is involved in BMP9-induced osteogenic differentiation coupled with angiogenesis in hAMSCs. In this investigation, we conducted a comprehensive study to identify the effect of Shn3 on BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs and analyze the responsible signaling pathway. The results from in vitro and in vivo experimentation show that Shn3 notably inhibits BMP9-induced early and late osteogenic differentiation of hAMSCs, expression of osteogenesis-related factors, and subcutaneous ectopic bone formation from hAMSCs in nude mice. Shn3 also inhibited BMP9-induced angiogenic differentiation, expression of angiogenesis-related factors, and subcutaneous vascular invasion in mice. Mechanistically, we found that Shn3 prominently inhibited the expression of BMP9 and activation of the BMP/Smad and BMP/MAPK signaling pathways. In addition, we further found activity on runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF), and the target genes shared by BMP and Shn3 signaling pathways. Silencing Shn3 could dramatically enhance the expression of Runx2, which directly regulates the downstream target VEGF to couple osteogenic differentiation with angiogenesis. To summarize, our findings suggested that Shn3 significantly inhibited the BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs. The effect of Shn3 was primarily seen through inhibition of the BMP/Smad signaling pathway and depressed expression of Runx2, which directly regulates VEGF, which couples BMP9-induced osteogenic differentiation with angiogenesis.

All-arthroscopic release for treating severe knee extension contractures could improve the knee range of motion and the mid-term functional outcomes.

PURPOSE: To evaluate the safety, feasibility, and effectiveness of an all-arthroscopic technique for the intra- and extraarticular release of severe knee extension contractures. METHODS: From 2012 to 2016, 25 patients with severe knee extension contractures (less than 45° range of flexion) were treated with an all-arthroscopic release technique. The patients underwent intra- and extraarticular arthroscopic release and arthroscopic-assisted mini-incision quadriceps plasty. The post-operative rehabilitation was initiated the first day after the procedures. Comprehensive clinical follow-up evaluations including the range-of-motion (ROM) assessment, the Lysholm score, and the International Knee Documentation Committee (IKDC) score were performed on all patients. RESULTS: The median follow-up time was 28 months (range 12-65 months). The ROM improved from 23.9° ± 7.5° pre-operatively to 105.9° ± 6.5° at the final follow-up (P < 0.001). In addition, the Lysholm score increased from 59.9 ± 5.2 pre-operatively to 89.7 ± 3.3 (P < 0.001). The IKDC score increased from 47.6 ± 3.4 pre-operatively to 91.7 ± 2.4 (P < 0.001). All patients were satisfied with their final ROM and functional outcomes. CONCLUSION: The all-arthroscopic release technique was a safe, feasible and effective method for treating severe knee extension contractures. The severe knee extension contractures may be successfully addressed by the all-arthroscopic release technique during our clinical practice. LEVEL OF EVIDENCE: IV.

Differentiation of Human Amniotic Mesenchymal Stem Cells into Human Anterior Cruciate Ligament Fibroblast Cells by In Vitro Coculture.

Anterior cruciate ligament injuries are common in humans, though cellular components of the knee have little regenerative or proliferation potential. This study investigated the differentiation of human amnion-derived mesenchymal stem cells (hAMSCs) into human anterior cruciate ligament fibroblasts (hACLFs) in vitro through induction with bFGF and TGF-ß1 with coculture systems. Groups A and B comprised hAMSCs at the 3rd passage cultured with and without bFGF and TGF-ß1, respectively; Groups C and D consisted of hAMSCs and hACLFs in monolayer coculture with and without bFGF and TGF-ß1, respectively; Groups E and F were composed of hAMSCs and hACLFs in Transwell coculture with and without bFGF and TGF-ß1, respectively. Cell morphology and proliferation were recorded. Protein expression and relative mRNA expression were evaluated in each group. Cell proliferation was significantly higher in the induced groups than in the noninduced groups. Protein expression increased over time with the highest expression observed in Group E. mRNA levels were significantly higher in Group E than in other groups. This study is the first to demonstrate the use of the Transwell coculture system for this purpose, and hAMSCs were successfully differentiated into hACLFs. Thus, hAMSCs may be a superior choice for hACLF differentiation via Transwell coculture.

Epidemiological, Clinical and Microbiological Characteristics of Patients with Post-Traumatic Osteomyelitis of Limb Fractures in Southwest China: A Hospital-Based Study.

Objective To determine the epidemiological, clinical and microbiological characteristics, of patients with post-traumatic osteomyelitis of extremity fractures, and provide evidence-based guidelines for early diagnosis and treatment, including empiric antibiotic therapy. Methods Human subject research was performed using institutional review board approved protocols. A retrospective chart review was conducted on 5,368 patients diagnosed with extremity traumatic fractures from January 1, 2012 to December 31, 2015, to identify osteomyelitis patients. Records from the Microbiology Department were reviewed, and patients with a positive wound culture, or bone biopsy culture, were selected for the study. Microbial suceptability was determined by the M-100-S22 protocol (Clinical & Laboratory Standards Institute® (CLSI) 2012 USA). Additional clinical information, including data on patients' baseline epidemiological, clinical, and microbiological records was collected from all available charts, and reviewed using a designed protocol. Results 84 (1.56%) patients were diagnosed with osteomyelitis based on a positive culture result. The most prevalent comorbidities in these patients were compartment syndrome, diabetes and hypertension. The most commonly involved infected site was the tibia-fibula (47.62%). 66 (78.57%) of these cases were monomicrobial, and 18 cases (21.43%) were polymicrobial. The infections were predominantly caused by Gram-positive bacteria (56, 53.85%). The most common Gram-positive bacteria were Staphylococcus aureus (39 cases, 37.50%) and S. epidermidis (6 cases, 5.77%), which were sensitive to ampicillin, synercid/ dalfopristin, linezolid, tigecycline, macrodantin, and vancomycin. S. aureus was the most common pathogen in both monomicrobial and polymicrobial cases. All 17 cases of MRSA infection were sensitive to Imezolid, ampicillin, synercid/ dalfopristin, linezolid, tigecycline, furadantin, piperacillin/yaz, rifampicin, and vancomycin, respectively. The most common Gram-negative bacteria were E. coli (16 cases, 15.38%) and Enterobacter cloacae (11 cases, 10.58%), which were sensitive to thienamycin. Conclusions In this study, the overall rate of post-traumatic osteomyelitis of limb fractures (1.56%) is lower than the national average rate (2.6-7.8%), for major medical centers in China. The main medical comorbidities were compartment syndrome, diabetes mellitus and hypertension. The most common infection was monomicrobial in lower extremities. S. aureus was the most common pathogen, which presented in 39 (37.50%) cases, and 17 of these (43.59%) were caused by MRSA. These findings can guide empiric antibiotic therapy in Southwest China for osteomyelitis in patients with traumatic limb fractures.