Lifeng Shen

Rapid Quantification of Tobramycin and Vancomycin by UPLC-TQD and Application to Osteomyelitis Patient Samples

Tobramycin and vancomycin are the most commonly used antibiotics for the treatment of osteomyelitis. A sensitive and rapid method was developed for the analysis of tobramycin and vancomycin in human drainage tissue fluid. The procedure involved a simple liquid-liquid extraction of tobramycin, vancomycin and atenolol (internal standard) and separation by ultra-high performance liquid chromatography on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with a mobile phase consisting of 0.1% (v/v) formic acid water solution and 0.1% (v/v) formic acid acetonitrile solution at a flow rate of 0.3 mL/min. Detection was performed by positive ion electrospray ionization in multiple reaction monitoring mode (m/z 468 → 163 transitions for tobramycin; m/z 725 → 144 for vancomycin; m/z 267 → 74 for the internal standard). The retention times of tobramycin, vancomycin, and the internal standard were 0.68, 3.62 and 3.03 min, respectively. The total analysis time was less than 10 min. Excellent linear relationships (correlation coefficient > 0.99) were demonstrated between the area under the peak ratios of tobramycin and vancomycin to the internal standard in the drainage tissue fluid, and the concentration ranges were 1.25-100.00 mg/L and 0.50-150.00 mg/L for tobramycin and vancomycin, respectively. The intra-day and inter-day accuracy and precision (coefficient of variation) acceptance criteria for each quality control was ≤ 7.8% and the mean accuracy values were < 5.0% for tobramycin and < 4.0% for vancomycin. All experiments suggested the high-throughput potential of the proposed method. The method was successfully applied to investigate local delivery of tobramycin and vancomycin in four calcaneal osteomyelitis patients who had accepted drug-loaded artificial bone implantation.

STUDY OF THE DIFFERENT RELEASE CHARACTERISTICS OF ANTIBIOTICS FROM PATIENTS: A DYNAMIC ELUTING SYSTEM TO INVESTIGATE DRUG RELEASE FROM ANTIBIOTIC-IMPREGNATED CALCIUM SULFATE DELIVERY

Background: Antibiotic-impregnated calcium sulfate delivery systems (ACDS) are commonly used to treat chronic osteomyelitis. Our research is to investigate drug release in vitro over a longer period, as a cautious predictor of in vivo release. Methods: The local release behavior of antibiotic in vitro was simulated. The consecutive dynamic eluting experiment was performed based on the pro-operative characteristic of osteomyelitis patients and the determined results of drug concentration in the human drainage tissue fluid (DTF). The concentration of each drug in the receiving solution was detected by ultra-performance liquid chromatography-tandem quadrupole detector mass spectrometry. The ACDS was reviewed by scanning electronic microscopy (SEM) after 48 h, and prepared to be eluted for another examination after 33 days. The mechanism of antibiotic release was analyzed by using the Ritger–Peppas and Weibull equations. Results: The cumulative release rate of vancomycin in a vancomycin-calcium sulfate delivery system (VCDS) was 77.50 % (3.0 mm diameter) and 72.43 % (4.8 mm diameter), while that of the tobramycin in a tobramycin-calcium sulfate delivery system (TCDS) was 88.0 % (3.0 mm diameter) and 84.55 % (4.8 mm diameter). At the 15th day, approximately 27.92% of vancomycin was and 29.35% of tobramycin was released from the local implant in vivo. Using SEM, numerous vancomycin and tobramycin particles were found to be attached to the columnar calcium sulfate crystals at the start of the experiment. The release behavior of the two antibiotics followed a combination of Fickian diffusion and Case II transport mechanisms within the first 48 h, and a Fickian diffusion mechanism during the subsequent time period. The correlation coefficient of tobramycin and vancomycin in vivo and in vitro was 0.9704–0.9949 and 0.9549–0.9782, respectively. Conclusion: A good correlation of the in vivo and in vitro cumulative release rates was observed by comparing the cumulative release rate of drugs in vitro by means of the dynamic eluting model, and in the DTF. Therefore, our study has proved that it is possible to use the dynamic eluting model as a cautious predictor of in vivo release.

Flavonoids of Herba Epimedii Enhances Bone Repair in a Rabbit Model of Chronic Osteomyelitis During Post‐infection Treatment and Stimulates Osteoblast Proliferation in Vitro

Flavonoids are the active component of the Herba Epimedii (H. Epimedii), which is commonly used in Asia. This study is to investigate the effect of H. Epimedii on bone repair after anti‐infection treatment in vivo. The bioactive‐composition group of H. Epimedii (BCGE) contained four flavonoids with the total content of 43.34%. Rabbits with chronic osteomyelitis in response to injection with Staphylococcus aureus were treated with BCGE of 242.70 mg/kg/day intragastrically after vancomycin‐calcium sulphate treatment. Micro‐computerd tomography (CT), morphology, blood biochemistry and osteocalcin levels were assessed for effect evaluation. In addition, the rat calvarial osteoblasts infected with S. aureus were treated with vancomycin and BCGE. Cell viability, alkaline phosphatase activity, bone morphogenetic protein 2, Runt‐related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor‐κB ligand mRNA levels and protein expression were assessed. Our results indicated that BCGE promoted bone repair via increasing the bone mass, the volume of bone, promoting osteocalcin secretion after vancomycin‐calcium sulfate treatment. BCGE enhanced the cell proliferation, by regulating bone morphogenetic protein 2, runt‐related transcription factor 2, and osteoprotegerin/receptor activator of nuclear factor κ‐B ligand mRNA and protein expression to maintain the balance between bone formation and bone resorption. Therefore, BCGE is a potential adjuvant herbal remedy for the post‐infection treatment of chronic osteomyelitis. Copyright

Efficient induction of antimicrobial activity with vancomycin nanoparticle-loaded poly(trimethylene carbonate) localized drug delivery system

Surgery and the local placement of an antibiotic are the predominant therapies to treat chronic osteomyelitis. Vancomycin-loaded N-trimethyl chitosan nanoparticles (VCM/TMC NPs) as a potential drug delivery system have high intracellular penetration and effective intracellular antibacterial activity. This study investigated the effects of a biocompatible material, poly(trimethylene carbonate) (PTMC), to increase the sustained effectiveness of an intracellular antibiotic and its potential application in antibiotic delivery. VCM/TMC NP-PTMC was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy to determine the morphology, stability and chemical interaction of the drug with the polymer. Further, the biodegradation, antibacterial activity, protein adsorption, cell proliferation and drug release characteristics were evaluated. In addition, a Staphylococcus aureus-induced osteomyelitis rabbit model was used to investigate the antibiotic activity and bone repair capability of VCM/TMC NP-PTMC. The results showed that the composite beads of VCM/TMC NPs followed a sustained and slow release pattern and had excellent antibacterial activity and a higher protein adsorption and cell proliferation rate than the VCM-PTMC in vitro. Furthermore, VCM/TMC NP-PTMC inhibits bacteria and promotes bone repair in vivo. Thus, VCM/TMC NP-PTMC might be beneficial in periodontal management to reduce the bacterial load at the infection site and promote bone repair.

Icariin Enhances Bone Repair in Rabbits with Bone Infection during Post-infection Treatment and Prevents Inhibition of Osteoblasts by Vancomycin

Vancomycin is an effective antibiotic for treatment of bone infection caused by Staphylococcus aureus, however, a high local concentration of vancomycin might induce a delay in bone union. Icariin has been reported to suppress osteoclastogenes and promote osteogenesis. Our study aimed to investigate the effect of icariin on bone repair after anti-infection treatment in vivo and to explore the resisting effect of icariin on rat calvarial osteoblasts (ROBs) inhibited with high doses of vancomycin. Rabbits with bone infection of S. aureus were treated with implanted vancomycin-calcium sulfate (VCS) and icariin at 10.86 mg/kg/day for consecutive 8 weeks. Micro-CT, morphology, blood biochemistry were evaluated. In addition, ROBs were treated with vancomycin and icariin at different doses. Cell proliferation and differentiation capabilities, BMP2, Runx2, OPG, RANKL mRNA levels and protein expression were assessed. The results indicated that high dose of vancomycin significantly decreased bone mass and inhibited osteocalcin secretion; icariin increased these indicators compared with the single vancomycin treatment. Over 0.1 mg/mL of vancomycin inhibited the proliferation and differentiation of ROBs, while icariin resisted the inhibition of vancomycin by regulating cell cycle and promoting the Alkaline phosphatase (ALP) activity. Moreover, icariin promote bone formation by up-regulating BMP2/Runx2 and OPG/RANKL pathways. Icariin exhibited osteoplastic properties on osteoblasts that had been inhibited with high doses of vancomycin. Therefore, icariin is helpful for post-infection treatment of bone infection.

Peroneal perforator pedicle propeller flap for lower leg soft tissue defect reconstruction: Clinical applications and treatment of venous congestion:

Objective To describe the characteristics of the perforator vessel in the peroneal artery of the lower leg and to explore the use of perforator pedicle propeller flaps to repair soft tissue defects in the lower leg, heel and foot. Methods This retrospective study enrolled patients with soft tissue defects of the distal lower leg, heel and foot who underwent surgery using peroneal perforator-based propeller flaps. The peroneal artery perforators were identified preoperatively by colour duplex Doppler ultrasound. The flap was designed based on the preoperatively-identified perforator location, with the posterior border of the fibula employed as an axis, and the perforator vessel as the pivot point of rotation. Patients were followed-up to determine the outcomes. Results The study analysed 36 patients (mean age, 39.7 years). The majority of the soft tissue defects were on the heel (20; 55.6%). The donor-site of the flap was closed in 11 patients by direct suturing and skin grafting was undertaken in 25 patients. Postoperative complications included venous congestion (nine patients), which was managed with delayed wound coverage and bleeding therapy. All wounds were eventually cured and the flaps were cosmetically acceptable. Conclusions The peroneal perforator pedicle propeller flap is an appropriate choice to repair soft tissue defects of the distal limbs.

Mean platelet volume: A critical factor relative to thrombosis in microsurgery

With technical and patient-selection advances, microvascular free-flap techniques have improved over the past two decades. Despite greater experience, improved technology, and development of anastomotic devices, some components of free-flap failure might be attributable to undiagnosed patient factors, among which thrombogenesis in anastomotic blood vessels is one of the primary contributing events. Central to this process is platelet activation and subsequent platelet-endothelial adhesion, platelet-platelet aggregation, activation of the coagulation cascade and the formation of a platelet-fibrin plug. It is traditionally recognised that thrombogenesis correlated with the total platelet count, that is, an elevated number of platelets leads to a hypercoagulable state. Thus, most surgeons recognise the total platelet count as a critical index in the perioperative period of free-flap surgery with microvascular anastomosis. Similar to total platelet count, platelet volume is also a marker of platelet function and activation, which is readily measured as the mean platelet volume (MPV) by clinical haematology analysers that use sodium citrate as the anticoagulant. Although MPV has been included for decades in routine complete blood-counting measurements, it has not been routinely involved as a clinical monitoring index in patients undergoing free-flap surgery. Established as a technique nearly 30 years ago, MPV quantification has only recently been fully appreciated as a biomarker of cardiovascular (CV) disease, and there are strong links between an elevated MPV and increased coronary artery disease (CAD) risk. Increasing evidence demonstrates that MPV is likely an indicator of platelet activation and elevated in acute coronary syndromes. Potential reasons for this finding might be that MPV correlates with platelet function, as well as the fact that larger platelets have greater mass and are both metabolically and enzymatically more active than smaller platelets. Hence, increased MPV likely leads to a hypercoagulable state, which is an important risk factor for CAD. The abovementioned data relevant to MPV from the field of cardiology could bring novel inspirations to microvascular surgery: MPV might be a more important clinical index reflecting blood coagulability than total platelet count, which is traditionally emphasised in patients scheduled for free-flap surgery. However, limited data clarify the relationship between thrombogenesis and MPV after microvascular anastomotic surgeries. Moreover, it is still unknown whether the patients with elevated MPV should receive prophylactic applications of anticoagulant during the perioperative period rather than the postoperative period. To date there are no unified standards for the indication of anticoagulant therapy among patients who need free-flap surgery. Furthermore, different hospitals e even various surgeons in the same department e may have divergent opinions. On the basis of these abovementioned studies, we hypothesise that MPV and its dynamic variation during the operative period may be a more efficient criterion compared to total platelet count when attempting to characterise blood coagulability. It is necessary to monitor the MPV value in each patient scheduled to undergo free-flap surgery with vascular anastomosis. It is also necessary to include ‘high MPV’ as an indication for anticoagulant therapy not only before but also after microvascular operations. Further clinical research efforts are needed to test this hypothesis.

Effects of Sanguis Draconis on Perforator Flap Survival in Rats

Sanguis draconis, a resin known to improve blood circulation, relieve pain, stimulate tissue regeneration, and heal wounds, is widely used in clinical practice. In this study, we prepared an ethanol extract of sanguis draconis (EESD) containing 75.08 mg/g of dracorhodin. The experiment was carried out on 20 rats that were divided into two groups, a control group (n = 10) and an EESD group (n = 10). All the rats underwent a perforator flap surgery, after which post-operative abdominal compressions of EESD were given to the EESD group for seven days, while the control group received saline. Flap survival percentages were determined after seven days, and were found to be significantly higher in the EESD group than in the control group. Results of laser Doppler flowmetry (LDF) showed that perforator flaps in the EESD group had higher perfusion values than those of the control group. The flap tissues were stained with hematoxylin and eosin, followed by immunohistochemical evaluation. Superoxide dismutase (SOD) expression and micro-vessel development markedly increased in the EESD group, while malondialdehyde (MDA) levels decreased. This is the first study to investigate the effect of sanguis draconis on perforator flap survival. Our results demonstrate that sanguis draconis can improve perforator flap survival in rats by promoting microvessel regeneration and blood perfusion.