In vitro elution of gentamicin from Plaster of Paris beads

OBJECTIVE: To determine the in vitro elution characteristics of gentamicin from Plaster of Paris-gentamicin (POP-gent) beads. STUDY DESIGN: In vitro, controlled, experimental study. METHODS: The POP-gent beads were made using a bead mold from 20 g calcium sulfate hemihydrate, 5 mL (500 mg) gentamicin solution, and 3 mL of phosphate buffered saline (PBS). Control beads were made similarly, using 30 g of dried powder and 8 mL of PBS. Beads were left in the mold overnight, gas-sterilized with ethylene oxide, and stored at room temperature for 5 months before testing. Bead chains were placed in sterile tubes containing porcine serum, and tubes were placed in a 37 degrees C incubator on a rocker. Serum was removed at intervals over 14 days and the concentration of gentamicin determined by fluorescent polarization immunoassay. Serum antibacterial activity was determined against an equine origin Escherichia coli. RESULTS: POP-gent beads released gentamicin for the 14-day sampling period. Eighty percent of the gentamicin incorporated in the beads was released over the first 48 hours. Eluent from POP-gent beads inhibited the growth of E coli at all time periods. No gentamicin was eluted from control beads and control eluent did not inhibit growth of E coli. CONCLUSIONS: In this experimental model, POP-gent beads released bactericidal drug for 14 days. Eighty percent of the gentamicin incorporated into the beads was released during the first 48 hours. The drug retains its bactericidal activity after ethylene oxide sterilization and storage at room temperature for up to 5 months. CLINICAL RELEVANCE: Pop-gent beads may be a useful repository device to deliver gentamicin locally in tissues.     

In Vitro Elution and Antibacterial Activity of Clindamycin,Amikacin, and Vancomycin from R‐gel Polymer

Objective: To characterize the in vitro elution and bioactivity of 2 formulations of antibiotics in a novel, dissolvable, cross‐linked dextran polymer matrix: Formulation 1—amikacin and clindamycin (AC); Formulation 2—amikacin, clindamycin, and vancomycin (ACV). Study Design: Prospective, in vitro, experimental study. Methods: Aliquots of the antibiotic impregnated polymer were incubated in PBS buffer for 10 days. PBS was changed every 24 hours and concentrations of the antibiotics eluted into saline were quantified. Antimicrobial activity of the eluent from each sampling period was tested for growth inhibition of Staphylococcus aureus. Results: Both formulations of R‐gel^? had a rapid initial release of antibiotics within the first 24 hours and then the concentrations decreased gradually over 10 days. The concentration of amikacin, clindamycin, and vancomycin remained above the breakpoint minimum inhibitory concentration of each drug for a minimum of 9 days. No significant difference (P=.9938, P=.9843) was present in the elution pattern or total amount of antibiotic eluted from clindamycin or amikacin, respectively. Eluent from both groups demonstrated bioactivity against S. aureus for the entire 10‐day study period. Conclusions: Amikacin and clindamycin together, or in combination with vancomycin, elute from R‐gel^? effectively and at gradually decreasing concentrations for at least 10 days. The antibiotics maintained their bioactivity following polymerization and elution from the R‐gel^?.     

Elution of antimicrobials from a cross‐linked dextran gel: In vivo Quantification

Reasons for performing study: Use of a novel, biodegradable, antimicrobial‐impregnated gel provides an alternative method of local treatment of infections in horses. Objectives: To determine in vivo elution of antimicrobial medications from antimicrobial‐impregnated cross‐linked dextran gel and to evaluate the effect on wound healing when implanted subcutaneously in horses. Methods: Amikacin‐, vancomycin‐ or amikacin/clindamycin‐impregnated gel was placed subcutaneously in 11 horses' necks, using 6 replicates with a 3 month washout between experiments. Capillary ultrafiltration probes for collection of interstitial fluid were placed 0 cm and 1.5 cm from the gel‐filled incisions. Samples were collected at 0, 4, 8 and 12 h, and on Days 1–10. Blood was collected on Days 0, 1 and 7. Amikacin and vancomycin samples were analysed via fluorescence polarisation immunoassay, and clindamycin samples via high‐performance liquid chromatography. Histology of biopsy samples was performed at the completion of the study. Differences in mean histomorphological scores between groups were assessed using Wilcoxon's signed ranks test. Results: Maximum antimicrobial concentrations were detected at 4 h (amikacin), and 8 h (vancomycin, and amikacin and clindamycin from the combination gel). Mean ± s.d. peak concentrations for amikacin, vancomycin, amikacin (amikacin/clindamycin) and clindamycin were 6133 ± 1461, 7286 ± 2769, 3948 ± 317 and 985 ± 960, respectively. Median number of days for which antimicrobial concentration remained above minimum inhibitory concentration for target microorganisms at implantation was ≥10 days for vancomycin, 9 days (± 1) for amikacin and 8 days (± 1) for clindamycin. Mean plasma amikacin and vancomycin concentrations were lower than detectable limits; mean serum clindamycin concentrations were 0.52 µg/ml and 0.63 µg/ml at 24 h and 7 days, respectively. There were no significant differences in histomorphological scores between treatment and control incisions (P≥0.22). Conclusions and potential relevance: Cross‐linked dextran gel is a safe, effective alternative local antimicrobial delivery method.     

Elution of metronidazole and gentamicin from polymethylmethacrylate beads

OBJECTIVE: To characterize the elution and bioactivity of metronidazole and gentamicin sulfate polymerized, individually and in combination, with polymethylmethacrylate (PMMA). STUDY DESIGN: In vitro experimental study. METHODS: PMMA beads containing metronidazole (3 concentrations), gentamicin sulfate, or metronidazole and gentamicin sulfate were immersed in 5 mL of phosphate-buffered saline in triplicate. Eluent was replaced at specified time intervals for 1 or 21 days, and antibiotic concentrations were measured by high-performance liquid chromatography. Changes in antibiotic bioactivity attributable to polymerization or copolymerization of the antibiotics with PMMA, ethylene oxide sterilization, and storage of AIPMMA beads containing metronidazole were evaluated. RESULTS: Antibiotic elution patterns were similar for all groups. Day 1 elution for groups containing metronidazole or gentamicin individually represented a mean 63%-66% and 79%, respectively, of the 21-day total. Approximately 50% of the day 1 elution occurred during the first hour. The elution of metronidazole was dose dependent. The elution of metronidazole (day 3-21) and gentamicin (all days) was significantly greater when metronidazole and gentamicin were combined (P <.05). The addition of metronidazole delayed polymerization of PMMA. Neither polymerization nor copolymerization of metronidazole and gentamicin with PMMA, gas sterilization, or 2-month storage of beads containing metronidazole significantly affected antimicrobial bioactivity. CONCLUSIONS: Metronidazole elution from PMMA was dose dependent. Copolymerization of metronidazole and gentamicin sulfate in PMMA resulted in increased rates of elution. Intraoperative preparation of metronidazole-impregnated PMMA beads is not practical, but sterilization and storage for 2 months should not affect efficacy. CLINICAL RELEVANCE: The local delivery of biologically active metronidazole and gentamicin by elution from PMMA is feasible.     

阿莫西林与阿米卡星对沙门氏菌的体外联合药敏试验

采用阿莫西林与阿米卡星联用对沙门氏菌进行药物敏感性试验,结果表明阿莫西林、阿米卡星联用对沙门氏菌呈现协同作用.     

In Vitro and In Vivo Evaluation of Ferric-Hyaluronate Implants for Delivery of Amikacin Sulfate to the Tarsocrural Joint of Horses

Objective— To assess the antimicrobial elution characteristics, toxicity, and antimicrobial activity of amikacin‐impregnated ferric‐hyaluronate implants (AI‐FeHAI) for amikacin delivery to the tarsocrural joint of horses. Study Design— Experimental study. Sample Population— AI‐FeHAI implants, equine cartilage, and synovium, and horses (n=6). Methods— In vitro study: Five AI‐FeHAI were placed in saline solution with daily replacement until implant degradation. Eluent was tested for amikacin concentration and bioactivity. Synovial and cartilage explants were incubated in the presence or absence of AI‐FeHAI for 72 hours and subsequently assessed for morphology, viability, and composition. Synovial explants were incubated with Staphylococcus aureus in the presence or absence of AI‐FeHAI. Spent medium was cultured daily and explants were assessed for morphology and viability after 96 hours. In vivo study: AI‐FeHAI were placed in 6 tarsocrural joints. Standard cytologic analysis and amikacin concentration (SFAC) were determined in synovia obtained regularly for 28 days thereafter. Similar analyses were conducted after a single intra‐articular injection of amikacin 6 months later. Results— In vitro study: Amikacin concentrations exceeded 16 μg/mL and inhibited S. aureus growth for 8 days. AI‐FeHAI had no effect on cartilage explants. AI‐FeHAI eliminated bacteria from synovial explants. In vitro study: After AI‐FeHAI placement, SFAC was highest (140.78+63.81 μg/mL) at first sampling time. By 24 hours SFAC was <16 μg/mL. After intra‐articular injection, SFAC was the highest (377.91 ± 40.15 μg/mL) at first sampling time. By 48 hours SFAC was <16 μg/mL. Conclusions— A single intra‐articular amikacin injection demonstrated superior pharmacokinetics than AI‐FeHAI prepared as described. Clinical Relevance— AI‐FeHAI cannot be recommended for clinical use.     

In vitro elution studies of amikacin and cefazolin from polymethylmethacrylate

OBJECTIVE: To compare the in vitro elution characteristics of amikacin and cefazolin from polymethylmethacrylate (PMMA) alone and in combination. STUDY DESIGN: A prospective, controlled, experimental study. METHODS: Three aliquots of 6 g sterile PMMA were measured and to them added (1) 750 mg amikacin; (2) 1050 mg cefazolin; and (3) 750 mg amikacin and 1050 mg cefazolin. Ten beads of each antimicrobial/PMMA combination were placed in 5 mL phosphate-buffered saline (PBS) at pH 7.4 and room temperature with constant agitation. PBS was sampled at 15 time points between 1 hour and 30 days. Amikacin concentrations were determined by fluorescence polarization immunoassay and cefazolin concentrations by high-performance liquid chromatography. RESULTS: Amikacin and cefazolin eluted at concentrations greater than 8 and 4 times, respectively, above the minimum inhibitory concentration (MIC) for susceptible bacteria over 30 days. Co-elution of the antibiotics resulted in a greater rate and proportion of antibiotic eluted. Concentrations of amikacin and cefazolin in the co-eluted fluid were not maintained sufficiently above the MIC for selected bacteria over 30 days. CONCLUSIONS: PMMA beads of only amikacin or cefazolin-eluted concentrations greater than the MIC for selected bacteria for 30 days. Co-elution of the antibiotics at the selected doses resulted in a significantly shorter duration of elution and may not be effective for treatment of wound infection. CLINICAL RELEVANCE: Co-elution of amikacin and cefazolin from PMMA at the selected doses cannot be recommended for sustained treatment of infection.     

In vitro migration responses of neutrophils from cows and calves

The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P less than or equal to 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P less than or equal to 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.     

The Effects of Plaster of Paris and Autogenous Cancellous Bone on the Healing of Cortical Defects in the Femurs of Dogs

Four one quarter inch evenly spaced circular defects were created bilaterally in the lateral femoral diaphysis of 12 clinically normal adult dogs. The defects were left unfilled (control), or were filled with one of the following: (1) plaster of Paris, (2) an equal-volume mixture of plaster of Paris and autogenous cancellous bone, and (3) autogenous cancellous bone. The degree of bone healing was evaluated radiographically and histologically at 2, 4, 6, 8, 10, and 12 weeks. Radiographically, no objective conclusions could be drawn due to the small size of the defects and limited amount of plaster of Paris implanted. Histologically, there was no inflammatory reaction to the plaster of Paris. No differences were determined in the degree of bone healing between autogenous cancellous bone, plaster of Paris, and a mixture of plaster of Paris and autogenous cancellous bone. All implants were superior to the control defect in degree of bone healing.     

大鼠大脑皮质星形胶质细胞体外分离培养和纯化

星形胶质细胞是胶质细胞中数量最多、分布最片的细胞亚群，担负着胶质细胞的大部分功能，参与多种神经系统疾病病理过程。研究星形胶质细胞的反应对阐明动物多种神经系统疾病的病理发生和分子作用机制具有重要意义。但星形胶质细胞在体内与其他细胞混杂在一起，研究其病理性反应，必须对其进行分离和纯化，     

In vitro characteristics of normal and dystrophic skeletal muscle from dogs

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (post-fusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.     

In vitro fermentation of feces from normal and chronically diarrheal horses

Feces from 13 healthy horses and 8 horses with chronic diarrhea were subjected to an in vetro fermentation procedure that had been developed for rumen fluid. Fermentations were conducted over 6 hours in a closed system, with and without an essential amino acid (EAA) mixture being added to the basic starch-buffer medium. The addition of EAA caused no significant difference in results of fermentation of feces from healthy horses. For diarrheic animals, there was a significant (P less than 0.01) increase in gas and total volatile fatty acids production whether EAA were present or not, and alpha-amino nitrogen was utilized in significantly (P less than 0.01) greater amounts only if EAA was present. Fermentations were repeated on feces from five of the eight diarrheal horses after they had been treated with oral iodochlorhydroxyquin for 1 week, and had shown desirable clinical response. A significant difference was not shown between pre- and posttreatment fermentations, except for decreased butyrate production. The results are consistent with the hypothesis that chronic equine diarrhea is primarily a colonic disease and indicates that colonic maldigestion may, in part, be responsible for excess fecal water.     

Minimum Inhibitory Concentration and Postantibiotic Effect of Amikacin for Equine Isolates of Methicillin-Resistant Staphylococcus aureus In Vitro

Objective— To report the minimum inhibitory concentration (MIC) of amikacin sulfate for equine clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and characterize the initial kill and duration of the postantibiotic effect (PAE) for selected strains.
Study Design— Experimental study.
Methods— Isolates of MRSA (n=35) had their amikacin MIC determined using the E-test agar diffusion method. Two isolates with MICs>256 μg/mL limit were further characterized using broth macrodilution. Six distinct isolates with amikacin MICs of 32, 48, 128 (2 isolates) and 500 (2 isolates) μg/mL had PAE determinations made over a range of amikacin concentrations from 31.25–1000 μg/mL using standard culture-based techniques.
Results— Median MIC of the 35 isolates was 32 μg/mL (range 2 to >256 μg/mL). Mean PAE of selected MRSA strains had an overall mean (all amikacin doses) of 3.43 hours (range 0.10–9.57 hours). PAE for MRSA exposed to amikacin at 1000 μg/mL was 6.18 hours (range 3.30–9.57 hours), significantly longer than that for all other concentrations ( P <.0001). There was no statistically significant effect of isolate MIC on PAE.
Conclusions— Isolates had a wide range of MIC; however, growth of all 6 selected strains were inhibited within the range of concentrations tested, including 2 strains with MICs of 500 μg/mL. PAE duration was not influenced by the MIC of amikacin but was significantly longer with treatment at 1000 μg/mL than at lower concentrations.
Clinical Relevance— Clinical isolates of MRSA are susceptible to amikacin at concentrations achieved by regional perfusion: however, the modest duration of PAE observed suggest that further laboratory and in vivo evaluation be conducted before recommending the technique for clinical use.     

陆川猪耳部成纤维细胞的体外培养

本研究建立陆川猪耳部成纤维细胞的体外培养体系,采用组织块培养法可以获得陆川猪耳部成纤维细胞。用0.25%胰蛋白酶＋0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,能很好的支持陆川猪耳部成纤维细胞的生长。传3代后,观察到培养的细胞形态逐渐均一,为典型的成纤维细胞,绝大部分呈梭形或不规则三角形...