Facilitating GL13K Peptide Grafting on Polyetheretherketone via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide: Surface Properties and Antibacterial Activity.

In the present work, the antimicrobial peptide (AMP) of GL13K was successfully coated onto a polyetheretherketone (PEEK) substrate to investigate its antibacterial activities against Staphylococcus aureus (S. aureus) bacteria. To improve the coating efficiency, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was mixed with a GL13K solution and coated on the PEEK surface for comparison. Both energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) data confirmed 30% greater peptide coating on PEEK/GL13K-EDC than PEEK without EDC treatment. The GL13K graft levels are depicted in the micrograms per square centimeter range. The PEEK/GL13K-EDC sample showed a smoother and lower roughness (Rq of 0.530 µm) than the PEEK/GL13K (0.634 µm) and PEEK (0.697 µm) samples. The surface of the PEEK/GL13K-EDC was more hydrophilic (with a water contact angle of 24°) than the PEEK/GL13K (40°) and pure PEEK (89°) samples. The pure PEEK disc did not exhibit any inhibition zone against S. aureus. After peptide coating, the samples demonstrated significant zones of inhibition: 28 mm and 25 mm for the PEEK/GL13K-EDC and PEEK/GL13K samples, respectively. The bacteria-challenged PEEK sample showed numerous bacteria clusters, whereas PEEK/GL13K contained a little bacteria and PEEK/GL13K-EDC had no bacterial attachment. The results confirm that the GL13K peptide coating was able to induce antibacterial and biofilm-inhibitory effects. To the best of our knowledge, this is the first report of successful GL13K peptide grafting on a PEEK substrate via EDC coupling. The present work illustrates a facile and promising coating technique for a polymeric surface to provide bactericidal activity and biofilm resistance to medical implantable devices.

Preclinical experiments on the release behavior of biodegradable nanofibrous multipharmaceutical membranes in a model of four-wall intrabony defect.

Guided tissue regeneration (GTR) therapy has been widely used to regenerate lost periodontium from periodontal disease. However, in terms of regenerative periodontal therapy, a multidrug-loaded biodegradable carrier can be even more promising in dealing with periodontal disease. In the current study, we fabricated biodegradable nanofibrous collagen membranes that were loaded with amoxicillin, metronidazole, and lidocaine by an electrospinning technique. The in vitro release behavior and the cytotoxicity of the membranes were investigated. A four-wall intrabony defect was created in rabbits for in vivo release analysis. The bioactivity of the released antibiotics was also examined. The experimental results showed that the drug-loaded collagen membranes could provide sustainable release of effective amoxicillin, metronidazole, and lidocaine for 28, 56, and 8 days, respectively, in vivo. Furthermore, the bioactivity of the released antibiotics remained high, with average bioactivities of 50.5% for amoxicillin against Staphylococcus aureus and 58.6% for metronidazole against Escherichia coli. The biodegradable nanofibrous multipharmaceutical membranes developed in this study may provide a promising solution for regenerative periodontal therapy.

The influence of storage temperature on the antibiotic release of vancomycin-loaded polymethylmethacrylate.

Periprosthetic joint infection is devastating and increases medical expenditure and socioeconomic burden. Antibiotic-loaded cement spacer is useful in the interim period before the reimplantation surgery. Prefabricated antibiotic-loaded cement spacers can decrease operation time but have been limitedly used clinically. In the literature, there is no clear recommendation on the storage temperature for the prefabricated cement spacers. We used an in vitro model to analyze whether the storage temperature at 25°C, 4°C, or -20°C for 2 weeks or 3 months could affect the release of vancomycin from the cement. We found that the storage temperature and time had no significant effects on the pattern and amount of vancomycin release. The patterns of vancomycin release from the cement stored at different temperatures were similar with an abrupt release in the first 3 days and steadily declined in the following period. This study provides a preliminary result to justify the storage of fabricating antibiotic-loaded cement spacer sterilely packed at room temperature. Further studies to examine the effects of storage temperature on the mechanical strength and the release pattern of other antibiotics should be done to provide more evidence to support the clinical use of prefabricated ready-to-use antibiotic-loaded cement spacer.

In vitro activities of daptomycin-, vancomycin-, and teicoplanin-loaded polymethylmethacrylate against methicillin-susceptible, methicillin-resistant, and vancomycin-intermediate strains of Staphylococcus aureus.

The objective of this study was to evaluate the antibacterial effects of polymethylmethacrylate (PMMA) bone cements loaded with daptomycin, vancomycin, and teicoplanin against methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-intermediate Staphylococcus aureus (VISA) strains. Standardized cement specimens made from 40 g PMMA loaded with 1 g (low-dose), 4 g (middle-dose) or 8 g (high-dose) antibiotics were tested for elution characteristics and antibacterial activities. The patterns of release of antibiotics from the cement specimens were evaluated using in vitro broth elution assay with high-performance liquid chromatography. The activities of broth elution fluid against different Staphylococcus aureus strains (MSSA, MRSA, and VISA) were then determined. The antibacterial activities of all the tested antibiotics were maintained after being mixed with PMMA. The cements loaded with higher dosages of antibiotics showed longer elution periods. Regardless of the antibiotic loading dose, the teicoplanin-loaded cements showed better elution efficacy and provided longer inhibitory periods against MSSA, MRSA, and VISA than cements loaded with the same dose of vancomycin or daptomycin. Regarding the choice of antibiotics for cement loading in the treatment of Staphylococcus aureus infection, teicoplanin was superior in terms of antibacterial effects.

In vitro and in vivo investigation of drug-eluting implants for the treatment of periodontal disease.

This paper developed solvent-free drug-eluting implants for metronidazole delivery for the treatment of periodontal disease and investigated the characteristics of the drug's release from the implants, both in vitro and in vivo, using an HPLC assay. The metronidazole exhibited a two-stage release behavior in vitro with an initial burst release followed by a diffusion-controlled release and then a secondary burst release. The accumulated drug release reached 100% on the 18th day, and the drug-eluting implant was totally dissolved on the same day. Additionally, the drug-eluting disks were implanted within the sub-gingival space of both lower incisors of six rabbits. The curve of in vivo drug release was smoother and showed a predominantly diffusion-controlled release. The implants were totally dissolved at 2 weeks after implantation. The concentration of metronidazole remained above the MIC(90) during the entire investigation.

Engineered periosteum-bone biomimetic bone graft enhances posterolateral spine fusion in a rabbit model.

BACKGROUND CONTEXT: Bone marrow derived mesenchymal stem cells (BMSCs) and periosteum-derived cells (PDCs) have shown great viability in terms of osteogenic potential and have been considered the major cellular source for skeletal tissue engineering. Using a PDCs-impregnated cell sheet to surround a BMSCs-impregnated tricalcium phosphate (TCP) scaffold might create a periosteum-bone biomimetic bone graft substitute to enhance spine fusion. PURPOSE: The purpose of this study was to determine the feasibility of using this newly tissue-engineered biomimetic bone graft for posterolateral spine fusion. STUDY DESIGN/SETTING: This study design was based on an animal model using adult male New Zealand White rabbits. METHODS: New Zealand White rabbits underwent operation and were divided into three groups based on the experimental material implanted in the bilateral L4-L5 intertransverse space. Group 1 was BMSCs-free TCP wrapped in a PDCs-free cell sheet. Group 2 was BMSCs-loaded-TCP wrapped in a PDCs-free cell sheet. Group 3 was BMSCs-loaded-TCP wrapped in a PDCs-loaded cell sheet. After 12 weeks, six rabbits from each group were euthanized for computed tomography scanning, manual palpation, biomechanical testing, and histology. Each group had 12 radiographic fusion areas for analysis because the right and left intertransverse fusion areas were collected separately. RESULTS: Radiographic union of 12 fusion areas for groups 1, 2, and 3 was 0, 3, and 9, respectively. Group 3 had significantly higher fusion success than groups 1 and 2 (p<.001). Solid fusion of six fusion segments in each group by manual palpation was 0, 1, and 5, accordingly. Group 3 had a higher successful solid fusion rate than groups 1 and 2 (p=.005). The average maximal torques at failure were 727±136 N mm, 627±91 N mm, and 882±195 N mm for groups 1, 2, and 3, accordingly. The maximal torque was significantly higher in group 3 than in group 2 (p=.028). Histological evaluation verified that new bone regeneration were greater in the group 3 samples. CONCLUSIONS: The results indicated the potential of using a PDCs-impregnated cell sheet to surround the BMSCs-impregnated TCP scaffold for creating a periosteum-bone biomimetic bone graft substitute to enhance bone regeneration and posterolateral fusion success.

Biodegradable drug-eluting nanofiber-enveloped implants for sustained release of high bactericidal concentrations of vancomycin and ceftazidime: in vitro and in vivo studies.

We developed biodegradable drug-eluting nanofiber-enveloped implants that provided sustained release of vancomycin and ceftazidime. To prepare the biodegradable nanofibrous membranes, poly(D,L)-lactide-co-glycolide and the antibiotics were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into biodegradable drug-eluting membranes, which were then enveloped on the surface of stainless plates. An elution method and a high-performance liquid chromatography assay were employed to characterize the in vivo and in vitro release rates of the antibiotics from the nanofiber-enveloped plates. The results showed that the biodegradable nanofiber-enveloped plates released high concentrations of vancomycin and ceftazidime (well above the minimum inhibitory concentration) for more than 3 and 8 weeks in vitro and in vivo, respectively. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The bioactivity ranged from 25% to 100%. In addition, the serum creatinine level remained within the normal range, suggesting that the high vancomycin concentration did not affect renal function. By adopting the electrospinning technique, we will be able to manufacture biodegradable drug-eluting implants for the long-term drug delivery of different antibiotics.

Novel biodegradable sandwich-structured nanofibrous drug-eluting membranes for repair of infected wounds: an in vitro and in vivo study.

BACKGROUND: The purpose of this study was to develop novel sandwich-structured nanofibrous membranes to provide sustained-release delivery of vancomycin, gentamicin, and lidocaine for repair of infected wounds. METHODS: To prepare the biodegradable membranes, poly(D, L)-lactide-co-glycolide (PLGA), collagen, and various pharmaceuticals, including vancomycin, gentamicin, and lidocaine, were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into sandwich-structured membranes with PLGA/collagen as the surface layers and PLGA/drugs as the core. An elution method and a high-pressure liquid chromatography assay were used to characterize in vivo and in vitro drug release from the membranes. In addition, repair of infected wounds in rats was studied. Histological examination of epithelialization and granulation at the wound site was also performed. RESULTS: The biodegradable nanofibrous membranes released large amounts of vancomycin and gentamicin (well above the minimum inhibition concentration) and lidocaine in vivo for more than 3 weeks. A bacterial inhibition test was carried out to determine the relative activity of the antibiotics released. The bioactivity ranged from 40% to 100%. The nanofibrous membranes were functionally active in treating infected wounds, and were very effective as accelerators in early-stage wound healing. CONCLUSION: Using the electrospinning technique, we will be able to manufacture biodegradable, biomimetic, nanofibrous, extracellular membranes for long-term delivery of various drugs.

Sustainable release of vancomycin, gentamicin and lidocaine from novel electrospun sandwich-structured PLGA/collagen nanofibrous membranes.

This study investigated the in vitro release of vancomycin, gentamicin, and lidocaine from novel electrospun sandwich-structured polylactide-polyglycolide (PLGA)/collagen nanofibrous membranes. For the electrospinning of biodegradable membranes, PLGA/collagen and PLGA/vancomycin/gentamicin/lidocaine were separately dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). They were then electrospun into sandwich structured membranes, with PLGA/collagen for the surface layers and PLGA/drugs for the core layer. After electrospinning, an elution method and HPLC assay were employed to characterize the in vitro release rates of the pharmaceutics over a 30-day period. The experiment showed that biodegradable nanofibrous membranes released high concentrations of vancomycin and gentamicin (well above the minimum inhibition concentration) for 4 and 3 weeks, respectively, and lidocaine for 2 weeks. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The bioactivity of vancomycin and gentamicin ranged from 30% to 100% and 37% to 100%, respectively. In addition, results indicated that the nanofibrous membranes were functionally active in responses in human fibroblasts. By adopting the electrospinning technique, we will be able to manufacture biodegradable biomimetic nanofibrous extracellular membranes for long-term drug delivery of various pharmaceuticals.

A double antigen bridging immunogenicity ELISA for the detection of antibodies to polyethylene glycol polymers.

INTRODUCTION: Polyethylene glycol (PEG) polymers attached to biotherapeutic molecules enhance in vivo delivery and stability of these large molecular weight drugs. However, these polymers may by themselves be immunogenic and elicit antibodies that can reduce the efficacy of the drug and contribute to potential patient morbidity. A double antigen bridging ELISA immunogenicity assay for the detection of anti-drug antibodies (ADAs) specific to PEG polymers of various sizes has been developed. METHODS: Hapten-labeled conjugate of 40kDa PEG polymer was synthesized and used in a double antigen bridging ELISA. The hapten-labeled PEG is incubated with the patient sample, then this mixture is added to a 96-well microplate precoated with 40kDa PEG, allowing PEG-specific ADA to form a bridge complex with the PEG conjugate and the PEG coated on the microplate. After incubation, the reaction mixture is removed and replaced by horseradish peroxidase (HRP)-labeled anti-hapten antibody. After sufficient incubation, the plate is washed and substrate reagent is added. Enzyme color development, directly proportional to ADA, is stopped after 20min with 2N sulfuric acid and the absorbance in each well is measured at 450/630nm. Dose response, drug tolerance, matrix effects, reproducibility, specificity/free drug depletion experiments and screening cut-point determination of 350 naïve normal human sera were performed. RESULTS: Using an anti-PEG mouse monoclonal IgM as a positive control, a reproducible dose response curve was demonstrated for the PEG Immunogenicity ELISA. Pre-existing PEG-specific antibodies which were proven to be highly specific to the PEG polymer structure were found in 15 human serum samples in a total population of 350 naïve donors. The assay exhibited no significant matrix effects and was shown to be highly reproducible. DISCUSSION: A double antigen bridging immunogenicity assay for the detection of antibodies to PEG in the typical polymer size ranges used in biotherapeutics has been successfully developed in ELISA format. The antibodies detected in positive samples displayed a diverse spectrum of specificities for different PEG polymer lengths and linking functional groups. The discovery of 15 confirmed positive samples among 350 naïve patient samples calls into focus the need for testing PEG-specific immunogenicity of PEGylated biotherapeutics.