Carbon quantum dots-embedded electrospun antimicrobial and fluorescent scaffold for reepithelialization in albino wistar rats.

A prosthetic scaffold development using fluorescent nanofiber is reported for an enhanced reepithelialization in wistar albino rats. In this study, a novel approach was followed to construct the biocompatible fluorescent nanofiber that will be helpful to monitor the tissue regeneration process. Here, a multifunctional carbon quantum dots (CQDs)-embedded electrospun polyacrylonitrile (PAN) nanofiber was fabricated and characterized using standard laboratory techniques. The biodegradation ability was assessed by simulated body fluid thereby analyzing porosity and water absorption capacity of the material. The fluorescent scaffold was tested for cytotoxicity and antimicrobial activity using bacterial and fibroblast cells and fluorescent stability was analyzed by bioimaging of animal and bacterial cells. Tissue regeneration capability of the developed scaffold was evaluated using wistar albino rats. Unlike biomicking scaffolds, the CQDs-embedded PAN-based substrate has given dual support by enhancing reepithelialization without growth factors and acted as an antimicrobial agent to provide contamination free tissue regeneration. Scaffolds were examined by using histostaining techniques and scanning electron microscopy to observe the reepithelialization in the regenerated tissues. The novel approach for developing infection free soft tissue regeneration was found to be phenomenal in scaffold development.

Evaluation of the temporary effect of physical vapor deposition silver coating on resistance to infection in transdermal skin and bone integrated pylon with deep porosity.

Periprosthetic infection via skin-implant interface is a leading cause of failures and revisions in direct skeletal attachment of limb prostheses. Implants with deep porosity fabricated with skin and bone integrated pylons (SBIP) technology allow for skin ingrowth through the implant's structure creating natural barrier against infection. However, until the skin cells remodel in all pores of the implant, additional care is required to prevent from entering bacteria to the still nonoccupied pores. Temporary silver coating was evaluated in this work as a means to provide protection from infection immediately after implantation followed by dissolution of silver layer in few weeks. A sputtering coating with 1 µm thickness was selected to be sufficient for fighting infection until the deep ingrowth of skin in the porous structure of the pylon is completed. In vitro study showed less bacterial (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) growth on silver coated tablets compared to the control group. Analysis of cellular density of MG-63 cells, fibroblasts, and mesenchymal stem cells (MSCs) showed that silver coating did not inhibit the cell growth on the implants and did not affect cellular functional activity. The in vivo study did not show any postoperative complications during the 6-month observation period in the model of above-knee amputation in rabbits when SBIP implants, either silver-coated or untreated were inserted into the bone residuum. Three-phase scintigraphy demonstrated angiogenesis in the pores of the pylons. The findings suggest that a silver coating with well-chosen specifications can increase the safety of porous implants for direct skeletal attachment. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 169-177, 2019.

Biomimetic 3D in vitro model of biofilm triggered osteomyelitis for investigating hematopoiesis during bone marrow infections.

In this work, we define the requirements for a human cell-based osteomyelitis model which overcomes the limitations of state of the art animal models. Osteomyelitis is a severe and difficult to treat infection of the bone that develops rapidly, making it difficult to study in humans. We have developed a 3D in vitro model of the bone marrow, comprising a macroporous material, human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Inclusion of biofilms grown on an implant into the model system allowed us to study the effects of postoperative osteomyelitis-inducing bacteria on the bone marrow. The bacteria influenced the myeloid differentiation of HSPCs as well as MSC cytokine expression and the MSC ability to support HSPC maintenance. In conclusion, we provide a new 3D in vitro model which meets all the requirements for investigating the impact of osteomyelitis. STATEMENT OF SIGNIFICANCE: Implant-associated osteomyelitis is a persistent bacterial infection of the bone which occurs in many implant patients and can result in functional impairments or even entire loss of the extremity. Nevertheless, surprisingly little is known on the triangle interaction between implant material, bacterial biofilm and affected bone tissue. Closing this gap of knowledge would be crucial for the fundamental understanding of the disease and the development of novel treatment strategies. For this purpose, we developed the first biomaterial-based system that is able to mimic implant-associated osteomyelitis outside of the body, thus, opening the avenue to study this fatal disease in the laboratory.

Understanding long-term silver release from surface modified porous titanium implants.

Prevention of orthopedic device related infection (ODRI) using antibiotics has met with limited amount of success and is still a big concern during post-surgery. As an alternative, use of silver as an antibiotic treatment to prevent surgical infections is being used due to the well-established antimicrobial properties of silver. However, in most cases silver is used in particulate form with wound dressings or with short-term devices such as catheters but not with load-bearing implants. We hypothesize that strongly adherent silver to load-bearing implants can offer longer term solution to infection in vivo. Keeping that in mind, the focus of this study was to understand the long term release study of silver ions for a period of minimum 6months from silver coated surface modified porous titanium implants. Implants were fabricated using a LENS™ system, a powder based additive manufacturing technique, with at least 25% volume porosity, with and without TiO2 nanotubes in phosphate buffer saline (pH 7.4) to see if the total release of silver ions is within the toxic limit for human cells. Considering the fact that infection sites may reduce the local pH, silver release was also studied in acetate buffer (pH 5.0) for a period of 4weeks. Along with that, the osseointegrative properties as well as cytotoxicity of porous titanium implants were assessed in vivo for a period of 12weeks using a rat distal femur model. In vivo results indicate that porous titanium implants with silver coating show comparable, if not better, biocompatibility and bonding at the bone-implant interface negating any concerns related to toxicity related to silver to normal cells. The current research is based on our recently patented technology, however focused on understanding longer-term silver release to mitigate infection related problems in load-bearing implants that can even arise several months after the surgery. STATEMENT OF SIGNIFICANCE: Prevention of orthopedic device related infection using antibiotics has met with limited success and is still a big concern during post-surgery. Use of silver as an antibiotic treatment to prevent surgical infections is being explored due to the well-established antimicrobial properties of silver. However, in most cases silver is used in particulate form with wound dressings or with short-term devices such as catheters but not with load-bearing implants. We hypothesize that strongly adherent silver to load-bearing implants can offer longer-term solution towards infection in vivo. Keeping that in mind, the focus of this study was to understand the long-term release of silver ions, for a period of minimum 6months, from silver coated surface modified porous titanium implants.

Atomic Layer Deposition of a Silver Nanolayer on Advanced Titanium Orthopedic Implants Inhibits Bacterial Colonization and Supports Vascularized de Novo Bone Ingrowth.

Joint replacement surgery is associated with significant morbidity and mortality following infection with either methicillin-resistant Staphylococcus aureus (MRSA) or Staphylococcus epidermidis. These organisms have strong biofilm-forming capability in deep wounds and on prosthetic surfaces, with 103 -104 microbes resulting in clinically significant infections. To inhibit biofilm formation, we developed 3D titanium structures using selective laser melting and then coated them with a silver nanolayer using atomic layer deposition. On bare titanium scaffolds, S. epidermidis growth was slow but on silver-coated implants there were significant further reductions in both bacterial recovery (p < 0.0001) and biofilm formation (p < 0.001). MRSA growth was similarly slow on bare titanium scaffolds and not further affected by silver coating. Ultrastructural examination and viability assays using either human bone or endothelial cells, demonstrated strong adherence and growth on titanium-only or silver-coated implants. Histological, X-ray computed microtomographic, and ultrastructural analyses revealed that silver-coated titanium scaffolds implanted into 2.5 mm defects in rat tibia promoted robust vascularization and conspicuous bone ingrowth. We conclude that nanolayer silver of titanium implants significantly reduces pathogenic biofilm formation in vitro, facilitates vascularization and osseointegration in vivo making this a promising technique for clinical orthopedic applications.

Competitive colonization of prosthetic surfaces by staphylococcus aureus and human cells.

Implantation of a biomaterial provides an adhesion substratum both to host cell integration and to contaminating bacteria. We studied simultaneous competitive adhesion of Staphylococcus aureus in serial 1:10 dilutions of 108 colony forming units (CFU)/mL and human osteogenic sarcoma (SaOS-2) or primary osteoblast (hOB) cells, both 1x105 cells/mL, to the surfaces of titanium, polydimethylsiloxane and polystyrene. The bacterial adherence and human cell proliferation, cytotoxicity and production of reactive oxygen species (ROS) were studied using fluorometric (fluorescent microscopy and flow cytometry) and colorimetric methods (MTT, LDH and crystal violet). The bacterial cell viability was also evaluated using the drop plate method. The presence of bacteria resulted in reduced adherence of human cells to the surface of the biomaterials, increased production of ROS, and into increased apoptosis. On the other hand, the presence of either type of human cells was associated with a reduction of bacterial colonization of the biomaterial with Staphylococcus aureus. These results suggest that increasing colonization of the biomaterial surface in vitro by one negatively affects colonization by the other. Host cell integration to an implant surface reduces bacterial contamination, which opens novel opportunities for the design of infection-resistant biomaterials in current implantology and future regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 62-72, 2017.

Brain abscess in a rhesus macaque (Macaca mulatta) with a cephalic implant.

We report a case of brain abscess after craniotomy and the placement of a recording chamber for electrophysiologic records in an adult rhesus macaque (Macaca mulatta) enrolled in visual research. Approximately 2 wk after surgery, the macaque presented with nonspecific gastrointestinal signs and showed no evidence of fever, neurologic deficits, increased intracranial pressure, suggestive alterations in the CBC, or abnormal changes in the recording chamber. The macaque responded to symptomatic and antibiotic treatment and showed no behavioral or abnormal clinical signs for 3 wk before collapsing suddenly. The macaque was euthanized, and pathologic evaluation revealed a large brain abscess immediately under the original craniotomy.

Endotoxin contamination delays the foreign body reaction.

Biomaterials are at continuous risk of bacterial contamination during production and application. In vivo, bacterial contamination of biomaterials delays the foreign body reaction (FBR). Endotoxins such as lipopolysaccharides (LPS), major constituents of the bacterial cell wall, are potent stimulators of the immune system in vitro and in vivo. In vitro, biomaterials contaminated with LPS induce the production of proinflammatory cytokines by adherent macrophages. This suggests that the presence of endotoxins on biomaterials will intensify the FBR. The effects of LPS on the course of the FBR have never been studied in vivo. In this study, the influence of LPS contamination on the FBR to subcutaneously implanted Puramatrix-loaded hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) disks was studied in rats. During the onset phase of the FBR, a massive influx of granulocytes was detected in LPS-contaminated disks, while their presence was prolonged. IL-10 production inside LPS-contaminated disks was increased at days 10 and 21. Macrophage densities were not affected by the presence of LPS. However, macrophage functionality was altered: giant cell formation and biomaterial degradation were delayed by LPS-contamination up to 21 days. On the basis of these results, we conclude that LPS delays the FBR. This finding indicates that endotoxin contamination has significant implications for the in vivo function of biomaterials and medical devices and emphasizes the importance of endotoxin testing.

Cyclodextrin and maltodextrin finishing of a polypropylene abdominal wall implant for the prolonged delivery of ciprofloxacin.

The aim of this work was to develop a polypropylene (PP) artificial abdominal wall implant for the prolonged release of ciprofloxacin (CFX). This sustained release effect was obtained by functionalization of the textile mesh with citric acid and hydroxypropyl-γ-cyclodextrin (HPγCD) or maltodextrin (MD). In both cases the textile finishing reaction yielded a cyclo- or malto-dextrin crosslinked polymer coating the fibers. The modified supports were characterized by thermogravimetric analysis (TGA), differential scanning calorimetry and scanning electron microscopy. The sorption capacities and the kinetics of CFX release were studied by batch tests coupled with spectrophotometric assays. Microbiological assays were carried out on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, while proliferation and viability tests used fibroblasts. The main results were as follows. (i) Due to the differences between the range of temperature of thermal degradation of the (cyclo)dextrins polymers and of the PP fibers TGA was a reliable method for quantifying the degree of functionalization of the textiles. (ii) Both modified supports showed improved sorption/desorption capacities for CFX, compared with the virgin mesh. The HPγCD-finished support showed an increased sorption capacity and a lower release rate of CFX compared with the MD modified support. (iii) Microbiological assays confirmed the latter result, with greater sustained antibacterial activity of the HPγCD treated support. These experiments have demonstrated the role of the cyclodextrin cavity in interactions with CFX: the antibiotic was not only adsorbed via hydrogen and acid-base interactions with the polyCTR-HPγCD network, but also via host-guest complexation. (iv) Biological tests revealed a slight decrease in fibroblast proliferation after 6 days on the modified supports, but cell viability tests showed that this was not due to toxicity of the (cyclo)dextrin polymer coatings.

The risk of biomaterial-associated infection after revision surgery due to an experimental primary implant infection.

The fate of secondary biomaterial implants was determined by bio-optical imaging and plate counting, after antibiotic treatment of biomaterials-associated-infection (BAI) and surgical removal of an experimentally infected, primary implant. All primary implants and tissue samples from control mice showed bioluminescence and were culture-positive. In an antibiotic treated group, no bioluminescence was detected and only 20% of all primary implants and no tissue samples were culture-positive. After revision surgery, bioluminescence was detected in all control mice. All the implants and 80% of all tissue samples were culture-positive. In contrast, in the antibiotic treated group, 17% of all secondary implants and 33% of all tissue samples were culture-positive, despite antibiotic treatment. The study illustrates that due to the BAI of a primary implant, the infection risk of biomaterial implants is higher in revision surgery than in primary surgery, emphasizing the need for full clearance of the infection, as well as from surrounding tissues prior to implantation of a secondary implant.

Effect of age on cytokine response in an experimental model of osteomyelitis.

To study the effect of age on cytokine response in an experimental model of osteomyelitis. Forty adult male Wistar rats received a stainless steel needle, intramedullarly in the left tibia. Young rats (3 months old) and old rats (22 months old) were allotted in: Group A: Sterile implant. Group B: Sterile implant + slime producing S. aureus. Rats were sacrificed 9 weeks after surgery. Determinations: Cytokines (ELISA) in blood and in tibia extract and the number of bacteria in tibia and implant. The Wilcoxon, Mann-Whitney U tests were used (P < or = 0.01 significant). Infection was detected in every old rat receiving S. aureus, and in 7 of 10 young rats. In blood: prior to surgery, old rats presented higher IL-2 and lower IL-4 levels. Surgery alone did not induce significant changes in old rats; surgery + S. aureus induced significant increases of IL-2 and IL-10 in young rats, and of IL-6 in old rats. Tibia analysis S. aureus group showed increased levels of: IL-10 in young rats, and IL-1beta in old rats. In experimentally induced osteomyelitis, significant differences were observed in cytokine response with regard to age.

Efficacy of titanium dioxide photocatalyst for inhibition of bacterial colonization on percutaneous implants.

The purpose of this study was to evaluate the efficacy of titanium dioxide photocatalyst in inhibition of bacterial colonization on percutaneous implants. Titanium dioxide photocatalyst was prepared by direct oxidization of pure titanium substrate, and a comparative study with pure titanium was performed. The bactericidal ability of the photocatalyst was examined using methicillin-resistant Staphylococcus aureus (MRSA) suspensions in a colony-forming assay according to the Japanese Industrial Standards committee standard. After exposing the MRSA suspension on sample plates to ultraviolet A (UVA) light, the number of surviving bacteria was estimated. Next, an animal model for inhibition of colonization was examined in vivo. Pins were inserted into the femurs of rabbits, were infected with 10(8) colony-forming units of MRSA suspension, and were illuminated with UVA light for 60 min daily; the number of colonizing bacteria was estimated after 7 days. The bactericidal ability of the photocatalyst was apparent after 60 min, when the bacteria had almost disappeared. The number of colonizing bacteria on photocatalytic pins was decreased significantly in vivo. The photocatalyst was effective even against resistant bacterial colonization. Clinically, the incidence of percutaneous implant infection such as pin tract infection in external fixation could be reduced using the titanium photocatalyst.

Pre-clinical in vitro and in vivo studies to examine the potential use of photodynamic therapy in the treatment of osteomyelitis.

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1000 neonates and 1 in 5000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regimens of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (S. aureus) grown onto kirschner wires (K-wire). S. aureus-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of S. aureus biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. S. aureus infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg kg(-1)) intraperitoneally followed 4 h later by light (635 +/- 10 nm; 75 J cm(-2)) delivered transcutaneously via an optical fiber placed onto the tibia and resulted in significant delay in bacterial growth. In vitro, MB and ALA displayed similar cell kill with > or =4 log(10) cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat S. aureus cultures grown in vitro or in vivo using an animal model of osteomyelitis.

In vitro and in vivo biocompatibility evaluation of a polyalkylimide hydrogel for soft tissue augmentation.

Injectable fillers are commonly used in Plastic and Reconstructive Surgery to correct serious and slight aesthetic defects due to their low invasiveness and an easy implant technique procedure. Synthetic hydrogels are proposed as filler materials for their similarity with soft tissue and to avoid many disadvantages of naturally derived materials such as short persistence, allergenicity, and immunogenicity. Our studies are focused on the biocompatibility evaluation of a polyacrylic hydrogel containing alkylimide-amide groups and pyrogen free water (96%) (Bio-Alcamid by means of the in vitro cytotoxicity and mutagenicity assays and the in vivo skin irritation, sensitization test, and subcutaneous implant. All tests conducted on Bio-Alcamid showed no toxicity. It is a substance easy to inject and remove; it does not migrate, and its safety allows it to be a suitable filler for the correction of slight and also very serious aesthetic defects.

Prophylactic efficacy of topical temporin A and RNAIII-inhibiting peptide in a subcutaneous rat Pouch model of graft infection attributable to staphylococci with intermediate resistance to glycopeptides.

BACKGROUND: Bacteria that adhere to implanted medical devices play an important role in industry and in modern medicine. Staphylococci are among the most common pathogens that cause biomaterial infections. Vascular prosthetic graft infection is one of the most feared complications that the vascular surgeon treats, frequently resulting in prolonged hospitalization, organ failure, amputation, and death. A rat model was used to investigate the topical efficacies of temporin A and the quorum-sensing inhibitor RNAIII-inhibiting protein (RIP) as prophylactic agents of vascular prosthetic graft infections caused by Staphylococcus aureus and Staphylococcus epidermidis with intermediate resistance to glycopeptides. METHODS AND RESULTS: Graft infections were established in the back subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses 1 cm2 followed by topical inoculation with 2x10(7) colony-forming units of bacterial strains. The study included, for each staphylococcal strain, a control group (no graft contamination), a contaminated group that did not receive antibiotic prophylaxis, and 6 contaminated groups that received grafts soaked with temporin A, RIP, rifampin, temporin A plus RIP, RIP plus rifampin, or temporin A plus RIP. The infection was evaluated by quantitative agar culture. When tested alone, temporin A and RIP showed comparable efficacies, and their efficacies were significantly higher than that of rifampin against both strains. All combinations showed efficacies significantly higher than that of each single compound. The combinations of temporin A and RIP exerted the strongest antistaphylococcal efficacies, eliminating infection by 100%. CONCLUSIONS: The results of the present study make these molecules potentially useful for antimicrobial chemoprophylaxis in vascular surgery.