Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection.

Pseudomonas aeruginosa (PA) is a highly, if not the most, versatile microorganism capable of colonizing diverse environments. One of the niches in which PA is able to thrive is the lung of cystic fibrosis (CF) patients. Due to a genetic aberration, the lungs of CF-affected patients exhibit impaired functions, rendering them highly susceptible to bacterial colonization. Once PA attaches to the epithelial surface and transitions to a mucoid phenotype, the infection becomes chronic, and antibiotic treatments become inefficient. Due to the high number of affected people and the severity of this infection, CF-chronic infection is a well-documented disease. Still, numerous aspects of PA CF infection remain unclear. The scientific reports published over the last decades have stressed how PA can adapt to CF microenvironmental conditions and how its surrounding matrix of extracellular polymeric substances (EPS) plays a key role in its pathogenicity. In this context, it is of paramount interest to present the nature of the EPS together with the local CF-biofilm microenvironment. We review how the PA biofilm microenvironment interacts with drugs to contribute to the pathogenicity of CF-lung infection. Understanding why so many drugs are inefficient in treating CF chronic infection while effectively treating planktonic PA is essential to devising better therapeutic targets and drug formulations.

An Antibiotic-Loaded Hydrogel Demonstrates Efficacy as Prophylaxis and Treatment in a Large Animal Model of Orthopaedic Device-Related Infection.

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x108, range 7x106-3x108 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x101, range 0 - 1x105 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x105, range 2x103- 9x106 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x102, range 0 - 7x104 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials.

Thermo-Responsive Antimicrobial Hydrogel for the In-Situ Coating of Mesh Materials for Hernia Repair.

The prophylactic coating of prosthetic mesh materials for hernia repair with antimicrobial compounds is commonly performed before implantation of the mesh in the abdominal wall. We propose a novel alternative, which is a rifampicin-loaded thermo-responsive hydrogel formulation, to be applied on the mesh after its implantation. This formulation becomes a gel in-situ once reached body temperature, allowing an optimal coating of the mesh along with the surrounding tissues. In vitro, the hydrogel cytotoxicity was assessed using rabbit fibroblasts and antimicrobial efficacy was determined against Staphylococcus aureus. An in vivo rabbit model of hernia repair was performed; implanted polypropylene meshes (5 × 2 cm) were challenged with S. aureus (106 CFU), for two study groups-unloaded (n = 4) and 0.1 mg/cm2 rifampicin-loaded hydrogel (n = 8). In vitro, antibacterial activity of the hydrogel lasted for 5 days, without sign of cytotoxicity. Fourteen days after implantation, meshes coated with drug-free hydrogel developed a strong infection and resulted in poor tissue integration. Coating meshes with the rifampicin-loaded hydrogel fully prevented implant infection and permitted an optimal tissue integration. Due to its great performance, this, degradable, thermo-responsive antimicrobial hydrogel could potentially be a strong prophylactic armamentarium to be combined with prosthesis in the surgical field.

Synergistic anti-fouling and bactericidal poly(ether ether ketone) surfaces via a one-step photomodification.

Implants of poly(ether ether ketone) (PEEK) are gaining importance in surgical bone reconstruction of the skull. As with any implant material, PEEK is susceptible to bacterial contamination and occasionally PEEK implants were removed from patients because of infection. To address this problem, a combination of anti-fouling and bactericidal polymers is grafted onto PEEK. The originality is that anti-fouling (modified poly(ethylene glycol)) and bactericidal (quaternized poly(dimethylaminoethyl acrylate)) moieties are simultaneously and covalently grafted onto PEEK via UV photoinsertion. The functionalized PEEK surfaces are evaluated by water contact angle measurements, FTIR, XPS and AFM. Grafting of anti-fouling and bactericidal polymers significantly reduces Staphylococcus aureus adhesion on PEEK surfaces without exhibiting cytotoxicity in vitro. This study demonstrates that grafting combinations of anti-fouling and bactericidal polymers synergistically prevents bacterial adhesion on PEEK implants. This approach shows clinical relevance as grafting is rapid, does not modify PEEK properties and can be conducted on pre-formed implants.

Orbital floor repair using patient specific osteoinductive implant made by stereolithography.

The orbital floor (OF) is an anatomical location in the craniomaxillofacial (CMF) region known to be highly variable in shape and size. When fractured, implants commonly consisting of titanium meshes are customized by plying and crude hand-shaping. Nevertheless, more precise customized synthetic grafts are needed to meticulously reconstruct the patients' OF anatomy with better fidelity. As alternative to titanium mesh implants dedicated to OF repair, we propose a flexible patient-specific implant (PSI) made by stereolithography (SLA), offering a high degree of control over its geometry and architecture. The PSI is made of biodegradable poly(trimethylene carbonate) (PTMC) loaded with 40 wt % of hydroxyapatite (called Osteo-PTMC). In this work, we developed a complete work-flow for the additive manufacturing of PSIs to be used to repair the fractured OF, which is clinically relevant for individualized medicine. This work-flow consists of (i) the surgical planning, (ii) the design of virtual PSIs and (iii) their fabrication by SLA, (iv) the monitoring and (v) the biological evaluation in a preclinical large-animal model. We have found that once implanted, titanium meshes resulted in fibrous tissue encapsulation, whereas Osteo-PMTC resulted in rapid neovascularization and bone morphogenesis, both ectopically and in the OF region, and without the need of additional biotherapeutics such as bone morphogenic proteins. Our study supports the hypothesis that the composite osteoinductive Osteo-PTMC brings advantages compared to standard titanium mesh, by stimulating bone neoformation in the OF defects. PSIs made of Osteo-PTMC represent a significant advancement for patients whereby the anatomical characteristics of the OF defect restrict the utilization of traditional hand-shaped titanium mesh.

Interaction of gentamicin sulfate with alginate and consequences on the physico-chemical properties of alginate-containing biofilms.

BACKGROUND: Alginate is one of the main extracellular polymeric substances (EPS) in biofilms of Cystic Fibrosis (CF) patients suffering from pulmonary infections. Gentamicin sulfate (GS) can strongly bind to alginate resulting in loss of pharmacological activity; however neither the mechanism nor its repercussion is fully understood. In this study, we investigated how GS modifies the alginate macromolecular network and its microenvironment. MATERIAL AND METHODS: Alginate gels of two different compositions (either enriched in guluronate units (G) or enriched in mannuronate units (M)) were crosslinked with Ca2+ and exposed to GS at varying times and concentrations. The complexes formed were characterized via turbidimetry, mechanical tests, swelling assay, calorimetry techniques, nuclear magnetic resonance, Ca2+ displacement, macromolecular probe diffusion and pH alteration. RESULTS: In presence of GS, the alginate network and its environment undergo a tremendous reorganization in terms of gel density, stiffness, diffusion property, presence and state of the water molecules. We noted that the intensity of those alterations is directly dependent on the polysaccharide motif composition (ratio M/G). CONCLUSION: Our results underline the importance of alginate as biofilm component, its pernicious role during antibiotherapy and could represent a potential macromolecular target to improve anti-infectious therapies.

A drug eluting poly(trimethylene carbonate)/poly(lactic acid)-reinforced nanocomposite for the functional delivery of osteogenic molecules.

BACKGROUND: Poly(trimethylene carbonate) (PTMC) has wide biomedical applications in the field of tissue engineering, due to its biocompatibility and biodegradability features. Its common manufacturing involves photofabrication, such as stereolithography (SLA), which allows the fabrication of complex and controlled structures. Despite the great potential of SLA-fabricated scaffolds, very few examples of PTMC-based drug delivery systems fabricated using photo-fabrication can be found ascribed to light-triggered therapeutics instability, degradation, side reaction, binding to the macromers, etc. These concerns severely restrict the development of SLA-fabricated PTMC structures for drug delivery purposes. METHODS: In this context, we propose here, as a proof of concept, to load a drug model (dexamethasone) into electrospun fibers of poly(lactic acid), and then to integrate these bioactive fibers into the photo-crosslinkable resin of PTMC to produce hybrid films. The hybrid films' properties and drug release profile were characterized; its biological activity was investigated via bone marrow mesenchymal stem cells culture and differentiation assays. RESULTS: The polymer/polymer hybrids exhibit improved properties compared with PTMC-only films, in terms of mechanical performance and drug protection from UV denaturation. We further validated that the dexamethasone preserved its biological activity even after photoreaction within the PTMC/poly(lactic acid) hybrid structures by investigating bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. CONCLUSION: This study demonstrates the potential of polymer-polymer scaffolds to simultaneously reinforce the mechanical properties of soft matrices and to load sensitive drugs in scaffolds that can be fabricated via additive manufacturing.

Dual-functional 3D-printed composite scaffold for inhibiting bacterial infection and promoting bone regeneration in infected bone defect models.

Infection is one of the pivotal causes of nonunion in large bone defect after trauma or tumor resection. Three-dimensional (3D) composite scaffold with multifunctional-therapeutic properties offer many advantages over allogenic or xenogenic bone grafting for the restoration of challenging infected bone defects. In the previous study, we demonstrated that quaternized chitosan (HACC)-grafted polylactide-co-glycolide (PLGA)/hydroxyapatite (HA) scaffold (PLGA/HA/HACC) via 3D-printing technique exhibited significantly improved antimicrobial and osteoconductive property in vitro, together with good biocompatibility in vivo. Hence, the present study further investigated whether such an innovative bone substitute could effectively inhibit the bacterial biofilm formation and promote bone regeneration in vivo. To evaluate the bone repairing effects of the 3D-printed scaffolds on infected cortical and cancellous bone defects scenarios, eighty female Sprague Dawley rats and thirty-six female New Zealand white rabbits were used to establish infected femoral shaft defect and condyle defect model, respectively. X-ray, micro-CT, microbiological and histopathological analyses were used to assess the anti-infection and bone repairing potential of the dual-functional porous scaffolds. We observed that HACC-grafted PLGA/HA scaffolds exhibited significantly enhanced anti-infection and bone regeneration capability in different infected bone defect models. In addition, the degradation rate of the scaffolds appeared to be closely related to the progress of infection, influencing the bone repairing potential of the scaffolds in infected bone defects models. In general, this investigation is of great significance as it demonstrates promising applications of the 3D-printed dual-functional PLGA/HA/HACC scaffold for repairing different types of bone defect under infection. STATEMENT OF SIGNIFICANCE: Currently, it is clinically urgent to exploit bone substitutes with potential of bacterial inhibition and bone regeneration. However, bone scaffolds with relatively low risks of bacterial resistance and tissue toxicity used for combating infected bone defects remain to be developed. We have reported that quaternized chitosan (HACC)-grafted 3D-printed PLGA/HA composite scaffold had enhanced in vitro antimicrobial and osteoconductive property, and well cytocompatibility in our published study. This continuing study further confirmed that HACC-grafted PLGA/HA scaffolds exhibited significantly enhanced anti-infection and bone regeneration efficacy in both cortical bone defect in rat and cancellous bone defect in rabbit under infection. Meanwhile, we also found that the degradation rate of the scaffolds seemed to be closely related to the progress of infection, influencing the bone repairing potential of the scaffolds in infected bone defects models. In conclusion, this study provides significant opportunities to develop a 3D-printed bone scaffold with dual functions used for infected bone defects in future plastic and orthopaedic surgery.

Anti-infective efficacy, cytocompatibility and biocompatibility of a 3D-printed osteoconductive composite scaffold functionalized with quaternized chitosan.

Contaminated or infected bone defects remain serious challenges in clinical trauma and orthopaedics, and a bone substitute with both osteoconductivity and antibacterial properties represents an improvement for treatment strategy. In this study, quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) was grafted to 3D-printed scaffolds composed of polylactide-co-glycolide (PLGA) and hydroxyapatite (HA), in order to design bone engineering scaffolds endowed with antibacterial and osteoconductive properties. We found that both the PLGA/HA/HACC and PLGA/HACC composite scaffolds decreased bacterial adhesion and biofilm formation under in vitro and in vivo conditions. Additionally, ATP leakage assay indicated that immobilizing HACC on the scaffolds could effectively disrupt microbial membranes. Using human bone marrow-derived mesenchymal stem cells (hBMSCs), we demonstrated that HA incorporated scaffolds, including PLGA/HA and PLGA/HA/HACC, favoured cell attachment, proliferation, spreading and osteogenic differentiation compared to HA-free PLGA or PLGA/HACC scaffolds. Finally, an in vivo biocompatibility assay conducted on rats, showed that HA incorporated scaffolds (including PLGA/HA and PLGA/HA/HACC scaffolds) exhibited good neovascularization and tissue integration. Taken together, our findings support the approach for developing porous PLGA/HA/HACC composite scaffold with potential clinical application in the treatment of infected bone. STATEMENT OF SIGNIFICANCE: Although plenty of conductive scaffold biomaterials have been exploited to improve bone regeneration under infection, potential tissue toxicity under high concentration and antibiotic-resistance are their main deficiencies. This study indicated that HACC-grafted PLGA/HA composite scaffold prepared using an innovative 3D-printing technique and covalent grafting strategy showed significantly enhanced antibacterial activities, especially against the antibiotic-resistant strains, together with good osteogenic activity and biocompatibility. Therefore, it provides an effective porous composite scaffold to combat the infected bone defect in clinic with decreased risks of bacterial resistance and open a feasible strategy for the modification of scaffold interfaces involved in the bone regeneration and anti-infection.

Emerging Trends in Abdominal Wall Reinforcement: Bringing Bio-Functionality to Meshes.

Abdominal wall hernia is a recurrent issue world-wide and requires the implantation of over 1 million meshes per year. Because permanent meshes such as polypropylene and polyester are not free of complications after implantation, many mesh modifications and new functionalities have been investigated over the last decade. Indeed, mesh optimization is the focus of intense development and the biomaterials utilized are now envisioned as being bioactive substrates that trigger various physiological processes in order to prevent complications and to promote tissue integration. In this context, it is of paramount interest to review the most relevant bio-functionalities being brought to new meshes and to open new avenues for the innovative development of the next generation of meshes with enhanced properties for functional abdominal wall hernia repair.

Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

Lower lumbar disc disorders pose a significant problem in an aging society with substantial socioeconomic consequences. Both inner tissue (nucleus pulposus) and outer tissue (annulus fibrosus) of the intervertebral disc are affected by such debilitating disorders and can lead to disc herniation and lower back pain. In this study, we developed an alginate-collagen composite porous scaffold with shape-memory properties to fill defects occurring in annulus fibrosus tissue of degenerated intervertebral discs, which has the potential to be administered using minimal invasive surgery. In the first part of this work, we assessed how collagen incorporation on preformed alginate scaffolds influences the physical properties of the final composite scaffold. We also evaluated the ability of annulus fibrosus cells to attach, migrate, and proliferate on the composite alginate-collagen scaffolds compared to control scaffolds (alginate only). In vitro experiments, performed in intervertebral disc-like microenvironmental conditions (low glucose and low oxygen concentrations), revealed that for alginate only scaffolds, annulus fibrosus cells agglomerated in clusters with limited infiltration and migration capacity. In comparison, for alginate-collagen scaffolds, annulus fibrosus cells readily attached and colonized constructs, while preserving their typical fibroblastic-like cell morphology with spreading behavior and intense cytoskeleton expression. In a second part of this study, we investigated the effects of alginate-collagen scaffold when seeded with bone marrow derived mesenchymal stem cells. In vitro, we observed that alginate-collagen porous scaffolds supported cell proliferation and extracellular matrix deposition (collagen type I), with secretion amplified by the local release of transforming growth factor-ß3. In addition, when cultured in ex vivo organ defect model, alginate-collagen scaffolds maintained viability of transplanted mesenchymal stem cells for up to 5 weeks. Taken together, these findings illustrate the advantages of incorporating collagen as a means to enhance cell migration and proliferation in porous scaffolds which could be used to augment tissue repair strategies.

Multilayer, degradable coating as a carrier for the sustained release of antibiotics: preparation and antimicrobial efficacy in vitro.

One of the most critical post-surgical complications is mesh-related infection. This paper describes how a commercially available polypropylene (PP) mesh was modified to minimize the risk of post-implantation infection. A dual drug-release coating was created around mesh filaments using an airbrush spray system. This coating was composed of three layers containing ofloxacin and rifampicin dispersed in a degradable polymer reservoir made up of [poly(ε-caprolactone) (PCL) and poly(DL-lactic acid) (PLA)]. Drug release kinetics were managed by varying the structure of the degradable polymer and the multilayer coating. In vitro, this new drug delivery polymer system was seen to be more rapidly invaded by fibroblasts than was the initial PP mesh. Active mesh showed excellent antibacterial properties with regard to microorganism adhesion, biofilm formation and the periprosthetic inhibition of bacterial growth. Sustained release of the two antibiotics from the coated mesh prevented mesh contamination for at least 72 h. This triple-layer coating technology is potentially of great interest for it can be easily extrapolated to other medical devices and drug combinations for the prevention or treatment of other diseases.

Permanent polymer coating for in vivo MRI visualization of tissue reinforcement prostheses.

The clinical advantage of MRI visualization of prostheses in soft tissue prolapses is very appealing as over 1,000000 MRI-transparent synthetic meshes are implanted annually, and postoperative complications such as mesh shrinkage and migration are frequent. Here, the synthesis of a new material composed of a DTPA-Gd complex grafted onto a backbone of PMA via a covalent bond is described (DTPA-Gd-PMA). This new polymer is sprayed onto meshes and gives an MR signal for a long period without any significant release of Gd. In vitro cytocompatibility tests on fibroblasts show limited cytotoxicity. Microscopic investigations indicate that vital cells rapidly colonize the material. Finally, coated meshes implanted in rats are easily recognizable using an MR imaging system.

New antibiotic-eluting mesh used for soft tissue reinforcement.

The surgical implantation of prostheses for soft tissue repair may be followed by post-operative mesh-related infection, a significant and dramatic complication, that is treated by mesh removal. A new antibiotic-eluting mesh has been manufactured on pre-existing polypropylene prostheses using an airbrush spraying technology. Among the degradable polymers tested as coating agents and drug reservoirs, poly(ε-caprolactone) (PCL), which is deposited after heating, provides a homogeneous, regular and smooth shell around the polypropylene filaments of the mesh without dramatically altering the biomechanical properties of the new modified mesh. An anti-infective drug (e.g. ofloxacin) is incorporated into this polymeric coating giving a limited burst effect followed by sustained drug diffusion for several days. An ofloxacin-eluting mesh has demonstrated excellent antibacterial activity in vitro on Escherichia coli adherence, biofilm formation and inhibitory diameter, even with low drug loads. Although further in vivo investigations are required to draw conclusions on the anti-infective effectiveness of the coated mesh, the airbrush coating of ofloxacin-PCL on existing prostheses is already potentially appealing in an effort to decrease post-operative infection.