Porous Precision-Templated 40 µm Pore Scaffolds Promote Healing through Synergy in Macrophage Receptor with Collagenous Structure and Toll-Like Receptor Signaling.

Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 µm pores have shown favorable healing outcomes and a reduced foreign body reaction (FBR). Macrophage receptor with collagenous structure (MARCO) and toll-like receptors (TLRs) have been identified as key surface receptors in the initial inflammatory phase of wound healing. However, the role of MARCO and TLRs in modulating monocyte and macrophage phenotypes within PTS remains uncharacterized. In this study, we demonstrate a synergetic relationship between MARCO and TLR signaling in cells inhabiting PTS, where induction with TLR3 or TLR4 agonists to 40 µm scaffold-resident cells upregulates the transcription of MARCO. Upon deletion of MARCO, the prohealing phenotype within 40 µm PTS polarizes to a proinflammatory and profibrotic phenotype. Analysis of downstream TLR signaling shows that MARCO is required to attenuate nuclear factor kappa B (NF-κB) inflammation in 40 µm PTS by regulating the transcription of inhibitory NFKB inhibitor alpha (NFKBIA) and interleukin-1 receptor-associated kinase 3 (IRAK-M), primarily through a MyD88-dependent signaling pathway. Investigation of implant outcome in the absence of MARCO demonstrates an increase in collagen deposition within the scaffold and the development of tissue fibrosis. Overall, these results further our understanding of the molecular mechanisms underlying MARCO and TLR signaling within PTS. Impact statement Monocyte and macrophage phenotypes in the foreign body reaction (FBR) are essential for the development of a proinflammatory, prohealing, or profibrotic response to implanted biomaterials. Identification of key surface receptors and signaling mechanisms that give rise to these phenotypes remain to be elucidated. In this study, we report a synergistic relationship between macrophage receptor with collagenous structure (MARCO) and toll-like receptor (TLR) signaling in scaffold-resident cells inhabiting porous precision-templated 40 µm pore scaffolds through a MyD88-dependent pathway that promotes healing. These findings advance our understanding of the FBR and provide further evidence that suggests MARCO, TLRs, and fibrosis may be interconnected.

Designed α-sheet peptides disrupt uropathogenic E. coli biofilms rendering bacteria susceptible to antibiotics and immune cells.

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed "α-sheet". Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 µm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 µm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 µm PTS compared to 100 µm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 µm PTS. When subcutaneously implanted in MyD88KO mice, 40 µm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 µm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

Monocytes contribute to a pro-healing response in 40 µm diameter uniform-pore, precision-templated scaffolds.

Porous precision-templated scaffolds (PTS) with uniformly distributed 40 µm spherical pores have shown a remarkable ability in immunomodulating resident cells for tissue regeneration. While the pore size mediated pro-healing response observed only in 40 µm pore PTS has been attributed to selective macrophage polarization, monocyte recruitment and phenotype have largely been uncharacterized in regulating implant outcome. Here, we employ a double transgenic mouse model for myeloid characterization and a multifaceted phenotyping approach to quantify monocyte dynamics within subcutaneously implanted PTS. Within 40 µm PTS, myeloid cells were found to preferentially infiltrate into the scaffold. Additionally, macrophage receptor with collagenous structure (MARCO), an innate activation marker, was significantly upregulated within 40 µm PTS. When 40 µm PTS were implanted in monocyte-depleted mice, the transcription of MARCO was significantly decreased and an increase in pro-inflammatory inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα) were observed. Typical of a foreign body response (FBR), 100 µm PTS significantly upregulated pro-inflammatory iNOS, secreted higher amounts of TNFα, and displayed a pore size dependent morphology compared to 40 µm PTS. Overall, these results identify a pore size dependent modulation of circulating monocytes and implicates MARCO expression as a defining subset of monocytes that appears to be responsible for regulating a pro-healing host response.

Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication.

Implanted porous precision templated scaffolds (PTS) with 40-µm spherical pores reduce inflammation and foreign body reaction (FBR) while increasing vascular density upon implantation. Larger or smaller pores, however, promote chronic inflammation and FBR. While macrophage (MØ) recruitment and polarization participates in perpetuating this pore-size-mediated phenomenon, the driving mechanism of this unique pro-healing response is poorly characterized. We hypothesized that the primarily myeloid PTS resident cells release small extracellular vesicles (sEVs) that induce pore-size-dependent pro-healing effects in surrounding T cells. Upon profiling resident immune cells and their sEVs from explanted 40-µm- (pro-healing) and 100-µm-pore diameter (inflammatory) PTS, we found that PTS pore size did not affect PTS resident immune cell population ratios or the proportion of myeloid sEVs generated from explanted PTS. However, quantitative transcriptomic assessment indicated cell and sEV phenotype were pore size dependent. In vitro experiments demonstrated the ability of PTS cell-derived sEVs to stimulate T cells transcriptionally and proliferatively. Specifically, sEVs isolated from cells inhabiting explanted 100 µm PTS significantly upregulated Th1 inflammatory gene expression in immortalized T cells. sEVs isolated from cell inhabiting both 40- and 100-µm PTS upregulated essential Treg transcriptional markers in both primary and immortalized T cells. Finally, we investigated the effects of Treg depletion on explanted PTS resident cells. FoxP3+ cell depletion suggests Tregs play a unique role in balancing T cell subset ratios, thus driving host response in 40-µm PTS. These results indicate that predominantly 40-µm PTS myeloid cell-derived sEVs affect T cells through a distinct, pore-size-mediated modality.

Injectable Biodegradable Chitosan-Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery.

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide-based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low-transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan-alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold-mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex-loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN-γ secretion are seen with in vivo scaffold-mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold-mediated mRNA lipoplex delivery, while protein-based immunization did not elicit IgG production until 2 weeks post-injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.

Scaffold-mediated delivery for non-viral mRNA vaccines.

mRNA is increasingly being recognized as a promising alternative to pDNA in gene vaccinations. Only recently, owing to the needs of cancer immunotherapies, has the biomaterials/gene delivery community begun to develop new biomaterial strategies for immunomodulation. Here, we report a novel way to use implantable porous scaffolds as a local gene delivery depot to enhance mRNA vaccine immunization in vitro, and in vivo when compared with conventional bolus injections. We first evaluated transfection efficiencies of single-stranded mRNA condensed and charge neutralized with two lipids (Lipofectamine Messenger MAXTM LM-MM and StemfectTM SF) and two cationic polymers (in vivo-jetPEI™, Poly (ß-amino ester)) as gene carriers. As SF demonstrated highest in vitro transfection and cell viability, it was selected for subsequent porous polymer scaffold-loading trials. Enhanced in vitro transfection of SF:mRNA nanoparticle-loaded poly (2-hydroxyethyl methacrylate) (pHEMA) scaffolds was also observed with a DC2.4 cell line. Improved sustained local release and local transgene expression were also demonstrated with SF:mRNA nanoparticle-loaded pHEMA scaffolds in vivo compared with bolus injections. Our results suggest that mRNA polyplex-loaded scaffolds may be a superior alternative to either repeated bolus immunizations or ex vivo transfection cell immunotherapies.

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms.

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

Chemical and Physical Variability in Structural Isomers of an l/d α-Sheet Peptide Designed To Inhibit Amyloidogenesis.

There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two de novo-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating l/d-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the ß-amyloid peptide implicated in Alzheimer's disease, in addition to neutralizing its cytotoxicity. In contrast, the all-l-amino acid isomer does not form α-sheet structure and is insoluble and inactive.

Precision-porous templated scaffolds of varying pore size drive dendritic cell activation.

Scaffold based systems have shown significant potential in modulating immune responses in vivo. While there has been much attention on macrophage interactions with tissue engineered scaffolds for tissue regeneration, fewer studies have looked at the effects of scaffold design on the response of immune cells-that is, dendritic cells (DCs). Here, we present the effects of varying pore size of poly (2-hydroxyethyl methacrylate) (pHEMA) and poly(dimethylsiloxane) (PDMS, silicone) scaffolds on the maturation and in vivo enrichment of DCs. We employ a precision templating method to make 3-D porous polymer scaffolds with uniformly defined and adjustable architecture. Hydrophilic pHEMA and hydrophobic PDMS scaffolds were fabricated in three pore sizes (20, 40, 90 µm) to quantify scaffold pore size effects on DCs activation/maturation in vitro and in vivo. In vitro results showed that both pHEMA and PDMS scaffolds could promote maturation in the DC cell line, JAWSII, that resembled lipopolysaccharide (LPS)-activated/matured DCs (mDCs). Scaffolds with smaller pore sizes correlate with higher DC maturation, regardless of the polymer used. In vivo, when implanted subcutaneously in C57BL/6J mice, scaffolds with smaller pore sizes also demonstrated more DCs recruitment and more sustained activation. Without the use of DC chemo-attractants or chemical adjuvants, our results suggested that DC maturation and scaffold infiltration profile can be modulated by simply altering the pore size of the scaffolds.

Artificial opsonin enhances bacterial phagocytosis, oxidative burst and chemokine production by human neutrophils.

Here, we describe the application of an 'artificial opsonin' to stimulate the innate immune response against Gram-positive bacteria. The artificial opsonin comprises a poly(L-lysine)-graft-poly(ethylene glycol) backbone displaying multiple copies of vancomycin and human IgG-Fc. The vancomycin targets bacteria by recognizing d-Ala-d-Ala-terminated peptides present in the bacterial cell wall. The human IgG-Fc antibody fragments serve as phagocyte recognition moieties that recognize the Fcγ cell surface receptors expressed by professional human phagocytes. Staphylococcus epidermidis RP62A, a biofilm-forming, methicillin-resistant strain, was utilized to investigate the effects of opsonization on phagocytosis, oxidative burst and IL-8 chemokine production by human neutrophils. Results show that opsonization of S. epidermidis RP62A with the artificial opsonin resulted in an ∼2-fold increase in neutrophil phagocytosis. Analysis of the cell supernatant found a 2- to 3-fold increase in neutrophil IL-8 secretion. The neutrophil oxidative burst was investigated using the oxidation-sensitive fluorophore dihydrorhodamine-123. Bacterial opsonization resulted in a 20% increase in fluorescence intensity, indicating a significant increase in the production of reactive oxygen species by the neutrophils. These studies suggest that artificial opsonins may be a novel immunostimulation therapeutic strategy to control infections caused by Gram-positive bacteria, particularly those that are known to be immune evasive and/or antibiotic resistant.

Designed α-sheet peptides suppress amyloid formation in Staphylococcus aureus biofilms.

Nosocomial infections affect hundreds of millions of patients worldwide each year, and ~60% of these infections are associated with biofilm formation on an implanted medical device. Biofilms are dense communities of microorganisms in which cells associate with surfaces and each other using a self-produced extracellular matrix composed of proteins, polysaccharides, and genetic material. Proteins in the extracellular matrix take on a variety of forms, but here we focus on functional amyloid structures. Amyloids have long been associated with protein misfolding and neurodegenerative diseases, but recent research has demonstrated that numerous bacterial species utilize the amyloid fold to fortify the biofilm matrix and resist disassembly. Consequently, these functional amyloids, in particular the soluble oligomeric intermediates formed during amyloidogenesis, represent targets to destabilize the extracellular matrix and interrupt biofilm formation. Our previous studies suggested that these amyloidogenic intermediates adopt a non-standard structure, termed "α-sheet", as they aggregate into soluble oligomeric species. This led to the design of complementary α-sheet peptides as anti-α-sheet inhibitors; these designs inhibit amyloidogenesis in three unrelated mammalian disease-associated systems through preferential binding of soluble oligomers. Here we show that these anti-α-sheet peptides inhibit amyloid formation in Staphylococcus aureus biofilms. Furthermore, they inhibit aggregation of pure, synthetic phenol soluble modulin α1, a major component of Staphylococcus aureus functional amyloids. As it aggregates phenol soluble modulin α1 adopts α-helix then α-sheet and finally forms ß-sheet fibrils. The binding of the designed peptide inhibitors coincides with the formation of α-sheet.

Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations.

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

Interruption of Electrical Conductivity of Titanium Dental Implants Suggests a Path Towards Elimination Of Corrosion.

Peri-implantitis is an inflammatory disease that results in the destruction of soft tissue and bone around the implant. Titanium implant corrosion has been attributed to the implant failure and cytotoxic effects to the alveolar bone. We have documented the extent of titanium release into surrounding plaque in patients with and without peri-implantitis. An in vitro model was designed to represent the actual environment of an implant in a patient's mouth. The model uses actual oral microbiota from a volunteer, allows monitoring electrochemical processes generated by biofilms growing on implants and permits control of biocorrosion electrical current. As determined by next generation DNA sequencing, microbial compositions in experiments with the in vitro model were comparable with the compositions found in patients with implants. It was determined that the electrical conductivity of titanium implants was the key factor responsible for the biocorrosion process. The interruption of the biocorrosion current resulted in a 4-5 fold reduction of corrosion. We propose a new design of dental implant that combines titanium in zero oxidation state for osseointegration and strength, interlaid with a nonconductive ceramic. In addition, we propose electrotherapy for manipulation of microbial biofilms and to induce bone healing in peri-implantitis patients.

Antibacterial activity of gold-titanates on Gram-positive cariogenic bacteria.

Objective: Gram-positive cariogenic bacteria are etiological agents in dental caries; therefore, strategies to inhibit these bacteria to reduce the incident of this disease have intensified. In this study, we investigated antibacterial activities of titanates and gold-titanates against Lactobacillus casei (Lc) and Streptococcus mutans (Sm). Materials and methods: Monosodium titanate (MST), nanomonosodium titanate (nMST) and amorphous peroxo-titanate (APT), which are inorganic compounds with high-binding affinity for specific metal ions, were used. Total bacterial proteins were measured to represent bacterial cell mass after 24 h incubation with gold-titanates. We further examined the effect of nMST-Au(III) concentrations (10,200,400 mg/L) on Lc and Sm cell viability over time via Live/Dead fluorescent staining and colony forming units (CFUs). Transmission electron microscopy (TEM) was used to determine specific locations on the bacterial cells affected by the nMST-Au(III). Results: We found all gold-titanates and APT alone reduced bacterial protein for Lc (p value <0.001) while only MST-Au(III) and nMST-Au(III) affected Sm growth (p value <0.001). Overall, nMST-Au(III) showed the most effectiveness against both Lc and Sm at 400 mg/L. The Live/Dead staining showed all concentrations of nMST-Au(III) affected Lc growth but only 200 and 400 mg/L nMST-Au(III) interrupted Sm growth. The growth curves based on CFUs/mL showed all nMST-Au(III) concentrations affected growth of both Lc and Sm. TEM images showed nMST-Au(III) attached to Lc and Sm cell wall and were internalized into both cells.Conclusions: nMST-Au(III) demonstrated potential antimicrobial activity against Gram-positive cariogenic bacteria. These results support further development of nMST-Au(III) as a potential novel material to prevent dental caries.

Precision-Porous PolyHEMA-Based Scaffold as an Antibiotic-Releasing Insert for a Scleral Bandage.

Combat-related penetrating ocular injuries have become a common form of battlefield injury in modern warfare and can lead to potentially devastating visual impairments. Prompt stabilization of the wounded globe via prevention of infection and fibrosis enhances the probability of a successful outcome after professional medical treatment. In this study, a norfloxacin-releasing poly(hydroxyethyl methacrylate)-based insert was designed and fabricated as a part of scleral bandage to prevent development of infection and scar formation. First, a sphere-templating technique was applied, during which 2-hydroxyethyl methacrylate monomer was photocopolymerized with acrylic acid and/or 4-fluorostyrene at different molar ratios to generate poly(hydroxyethyl methacrylate)-based porous scaffolds of various compositions with interconnected, monodisperse, 38 µm diameter pores. The scaffolds were then loaded with norfloxacin via swelling in drug-saturated solutions of various solvents, such as water, acetone, chloroform and ethanol, and the effect of the scaffold composition and the swelling solvent on norfloxacin uptake was explored. An in vitro drug release study was then conducted to explore the release kinetics of norfloxacin from the drug-loaded scaffolds, with the aim to find the optimal scaffold composition to provide release of norfloxacin over a 1 week period. The antibacterial potential of the optimal composition norfloxacin-loaded scaffold to inhibit the growth of the relevant clinical pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa during a 1 week period was evaluated in vitro using a continuous culture flow cell system and a soft agar overlay plate assay.

A Single B-repeat of Staphylococcus epidermidis accumulation-associated protein induces protective immune responses in an experimental biomaterial-associated infection mouse model.

Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine's efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10(6) CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova)-immunized mice.

Development of a poly(ether urethane) system for the controlled release of two novel anti-biofilm agents based on gallium or zinc and its efficacy to prevent bacterial biofilm formation.

Traditional antibiotic therapy to control medical device-based infections typically fails to clear biofilm infections and may even promote the evolution of antibiotic resistant species. We report here the development of two novel antibiofilm agents; gallium (Ga) or zinc (Zn) complexed with protoporphyrin IX (PP) or mesoprotoporphyrin IX (MP) that are both highly effective in negating suspended bacterial growth and biofilm formation. These chelated gallium or zinc complexes act as iron siderophore analogs, supplanting the natural iron uptake of most bacteria. Poly (ether urethane) (PEU; Biospan®) polymer films were fabricated for the controlled sustained release of the Ga- or Zn-complexes, using an incorporated pore-forming agent, poly(ethylene glycol) (PEG). An optimum formulation containing 8% PEG (MW=1450) in the PEU polymer effectively sustained drug release for at least 3months. All drug-loaded PEU films exhibited in vitro ≥ 90% reduction of Gram-positive (Staphylococcus epidermidis) and Gram-negative (Pseudomonas aeruginosa) bacteria in both suspended and biofilm culture versus the negative control PEU films releasing nothing. Cytotoxicity and endotoxin evaluation demonstrated no adverse responses to the Ga- or Zn-complex releasing PEU films. Finally, in vivo studies further substantiate the anti-biofilm efficacy of the PEU films releasing Ga- or Zn- complexes.

Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.

Methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and PCR. However, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. We have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. The probability of plasmid segregational loss in planktonic and biofilm cultures of Pseudomonas putida carrying the TOL plasmid (pWWO::gfpmut3b) was determined directly in situ, in the absence of any applied selection pressure. Compared to suspended liquid culture, we report that the biofilm mode of growth enhances plasmid segregational loss. Results based on a biofilm-averaged analysis reveal that the probability of plasmid loss in biofilm cultures (0.016 ± 0.004) was significantly greater than that determined in planktonic cultures (0.0052 ± 0.0011). Non-invasive assessments showed that probabilities of plasmid segregational loss at different locations in a biofilm increased dramatically from 0.1% at the substratum surface to 8% at outside layers of biofilm. Results suggest that higher nutrient concentrations and subsequentially higher growth rates resulted in higher probability of plasmid segregational loss at the outer layers of the biofilm.

Giant extracellular matrix binding protein expression in Staphylococcus epidermidis is regulated by biofilm formation and osmotic pressure.

Staphylococcus epidermidis is an opportunistic bacterium that thrives as a commensal cutaneous organism and as a vascular pathogen. The S. epidermidis extracellular matrix binding protein (Embp) has been reported to be a virulence factor involved in colonization of medical device implants and subsequent biofilm formation. Here, we characterize the expression patterns of Embp in planktonic and biofilm cultures, as well as under high osmotic stresses that typify the commensal environment of the skin. Embp expression without osmotic stress was similar for planktonic and adherent cultures. Addition of osmotic stress via NaCl caused slight increases in embp expression in planktonic cultures. However, in adherent cultures a 100-fold increase in embp expression with NaCl versus controls occurred and coincided with altered biofilm morphology. Results suggest that the central role of Embp lies in commensal skin colonization, stabilizing the cell wall against osmotic stresses, rather than as a virulence factor promoting adhesion.