Antibiotic-eluting resorbable bone-void filler evaluated in a large animal infection prevention model.

Periprosthetic infection in total knee arthroplasty is a difficult-to-treat complication. Current implant revision procedures use non-degradable, antibiotic-loaded bone cement for local antimicrobial delivery. As a permanent foreign body, antibiotic-loaded bone cement is susceptible to bacterial colonisation after antibiotic release. In this first step, of a multi-study approach, an infection prevention model assessed a resorbable, antibiotic-eluting bone-void filler for preventing infection in a large animal model. Four groups of sheep were utilised to monitor antibiotic-eluting bone-void filler-induced osteoconductivity, infection prevention, and implant resorption. Explanted bone and surrounding tissues were evaluated using quantitative microbiology, backscattered electron microscopy, bone mineral apposition, and Sanderson's staining at the 12-week endpoint. Control groups received commercially available bone-void filler, implanted into a surgically created defect on the right medial femoral condyle. Experimental groups received six antibiotic-eluting bone-void filler devices placed into identically sized defects. One control and one experimental group tested osteoconductivity. An additional control and experimental group were each inoculated with 5 × 105 colony forming units/mL Staphylococcus aureus during implant placement for bactericidal effects. Osteoconductivity was confirmed for both antibiotic-eluting bone-void filler and commercially available bone-void filler. The experimental group inoculated with S. aureus showed no detectable bacteria at the study's 12-week endpoint, while infection controls required euthanasia 6-11 d post-inoculation due to infection. This large animal study validated this antibiotic-eluting bone-void filler as osteoconductive, in situ degradable, and bactericidal. All groups, except the infection control, exhibited bone formation comparable to commercial filler ProOsteon®500R.

Challenges in linking preclinical anti-microbial research strategies with clinical outcomes for device-associated infections.

Infections related to implanted medical devices have become a significant health care issue in recent decades. Increasing numbers of medical devices are in use, often in an aging population, and these devices are implanted against a background of increasing antibiotic-resistant bacterial populations. Progressively more antibiotic resistant infections, requiring ever more refined treatment options, are therefore predicted to emerge with greater frequency in the coming decades. Improvements in the prevention, diagnosis and treatment of these device-associated infections will remain priority targets both for clinicians and the translational research community charged with addressing these challenges. Preclinical strategies, predictive of ultimate clinical efficacy, should serve as a control point for effective translation of new technologies to clinical applications. The development of new anti-infective medical devices requires a validated preclinical testing protocol; however, reliable validation of experimental and preclinical antimicrobial methodologies currently suffers from a variety of technical limitations. These include the lack of agreement or standardisation of experimental protocols, a general lack of correlation between in vitro and in vivo preclinical results and lack of validation between in vivo preclinical implant infection models and clinical (human) results. Device-associated infections pose additional challenges to practicing clinicians concerning diagnosis and treatment, both of which are complicated by the biofilms formed on the medical device. The critical challenges facing both preclinical research and clinical laboratories in improving both diagnosis and treatment of medical device-associated infections are the focus of this review.

Local release of masitinib alters in vivo implantable continuous glucose sensor performance.

Continuous glucose monitoring (CGM) sensors are often advocated as a clinical solution to improve long-term glycemic control in the context of diabetes. Subcutaneous sensor inflammatory response, fouling and fibrous encapsulation resulting from the host foreign body response (FBR) reduce sensor sensitivity to glucose, eventually resulting in sensor performance compromise and device failure. Several combination device strategies load CGM sensors with drug payloads that release locally to tissue sites to mitigate FBR-mediated sensor failure. In this study, the mast cell-targeting tyrosine kinase inhibitor, masitinib, was released from degradable polymer microspheres delivered from the surfaces of FDA-approved human commercial CGM needle-type implanted sensors in a rodent subcutaneous test bed. By targeting the mast cell c-Kit receptor and inhibiting mast cell activation and degranulation, local masitinib penetration around the CGM to several hundred microns sought to reduce sensor fibrosis to extend CGM functional lifetimes in subcutaneous sites. Drug-releasing and control CGM implants were compared in murine percutaneous implant sites for 21 days using direct-wire continuous glucose reporting. Drug-releasing implants exhibited no significant difference in CGM fibrosis at implant sites but showed relatively stable continuous sensor responses over the study period compared to blank microsphere control CGM implants.

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy.

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

Small unilamellar liposomes from mixed natural and polymeric phospholipids: stability and susceptibility to phospholipase A2.

The concept of the uncorkable liposome composed of phase-separated mixtures of a polymerized phospholipid and an enzymically digestible phospholipid has been investigated, using small unilamellar vesicles composed of mixtures of (polymerized) dienoylphosphatidylcholine (DENPC) and dimyristoylphosphatidylcholine (DMPC). Mixed liposomes, even those containing only 10% DENPC, were much more stable than DMPC liposomes, as indicated by the release of entrapped [3H]inulin or [14C]glucose. DMPC liposomes released entrapped solute on exposure to phospholipase A2, whereas mixed vesicles were resistant. The results are compared with those of an earlier study on monolayers of similar compositions. It is concluded that the liposomes, like the monolayers, are phase-mixed, and that uncorkable liposomes cannot be constructed from the phospholipid mixture employed. It is proposed that, until further experimental evidence is produced, the enzymatically uncorkable liposome must be regarded as a theoretical construct.

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

Mixed monolayers of natural and polymeric phospholipids: structural characterization by physical and enzymatic methods.

This study has focused on physical characterization and enzymatic hydrolysis of mixed monolayers of a natural phospholipid substrate and a polymerizable phospholipid analogue. Such a mixed system presents the possibility to stabilize model biomembranes, vary the molecular environment within the layer through polymerization and simultaneously examine these influences on monolayer structure. Phospholipase A2 was used here as a sensitive probe of the molecular environment within these mixed, polymerizable monolayers to complement information obtained from isotherm and isobar data. The results clearly show a strong influence of molecular environment on phospholipase A2 activity, even if differences in the physical state of mixed monolayers are not detectable with isotherm and isobar measurements. Physical characterization indicated that both monomeric and polymeric mixed monolayers were phase-mixed. Enzyme hydrolysis, however, showed large differences in the ability of the enzyme to selectively hydrolyze the natural phosphatidylcholine component from the monomeric as opposed to the polymeric mixtures. This demonstrates a high sensitivity of phospholipase A2 to distinguish subtle differences in molecular arrangement within mixed monolayers on a molecular level.

Design strategies to improve soluble macromolecular delivery constructs.

Macromolecular therapeutics provide numerous benefits for the delivery of cytotoxic or poorly soluble drugs in vivo. However, these constructs often encounter barriers for drug delivery on both the systemic and subcellular level. Many soluble polymer carriers have been designed to surmount specific physiological barriers individually, but less work has been dedicated to designing an all-encompassing construct that addresses multiple therapeutic barriers at once. Incorporation of multiple agents already individually known to increase effectiveness into one carrier could further improve current drug delivery technology. Recent developments in subcellular delivery of therapeutic agents in soluble macromolecular carriers are discussed in the context of the future possibility for the design of an all-encompassing soluble multi-functional drug delivery vehicle.

RhoA-induced changes in fibroblasts cultured on organic monolayers.

Substantial previous work indicates that adherent cell morphology in culture is modulated by surface chemistry. Activation of the intracellular small molecular weight GTPase, RhoA, has recently been shown to play an essential role in controlling initiation of key integrin-mediated events in surface adhesion and proliferation. RhoA is interconvertible between an active, membrane-bound form and an inactive, cytosolic RhoGDI-bound form in response to integrin stimulation. This study reports the use of self-assembled functionalized organic alkylthiol monolayers (SAMs) as well-defined cell culture substrates to investigate the relationships between surface chemistry, RhoA activation and subsequent cell morphological and molecular level signal transduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on -CH3 terminated SAMs, which normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by fluorescence microscopy. Results support surface chemistry influences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state.

Surgical irrigation with pooled human immunoglobulin G to reduce post-operative spinal implant infection.

A multiple-site, nonlethal rabbit surgical model of spinal implant infection was used to assess the efficacy of a spinal wound lavage to reduce post-operative infection from methicillinresistant Staphylococcus aureus (MRSA). Multiple aqueous lavages of isotonic saline were compared to the same procedure using 1wt% pooled human immunoglobulin G (IgG) applied directly to the surgical implant sites. Visually observed clinically relevant signs of infection (e.g. , swelling, erythema, pus) were supported by bacterial enumeration from multiple biopsied tissue and bone sites post-mortem at 7 and 28 days post-challenge. Clinical signs of infection were significantly reduced in IgG-lavaged infected spinal sites. Bacterial enumeration also exhibited statistically significant reductions in soft tissues, bone and on K-wire spinal implants using IgG lavage compared with saline. Complete healing of all surgical wounds was seen after 28 days, although isolated fibrosed abscesses were observed in autopsied sites treated with both IgG and saline lavages. Local use of IgG wound lavage is proposed as supplementary infection prophylaxis against antibiotic resistant implant-centered or surgical wound infection.

Polyurethane coatings release bioactive antibodies to reduce bacterial adhesion.

This study describes the formulation of a biomedical grade polyurethane hydrogel coating containing solid dispersed bioactive antibodies cast from an organic solvent onto a model polymer biomaterial substrate. A prepolymer dispersion in anhydrous isopropanol containing a uniformly distributed slurry of 22 microm sieved commercial lyophilized polyclonal pooled human immunoglobulin G (IgG) solids was coated onto polymer substrates by simple immersion. Maximum antibody release was approximately 50 microg/cm(2) from a 15% w/w IgG polymer coating. In vitro antimicrobial studies utilized Escherichia coli to compare performance of bare uncoated tubing, hydrogel-coated tubing with added aqueous phase antibodies, and antibody-dispersed hydrogel-coated tubing. Bacterial adhesion was reduced significantly (p<0.05) in the presence of antibodies with the greatest reduction seen with the antibody releasing coating. The presence of antibody also significantly enhanced the killing of the bacteria in an in vitro opsonophagocytic assay using freshly isolated blood neutrophils over 2 h indicating that antibody bioactivity is maintained. This controlled release polyurethane hydrogel coating imparts infection resistance by exploiting the low adhesive properties of the biomedical grade hydrogel and the intrinsic bioactive role of the antibodies to reduce bacterial adhesion and promote clearance via natural immune mechanisms.

Phase separated anionic domains in ternary mixed lipid monolayers at the air-water interface.

A series of ternary mixed monolayers containing varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and equimolar additions of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LYSO-PC) and palmitic acid (PA) were studied at the air-water interface. These mixed monolayers were used to model phospholipid biomembrane interfaces resulting from phospholipase A2 (PLA2) hydrolysis. Recent work [D.W. Grainger A. Reichert, H. Ringsdorf and C. Salesse (1989) Biochim. Biophys. Acta. 1023, 365-379] has shown that PLA2 hydrolysis of pure phospholipid monolayers results in formation of large PLA2 domains at the air-water interface. These domains are proposed to result from PLA2 adsorption to phase separated regions in the hydrolyzed monolayer. To elucidate the phase behaviour in these monolayer systems, surface pressure-area isotherms were measured for the ternary mixtures on pure water and buffered subphases. Fluorescence microscopy at the air-water interface was used to image fluorescent probe-doped monolayer mixtures during isothermal compressions. A water-soluble cationic carbocyanine dye was used to probe the interfacial properties of the mixed monolayers. Isotherm data do not provide unambiguous evidence for either phase separation or ideal mixing of monolayer components. Fluorescence microscopy is more revealing, showing that lateral phase separation of microstructures containing palmitic acid occurred only when monolayer subphases contained Ca2+ ions at alkaline pH. At either low pH or on Ca(2+)-free subphases, phase separation was not observed.

A novel spinal implant infection model in rabbits.

STUDY DESIGN: A new spinal implant model was designed to study device-centered infection with methicillin-resistant Staphylococcus aureus in multiple noncontiguous surgical sites in the lumbar spine region of a rabbit. OBJECTIVE: To develop a multiple-site spinal implant device-centered infection model in rabbits. SUMMARY OF BACKGROUND DATA: Results in many recent studies show that postoperative wound infection after spinal implant surgery and the increase in antibiotic-resistant bacteria are a concern. Anti-infection strategies must be tested in relevant animal models that will lead to appropriate clinical studies. METHODS: Eight anesthetized New Zealand White rabbits underwent completely isolated partial laminectomy and subsequent stainless steel Kirschner wire implantation directly into the transverse processes of vertebrae T13, L3, and L6. The middle sites (L3) were used as sterile control sites, and the outer sites (T13, L6) were challenged with different amounts of methicillin-resistant Staphylococcus aureus. Rabbits were killed after 7 days, and biopsies were performed to provide evidence for device-centered infection. Bacterial growth on the implant surfaces and in surrounding tissues and bone was assayed. RESULTS: Overall device-centered infection was established after 7 days in 100% of the sites challenged with 10(3) colony-forming units methicillin-resistant Staphylococcus aureus or higher. No infection was seen in any of the control sites located between infected vertebrae. Multiple blood and liver samples showed that the separate localized infections did not become systemic after 7 days. CONCLUSIONS: This new animal model demonstrates that multiple biomaterial implants can be evaluated in the same animal and provides a technique for investigating postoperative device-centered infection of the spine. Infection was demonstrated in noncontiguous lumbar sites of the spine, whereas adjacent control sites remained sterile. Because there was no cross contamination or systemic spread of the infection, multiple anti-infection strategies or implant materials can now be tested for efficacy in a single animal to combat dramatic and costly postoperative implant infections.

Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa infection in a murine burn wound model.

The leading cause of morbidity and mortality in severe burn wound patients is infection. Treatment of burn wound infection is complicated by the emergence of antibiotic resistant organisms. A potential therapeutic alternative to antibiotic drugs is the local administration of polyclonal antibodies, termed passive local immunotherapy (PLI), directly to the burned tissue. A mouse burn wound infection model to simulate full thickness burn wound infection was used to evaluate the efficacy of passive local immunotherapy as a viable prophylactic or therapeutic agent. Pooled human immunoglobulins (IgG), delivered locally to the site of infection, are shown to be more effective at preventing fatal burn wound sepsis than treatment by intravenous infusion of IgG. A single 10 mg dose of human IgG administered locally to the burned, infected tissue site, either 24 hours prior to bacterial challenge, or within 3 hours after bacterial challenge, enhanced animal survival significantly (P < 0.001 and P < 0.05 respectively) compared to control animals. In addition, reduced levels of bacteria were found in local and systemic tissues of IgG-treated mice compared to control mice (P < 0.05). These data support the local use of polyclonal immunoglobulin preparations as an efficacious and cost effective means to prevent and treat burn wound infections.

Foreign body response to subcutaneous biomaterial implants in a mast cell-deficient Kit(w-Sh) murine model.

Mast cells (MCs)_are recognized for their functional role in wound-healing and allergic and inflammatory responses - host responses that are frequently detrimental to implanted biomaterials if extended beyond acute reactivity. These tissue reactions impact especially on the performance of sensing implants such as continuous glucose monitoring (CGM) devices. Our hypothesis that effective blockade of MC activity around implants could alter the host foreign body response (FBR) and enhance the in vivo lifetime of these implantable devices motivated this study. Stem cell factor and its ligand c-KIT receptor are critically important for MC survival, differentiation and degranulation. Therefore, an MC-deficient sash mouse model was used to assess MC relationships to the in vivo performance of CGM implants. Additionally, local delivery of a tyrosine kinase inhibitor (TKI) that inhibits c-KIT activity was also used to evaluate the role of MCs in modulating the FBR. Model sensor implants comprising polyester fibers coated with a rapidly dissolving polymer coating containing drug-releasing degradable microspheres were implanted subcutaneously in sash mice for various time points, and the FBR was evaluated for chronic inflammation and fibrous capsule formation around the implants. No significant differences were observed in the foreign body capsule formation between control and drug-releasing implant groups in MC-deficient mice. However, fibrous encapsulation was significantly greater around the drug-releasing implants in sash mice compared to drug-releasing implants in wild-type (e.g. MC-competent) mice. These results provide insights into the role of MCs in the FBR, suggesting that MC deficiency provides alternative pathways for host inflammatory responses to implanted biomaterials.

Adsorbed serum albumin is permissive to macrophage attachment to perfluorocarbon polymer surfaces in culture.

Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive because of insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces. Bone marrow macrophages (BMMO) and IC-21, RAW 264.7, and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pretreated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking were used to assess integrins critical for monocyte/macrophage adhesion in vitro. Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures preadsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared with Teflon AF, plasma-polymerized FC was less permissive to extended cell proliferation. The beta(2) integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of preadsorbed protein biasing, with respect to adhesion, cell morphology, motility, and proliferation.

Polymer Brushes Containing Sulfonated Sugar Repeat Units: Synthesis, Characterization and In Vitro Testing of Blood Coagulation Activation.

A new polymer brush chemistry containing sulfonated carbohydrate repeat units has been synthesized from silicon substrates using ATRP methods and characterized both in bulk and using surface analysis. The polymer brush was designed to act as a mimic for the naturally occurring sulfonated glycosaminoglycan, heparin, commonly used for modifying blood-contacting surfaces both in vitro and in vivo. Surface analysis showed conversion of brush saccharide precursor chemistry to the desired sulfonated polymer product. The sulfonated polymer brush surface was further analyzed using three conventional in vitro tests for blood compatibility -- plasma recalcification times, complement activation, and thrombin generation. The sulfonated polymer brush films on silicon oxide wafers exhibited better assay performance in these blood component assays than the unsulfonated sugar functionalized polymer brush in all tests performed.

Immunologic responses of bison to vaccination with Brucella abortus strain RB51: comparison of parenteral to ballistic delivery via compressed pellets or photopolymerized hydrogels.

This study compared responses of bison calves to 10(10)CFU of Brucella abortus strain RB51 (SRB51) delivered by parenteral or ballistic methods. Two types of biobullet payloads were evaluated; compacted SRB51 pellets or SRB51 encapsulated in photopolymerized poly(ethylene glycol) hydrogels. Bison were vaccinated with saline, parenteral SRB51 alone, or in combination with Spirovac, or ballistically with compressed SRB51 or hydrogel biobullets. Bison parenterally vaccinated with SRB51 had greater (P<0.05) immunologic responses when compared to control bison. Co-administration of Spirovac as an adjuvant did not influence immunologic responses. As compared to compressed SRB51 biobullets, ballistic vaccination with hydrogel biobullets increased cellular immune responses at some sampling times. Our data suggest that hydrogel formulations of SRB51 may be a superior alternative to compressed SRB51 tablets for ballistic vaccination of bison. Although preliminary, data suggests that immunologic responses of bison to SRB51 hydrogel bullets are similar to responses after parenteral vaccination with SRB51.

Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures.

The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-l-lactide and Teflon(®) AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly-l-lactide surfaces after several days, but absent from those grown on apolar polystyrene and Teflon(®) AF, indicating some surface influence on this GTPase in serum-containing cultures.

Photopolymerized hydrogel carriers for live vaccine ballistic delivery.

Photopolymerized poly(ethylene glycol) (PEG)-crosslinked hydrogels were assessed for their ability to serve as a payload vehicle to deliver a viable bacterial vaccine (Brucella abortus strain RB51 (RB51) to bison in Yellowstone National Park) ballistically using thermoplastic degradable Biobullets. PEG modified with degradable glycolide or lactide oligomers capped with photopolymerizable methacrylate groups served to crosslink the hydrogel vaccine carrier inside commercial hydroxypropylcellulose Biobullets. Release of 1 microm diameter model fluorescent particles from hydrogels followed known degradation trends for glycolide- and lactide-modified PEG hydrogels. All particles were released from PEG-co-glycolide hydrogels after approximately 10 days and PEG-co-lactide hydrogels after approximately 45 days following gel degradation. Minimal particle release was observed from pure PEG dimethacrylate hydrogels over 40 days. P. aeruginosa (strain PAO1) and RB51 live vaccines exhibit excellent viability following exposure to photopolymerization encapsulation within these gel matrices. Hydrogels photopolymerized into the payload chamber of Biobullets exhibit similar ballistic properties to commercially available Biobullets and penetrate and remain intact when fired intramuscularly into live elk for release of their gel payload in the host.