<OL> <LI>Explore the Influence of Nanocomposite Composition on Adult, Adipose Derived Stem Cells (ASC). Advance our understanding of nanomaterial toxicity as it applies to ASC. Help to determine the role of particle morphology and composite content in ASC function in osteoinductive and non-inductive condition, and characterize the impact of nanomaterial morphology and content on the evolution oriented polymer scaffolds. The methods validated in this study could be utilized in other scaffold-stem cell systems to generate a more complete picture of how materials interact with stem cells and potentially predict in-vivo response. A correlation between nanoparticle size and content with the evolution of particular scaffold nano and microstructure could allow for the determination of the underlying mechanisms of composite formation leading to the capability to accurately predict structures, rationally design scaffolds and synthesis processes and model and test outcomes. <LI>Synthesis and Characterization of Self-Assembled Antimicrobial Coatings for Indwelling Orthopaedic Devices. We will develop a novel antimicrobial delivery vehicle for musculoskeletal implants using nano technology. Nano-composite surfaces composed of bioresorbable polymers containing a silver nanoparticle (SNP) based antimicrobial component as well as an ionic liquid antibiotic formulation will provide a flexible, cost effective and safe platform technology that is compatible with a variety of indwelling orthopedic devices and implantation sites. In addition to substantially decreased infection rates and better treatment outcomes, our nano/microstructured biopolymer composite materials will result in earlier return to functionality and improved quality of life after surgery, as well as decrease the associated short and long term healthcare costs of trauma related orthopedic surgery. <LI>Exploration of Nanoparticle Toxicity, Transport, and Fate in Aquatic Systems. An In-vitro exploration of particle structure/function relationships utilizing a neuronal cell type derived from crayfish. Laboratory prepared and commercially sourced nanomaterials (silver and polymers) will be examined to determine the relationship between nanomaterial composition and bioactivity, intracellular transport and fate. An In-vivo crayfish model will be developed to examine the toxicity, accumulation, transport and transformation of nanomaterials under both acute and chronic exposure conditions. Experiments to address this objective will be conducted at the laboratory scale in a custom built, controlled microenvironment. Additionally, an engineered mesocosm environment will be used to explore the impact, on crayfish and the aquaculture environment, of acute and chronic nanomaterial exposure at sub-lethal concentrations.
Non-Technical Summary: Nanotechnology, as defined by the National Cancer Institute is "The field of research that deals with the engineering and creation of things from materials that are less than 100 nanometers (one-billionth of a meter) in size, especially single atoms or molecules." These technologies and materials once associated with the high technology industries of computing, lasers and optics have recently begun to appear in variety of consumer applications ranging from textiles and food additives to sunscreen. This rapidly expanding nanotechnology enabled market is expected to top $1 trillion annually in 2015 and the resulting increase in production, use and disposal of nanomaterials has drawn the attention of advocacy groups, academic scientists, representatives from industry and governmental regulatory groups, who share a concern that current regulations based on dated data may be inadequate to protect the humans, animals and environment. Developing a more complete understanding of how nanomaterials interact with biological systems will enable the development of a new generation of safe and effective medical devices, food additives, chemical catalysts and drugs. Louisiana as a leading producer of energy, commodity and specialty chemicals, agricultural products and sea food will be both a prime manufacturer and consumer of nano-enabled products. The development of domain expertise in novel nanomaterial manufacturing processes, characterization and biological interactions will be integral to establishing Louisiana businesses as leaders in the global nano market. The studies outlined in this proposal will explore new manufacturing methods for biocompatible nanocomposites and interactions of those materials with biological systems. The findings will be disseminated through peer-reviewed journal publications, public presentations at scientific conferences and extension personnel serving relevant stakeholders. The studies outlined in this proposal will explore new manufacturing methods for biocompatible nanocomposites and interactions of those materials with biological systems. There are three objectives for this program: 1. Explore the Influence of Nanocomposite Composition on Adult, Adipose Derived Stem Cells (ASC). 2. Synthesis and Characterization of Self-Assembled Antimicrobial Coatings for Indwelling Orthopaedic Devices. 3. Exploration of Nanoparticle Toxicity, Transport, and Fate in Aquatic Systems. Outcomes: These studies will advance our understanding of nanomaterial toxicity, determine the role of particle morphology and composite content in stem cell function and provide a flexible platform for imparting antimicrobial properties to orthopaedic implants. It is expected that the methods validated in this study could be utilized in other nanomaterial systems to generate a more complete picture of how materials interact with stem cells, the food web and the environment. <P> Approach: Experimental Methods Objective 1. Poly-lactic acid scaffolds containing TCP particles of two sizes, ~70nm and ~2mm, at concentrations of 0% (control), 10%, 30%, 45% and 60% (wt/wt) will be synthesized by unidirectional precipitation and phase separation. The scaffolds will be evaluated to determine: 1)Pore morphology (SEM), 2)Particle content and distribution (TGA and SEM) and 3)Rheology. Stem cell experiments will be conducted with 1 cm cylindrical sections of scaffolds loaded with human ASC from pooled donors (minimum 3) and exposed to either osteoinductive or non-inductive culture conditions. The following outcomes will be measured;1)ALP activity at day 1, 7 and 14 (enzyme fluorescent assay). 2)Cell viability at day 1, 7 and 14 (live-dead flow cytometry). 3)Cytoskelatal organization at day 14 (confocal microscopy). 4)Proinflammatory marker expression IL-6, 8 and 11 B- actin at 24 hours (RT-PCR). 5)Proinflammatory marker expression IL-6, 8 and 11 at 24 hours (ELISA). Objective 2.Antimicrobial nanomaterials and associated composite coatings will be developed and applied to titanium disks and interlocking nails. Antimicrobial composite coatings applied to titanium will be tested for their ability to inhibit or prevent bacterial colonization (CFUs of methicillin-resistant Staphylococcus aureus [MRSA], coagulase negative staphylococcus, Pseudomonas aeruginosa, and Acinetobacter baumannii) after both repeated challenge and in a high protein environment. Implant resistance to microbial colonization will be evaluated in an established lapin femoral osteotomy fracture model. Outcome measures including limb function, radiographic fracture healing, light microscopy, and bacterial load will be assessed at regular intervals up to 6 weeks after fracture stabilization. Objective 3. Live-dead assays will provide information on gross toxicity while growth assays and experiments which probe the oxidative effects of nanomaterials may reveal what specific pathways are perturbed by exposure. A modification of the 96 hour acute toxicity assay, previously established in our laboratories, will be used to determine simple acute toxicity and to explore the influence on toxicity of environmental organic matter/nanomaterial interactions. Chronic, 14 day exposures will be conducted at sub-LD50 concentrations (96 hr) to examine the transport, accumulation, biotransformation and toxicity of nanoparticles under conditions likely to be found in general crayfish aquaculture. Fluorescent microscopy, EM, XPS and OES analysis will be applied to dissected tissues to determine the concentration, distribution and chemical state of the nanomaterials. The mesocosm exposure methodology creates an environment approximating commercial aquaculture conditions for crayfish in southwest Louisiana. This component of the study will examine both the survival and the impact on reproduction of crayfish exposed to acute concentrations of nanomaterials. Exposures will be sub LD50 (96 hr) and water concentrations will be maintained as stable as possible by monitoring and repletion.