A Modified-Herringbone Micromixer for Assessing Zebrafish Sperm (MAGS).

Sperm motility analysis of aquatic model species is important yet challenging due to the small sample volume, the necessity to activate with water, and the short duration of motility. To achieve standardization of sperm activation, microfluidic mixers have shown improved reproducibility over activation by hand, but challenges remain in optimizing and simplifying the use of these microdevices for greater adoption. The device described herein incorporates a novel micromixer geometry that aligns two sperm inlet streams with modified herringbone structures that split and recombine the sample at a 1:6 dilution with water to achieve rapid and consistent initiation of motility. The polydimethylsiloxane (PDMS) chip can be operated in a positive or negative pressure configuration, allowing a simple micropipettor to draw samples into the chip and rapidly stop the flow. The device was optimized to not only activate zebrafish sperm but also enables practical use with standard computer-assisted sperm analysis (CASA) systems. The micromixer geometry could be modified for other aquatic species with differing cell sizes and adopted for an open hardware approach using 3D resin printing where users could revise, fabricate, and share designs to improve standardization and reproducibility across laboratories and repositories.

An automated modular open-technology device to measure and adjust concentration of aquatic sperm samples for cryopreservation.

Repositories for aquatic germplasm are essential for safeguarding valuable genetic diversity for species relevant to aquaculture, biomedical research, and conservation. Development of aquatic germplasm repositories is impeded by a lack of standardization within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as cell concentration, that strongly affect the success and reproducibility of cryopreservation. The wide dissemination and use of specialized tools and devices as open hardware can improve processing reliability and save costs. The goal of the present work was to develop and prototype a modular and open-technology approach to help to standardize the cell concentration of germplasm samples prior to cryopreservation. The specific objectives were to: 1) design and fabricate prototypes of the automated concentration measurement and adjustment system (CMAS), incorporating custom peristaltic pumps and optical evaluation modules, and 2) evaluate the performance of the CMAS with biological samples. Linear regression models were obtained for estimation of aquatic sperm concentration >108 cells/mL and for algae concentration > (3 × 105) cells/mL. Algae were diluted with extender medium by an automated process, resulting in a dilution precision of ±12.6% and ±6.7% in two trials, attaining means of 89% and 71% of the target cell concentration. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm and can invite new users, makers, and developers into the open-technology community. This will increase the reach and capabilities of much-needed aquatic germplasm repositories.

Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation.

Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.

Photoactivated miR-148b-nanoparticle conjugates improve closure of critical size mouse calvarial defects.

Inducible systems providing temporal control of differentiation have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. The aim of this study was to demonstrate that nanoformulated microRNA (miRNA) conjugates activated via photo exposure can lead to the induced osteogenic differentiation of human adipose-derived stromal/stem cells (hASCs) in vivo. The conjugate PC-miR-148b-SNP, a mimic of miRNA-148b tethered to silver nanoparticles (SNPs) via a photolabile linker, was used to modulate gene expression for improved closure of a critical size defect drilled on the right parietal bone of male CD-1 nude homozygous mice. The PC-miR-148b-SNP conjugates added to hASCs and loaded to either Matrigel or polycaprolactone (PCL) scaffolds resulted in different levels of healing of the defect. After 4 and 12weeks, 3-D micro-computed tomography reconstructed images indicate statistically significant defect closure from 3.83±1.19% to 5.46±2.01% and 6.54±4.28% to 32.53±8.3% for non-photoactivated and photoactivated conjugates, respectively, in the PCL scaffolds. The results were confirmed with H&E and Masson's Trichrome stains in the transverse sections of photoactivated conjugates. Collagen fiber staining was greatest at 12weeks when it reached approximately the same density and thickness as the native calvarium. This technology provides a platform that can be used with other miRNAs that actively govern the pathways responsible for regenerative and wound healing processes.

Photothermolysis of lymphatic endothelial cells by gold nanoshell-mediated hyperthermia.

Tumor-associated lymphatics and lymphangiogenesis have been shown to play important roles in promoting tumor growth and metastasis. However, the lymphatic system has received much less attention as a target of intervention in cancer treatment compared to the blood vascular system. In this study, we explored the feasibility of photothermal therapy targeting the lymphatic system as a strategy for inhibiting lymphatics-mediated tumor metastasis. Specifically, photothermolysis of lymphatic endothelial cells (LECs) via gold nanoshell-mediated hyperthermia was investigated. Near-Infrared-absorbing Au nanoshells (AuNSs) were synthesized and used as the photothermal coupling agent. After 24-hr incubation, significant amount of the AuNSs were taken up by murine simian virus lymphatic endothelial cells with minimal cytotoxicity. Thermally-induced injury to LECs was found to occur above a threshold temperature of 46 degrees C. Preliminary data also suggested apoptosis as the mechanism of thermally-induced cell death in this temperature range. In a proof-of-concept experiment, AuNS-mediated photothermal heating was found to induce cell death in statistically higher percent of LECs incubated with AuNSs after 15-min laser irradiation compared to the controls. We believed that the findings in this study represent the first step in developing AuNS-mediated photothermal therapy as a potential strategy to disrupt tumor-associated lymphatics.

Antimicrobial biocompatible bioscaffolds for orthopaedic implants.

Nationally, nearly 1.5 million patients in the USA suffer from ailments requiring bone grafts and hip and other joint replacements. Infections following internal fixation in orthopaedic trauma can cause osteomyelitis in 22-66% of cases and, if uncontrolled, the mortality rate can be as high as 2%. We characterize a procedure for the synthesis of antimicrobial and biocompatible poly-l-lactic acid (PLLA) and poly-ethyleneglycol (PEG) bioscaffolds designed to degrade and absorb at a controlled rate. The bioscaffold architecture aims to provide a suitable substrate for the controlled release of silver nanoparticles (SNPs) to reduce bacterial growth and to aid the proliferation of human adipose-derived stem cells (hASCs) for tissue-engineering applications. The fabricated bioscaffolds were characterized by scanning transmission microscope (SEM) and it showed that the addition of tncreasing concentrations of SNPs results in the formation of dendritic porous channels perpendicular to the axis of precipitation. The antimicrobial properties of these porous bioscaffolds were tested according to a modified ISO 22196 standard across varying concentrations of biomass-mediated SNPs to determine an efficacious antimicrobial concentration. The bioscaffolds reduced the Staphylococcus aureus and Escherichia coli viable colony-forming units by 98.85% and 99.9%, respectively, at an antimicrobial SNPs concentration of 2000 ppm. Human ASCs were seeded on bioscaffolds and cultured in vitro for 20 days to study the effect of SNPs concentration on the viability of cells. SEM analysis and the metabolic activity-based fluorescent dye, AlamarBlue®, demonstrated the growth of cells on the efficacious antimicrobial bioscaffolds. The biocompatibility of in vitro leached silver, quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES), proved non-cytotoxic when tested against hASCs, as evaluated by MTT assay.

Novel anterior cruciate ligament graft fixation device reduces slippage.

Clinically significant laxity occurs in 10%-30% of knees after anterior cruciate ligament reconstruction. Graft slippage and tension loss at the hamstring graft tibial fixation site during and after reconstruction surgery contribute to postoperative joint laxity and are detrimental to long-term knee stability and graft properties. Limiting graft slippage will reduce associated complications. We sought to compare the in vitro mechanical properties and in vivo joint stabilization, postoperative limb use, and graft incorporation of the novel GraftGrab™ (GG) device designed to reduce hamstring graft tibial fixation slippage with the commercially available bioabsorbable Bio-Post™ and spiked washer (BP). Mechanical testing was performed on canine tibia-hamstring graft constructs to quantify initial fixation properties. In vivo joint stabilization, postoperative limb use and graft incorporation of hamstring graft reconstructions were determined in a canine model. Outcomes included tibial translation and ground reaction forces preoperatively and 4 and 8 weeks postoperatively, three-dimensional graft and bone tunnel dimensions at the latter two time points, and graft-bone microstructure, as well as mechanical properties 8 weeks after implantation. Immediately after fixation, all grafts slipped from the BP constructs versus about 30% of GG constructs. In vivo limb use remained low, and tibial translation increased with time in the BP cohort. These results together confirm that initial graft slippage is lower with GG versus BP extracortical hamstring graft tibial fixation. In addition, postoperative recovery and joint stability are more consistent with the GG. This information supports the GG as an alternative to extracortical tibial hamstring graft fixation that has procedural advantages over current implants and reduces graft failure from slippage.

Silver nanoscale antisense drug delivery system for photoactivated gene silencing.

The unique photophysical properties of noble metal nanoparticles contribute to their potential as photoactivated drug delivery vectors. Here we demonstrate the synthesis and characterization of 60-80 nm silver nanoparticles (SNPs) decorated with thiol-terminated photolabile DNA oligonucleotides. In vitro assays and fluorescent confocal microscopy of treated cell cultures show efficient UV-wavelength photoactivation of surface-tethered caged ISIS2302 antisense oligonucleotides possessing internal photocleavable linkers. As a demonstration of the advantages of these novel nanocarriers, we investigate properties including: enhanced stability to nucleases, increased hybridization activity upon photorelease, and efficient cellular uptake as compared to commercial transfection vectors. Their potential as multicomponent delivery agents for oligonucleotide therapeutics is shown through regulation of ICAM-1 (Intracellular Adhesion Molecule-1) silencing. Our results suggest a means to achieve light-triggered, spatiotemporally controlled gene silencing via nontoxic silver nanocarriers, which hold promise as tailorable platforms for nanomedicine, gene expression studies, and genetic therapies.

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish.

Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 µL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 µL/h, there was a significant difference in mixing efficiency between 3.5 mm (75±4%; mean±SD) and 7 mm (92±2%; P=0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56±4%) than manual activation (45±7%; n=5, P=0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation.

A Planar Microfluidic Mixer Based on Logarithmic Spirals.

A passive, planar micromixer design based on logarithmic spirals is presented. The device was fabricated using polydimethylsiloxane soft photolithography techniques, and mixing performance was characterized via numerical simulation and fluorescent microscopy. Mixing efficiency initially declined as Reynolds number increased, and this trend continued until a Reynolds number of 15 where a minimum was reached at 53%. Mixing efficiency then began to increase reaching a maximum mixing efficiency of 86% at Re = 67. Three-dimensional simulations of fluid mixing in this design were compared to other planar geometries such as the Archimedes spiral and Meandering-S mixers. The implementation of logarithmic curvature offers several unique advantages that enhance mixing, namely a variable cross-sectional area and a logarithmically varying radius of curvature that creates 3-D Dean vortices. These flow phenomena were observed in simulations with multilayered fluid folding and validated with confocal microscopy. This design provides improved mixing performance over a broader range of Reynolds numbers than other reported planar mixers, all while avoiding external force fields, more complicated fabrication processes, and the introduction of flow obstructions or cavities that may unintentionally affect sensitive or particulate-containing samples. Due to the planar design requiring only single-step lithographic features, this compact geometry could be easily implemented into existing micro-total analysis systems requiring effective rapid mixing.

Ex vivo comparison of three surgical techniques to stabilize canine cranial cruciate ligament deficient stifles.

OBJECTIVE: To quantify and compare canine stifle stability after 3 stabilization techniques. STUDY DESIGN: Randomized controlled study. SAMPLE POPULATION: Adult canine cadaveric pelvic limbs. METHODS: Total craniocaudal (CrCa) tibial translation quantified in stifles with the cranial cruciate ligament (CrCL) intact, transected, and stabilized with 1 of 3 techniques: (1) hamstring graft (HG); (2) modified retinacular imbrication (MRIT); (3) anatometric fascia lata translocation (AFLT). Tibial translation was quantified from radiographs generated during application of cranial and caudal forces to the tibia. After removal of all soft tissues except periarticular ligaments and fixation, CrCa tibial translation, as before, and medial-lateral rotation, via torsional loading, was quantified with an active motion analysis system. Total tibial translation was evaluated for effect of technique and cruciate status using mixed effect linear model with significance considered at P-value <.05. RESULTS: CrCa translation was not significantly different across stabilization techniques with CrCLs intact, transected, or after stabilization. Poststabilization translation was significantly less than posttransection for all techniques. Compared with the intact CrCL, CrCa translation poststabilization after HG was significantly greater whereas poststabilization after MRIT and AFLT was not significantly different. Tibial rotation exceeded instrumentation limits in 62.5% HG limbs, 20% MRIT limbs, and 60% AFLT limbs. CONCLUSIONS: All 3 stifle stabilization techniques confer comparable CrCa translational stability after CrCL disruption with that provided by the MRIT and AFLT techniques comparable to the intact CrCL. CLINICAL RELEVANCE: The extra- and intracapsular techniques evaluated in this study reduced CrCa tibial translation in CrCL deficient stifles to varying amounts.

Characterization and applications of serum-free induced adhesion in Jurkat suspension cells.

In this study, we demonstrate that the presence of serum in different media plays an important role in inducing transient and reversible adhesion in Jurkat suspension cells. Attachment of Jurkat cells in two distinct media formulations (serum-fortified and serum-free) to untreated polystyrene (PS), plasma-treated PS, and fibronectin-coated PS was compared. Additional analysis characterized the occurrence of this transient cell adhesion, including attachment rate, reversibility of attachment, and viability and preservation of phenotype in cells during and after attachment. As a demonstration of the utility of this technique, a few applications of transiently adhering Jurkat cells are shown which would be otherwise difficult with freely suspended cells, such as increased gene delivery, confocal-based apoptosis detection, and real-time electric-field effect monitoring in Jurkat cells.

Caged siRNAs for spatiotemporal control of gene silencing.

Various strategies have been employed to achieve control over delivery of siRNA molecules to intended target cells. Photocaging is one specific class of modifications for silencing oligonucleotides that block their bioactivity until exposure to near-ultraviolet light. These caged RNAi effectors enable both spatial and temporal targeting of a dosed release of gene silencing agents by directed light exposure that photocleaves the cage moieties. Herein we compare the photochemical properties of cage compounds and strategies for their use, attached either randomly or site-specifically, to demonstrate various forms of gene expression regulation in vitro and in vivo. This light-controllable strategy has potential applications for precisely probing developing biological systems and eventually enabling targeted gene-silencing therapeutics.

Photoinduced RNA interference using DMNPE-caged 2'-deoxy-2'-fluoro substituted nucleic acids in vitro and in vivo.

Various chemical modifications to RNA have been incorporated in attempts to improve their pharmacological properties for RNAi interference (RNAi). Recent studies have shown that small interfering RNA (siRNA) containing 2'-fluoro modifications can elicit gene silencing through RNAi. Despite developments in using chemical modifications for increased stability, safety, and efficiency of these therapeutics, they still face challenges of spatial and temporal targeting. One potential targeting strategy is to use photocaging techniques, which involve the covalent attachment of photolabile compounds to the effector nucleic acid species that block bioactivity until exposed to near UV light. In this study we demonstrate that fully 2'-fluorinated nucleic acids (FNAs) can be caged for photoactivated gene silencing in cell culture and in zebrafish embryos. This strategy combines the improvement in chemical and enzymatic stability associated with 2'-substitutions with the targeting ability of a photoinducible trigger. Statistical alkylation of FNAs with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) improved resistance to enzymatic degradation, reduced RNAi effectiveness, and protected the biological system from toxic doses of the effector. Photo-exposure to 365 nm light partially restored the silencing activity of the 2'-fluoro siRNAs. These results suggest that photocaging may offer control over RNAi therapeutics for spatially and temporally directed activation, while improving enzymatic stability and potentially enabling therapeutic dosing via light dose intensity.

Oxygen electrode-based single antibody amperometric biosensor for qualitative detection of E. coli and bacteria in water.

Design and performance of an amperometric biosensor for E. coli O157:H7 that is based on a common dissolved oxygen probe is discussed. Anti-E. coli O157:H7 antibody was conjugated to horseradish peroxidase and immobilized on a nitrocellulose membrane that was placed over the oxygen probe membrane using a custom-fabricated polyvinyl chloride (PVC) insert. Upon bacterial cell binding, a decrease in enzyme activity resulted in a change in oxygen concentration that was detected with a Clark-type oxygen electrode probe. Validation experiments determined the effect of the outer membrane and insert on the Clarke electrode performance and linearity, and the effects of stirring on sensor response. The mechanism of enzymatic disruption is presumably steric hindrance due to binding of the bacterial cell and conformational change in antibody structure. Sampling various dilutions of heat-sterilized E. coli O157:H7 cells in water, as little as 50 bacterial cells/mL could be detected in approximately 20 minutes of sampling and processing procedures. Bacterial concentrations from 0 to 5000 cells/mL were tested, with 2.52 mg/L +/- 0.37 mg/L equivalents of oxygen produced from as few as 50 cells/mL, versus 6.26 +/- 0.64 mg/L when no cells were present in solution. Overall, the developed amperometric biosensor technology offered an efficient means of detection primarily due to its ease of use, cost-effectiveness, portability, and amenability to incorporation at existing water quality gaging stations.

Photobiological effects of UVA and UVB light in zebrafish embryos: evidence for a competent photorepair system.

The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6-7, 12, and 24h post-fertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebrafish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD(50) of 850 J/cm(2)) to UVA, confirming their suitability for photoactivation and photorepair studies.

Fully 2'-deoxy-2'-fluoro substituted nucleic acids induce RNA interference in mammalian cell culture.

RNA interference is a phenomenon in which RNA molecules elicit potent and sequence-specific post-transcriptional gene silencing. Recent studies have shown that small interfering RNA containing pyrimidine 2'-fluoro modifications elicit RNAi. In this study, we demonstrate that fully-2'-fluorinated nucleic acids can be generated for RNAi studies through either custom solid-phase synthesis or in vitro transcription using a mutated polymerase and fluorinated nucleoside triphosphates. Single-stranded and hybridized fully-2'-fluorinated nucleic acids were subjected to a ribonuclease to assess their resistance to digestion. Duplex siFNA and antisense fully-2'-fluorinated nucleic acids were evaluated for their ability to knockdown green fluorescent protein expression in mammalian cell culture. Based on the results, fully-2'-fluorinated nucleic acids can be successfully generated, and fully-2'-fluorinated nucleic acids products show superior resistance to digestion over native RNA. Melt curve analysis suggests that transcribed fully-2'-fluorinated nucleic acids may contain base miscoding errors or early termination products. Small interfering fluoronucleic acid can induce RNAi and the silencing efficiency is nearly equivalent to the unmodified small interfering RNA species. Silencing from antisense fully-2'-fluorinated nucleic acids was greatly reduced relative to the duplex form. The lack of silencing activity from single-stranded fully-2'-fluorinated nucleic acids, combined with reverse transcription polymerase chain reaction data showing that mRNA decreases following siFNA treatment, suggests that knockdown from siFNA is likely enzymatically driven as opposed to simple translational arrest.

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures.

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.

Scarless skin repair in immunodeficient mice.

Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude-nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude-nu, wild-type controls (C57BL/6J), severe-combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude-nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Masson's trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro-scarring cytokines platelet-derived growth factor-B and transforming growth factor beta1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8-positive cells in all studied animals except nude-nu mice. We conclude that scarless skin healing in athymic nude-nu mice provides a new model to study the influence of the immune system on tissue regeneration.

Functionalization of gold and glass surfaces with magnetic nanoparticles using biomolecular interactions.

Advances in nanotechnology have enabled the production and characterization of magnetic particles with nanometer-sized features that can be functionalized with biological recognition elements for numerous applications in biotechnology. In the present study, the synthesis of and interactions between self-assembled monolayers (SAMs) on gold and glass surfaces and functionalized magnetic nanoparticles have been characterized. Immobilization of 10-15 nm streptavidin-functionalized nanoparticles to biotinylated gold and glass surfaces was achieved by the strong interactions between biotin and streptavidin. Fluorescent streptavidin-functionalized nanoparticles, biotinylated surfaces, and combinations of the two were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electron and fluorescent microscopy to confirm that little or no functionalization occurred in nonbiotinylated regions of the gold and glass surfaces compared to the biotinylated sites. Together these techniques have potential use in studying the modification and behavior of functionalized nanoparticles on surfaces in biosensing and other applications.