Authors would like to thank Dr. Andre Walder and Nicolas Perrin for manufacturing titanium implants, K. Benmlih for the build-up of the Teflon molds and Dr. G. Prevost for his help with animal experiments. We also acknowledge the Region Alsace and PMNA (Pole Materiaux et Nanosciences d'Alsace) for financial contribution.

1. Preparation of Micropore Gradients in Macroporous Metallic Implants
<ol>
	<li>Clean the implants (such as implants made of medical grade titanium beads with a size range of 400-500 &mu;m, Neyco SAS, France) with ethanol and then sonicate in acetone for 15 min.</li>
	<li>Design and manufacture Teflon molds according to the implant size and shape (For standard experiments, cylindrical molds of 1.5 cm diameter with a height of 2 cm are used). Molds should be modular, so that the certain parts can be removed during extraction. Such a mold design ensures the control over the extraction process by forcing the movement of the extraction fluid in a directed manner.</li>
	<li>For tubular implants designed to replace trachea, the structure should be composed of three pieces: i) a bottom part to determine the size of the implant, ii) a mandrel and iii) an outer core which is removable. This way, the pore size gradient can be formed from outside towards inside.</li>
	<li>Prepare the synthetic polymer (PLLA) solution: For freeze/extraction PLLA solution needs to be prepared in a binary mixture of dioxane and water (87:13%, v:v).</li>
	<li>Heat the mixture to 60 &deg;C in order to obtain a homogenous solution. 60 &deg;C selected as it is the higher limit of the temperature resistance for the precision glass syringes that has to be used for the introduction of the solution into the implants.</li>
	<li>If high concentration (&ge;6%), high molecular weight PLLA solutions are used, introduce the solution into the implant after heating immediately. Otherwise gelation of the solution occurs without full immersion.</li>
	<li>Calculate the volume of the necessary polymer solution with respect to the porosity of the implant, for accuracy change in the volume of the frozen solution can be taken into account.</li>
	<li>Introduce the solution into the implants with precision glass syringes with 0.1 &mu;l accuracy. The lower limit for the polymer concentration is 3% for reproducible pore gradient formation whereas it becomes hard to obtain homogenous distribution in the thick samples above 6%. However, for specific applications other concentrations can be used.</li>
	<li>Freeze the samples either directly at -80 &deg;C or with a prior incubation period of 30 min at room temperature. Freezing conditions determine partially the pore formation, thus the freezing conditions can be adjusted according to the porosity aimed. Keep the samples overnight at -80 &deg;C.</li>
	<li>Extraction: Immerse the implants in 80% pre-chilled EtOH. Carry out the extraction at -20 &deg;C overnight. To obtain porosity gradients, remove all the mold parts except the mandrel for the tubular implants and all the parts except the bottom part for disk shaped implants. Use a pre-chilled scalpel for easier separation of the mold.</li>
	<li>After extraction at -20 &deg;C overnight 19, remove the remaining mold parts and air dry the implants. For characterization of the overall porosity of the structure mercury porosimeter analysis is necessary. Mercury Porosimeter measurements showed distinct peaks that correspond to the pores on the both sides of the implant and the smaller interdispersed pores. However the more crucial data is the difference between the porosities of intraluminal and extraluminal surfaces, which can be analyzed by Image J for pore size distribution with a scanning electron microscope (SEM) 20. For verification of the pore gradient, freeze-fracture the samples and observe the cross-section with SEM.</li>
	<li>Due to the open porous nature of the implants used, and the light reflecting capacity of Titanium, it is possible to do z-stacks of labeled cells within the porous implants. Label the cells with PKH26 or Calcein-AM and visualize the implants with confocal laser microscopy.</li>
</ol>
2. Surface Coating of Porous Metallic Implants with Collagen/Alginate Multilayers
<ol>
	<li>For build-up of multilayers, highest reproducibility is obtained with dipping robots. However, if a dipping robot is not available these steps can be done manually.</li>
	<li>Use medical grade collagen type I and sodium alginate. The optimized concentrations are 0.5 g/L for each in 150 mM NaCl in citrate buffer at pH 3.8.</li>
	<li>Dissolve the collagen solution overnight to ensure the homogeneity of the solution. Acidic pH of 3.8 is necessary for stable build-up of the layers as the structure is unstable before crosslinking in neutral pH.</li>
	<li>Deposit the layers by a dipping robot system by immersing the implants into collagen and alginate solutions alternatively. Deposition time is 15 min for each subsequent layer. Rinse the structure in between deposition steps with 150 mM NaCl at pH 3.8 for 5 min.</li>
	<li>Design specific holder for utilization of the implants with dipping robots used in polyelectrolyte multilayer production. Deposit the layers on the surface of either titanium only implants or implants modified as described in section 1.</li>
	<li>Stabilization of the basement membrane mimic with genipin: Prepare the crosslinking solution in a Dimethylsulfoxide (DMSO)/citrate buffer (150 mM NaCl, pH 3.8) at 1:4 v:v ratio. A wide range of concentrations can be used and 100 mM is adequate for crosslinking. Dissolve genipin first in the DMSO component and add the water component later to avoid clumping.</li>
	<li>Crosslink the samples by the immersion in the crosslinking solution between 12-24 hr. Afterwards rinse with copious amount of citrate buffer (pH 3.8).</li>
	<li>After washing steps, sterilize the samples either with UV treatment (30 min) or an antibiotic/antifungal bath (Penicillin/Streptomycin, Fungizone).</li>
	<li>The main parameters that determine the quality of the basement membrane mimic are its thickness and the diameter of the fibers. Calculate the fiber diameters using Atomic Force Microscopy (AFM) images obtained in contact mode. Dry the samples with a nitrogen flow before imaging. Quantify the thickness of at least 10 fibers per image to determine the average fibril thickness with Image J software.</li>
	<li>The thickness of the films can be determined by scratch tests using AFM. Dry (COL/ALG)24 / COL multilayer films. Use a syringe needle to scratch the film. After localization of the scratch with a light microscope, obtain images with AFM on 10 x 10 &mu;m2 surfaces at the boundary of the scratch. Calculate the heights from the profiles obtained with the AFM software, which provides the thickness of the film layer.</li>
</ol>
3. Indirect Monitoring of Implant Integration In vivo by Analysis of Blood Plasma
<ol>
	<li>All the necessary committee approvals should be taken for animal experimentation according to the governing rules for each country 21. In our case the Guide for the Care and Use of Laboratory Animals (National Research Council, 2010) is followed and the approval of the University of Strasbourg ethic committee is obtained.</li>
	<li>Carry out the implantation at the target site. The blood monitoring protocol given here was used for tracheal replacement in New Zealand white rabbits of a 15 mm tracheal resection.</li>
	<li>Following implantation a daily follow-up is necessary such as montioring the general well-being of the animals (healing around the surgical sites, rate of breathing) and recording of their weight.</li>
	<li>To validate the blood test, use a well-established method such as ELISA tests for blood CRP levels. CRP tests for many animals are available and the specific test used for rabbits is listed in Table 1. Similarly, use western blotting for the determination of CGA levels. Monoclonal anti-CGA antibodies (anti-CGA47-68) were used in this protocol.</li>
	<li>For plasma characterization, obtain blood samples from the auricular veins of the rabbits. Centrifuge at 5,000 rpm for 20 min at 4 &deg;C. Use the supernatant obtained for analysis. In our procedure, these tests are done on a weekly basis, but more frequent tests are also possible.</li>
	<li>Reverse phase HPLC purification of the Plasma protein content: Extract the rabbit plasma with 0.1% of trifluoroacetic acid (1:1; v:v). Purify the extract by using a Dionex HPLC system (Ultimate 3000; Sunnyvale, CA USA) on a nucleosil reverse-phase 300-5C18-column (4 x 250 mm; particle size 5 &mu;m; porosity, 300 &Aring;).</li>
	<li>Record the absorbance at 214 and 280 nm. The solvent system used is i)Solvent A: 0.1% (v/v) Trifluoroacetic acid (TFA) in water and ii) Solvent B: 0.09% (v/v) TFA in 70% (v/v) acetonitrile-water.</li>
	<li>Use a flow rate of 700 &mu;l/min using gradients for elutions. Collect the peak fractions. Concentrate the fractions by evaporation by speed-vacuum application. It is important to stop the speed-vacuum before complete dryness.</li>
	<li>Correlate the peaks obtained at different time-points over the course of the implantation period. Use the purified peptides that are showing consistent trends during the course of implantation for identification by automatic Edman sequencing.</li>
	<li>Automatic Edman sequencing of the peptides: Determine the N-terminal sequence of the purified peptides by automatic Edman degradation using a Procise microsequencer. Load the sample to polybrene-treated glass-fibre filters. Next step is the identification of Phenylthiohydantoin-amino acids (Pth-Xaa) by chromatography on a C18 column (PTH C-18, 2.1 x 200 mm) 22. After the sequence is obtained, it can be identified by Blast software using SWISS-Prot database.</li>
</ol>

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NE Vrana is an employee of Protip SAS.

Pore gradients are important tools in interface tissue engineering and the system described here can be used alone or in conjunction with metallic implants to form pore gradient to study cell migration. The system does not necessitate any extra setting or extra equipment except a chemical fume hood to handle organic solvents, thus it can be applied in biology laboratories. Similar polymers such as Poly(glycolic acid) (PGA), Poly(lactic-co-glycolic) acid (PLGA) and Poly(caprolactone) (PCL) can be used with slight modifica...

Currently available metallic implants are suitable for load-bearing applications, but their non-degradability necessitates designs which ensure a strong interface with the tissue surrounding them 1. By providing structures that facilitate cellular in-growth and colonization in vivo, the lifetime of metallic implants can be prolonged 2. Openly porous metallic implants are promising materials for tissue interface engineering and also for ensuring good colonization of the implants. They have been actively used as orthopedic implants and also as tracheal implants 3-5. However, there are still problems that need to be solved such as the precise control over cell movement in the pore areas. Failure to control this process might lead to incomplete colonization in one end and restenosis in the other. Also further functionalization of these implants is necessary for achieving higher functions such as, delivery of growth factors, directed vascularization and simultaneous movement of different cell types 6-8. For tracheal implants, this is crucial as the colonization of the implant by a vascularized tissue is desirable. However, the uncontrolled tissue in-growth to the lumen of trachea is undesirable because it decreases implant patency.
One possibility to control cell movement is size exclusion. By knowing the size of the target cells and their ability to interact with a given synthetic polymer it is possible to develop gradients of pores which can effectively determine the depth of cell movement. For example by creating a pore architecture that is large enough for the entry of connective tissue cells such as fibroblasts extraluminally, but small enough (less than 10 &mu;m) to prevent their movement intraluminally an effective control over colonization of a tubular implant can be achieved.
From available pore creation methods such as freeze-drying, particle leaching, gas foaming 9,10; the easiest to adapt method for fast formation of pore gradients with minimal amount of necessary equipments is freeze-extraction 11. In this method, a polymer solution is frozen in a binary mixture of an organic solvent and water. Afterwards, the solvent is exchanged via extraction by a miscible pre-chilled liquid such as ethanol. Freezing and extraction conditions determine the shape and size of the pores and if the extraction is done in a way where the movement of the extraction solution can be controlled, pore size and shape can be directionally modulated.
Second step for multicellular tissues is the formation of porous barriers between different cell types to control their interaction. This is also necessary for the availability of different microenvironments for different cell types depending on their requirements 12,13. Trachea is a tubular organ that connects larynx with bronchi. It has an inner pseudostratified ciliary epithelium lining with interdispersed goblet cells which produce mucus. The 3D structure and stability of trachea is maintained by cartilage in the shape of C-rings. Thus, in an artificial trachea there should be a defined junction between the connective tissue and the ciliary epithelial layer. While a 3D structure is necessary for the connective tissue part, the migration of epithelial cells requires a basement membrane-like surface to achieve directional movement and closure of the wound. Polyelectrolyte multilayer films (PEMs) are one possible option to obtain basement membrane mimics. Layer-by-layer method (LbL) is a versatile process to obtain thin and functional surface coatings. It is based on electrostatic interactions of two oppositely charged polyelectrolytes and their build-up in a sequential manner to obtain nanoscale surface coatings whose properties can be varied by simply changing variables such as polyelectrolyte species, pH, layer number, addition of a capping layer, crosslinking etc. One of the main advantages of the LbL method is its ability to conform to the topography of the underlying substrate. Thus, under controlled conditions this method can also be used for obtaining surface coverage of porous structures. If collagen is used as one of the polyelectrolytes it is possible to obtain nanofibrillar structures that can mimic the surface of basement membrane. The hydrophobicity of titanium enables development of such structures and fibrillarity can be preserved in thick coatings 14. This way attachment and movement of cell on the surface can also be controlled. By using freeze-extraction and LbL film coating sequentially, a structure where cell movement can be controlled laterally, longitudinally and circumferentially can be obtained 15.
Here we describe two novel modification methods for titanium implants by using their hydrophobic behavior which can be extended to modification of various porous implants: i) formation of gradients of micropores within the macroporous titanium implants with hydrophobic, synthetic polymers ii) formation of a thick polymeric film layer on the implant surface that supports cell growth and lining formation by polyelectrolyte multilayers. These methods can be used sequentially or separately. They provide structures that ensure controlled migration and spatial organization of different cell types in multicellular tissues 16,17. For the specific case of trachea, the desired outcome for the implant would be the colonization by fibrovascular tissue within the micropore gradients without restenosis and the formation of the inner lining of ciliated epithelial cells on the polyelectrolyte multilayers.
One way of controlling integration of implants is to do small surgical interventions during the period of their integration with the host in situ. In order to be able to decide on the timing of the interventions, it is important to have information on the systemic effects of the implant. C-Reactive Protein (CRP) has been used for monitoring of infection and inflammatory response in clinical settings. Chromogranin A (CGA) can also be used in a similar manner and might provide more accurate results to observe the level of inflammation 18. As a possible way of observing metallic implant integration in vivo, we present a continuous monitoring procedure of implant systemic effects by characterization of animal blood samples with High Pressure Liquid Chromatography (HPLC) and subsequent protein sequencing. Elaboration of this method can be used to evade regular end-point histological analysis. Histological cutting of metallic implants is a long, cumbersome and expensive process and can be only undertaken at specific time points. Because of this reason, well-designed blood tests providing robust information about the implant health would be possible routes to decrease animal experiments as mandated by the recent EU rules concerning animal experiments.
The methods presented here can be used to improve the performance of metallic implants via functionalization or to have an alternative way of monitoring the existing implants.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Dioxane</td>
 <td>Sigma-Aldrich</td>
 <td>360481</td>
 <td>Toxic material, Strictly under chemical hood</td>
 </tr>
 <tr>
 <td>PLLA 
 i. Poly(L-lactide) inherent viscosity ~0.5 dl/g 
 ii. Poly(L-lactide) inherent viscosity ~2.0 dl/g 
 </td>
 <td>Sigma-Aldrich</td>
 <td>94829, 81273</td>
 <td>The choice of molecular weight and inherent viscosity is application dependent.</td>
 </tr>
 <tr>
 <td>PRONOVA UP LVG (Sodium Alginate)</td>
 <td>Novamatrix</td>
 <td>4200006</td>
 <td>Low viscosity(20-200 mPa.s)</td>
 </tr>
 <tr>
 <td>Collagen type I (Bovine)</td>
 <td>Symatese</td>
 <td>CBPE2US100</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen/Strep, Fungizone </td>
 <td>Promocell</td>
 <td>C42020</td>
 <td></td>
 </tr>
 <tr>
 <td>Genipin</td>
 <td>Wako</td>
 <td>0703021</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicon nitride probes with aspring constant of 0.03 N.m-1. </td>
 <td>Bruker </td>
 <td>MSCT </td>
 <td></td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid for HPLC ,&ge;99.0% </td>
 <td>Sigma-Aldrich</td>
 <td>302031</td>
 <td>Hazardous Material, Please follow MSDS carefully</td>
 </tr>
 <tr>
 <td>Acetonitrile, for HPLC ,&ge;99.9% </td>
 <td>Sigma-Aldrich</td>
 <td>34998</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein-AM</td>
 <td>Invitrogen</td>
 <td>C3100MP </td>
 <td></td>
 </tr>
 <tr>
 <td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling </td>
 <td>Sigma-Aldrich</td>
 <td>PKH26GL</td>
 <td></td>
 </tr>
 <tr>
 <td>Rabbit C-Reactive Protein (CRP) ELISA kit</td>
 <td>Genway Bio</td>
 <td>GWB-9BF960 </td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO, Bioreagent, &ge;99.7% </td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Multimode Nanoscope IV Atomic Force microscope</td>
 <td>Bruker</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Procise microsequencer </td>
 <td>Applied Biosystems </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultima 3000 HPLC system </td>
 <td>Dionex</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope Hitachi TM 100</td>
 <td>Hitachi </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal Scanning Laser Microscope Zeiss LSM 510 </td>
 <td>Zeiss </td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>
Table 1. List of Materials and Reagents.

multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Formation of pore gradients
By changing the concentration of the PLLA solution, it is possible to control the size of the pores on the extraluminal side of the implants. Pore size and shape was significantly affected by the presence of titanium implants (Figures 1a and 1b). Pore sizes ranged from 40-100 &mu;m and utilization of lower concentrations resulted in smaller pores. Whereas, in the intraluminal side pore size was governed by the restricted extraction and...

Watch this Scientific Journal Video about Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo at JoVE.com

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

multi-scale-modification-metallic-implants-with-pore-gradients

Research

JoVE Journal

Bioengineering

10.8K Views.  INSERM. In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Video: Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...