Name: fabricating superhydrophobic polymeric materials for biomedical applications
Uploaded: 2015-08-28T14:00:00
Description: Watch this Scientific Journal Video about Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications at JoVE.com

Funding was provided in part by BU and the NIH R01CA149561. The authors wish to thank the electrospinning/electrospraying team including Stefan Yohe, Eric Falde, Joseph Hersey, and Julia Wang for their helpful discussions and contributions to the preparation and characterization of superhydrophobic biomaterials.

1. Synthesizing Functionalizable poly(1,3-glycerol carbonate-co-caprolactone)29 and poly(1,3-glycerol carbonate-co-lactide)27,28.
<ol>
	<li>Monomer synthesis.
	<ol>
		<li>Dissolve cis-2-Phenyl-1,3-dioxan-5-ol (50 g, 0.28 mol, 1 eq.) in 500 ml dry tetrahydrofuran (THF) and stir on ice under nitrogen. Add potassium hydroxide (33.5 g, 0.84 mol, 3 eq.), finely crushed with a mortar and pestle. Place flask in ice bath.</li>
		<li>Add 49.6 ml benzyl bromide (71.32 g, 0.42 mol,...

Our approach to constructing superhydrophobic materials from biomedical polymers combines synthetic polymer chemistry with the polymer processing techniques of electrospinning and electrospraying. These techniques provide either fibers or particles, respectively. Specifically, polycaprolactone and poly(lactide-co-glycolide) based superhydrophobic materials are prepared using this strategy. By varying the hydrophobic copolymer composition, percent copolymer in the final polymer blend, fiber/particle size, overall...

Superhydrophobic surfaces are generally categorized as exhibiting apparent water contact angles greater than 150&#176; with low contact angle hysteresis. These surfaces are fabricated by introducing high surface roughness on low surface energy materials to establish a resulting air-liquid-solid interface that resists wetting1-6. Depending on the fabrication method, thin or multilayered superhydrophobic surfaces, multilayered superhydrophobic substrate coatings, or even bulk superhydrophobic structures can be prepared. This permanent or semi-permanent water repellency is a useful property that is employed to prepare self-cleaning surfaces7, microfluidic devices8, anti-fouling cell/protein surfaces9,10, drag-reducing surfaces11, and drug delivery devices12-15. Recently, stimuli-responsive superhydrophobic materials are described where the non-wetted to wetted state is triggered by chemical, physical, or environmental cues (e.g., light, pH, temperature, ultrasound, and applied electrical potential/current)14,16-20, and these materials are finding use for additional applications21-25.
The first synthetic superhydrophobic surfaces were prepared by treating material surfaces with methyldihalogenosilanes26, and were of limited value for biomedical applications, as the materials used were not suitable for in vivo use. Herein we describe the preparation of surface and bulk superhydrophobic materials from biocompatible polymers. Our approach entails electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate)27-30. The fabrication techniques provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively, while the use of a copolymer dopant provides a low surface energy polymer that blends with the polyester and can be stably electrospun or electrosprayed27,31,32.
Aliphatic biodegradable polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), and polycaprolactone (PCL) are polymers used in clinically-approved devices and prominent in biomedical materials research because of their non-toxicity, biodegradability, and ease of synthesis33. PGA and PLGA debuted in the clinic as bioresorbable sutures in the 1960&#8217;s and early 1970&#8217;s, respectively34-37. Since then, these poly(hydroxy acids) have been processed into a variety of other application-specific form factors, such as micro-38,39 and nanoparticles40,41, wafers/discs42, meshes27,43, foams44, and films45.
Aliphatic polyesters, as well as other polymers of biomedical interest, can be electrospun to produce nano- or microfiber mesh structures possessing a high surface area and porosity as well as tensile strength. Table 1 lists the synthetic polymers electrospun for various biomedical applications and their corresponding references. Electrospinning and electrospraying are rapid and commercially-scalable techniques. These two similar techniques rely on applying high voltage (electrostatic repulsion) to overcome surface tension of a polymer solution/melt in a syringe pump setup as it is directed toward a grounded target46,47. When this technique is used in conjunction with low surface energy polymers (hydrophobic polymers such as poly(caprolactone-co-glycerol monostearate)), the resulting materials exhibit superhydrophobicity.
To illustrate this general synthetic and materials processing approach to constructing superhydrophobic materials from biomedical polymers, we describe the synthesis of superhydrophobic polycaprolactone- and poly(lactide-co-glycolide)-based materials as representative examples. The respective copolymer dopants poly(caprolactone-co-glycerol monostearate) and poly(lactide-co-glycerol monostearate) are first synthesized, then blended with polycaprolactone and poly(lactide-co-glycolide), respectively, and finally electrospun or electrosprayed. The resulting materials are characterized by SEM imaging and contact angle goniometry, and tested for in vitro and in vivo biocompatibility. Finally, bulk wetting through three-dimensional superhydrophobic meshes is examined using contrast-enhanced microcomputed tomography.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Silicone oil</td>
			<td>Sigma-Aldrich</td>
			<td>85409</td>
			<td></td>
		</tr>
		<tr>
			<td>Cis-2-Phenyl-1,3-dioxan-5-ol</td>
			<td>Sigma-Aldrich</td>
			<td align="right">13468</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl bromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Toxic, lacrymator/eye irritant, use in chemical fume hood</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">221473</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Buchi</td>
			<td>R-124</td>
			<td></td>
		</tr>
		<tr>
			<td>High-vacuum pump</td>
			<td>Welch</td>
			<td align="right">8907</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen, ultra high purity</td>
			<td>Airgas</td>
			<td>NI UHP300</td>
			<td>Compressed gas</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran, stabilized with BHT</td>
			<td>Pharmaco-Aaper</td>
			<td align="right">346000</td>
			<td>Flammable. Dried through column of XXX</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Pharmaco-Aaper</td>
			<td align="right">313000</td>
			<td>Flammable, toxic.</td>
		</tr>
		<tr>
			<td>Separatory funnel (1 L)</td>
			<td>Fisher Scientific</td>
			<td>13-678-606</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">239313</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute</td>
			<td>Pharmaco-Aaper</td>
			<td>111USP200</td>
			<td>Flammable, toxic.</td>
		</tr>
		<tr>
			<td>Buchner funnel</td>
			<td>Fisher Scientific</td>
			<td>FB-966-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Pharmaco-Aaper</td>
			<td>339000ACS</td>
			<td>Flammable, toxic.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td align="right">320331</td>
			<td>Corrosive. Diluted to 2N in distilled water.</td>
		</tr>
		<tr>
			<td>Ethyl chloroformate, 97%</td>
			<td>Sigma-Aldrich</td>
			<td align="right">185892</td>
			<td>Toxic, flammable, harmful to environment</td>
		</tr>
		<tr>
			<td>Triethylamine (anhydrous)</td>
			<td>Sigma-Aldrich</td>
			<td align="right">471283</td>
			<td>Toxic, flammable, harmful to environment</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Pharmaco-Aaper</td>
			<td>373ANHACS</td>
			<td>Highly flammable. Purified through XXX column.</td>
		</tr>
		<tr>
			<td>3,6-Dimethyl-1,4-dioxane-2,5-dione (D,L-lactide)</td>
			<td>Sigma-Aldrich</td>
			<td align="right">303143</td>
			<td></td>
		</tr>
		<tr>
			<td>Tin (II) ethylhexanoate</td>
			<td>Sigma-Aldrich</td>
			<td>S3252</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>&#949;-caprolactone (97%)</td>
			<td>Sigma-Aldrich</td>
			<td align="right">704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td align="right">244511</td>
			<td>Flammable, toxic.</td>
		</tr>
		<tr>
			<td>Glass syringe</td>
			<td>Hamilton Company</td>
			<td>1700-series</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterated chloroform</td>
			<td>Cambridge Isotopes Laboratories, Inc.</td>
			<td>DLM-29-10</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Nuclear magnetic resonance instrument</td>
			<td>Varian</td>
			<td>V400</td>
			<td></td>
		</tr>
		<tr>
			<td>Palladium on carbon catalyst</td>
			<td>Strem Chemicals, Inc.</td>
			<td>46-1707</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogenator unit</td>
			<td>Parr</td>
			<td align="right">3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogenator shaker vessel</td>
			<td>Parr</td>
			<td>66CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen</td>
			<td>Airgas</td>
			<td>HY HP300</td>
			<td>Highly flammable.</td>
		</tr>
		<tr>
			<td>Diatomaceous earth</td>
			<td>Sigma-Aldrich</td>
			<td align="right">22140</td>
			<td></td>
		</tr>
		<tr>
			<td>2H,2H,3H,3H-perflurononanoic acid</td>
			<td>Oakwood Products, Inc.</td>
			<td align="right">10519</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>Stearic acid</td>
			<td>Sigma-Aldrich</td>
			<td>S4751</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8217;-dicyclohexylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D80002</td>
			<td>Toxic, irritant.</td>
		</tr>
		<tr>
			<td>4-(dimethylamino) pyridine</td>
			<td>Sigma-Aldrich</td>
			<td align="right">107700</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>Pharmaco-Aaper</td>
			<td>359000ACS</td>
			<td>Toxic, flammable.</td>
		</tr>
		<tr>
			<td>Gel permeation chromatography (GPC) system</td>
			<td>Rainin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GPC column</td>
			<td>Waters</td>
			<td>WAT044228</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential scanning calorimeter</td>
			<td>TA Instruments</td>
			<td>Q100</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Pharmaco-Aaper</td>
			<td>309000ACS</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">227056</td>
			<td>Toxic, flammable.</td>
		</tr>
		<tr>
			<td>Polycaprolactone, MW 70-90 kg/mol</td>
			<td>Sigma-Aldrich</td>
			<td align="right">440744</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(lactide-co-glycolide), MW 136 kg/mol</td>
			<td>Evonik Industries</td>
			<td>LP-712</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL glass syringe</td>
			<td>Hamilton Company</td>
			<td align="right">81620</td>
			<td></td>
		</tr>
		<tr>
			<td>18 AWG blunt needle</td>
			<td>BRICO Medical Supplies</td>
			<td>BN1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrospinner enclosure box</td>
			<td>Custom-built</td>
			<td>N/A</td>
			<td>Made of acrylic panels</td>
		</tr>
		<tr>
			<td>High voltage DC supply</td>
			<td>Glassman High Voltage, Inc.</td>
			<td>PS/EL30R01.5</td>
			<td>High voltages, electrocution hazard</td>
		</tr>
		<tr>
			<td>Linear (translating) stage</td>
			<td>Servo Systems Co.</td>
			<td>LPS-12-20-0.2</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Programmable motor &#38; power supply</td>
			<td>Intelligent Motion Systems, Inc.</td>
			<td>MDrive23 Plus</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>24V DC motor &#38; power supply</td>
			<td>McMaster-Carr</td>
			<td>6331K32</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Aluminum collector drum</td>
			<td>Custom-built</td>
			<td></td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Fisher Scientific</td>
			<td>78-0100I</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted optical microscope</td>
			<td>Olympus</td>
			<td>IX70</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Carl Zeiss</td>
			<td>Supra V55</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductive copper tape</td>
			<td>3M</td>
			<td align="right">16072</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum SEM stubs</td>
			<td>Electron Microscopy Sciences</td>
			<td align="right">75200</td>
			<td></td>
		</tr>
		<tr>
			<td>Contact angle goniometer</td>
			<td>Kruss</td>
			<td>DSA100</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>W294004</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td align="right">324558</td>
			<td>Toxic.</td>
		</tr>
		<tr>
			<td>Ioxaglate</td>
			<td>Guerbet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>American Type Culture Collection</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-computed tomography instrument</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software (Analyze)</td>
			<td>Mayo Clinic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tensile tester</td>
			<td>Instron</td>
			<td align="right">5848</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrometer</td>
			<td>Multitoyo</td>
			<td>293-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Calipers</td>
			<td>Fisher Scientific</td>
			<td>14-648-17</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabricating superhydrophobic polymeric materials for biomedical applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Through a series of chemical transformations, the functional carbonate monomer 5-benzyloxy-1,3-dioxan-2-one is synthesized as a white crystalline solid (Figure 1A). 1H NMR confirms the structure (Figure 1B) and mass spectrometry and elemental analysis confirm the composition. This solid is then copolymerized with either D,L-lactide or &#949;-caprolactone using a tin-catalyzed ring opening reaction at 140 &#176;C. After purification by precipitation, the polymer compos...

Watch this Scientific Journal Video about Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications at JoVE.com

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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Unit 1

Synthetic Polymers

SCIENTISTS IN ACTION

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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 ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...