This work was supported by an EPSRC CASE studentship in collaboration with Ceramisys Ltd. and is also associated with MeDe Innovation, the EPSRC Centre for Innovative Manufacturing in Medical Devices [grant number EP/K029592/1]. The authors would also like to thank Robert Burton at Sheffield Hallam University for XRF analysis.

<img alt="Figure 1" src="/files/ftp_upload/55343/55343fig1.jpg" /> 
Figure 1. Schematic diagram of rapid mix preparation of bioinspired nanoscale hydroxyapatite. The phosphoric acid solution was poured into the calcium hydroxide suspension. After the suspension settled overnight, the nHA was washed with deionized water before being dried at 60 to 80&#176;C. The nHA was then ground in an agate mortar and pestle and sintered to investigate the thermal stability of the nHA product. <a href="http://cloudfront.jove.com/files/ftp_upload/55343/55343fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Rapid Mix Production of Nanoscale Hydroxyapatite
<ol>
	<li>Preparation of calcium and phosphorus solutions to prepare 5 g of nanoscale hydroxyapatite using a calcium to phosphorus molar ratio of 1.67.
	<ol>
		<li>Add 3.705 g of calcium hydroxide to 500 mL deionized water and stir on a magnetic stirrer plate for 1 h at 400 rpm.</li>
		<li>In a separate beaker, dissolve&#160;3.459 g of phosphoric acid (85%) in&#160;250 mL deionized water.</li>
	</ol>
	</li>
	<li>Pour the phosphorus solution into the stirring calcium hydroxide suspension at a rate of approximately 100 mL/s. Cover beaker with Parafilm (Bemis, USA).</li>
	<li>Leave the suspension to stir for 1 h at 400 rpm.</li>
	<li>Take the beaker off the stirrer plate and leave to settle overnight.</li>
	<li>Wash the suspension by pouring off the supernatant and adding 500 mL deionized water and stirring for 1 min at 400 rpm. Repeat this step three times in total, with 2 h between each wash.</li>
	<li>Leave nHA suspension to settle overnight.</li>
	<li>Pour off the clear supernatant and place the settled nHA suspension in a drying oven set at 60 to 80 &#176;C.</li>
	<li>When dry, place the dried nHA into an agate mortar and pestle and grind until fine.</li>
	<li>Place 2.5 g of produced nHA powder in an alumina crucible and sinter powder at 1,000 &#176;C for 2 h using a ramp rate of 10 &#176;C/min. After the heat treatment, leave the nHA to cool in the furnace.</li>
	<li>Store powders in a vacuum desiccator.</li>
</ol>
2. Characterization of Nanoscale Hydroxyapatite
<ol>
	<li>X-ray diffraction (XRD) using transmission mode diffractometers
	<ol>
		<li>Place a small amount (i.e. less than 200 &#181;L) of poly(vinyl alcohol) (PVA) glue on acetate film and mix with a small amount (i.e. less than 100 mg) of nHA powder.</li>
		<li>Treat with a hot air gun until dry.</li>
		<li>Mount the sample into a sample holder and load onto a transmission mode X-ray diffractometer with Cu K&#945; radiation.</li>
		<li>Use diffractometer settings of 40 kV and 35 mA, with a 2&#952; range of 10-70&#176;.</li>
		<li>Analyze the resultant XRD patterns.</li>
		<li>Use the following XRD cards for phase identification: Hydroxyapatite: 9-432. &#946;-tricalcium phosphate: 04-014-2292.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Place a small amount of powder (i.e. less than 10 mg) in a bijou and add approximately 3 mL ethanol.</li>
		<li>Ultra-sonicate sample for 15 - 30 minutes until a homogenous suspension is observed.</li>
		<li>Pipette a small amount of solution (i.e. less than 1 mL) onto a 400 mesh copper grid with carbon film, and allow to dry.</li>
		<li>Image samples at an accelerating voltage of 80 kV.</li>
	</ol>
	</li>
	<li>X-ray fluorescence (XRF) service by the Materials and Engineering Research Institute (MERI) at Sheffield Hallam University
	<ol>
		<li>Combine 0.8 g nHA powder with 8 g of lithium tetraborate.</li>
		<li>Melt mixture in a platinum-gold alloy crucible using a furnace set to 1,200 &#176;C.</li>
		<li>Analyze resultant samples in an XRF spectrometer to determine the elemental composition of the samples.</li>
	</ol>
	</li>
	<li>Fourier-transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR)
	<ol>
		<li>Perform 64 background scans from 4,000 - 500 cm-1 with a resolution of 4 cm-1.</li>
		<li>Place a small amount (i.e. less than 100 mg) of nHA powder on top of the diamond in the attenuated total reflectance mode adapter and compress onto the surface of the diamond using the screw top.</li>
		<li>Perform 32 scans from 4,000 - 500 cm-1 with a resolution of 4 cm-1 with the background scans subtracted from the sample scans.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Natural apatite is composed of nanoscale particles of non-stoichiometric carbonated hydroxyapatite with the approximate chemical formula of Ca10-x-y[(HPO4)(PO4)]6-x(CO3)y(OH)2-x. The production of biomaterials with close chemical similarity to naturally occurring mineral has been reported to promote optimal biological responses. For instance, research on biomimetic calcium-deficient carbonated nHA has shown it is able to stimulate proliferation and the alkaline phosphatase activity of murine preosteoblast cells to a greater degree than conventional nHA19.
In this study, the precipitation of HA which showed partial thermal decomposition at 1,000 &#176;C (Figure 2) suggested the formation of a calcium-deficient HA. This was supported by the lower than stoichiometric Ca:P ratio (1.63) obtained with the XRF data (Table 1). It is understood that a reduced Ca:P ratio is associated with a lower thermal stability20,21,22,23. In this method, the rapid addition of the phosphoric acid solution rapidly lowered the pH of the reaction suspension to generate HPO4 ions. The presence of HPO4 groups facilitated the precipitation of calcium deficient HA, with the molecular formula: Ca10-x(HPO4) x(PO4)6-x(OH)2-x, where 0&#60;x&#60;1.
The rapid addition of the phosphoric acid therefore had a marked effect on the precipitation kinetics of the reaction. As described previously, titration reactions involving calcium hydroxide and phosphoric acid carried out at room temperature tended to yield particles with a high aspect ratio13. For titration reactions involving these reactants, it was necessary to use an elevated temperature to produce particles with a lower aspect ratio which are more similar to biological apatite13. High aspect ratio particles are produced when the crystal nucleation rate is slower than the crystal growth rate24. For the new method developed in this study, the rapid addition of the phosphoric acid solution may have provided a larger number of nucleation sites which resulted in the increased presence of small rounded particles as opposed to fewer particles with a larger aspect ratio. As the authors have not fully investigated the effects of slowly pouring the phosphoric acid into the calcium hydroxide suspension, in order to achieve consistent results we recommend that the phosphoric acid is poured at a rate commensurate with that shown in the video (approximately 100 mL/s).
During the development of this method, the authors investigated a number of incremental changes to the nHA preparation method based on Prakash et al.13 including the comparison of products produced with the slow titration and the rapid addition of the phosphoric acid solution25. It was found that the slow titration of phosphoric acid into the calcium hydroxide suspension resulted in a product with a calcium hydroxide residue. We propose that the pH change caused by the rapid addition of phosphoric acid encouraged the dissolution of the calcium hydroxide and therefore allowed for the successful conversion of the reactants into hydroxyapatite. A comparison of products prepared using the rapid mixing method at room and elevated temperatures (60 &#176;C) found that an elevated temperature resulted in a higher conductivity after the reaction was completed. This suggested that residual calcium hydroxide was present which was likely to be due to the lower solubility of calcium hydroxide at increased temperatures. The presence of residual calcium hydroxide was undesirable as the basic nature of this compound could compromise biocompatibility.
FTIR detected the characteristic phosphate and hydroxyl group activity associated with HA (Figure 3). It was noted that the spectrum for the sintered product showed sharper phosphate and hydroxyl peaks. These changes have been associated with a greater product crystallinity26,27.The unsintered spectrum provided evidence for B-type carbonate substitution where carbonate ions have substituted for phosphate groups. This is in contrast to A-type substitution where carbonate ions may substitute for hydroxyl groups17. It has been reported that B-type carbonate substitution occurs in biological apatite3. However, Tampieri et al. reported that whilst B-type substitution was predominant in young bones, A-type carbonate substitution was increasingly present in bones of older individuals28. Carbonate substitution has been found to decrease the crystallinity and thermal stability of the nHA whilst increasing its solubility. These changes have been proposed to contribute to the increased bioactivity of carbonate-substituted HA29. Biological HA is also known to contain some of the other elements recorded in the XRF analysis (Table 1), such as magnesium, sodium and strontium30. The presence of these elements may also contribute to increased biological efficacy. Future work should be directed at the preparation of these nanoscale substituted apatites, and also products with increased biofunctionality such as silver-doped nHA31. In order to prepare substituted nHA, the element may be introduced with a corresponding reduction of the intended element to substitute for, e.g. a reduction in the amount of the calcium compound when strontium, magnesium or zinc substitution is attempted32. Alternatively, another approach may be to add elements with the intention of providing &#39;doped&#39; ions which are present on the surface of the nHA without necessarily intending to substitute the element into the HA crystal lattice31. For these modifications to the method it is possible to prepare mixed solutions such as calcium hydroxide and silver nitrate, and to carry out the reaction in the same manner as described here.
In conclusion, this paper reports a novel rapid and substantially improved method for the preparation of bioinspired nHA. For this method, the rapid mixing of the chemicals takes less than 5 seconds which is a marked reduction in time compared to titrations reactions typically requiring hours of careful monitoring. It has great potential for use in biomaterial development due to its relative simplicity and low cost compared to currently used industrial nHA manufacturing methods where the inherent complexity of current commercial systems results in lengthy research and development times, and substantially increased manufacturing costs. In particular, this new method is superior to continuous flow processes or hydrothermal techniques due to significantly lower start-up equipment investment requirements.

There is a great clinical need for advanced biomaterials with enhanced functionality in order to improve quality of life of patients and to reduce the healthcare burden of a global aging population. Hydroxyapatite has been widely used in medical applications for many years due to its good biocompatibility. Recently, there has been an increased interest in the use of nanoscale hydroxyapatite (nHA), particularly for mineralized tissue regeneration in medicine and dentistry. The mineral found in bone and tooth enamel is calcium-deficient, multi-substituted, nanoscale hydroxyapatite. Estimates for the size of biological nHA platelets report dimensions of 50 nm x 30 nm x 2 nm1, with even smaller structures described in immature bone2. Contrastingly, the mineral in tooth enamel is 10 to 100 times larger than that found in bone tissue in both length and width3,4. Synthetic nHA might be better termed bioinspired rather than biomimetic, as we are seeking to translate observations regarding the characteristics of natural materials into medical technologies with improved performance. It has been suggested that bioinspired nHA may be more favorable in bone and tooth tissue regeneration applications due to its similarity to naturally occurring mineral5.
There are various methods which have been reported to prepare nHA including hydrothermal6, spray-dry7 and sol-gel8 techniques. Of these, the wet precipitation method is considered a relatively convenient method for the production of nHA. The published nHA wet precipitation methods generally include a titration step when mixing calcium and phosphorus chemical precursors9,10,11,12,13,14. However, these approaches are associated with a number of disadvantages including lengthy and complex processes combined in some cases with the need for expensive equipment. Commercial production may be even more complex, with patents describing sophisticated reactors for manufacture of high quality medical grade nHA15. Despite this, the neutralization reaction between calcium hydroxide and phosphoric acid is advantageous due to the lack of noxious chemical by-products.
The relationship between processing conditions and the morphology of the nHA product has been reported for slow titration reactions. Specifically, for titration methods involving calcium hydroxide and phosphoric acid, an elevated temperature appeared to favor the preparation of particles with a low aspect ratio13. This work was extended considerably by Gentile et al.16 who demonstrated the relationship between temperature and other processing conditions on the quality of nHA products from a wide range of methods. He concluded that the wet chemical precipitation method of Prakash13 made the highest quality products, but it should be noted that the results were dependent upon technically challenging and slow/ mixing processes. The original Prakash titration step takes over one hour. However, longer titration times may be required for larger batches to be prepared.
To summarize, while the influence of several factors including temperature have now been studied extensively, almost no attention has been directed at reducing the complexity and associated time needed to perform titration-based methods. The aim of this study was therefore to investigate the effects of applying a rapid mix approach to the manufacture of a bioinspired nHA, and to fully characterize the resulting materials. If successful, a simplified rapid mix approach would have great benefits for laboratory researchers and industry alike where costs of manufacture could be substantially reduced without comprising quality.

<table border="0" cellpadding="0" cellspacing="0" width="1375">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Calcium hydroxide (purity of &#8805; 96%)</td>
			<td style="width:168px;">Sigma Aldrich UK</td>
			<td align="right" style="width:115px;">31219</td>
			<td style="width:695px;">Good laboratory practise should be used at all times including the use of appropriate personal protective equipment.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphoric acid (85 %)</td>
			<td>Sigma Aldrich UK</td>
			<td align="right">345245</td>
			<td>Safety goggles and a faceshield should be used when handling this product (see safety data sheet from Sigma Aldrich for further information).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STOE IP x-ray diffractometer</td>
			<td>Phillips</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">International centre for diffraction data (ICDD) PDF4+ database</td>
			<td colspan="2">International Centre for Diffraction Data</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Holey carbon films on 300 mesh grids</td>
			<td>Agar Scientific</td>
			<td>S147-3H&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tecnai G2 Spirit transmission electron microscope</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithium tetraborate</td>
			<td>ICPH, Malz&#233;ville, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PW2440 XRF spectrometer&#160;</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ThermoScientific Nikolett Spectrometer</td>
			<td>Unicam Ltd</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

XRD patterns (Figure 2) showed the precipitation of a pure HA phase with broad peaks, indicating a relatively small crystallite size and/or amorphous nature. After high temperature sintering, &#946;-tricalcium phosphate (&#946;-TCP) was detected, alongside a main phase of HA. The sharpening of the diffraction peaks, i.e. a reduction in the full width half maximum, indicated an increase in the crystallite size after sintering.
<img alt="Figure 2" src="/files/ftp_upload/55343/55343fig2.jpg" /> 
Figure 2. Crystal phase analysis of products. X-ray diffraction (XRD) patterns of unsintered nanoscale hydroxyapatite (nHA) powder and nHA powder sintered at 1,000 &#176;C for 2 h. Peak labels: &#9660; hydroxyapatite peaks, &#9632; &#946;-tricalcium phosphate peaks. <a href="http://cloudflare.jove.com/files/ftp_upload/55343/55343fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
FTIR-ATR spectra (Figure 3) confirmed the formation of a HA phase by the characteristic phosphate and hydroxyl bands17,18. In detail the bands were assigned as follows: 3,750 cm-1 (OH- stretch &#957;OH); 1,086 and 1,022 cm-1 (PO43- &#957;3); 962 cm-1 (PO43- &#957;1); 630 cm-1 (OH- libration &#948;OH); 600 and 570 cm-1 (PO43- &#957;4). In the unsintered sample the additional peaks were assigned as follows: broad peak centered around 3,400 cm-1 (absorbed water molecules); 1,455 and 1,410 cm-1 (CO32- &#957;3); 880 cm-1 (CO32- &#957;2). The absorbed water and carbonate groups observed in the unsintered powder were removed during the high temperature sintering stage. The sintering process also sharpened the hydroxyl and phosphate bands which was manifested by a greater peak to trough distance.
<img alt="Figure 3" src="/files/ftp_upload/55343/55343fig3.jpg" /> 
Figure 3. Infrared spectra of products. Fourier transform infrared in attenuated total reflectance mode (FTIR-ATR) spectra of unsintered nanoscale hydroxyapatite (nHA) powder and nHA powder sintered at 1,000 &#176;C for 2 h. <a href="http://cloudflare.jove.com/files/ftp_upload/55343/55343fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
TEM images (Figure 4) showed the formation of nanoscale particles with approximate dimensions of 50 nm by 30 nm. The particles had a low aspect ratio (particle length / particle width) of around 1.7. The size and shape of the nanoscale products were of similar dimensions to biological apatite1.
<img alt="Figure 4" src="/files/ftp_upload/55343/55343fig4.jpg" /> 
Figure 4. Nanoscale morphology of product. Transmission electron micrographs (TEM) of nanoscale hydroxyapatite (nHA) prepared using the rapid mixing method at two magnifications. <a href="http://cloudflare.jove.com/files/ftp_upload/55343/55343fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Quantitative chemical analysis of the nHA powder by XRF (Table 1) allowed the calcium: phosphorus ratio to be calculated as 1.63, which is slightly lower than the stoichiometric HA which has a calcium: phosphorus ratio of 1.67. XRF also showed the high purity of the nHA product with only trace amounts of other elements recorded.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Weight %</td>
		</tr>
		<tr>
			<td>CaO</td>
			<td>51.52</td>
		</tr>
		<tr>
			<td>P2O5</td>
			<td>39.89</td>
		</tr>
		<tr>
			<td>MgO</td>
			<td>0.46</td>
		</tr>
		<tr>
			<td>Na2O</td>
			<td>0.13</td>
		</tr>
		<tr>
			<td>Y2O3</td>
			<td>0.07</td>
		</tr>
		<tr>
			<td>Al2O3</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>SiO2</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>Mn3O4</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>SrO</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>TiO2</td>
			<td>0.01</td>
		</tr>
	</tbody>
</table>
Table 1. Quantitative chemical analysis of product. X-ray fluorescence (XRF) results for unsintered nHA powder showed &#62;99% purity by weight.

Watch this Scientific Journal Video about Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications at JoVE.com

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical 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 ...

rapid-mix-preparation-bioinspired-nanoscale-hydroxyapatite-for

Research

JoVE Journal

Bioengineering

19.1K Views.  University of Sheffield. 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. 

Video: Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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, ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

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.

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 ...

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 ...

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 ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

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 ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...