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><tbody><tr><td>Calcium hydroxide (purity of &amp;ge; 96%)</td><td>Sigma Aldrich UK</td><td>31219</td><td>Good laboratory practise should be used at all times including the use of appropriate personal protective equipment.</td></tr><tr><td>Phosphoric acid (85%)</td><td>Sigma Aldrich UK</td><td>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><td>STOE IP X-ray diffractometer</td><td>Phillips</td><td></td><td></td></tr><tr><td>International centre for diffraction data (ICDD) PDF4+ database</td><td>International Centre for Diffraction Data</td><td></td><td></td></tr><tr><td>Holey carbon films on 300 mesh grids</td><td>Agar Scientific</td><td>S147-3H&amp;nbsp;</td><td></td></tr><tr><td>Tecnai G2 Spirit transmission electron microscope</td><td>FEI</td><td></td><td></td></tr><tr><td>Lithium tetraborate</td><td>ICPH, Malz&amp;eacute;ville, France</td><td></td><td></td></tr><tr><td>PW2440 XRF spectrometer&amp;nbsp;</td><td>Philips</td><td></td><td></td></tr><tr><td>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

19.2K 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

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Chemistry

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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 Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

Biology

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been fabricated with hydroxyapatite to facilitate cell adherence, proliferation, and osteogenesis. In this study, hybrid nanocomposites were successfully developed using graphene nanoribbons (GNRs) and nanoparticles of hydroxyapatite (nHAPs), that when employed in bioactive scaffolds may potentially improve bone tissue regeneration. These nanostructures can be biocompatible. Here, two approaches were used for preparing the novel materials. In one approach, a co-functionalization strategy was used where nHAP was synthesized and conjugated to GNRs simultaneously, resulting in nanohybrids of nHAP on GNR surfaces (denoted as nHAP/GNR). High-resolution transmission electron microscopy (HRTEM) confirmed that the nHAP/GNR composite is comprised of slender, thin structures of GNRs (maximum length of 1.8 &#181;m) with discrete patches (150-250 nm) of needle-like nHAP (40-50 nm in length). In the other approach, commercially available nHAP was conjugated with GNRs forming GNR-coated nHAP (denoted as GNR/nHAP) (i.e., with an opposite orientation relative to the nHAP/GNR nanohybrid). The nanohybrid formed using the latter method exhibited nHAP nanospheres with a diameter ranging from 50 nm to 70 nm covered with a network of GNRs on the surface. Energy dispersive spectra, elemental mapping, and Fourier transform infrared (FTIR) spectra confirmed the successful integration of nHAP and GNRs in both nanohybrids. Thermogravimetric analysis (TGA) indicated that the loss at elevated heating temperatures due to the presence of GNRs was 0.5% and 0.98% for GNR/nHAP and nHAP/GNR, respectively. The nHAP-GNR nanohybrids with opposite orientations represent significant materials for use in bioactive scaffolds to potentially promote cellular functions for improving bone tissue engineering applications.

Novel nanocomposites of graphene nanoribbons and hydroxyapatite nanoparticles were prepared using solution-phase synthesis. These hybrids when employed in bioactive scaffolds can exhibit potential applications in tissue engineering and bone regeneration.

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

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Rozdziały w tym wideo

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Preparation of Functional Silica Using a Bioinspired Method

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

Procedure for Fabricating Biofunctional Nanofibers

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering