Name: fabrication of mechanically tunable and bioactive metal scaffolds for biomedical applications
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This research was supported by the Technology Innovation Program (Contract grant No. 0037915, WPM Biomedical Materials-Implant Materials) and Industrial Strategic Technology Development Program (Contract grant No. 10045329, Development of customized implant with porous structure for bone replacement), funded by the Ministry of Trade, industry &#38; Energy (MI, Korea), and BK21 PLUS SNU Materials Division for Educating Creative Global Leaders (Contract grant No. 21A20131912052).

1. Fabrication of Porous Metal Scaffolds
<ol>
	<li>Prepare Ti-camphene slurries by mixing commercially available Ti powder, camphene, and KD-4 after weighing the appropriate amounts of materials as described in Table 1 for porous Ti scaffolds with four initial porosities (40, 50, 60, and 70). Pour the slurries into 500 ml polyethylene (PE) bottles and rotate the bottles at 55 &#176;C for 30 min in a ball-mill oven at 30 rpm.</li>
	<li>Pour the slurries from the PE bottles into cylindrical aluminum (Al)...

<ol>
	<li>Long, M., Rack, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+alloys+in+total+joint+replacement-a+materials+science+perspective.">Titanium alloys in total joint replacement-a materials science perspective.</a> Biomaterials. 19 (18), 1621-1639 (1998).</li><li>Niinomi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+metallic+materials+for+biomedical+applications.">Recent metallic materials for biomedical applications.</a> Metall. Mater. Trans. A. 33 (3), 477-486 (2002).</li><li>Frosch, K. H., St&#252;rmer, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallic+biomaterials+in+skeletal+repair.">Metallic biomaterials in skeletal repair.</a> Eur. J. 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Lett. 120 (1), 228-231 (2014).</li><li>Baas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pretreating+morselized+allograft+bone+with+rhBMP-2+and/or+pamidronate+on+the+fixation+of+porous+Ti+and+HA-coated+implants.">The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants.</a> Biomaterials. 29 (19), 2915-2922 (2008).</li><li>Peng, L., Bian, W. -. G., Liang, F. -. H., Xu, H. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanting+hydroxyapatite-coated+porous+titanium+with+bone+morphogenetic+protein-2+and+hyaluronic+acid+into+distal+femoral+metaphysis+of+rabbits.">Implanting hydroxyapatite-coated porous titanium with bone morphogenetic protein-2 and hyaluronic acid into distal femoral metaphysis of rabbits.</a> Chin. J. Traumatol. (English Edition). 11 (3), 179-185 (2008).</li><li>Reiner, T., Kababya, S., Gotman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+incorporation+within+Ti+scaffold+for+bone+ingrowth+using+Sol-gel+SiO2+as+a+slow+release+carrier.">Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier.</a> J. Mater. Sci. - Mater. Med. 19, 583-589 (2008).</li><li>Lee, J. H., Kim, H. E., Shin, K. H., Koh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+strength+and+biocompatibility+of+porous+titanium+scaffolds+by+creating+elongated+pores+coated+with+a+bioactive,+nanoporous+TiO2+layer.">Improving the strength and biocompatibility of porous titanium scaffolds by creating elongated pores coated with a bioactive, nanoporous TiO2 layer.</a> Mater. Lett. 64, 2526-2529 (2010).</li><li>Li, J. C., Dunand, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+directionally+freeze-cast+titanium+foams.">Mechanical properties of directionally freeze-cast titanium foams.</a> Acta Mater. 59 (1), 146-158 (2011).</li><li>Chino, Y., Dunand, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directionally+freeze-cast+titanium+foam+with+aligned,+elongated+pores.">Directionally freeze-cast titanium foam with aligned, elongated pores.</a> Acta Mater. 56 (1), 105-113 (2008).</li><li>Kim, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+porous+titanium+scaffold+with+controlled+porous+structure+and+net-shape+using+magnesium+as+spacer.">Fabrication of porous titanium scaffold with controlled porous structure and net-shape using magnesium as spacer.</a> Mater. Sci. Eng. C. 33 (5), 2808-2815 (2013).</li><li>Brentel, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphometric+analysis+of+pure+titanium+implants+with+porous+surface+versus+rough+surface.">Histomorphometric analysis of pure titanium implants with porous surface versus rough surface.</a> J. Appl. Oral Sci. 14 (3), 213-218 (2006).</li><li>Buser, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+surface+characteristics+on+bone+integration+of+titanium+implants.+A+histomorphometric+study+in+miniature+pigs.">Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs.</a> J. Biomed. Mater. Res. 25 (7), 889-902 (1991).</li><li>Cochran, D., Schenk, R., Lussi, A., Higginbottom, F., Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+response+to+unloaded+and+loaded+titanium+implants+with+a+sandblasted+and+acid-etched+surface:+a+histometric+study+in+the+canine+mandible.">Bone response to unloaded and loaded titanium implants with a sandblasted and acid-etched surface: a histometric study in the canine mandible.</a> J. Biomed. Mater. Res. 40 (1), 1-11 (1998).</li><li>Young, D. R., Robb, R. A., Rock, M. G., Chao, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+periprosthetic+tissue+formation+around+a+porous+titanium+endoprosthesis+using+CT-based+spatial+reconstruction.">Analysis of periprosthetic tissue formation around a porous titanium endoprosthesis using CT-based spatial reconstruction.</a> J. Comput. Assist. Tomo. 18 (3), 461-468 (1994).</li><li>Spoerke, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioactive+titanium+foam+scaffold+for+bone+repair.">A bioactive titanium foam scaffold for bone repair.</a> Acta Biomater. 1 (5), 523-533 (2005).</li><li>Jung, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+aligned+porous+Ti+scaffold+coated+with+bone+morphogenetic+protein-loaded+silica/chitosan+hybrid+for+enhanced+bone+regeneration.">Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone regeneration.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 102 (5), 913-921 (2013).</li><li>Ryan, G. E., Pandit, A. S., Apatsidis, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+titanium+scaffolds+fabricated+using+a+rapid+prototyping+and+powder+metallurgy+technique.">Porous titanium scaffolds fabricated using a rapid prototyping and powder metallurgy technique.</a> Biomaterials. 29 (27), 3625-3635 (2008).</li><li>Vasconcellos, L. M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+titanium+scaffolds+produced+by+powder+metallurgy+for+biomedical+applications.">Porous titanium scaffolds produced by powder metallurgy for biomedical applications.</a> Mater. Res. 11 (3), 275-280 (2008).</li><li>Jung, H. D., Yook, S. W., Kim, H. E., Koh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+titanium+scaffolds+with+porosity+and+pore+size+gradients+by+sequential+freeze+casting.">Fabrication of titanium scaffolds with porosity and pore size gradients by sequential freeze casting.</a> Mater. Lett. 63 (17), 1545-1547 (2009).</li><li>Jung, H. -. D., Jang, T. -. S., Wang, L., Kim, H. -. E., Koh, Y. -. H., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+strategy+for+mechanically+tunable+and+bioactive+metal+implants.">Novel strategy for mechanically tunable and bioactive metal implants.</a> Biomaterials. 37, 49-61 (2015).</li><li>Tarng, Y. S., Ma, S. C., Chung, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+optimal+cutting+parameters+in+wire+electrical+discharge+machining.">Determination of optimal cutting parameters in wire electrical discharge machining.</a> Int. J. Mach. Tools Manufact. 35 (12), 1693-1701 (1995).</li><li>Jung, H. -. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Freeze+Casting+for+the+Production+of+Porous+Titanium+(Ti)+Scaffolds.">Dynamic Freeze Casting for the Production of Porous Titanium (Ti) Scaffolds.</a> Mater. Sci. Eng. C. 33 (1), 59-63 (2013).</li><li>Lee, J. -. H., Kim, H. -. E., Shin, K. -. H., Koh, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+strength+and+biocompatibility+of+porous+titanium+scaffolds+by+creating+elongated+pores+coated+with+a+bioactive,+nanoporous+TiO2.">Improving the strength and biocompatibility of porous titanium scaffolds by creating elongated pores coated with a bioactive, nanoporous TiO2.</a> Mater. Lett. 64 (22), 2526-2529 (2010).</li><li>Jung, H. -. D., Yook, S. -. W., Kim, H. -. E., Koh, Y. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+titanium+scaffolds+with+porosity+and+pore+size+gradients+by+sequential+freeze+casting.">Fabrication of titanium scaffolds with porosity and pore size gradients by sequential freeze casting.</a> Mater. Lett. 63 (17), 1545-1547 (2009).</li><li>Yook, S. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+freeze+casting:+A+new+method+for+fabricating+highly+porous+titanium+scaffolds+with+aligned+large+pores.">Reverse freeze casting: A new method for fabricating highly porous titanium scaffolds with aligned large pores.</a> Acta Biomater. 8 (6), 2401-2410 (2012).</li><li>Yook, S. W., Yoon, B. H., Kim, H. E., Koh, Y. H., Kim, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+titanium+(Ti)+scaffolds+by+freezing+TiH2/camphene+slurries.">Porous titanium (Ti) scaffolds by freezing TiH2/camphene slurries.</a> Mater. Lett. 62 (30), 4506-4508 (2008).</li><li>Yook, S. W., Kim, H. E., Koh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+porous+titanium+scaffolds+with+high+compressive+strength+using+camphene-based+freeze+casting.">Fabrication of porous titanium scaffolds with high compressive strength using camphene-based freeze casting.</a> Mater. Lett. 63 (17), 1502-1504 (2009).</li></ol>

While biometal systems have been widely used for biomedical applications, particularly, as load-bearing materials, high stiffness and low bioactivity of metals have been regarded as major challenges. In this study, we established the fabrication method of a new metal system, a densified porous metal scaffold which has biomimetic mechanical properties as well as bioactive surface with sustainable release behavior. The major advantages of our fabrication method include 1) no change in the previous dynamic freezing casting ...

While metallic biomaterials have been widely used as load-bearing implants and internal fixation devices because of their excellent mechanical strength and resilience,1-3 they involve two critical challenges: 1) mechanical mismatch because metals are much stiffer than biological tissues, causing undesirable damages to the surrounding tissues and 2) low bioactivity that often results in poor interface with biological tissues, often provoking foreign body reactions (e.g., inflammation or thrombosis).4-6 Porous metallic scaffolds have been proposed to promote bone ingrowth in the structures, improving bone-implant contact while the stress shield effects are suppressed because of their reduced stiffness.7-9 Moreover, various surface modifications have been applied to enhance the biological activities of metallic implants; such modifications include coating the metal surface with bioactive molecules (e.g., growth factors) or drugs (e.g., vancomycin, tetracycline).10-12 However, problems such as reduced mechanical properties of porous metal scaffolds, decreased stiffness and the fast release of the bioactive coating layers remain unresolved.13-16
In particular, titanium (Ti) and Ti alloys are one of the most popular biometal systems because of their excellent mechanical properties, chemical stability, and good biocompatibility.13,17-19 Their foam-shaped applications have also attracted increasing interest because the 3D porous networks promote bone ingrowth in addition to bone-like mechanical properties.20-22 Efforts have been made to improve the mechanical properties by developing new manufacturing techniques including replication of polymeric sponge, sintering of metal particles, rapid prototyping (RP) method, and space holder method in order to control the various features of the pores (e.g., pore fraction, shape, size, distribution, and connectivity) and material properties (e.g., metallic phase and impurity).23-25 Recently, the freeze casting of water-based metal slurry has gained considerable attention to produce mechanically enhanced Ti forms with well-aligned pore structures by utilizing the unidirectional ice dendrite growth during solidification; however, oxygen contamination caused by contact of metal powders with water requires special care to minimize the embrittlement of Ti scaffolds.14,15
Therefore, we have developed a new approach towards fabricating bioactive and mechanically tunable porous Ti scaffolds.25 The scaffolds initially have porous structures with a porosity of more than 50%. The fabricated porous scaffolds were coated with bioactive molecules and then compressed using a mechanical press during which the final porosity, mechanical properties and drug release behavior were controlled by the applied strain. The densified porous Ti implants have shown low porosity with good strength in spite of the low stiffness comparable to that of bone (3-20 GPa).2 Because of the coating layer, the bioactivity of the densified porous Ti was significantly improved. Moreover, because of the unique flat pore structures induced by the densification process, the coated bioactive molecules were seen to be gradually released from the scaffold, maintaining their efficacy for a prolonged period.
In this study, we introduced our established method to fabricate densified porous Ti scaffolds for potential use in biomedical applications. The protocol includes dynamic freezing casting with metal slurries and densification of porous scaffolds. First, to fabricate porous Ti scaffolds with good ductility the dynamic freeze casting method was introduced as shown in Figure 1A. Ti powder was dispersed in liquid camphene; then, by decreasing the temperature, the liquid phase was solidified, resulting in the phase separation between the Ti powder network and solid camphene crystals. Subsequently, the solidified Ti-camphene green body was sintered in which Ti powders were condensed with continuous Ti struts, and the camphene phase was completely removed to obtain a porous structure. The coating and densification process with the obtained porous scaffolds was employed, varying the degree of densification and initial porosity. The coating layer and its release behavior were visualized and quantified using the green fluorescent protein (GFP)-coated porous Ti with and without densification compared to the GFP-coated dense Ti. Finally, functionally graded Ti scaffolds that have two different porous structures were proposed and demonstrated by varying the degree of densification of the inner and outer parts of the porous scaffolds.

<table>
	<tbody>
		<tr>
			<td>Titanium powder</td>
			<td>Alfa Aesar</td>
			<td>#42624</td>
			<td>-325 mesh, 99.5% (metals basis)</td>
		</tr>
		<tr>
			<td>Camphene</td>
			<td>SigmaAldrich</td>
			<td>#456055</td>
			<td>95%, C10H16</td>
		</tr>
		<tr>
			<td>KD-4</td>
			<td>Croda</td>
			<td>&#173;</td>
			<td>Hypermer, polymeric dispersant</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution (PBS)</td>
			<td>Welgene</td>
			<td>ML 008-01</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>Green Fluorescent Protein (GFP)</td>
			<td>Genoss Co.</td>
			<td>-</td>
			<td>&#62;98% purity, 1mg/ml</td>
		</tr>
		<tr>
			<td>Ball mill oven</td>
			<td>SAMHENUG ENERGY</td>
			<td>SH-BDO150</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>Ilshin Lab.</td>
			<td>PVTFD50A</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>Cold isostatic pressing (CIP) machine</td>
			<td>SONGWON SYSTEMS</td>
			<td>CIP 42260</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>Vaccum furnace</td>
			<td>JEONG MIN INDUSTRIAL</td>
			<td>JM-HP20</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>electical chaege machine</td>
			<td>FANUC robocut</td>
			<td>0iB</td>
			<td>External use</td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>CG&#38;S</td>
			<td>AJP-200</td>
			<td>&#173;</td>
		</tr>
		<tr>
			<td>Confocal laser scanning spectroscopy (CLSM)</td>
			<td>Olympus</td>
			<td>FluoView FV1000</td>
			<td>External use</td>
		</tr>
	</tbody>
</table>

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.

The fabrication process used to produce porous Ti scaffolds is illustrated in Figure 1A. Ti powder is kept dispersed homogeneously in camphene by continuous rotation of the container at 44 &#176;C for 12 hr and, while liquid camphene is fully solidified, any sediments of relatively heavy Ti powder can be minimized. As a result, the homogeneous Ti-camphene green body was produced using the dynamic freeze casting process as shown in Figure 1B, in which 3-dimensionally interconnected large ...

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Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

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 ...

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

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-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 ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...