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. Trauma. 32 (2), 149-159 (2006).</li><li>Huiskes, R., Weinans, H., Van Rietbergen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+stress+shielding+and+bone+resorption+around+total+hip+stems+and+the+effects+of+flexible+materials.">The relationship between stress shielding and bone resorption around total hip stems and the effects of flexible materials.</a> Clin. Orthop. Relat. Res. 274, 124-134 (1992).</li><li>Kanayama, M., 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=In+vitro+biomechanical+investigation+of+the+stability+and+stress-shielding+effect+of+lumbar+interbody+fusion+devices.">In vitro biomechanical investigation of the stability and stress-shielding effect of lumbar interbody fusion devices.</a> J. Neurosurg. Spine. 93 (2), 259-265 (2000).</li><li>Jung, H. -. D., 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=Production+and+evaluation+of+porous+titanium+scaffolds+with+3-dimensional+periodic+macrochannels+coated+with+microporous+TiO2.">Production and evaluation of porous titanium scaffolds with 3-dimensional periodic macrochannels coated with microporous TiO2.</a> 135, 897-902 (2012).</li><li>Jones, A. C., 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=Assessment+of+bone+ingrowth+into+porous+biomaterials+using+MICRO-CT.">Assessment of bone ingrowth into porous biomaterials using MICRO-CT.</a> Biomaterials. 28 (15), 2491-2504 (2007).</li><li>Li, J. P., 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=Bone+ingrowth+in+porous+titanium+implants+produced+by+3D+fiber+deposition.">Bone ingrowth in porous titanium implants produced by 3D fiber deposition.</a> Biomaterials. 28 (18), 2810-2820 (2007).</li><li>Ahn, M. -. K., Jo, I. -. H., Koh, Y. -. H., Kim, H. -. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+highly+porous+titanium+(Ti)+scaffolds+by+vacuum-assisted+foaming+of+titanium+hydride+(TiH2)+suspension.">Production of highly porous titanium (Ti) scaffolds by vacuum-assisted foaming of titanium hydride (TiH2) suspension.</a> Mater. 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. 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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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10.6K Views.  Korea Institute of Industrial Technology. 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.

Video: Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

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 ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

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

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

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

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

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 a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

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

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

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

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

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

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

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

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

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

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 prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

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