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) molds with a diameter of 60 mm and a height of 60 mm. Seal each Al mold with the corresponding Al cover slip and rotate the molds in a ball-mill oven at a speed of 30 rpm at 55 &#176;C for 10 min.
	<ol>
		<li>Subsequently, decrease the temperature of the ball-mill oven to 44 &#176;C, and continuously rotate the molds at a speed of 30 rpm at the constant temperature of 44 &#176;C for 12 hr.</li>
	</ol>
	</li>
	<li>Take out the mold from the ball-mill oven after additionally rotating the molds at RT for 1 hr for the cooling process. Remove the solidified titanium/camphene green body from the Al mold using an Al plunger.</li>
	<li>Place the solidified titanium/camphene green body in a rubber bag by hand and completely seal the rubber bag by tying the mouth of the bag with a string. Place the rubber bag in the water tank of a cold isostatic pressing (CIP) machine and apply an isostatic pressure of 200 MPa for 10 min. Remove the compressed green body from the rubber bag.</li>
	<li>Transfer the Ti-camphene green body on to an alumina crucible by hand and place the crucible in the freeze-dryer machine. Freeze-dry the green body to sublimate the camphene phase in the green body at - 40 &#176;C for 24 hr.</li>
	<li>Subsequently, close the crucible with an alumina cover slip and place the closed crucible in a vacuum furnace (below 10-6 Torr) at RT. Then, increase the temperature of the furnace to 1,300 &#176;C at a heating rate of 5 &#176;C/min and hold the temperature at 1,300 &#176;C for 2 hr.</li>
	<li>After the heat treatment, keep the sintered porous Ti in the furnace for 6-7 hr until the furnace is fully cooled to RT. 
	Note: During 6 hr of the cooling process, the average cooling rate of the furnace above 400 &#176;C is ~15 &#176;C/min and the average cooling rate of the furnace below 400 &#176;C is ~2 &#176;C/min.</li>
	<li>If necessary, cut the block of sintered porous Ti into disc-shaped samples with a diameter of 16 mm through electrical discharge machining (EDM).27 
	Note: Depending on the size of the Al molds, the size of the sintered porous Ti needs to be modified through the machining process (Figure 2A).</li>
	<li>Place a glass beaker with the porous Ti samples in an autoclave and sterilize the samples at 121 &#176;C for 15 min. Remove the samples from the autoclave. Wash the porous Ti samples with distilled water twice and then with 70% ethanol twice. Finally, leave the porous Ti into a Petri dish and air-dry the samples at RT on a clean bench under UV light.</li>
</ol>
2. Dip Coating of Scaffolds with Bioactive Agents
<ol>
	<li>Dilute the commercial Green Fluorescence Protein (GFP) from 1 mg/ml to 100 &#956;g/ml in a clean bench by mixing 1 ml of GFP with 9 ml of Dulbecco&#8217;s Phosphate Buffered Saline (DPBS, pH 7.4) solution in a 10 ml-sterilized polystyrene (PS) tube as indicated in Table 1.</li>
	<li>Immerse the sterilized dense or porous Ti in 10 ml of diluted GFP solution (100 &#181;g/ml) by placing the Ti samples into the PS tube with the GFP solution at RT and placing on a clean bench.</li>
	<li>Place the PS tube in a vacuum desiccator and evacuate the desiccator for 10 min to ensure the GFP solution penetrates the pores of the porous Ti more effectively.</li>
	<li>Remove the porous titanium from the PS tube using tweezers. Place the GFP-coated porous Ti into a 10 cm diameter Petri dish and air-dry O/N at RT on a clean bench.</li>
	<li>Rinse the porous Ti twice with 10 ml of Dulbecco&#39;s Phosphate Buffered Saline (DPBS) in a glass beaker, and move the porous Ti into a 10 cm diameter Petri dish using tweezers and air-dry at RT on a clean bench.</li>
</ol>
3. Densification of Porous Scaffolds
<ol>
	<li>Place the GFP-coated porous Ti samples with various heights in a cylindrical steel die, and insert a set of punches into the top and bottom holes of the steel die (Figure 3A).</li>
	<li>Compress the porous Ti within the steel die assembly at RT in the z direction of the sample (Figure 3A) using a press machine at intermediate strain rates of 0.05 ~ 0.1 sec-1 against the predetermined applied strains shown in Table 2. Hold the pressure for 1 min before unloading.</li>
	<li>Remove the densified Ti samples from the steel die. Wash the densified samples twice with 10 ml of DPBS in a beaker and air-dry O/N at RT on a clean bench.</li>
</ol>
4. Release Test of GFP-coated Scaffolds
<ol>
	<li>Immerse three types of specimens (GFP-coated dense Ti (after steps 2), GFP-coated porous Ti (after steps 1 and 2) and GFP-coated densified porous Ti (after steps 1-3)) in 5 ml DPBS (pH 7.4) solution contained in a 10 ml sterilized PS tube at 37 &#176;C on a clean bench.</li>
	<li>Suction out all the DPBS solution from each PS tube with the GFP-coated sample and replenish with a new 5 ml DPBS solution (pH 7.4) using a pipette according to the predetermined times of 1, 2, 3, 5, 8, 12, 15, 22 and 29 days after immersion.</li>
	<li>Take the fluorescence images of the GFP-coated samples before immersion (day 0) and after 22 day-immersion using confocal laser scanning spectroscopy (CLSM).</li>
	<li>Measure the fluorescence signal intensity of the released GFP in 1 ml solution from a total of 5 ml DPBS solution drawn from each PS tube in section 4.2 using UV spectroscopy at a wavelength of 215 nm. Convert the intensity value into the concentration of the GFP solution using the standard curve. 
	Note: Before the measurement, draw the standard curve of GFP solution by measuring the fluorescence signal intensity of the GFP solution in the concentration range of 0 ng/ml - 10 &#181;g/ml.</li>
</ol>
5. Fabrication of Graded Porous Ti Scaffolds
<ol>
	<li>Produce a block of the sintered porous Ti by repeating step 1.1 to step 1.7.</li>
	<li>Machine the sintered porous Ti block according to the predetermined structure designs (e.g., Figure 5a and 5d) by EDM.</li>
	<li>Place the machined Ti samples with height distribution in a steel die where the diameter of porous Ti is ~0.1 mm smaller than the diameter of the die and insert a set of punches into the top and bottom holes of the steel die.</li>
	<li>Perform steps 3.2 and 3.3.</li>
</ol>
6. Porosity Measurement of Ti Scaffolds
<ol>
	<li>Measure the mass (ms) of Ti scaffolds.</li>
	<li>Calculate the apparent volume (Vs) by measuring length, width and height of Ti scaffolds.</li>
	<li>Compute the porosity using the following equation: 
	&#160; <img alt="Equation 1" src="/files/ftp_upload/53279/53279eq1.jpg" /> 
	where P is the total porosity percentage, &#961;Ti is the theoretical density of the titanium and mS/VS is the measured density of the sample. 
	Note: The porosity of Ti samples can be directly retrieved from microCT images after microCT imaging is carried out using a micro-computed tomography scanner.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 method which we already developed,28 2) control of one parameter-degree of densification-to achieve both the mechanical enhancement and sustainable release behavior of biomolecules from porous metal scaffolds and 3) potential application to functionally graded materials.
One of the critical steps needed to produce the densified porous metal is the fabrication of porous Ti, which possesses two important features: 1) ductility to control the release rate of bioactive molecules and the mechanical properties and 2) high pore interconnectivity to load and release biomolecules. However, previously reported porous titanium scaffolds produced using the space holder method, sponge template method, and powder metallurgy have shown either limited pore interconnectivity or ductility.14,24,29 In particular, the impurities created by the reaction of metal powders with other surrounding materials during the heat treatment process are known to significantly reduce the ductility of the material because metal powders are in contact with the second materials (e.g., space holder or polymer template), resulting in brittle failure under mechanical tests.14,24,29 Thus, to fabricate densified porous metal, the impurities need to be minimized for most of the conventional fabrication methods. To avoid this complication, we investigated the porous morphology and mechanical behaviors of porous titanium scaffolds fabricated using the freeze casting method with camphene in order to minimize the interaction between metal powders and the liquid phase.26,28,30-33
A disadvantage of the conventional freeze casting method is that it often results in directional pore channels (Figure 2B, top image). On the other hand, with dynamic freeze casting, the pore shape and size were found to be more uniform than those of the conventional freeze casting and the pore distribution within the scaffold is almost random. These structural features of porous scaffolds from dynamic freeze casting show isotropic mechanical behavior, thus allowing densification in a confined mold under uniaxial pressure.26,28 During dynamic freeze casting, two major events occur within the metal slurry: 1) crystal growth of camphene phase and 2) redistribution of metal powders and solidified camphene within the remaining liquid phase avoiding sedimentation. Gravity causes the metal powders to segregate until the liquid camphene is fully solidified. The continuous rotation of the slurry near the melting temperature of camphene gives sufficient time for spherical camphene crystals to grow homogeneously, allowing the random and uniform distribution of Ti powders and camphene crystals as shown in Figure 1C.
Following the cooling process, fully solidified Ti-camphene biphasic green body (Figure 1B) was obtained. In order to completely remove the camphene from the solidified green body without the structure collapsing, camphene was sublimed in a vacuum desiccator at -20 &#176;C. After removal of the camphene phase, the green body became porous, consisting of only Ti powder. Since there is no interaction among Ti particles, the porous Ti green body is fragile so that careful handling is required. To avoid any direct handling of the green body with hands before heat treatment, a ceramic crucible was chosen for the container of the green body for freeze-drying and sintering. The container with the green body was placed in a vacuum furnace immediately after the freeze-drying and heat treatment at 1,300 &#176;C, which allows the green body to be fully densified without significant defects in the metal struts. For the evaluation of the samples, porous Ti blocks were cut into smaller porous Ti cylinders because the geometry and size of the porous samples should be identical (Figure 2A). All the specimens were successfully machined without any significant defects (Figures 2B and 2C). Depending on the amount of Ti power in the slurry, Ti scaffolds with different porosities were obtained with spherical shapes and randomly distributed pores (Figure 2C).
After the porous Ti scaffolds were obtained using the dynamic freeze casting method as reported in our previous study,28 the biomolecules were coated on the Ti surface and densification of the coated porous Ti was performed as illustrated in Figure 3A. In order to avoid any contamination or denaturation of the biomolecules, the coating process was conducted on a clean bench at RT within 24 hr after the porous scaffolds were autoclaved and carefully cleaned out. To minimize the loss of the coated biomolecules before densification, the cleaning process was minimized after the coating process was performed. The densification process was controlled by the applied deformation of the porous Ti samples in the z-direction, converted into strain, &#949;zz.26 Depending on the initial porosity of the Ti scaffolds, the applied strain and corresponding final porosity were varied (Table 2). In order to ensure the densified porous scaffolds with different initial porosities had identical final geometries and sizes, the applied strain of the individual scaffolds was calculated and the total sample height (length in z direction) of each sample was then predicted before densification. Figure 3D indicates that different heights of individual porous samples with varying porosity could lead to the densified porous sample with identical final height at the same final porosity.
By controlling the degree of densification, the densified porous scaffolds have unique mechanical behavior with prolonged release of the coated biomolecules. The applied strain changes two important parameters of the porous Ti scaffolds: final porosity and pore size. The porous scaffolds with lower porosity show higher stiffness and strength. Our previous study reported the stress-strain behavior of densified porous scaffolds with improved strength compared to porous Ti (Table 4) as well as significantly reduced stiffness compared to commercial dense Ti.26 In this study, we also observed the release behavior of densified porous Ti compared with both dense and porous Ti through visualized detection of the GFP-coating layer as shown in Figure 4. The results were consistent with our previous study,26 in which the densified porous scaffolds possess significantly improved release behavior of coated materials, prolonging the release time by up to four months due to increased torturosity of the scaffolds with decreased pore sizes. The current 30 day-release test clearly shows the remaining GFP on the surface of the densified porous Ti in contrast to no GFP remnant on either dense or porous Ti surfaces.
Finally, the densification method was applied to the production of functionally graded porous scaffolds in which the inner and outer parts have different porosities. For the cylindrical scaffold, differentiating the z-heights of the inner and outer parts can easily lead to graded porous scaffolds as shown in Figure 5. The applied strain (&#949;zz) on the inner part in the porous Ti scaffold shown in Figure 5B was ~0.27, which resulted in the final porosity of ~ 57%, while no strain was applied to the outer part. On the other hand, the applied strain (&#949;zz) on the outer part in the porous Ti scaffold in Figure 5B was ~0.33, which resulted in the final porosity of ~ 45% while the inner part was almost intact, preserving the initial porosity (Table 3). However, two major challenges for the graded porous scaffolds were observed from this experiment. First, the continuous inner and outer parts induced inconsistent stress and strain distribution within the scaffold; thus, the densification occurred inhomogeneously, where the regions around the top and bottom surfaces were denser than those around the inner surface. This tendency was critical as the height difference of the two parts increased. Moreover, the graded porous scaffold with the denser inner part was more difficult to produce than the scaffold with the denser outer part because the densification of the inner part should be carried out, being confined with the outer part, which resulted in inhomogeneous deformation within the two parts. To resolve the inhomogeneous densification of the graded scaffold, we developed two separate parts that can be assembled during the densification process. Even though in this paper, the optimal condition to produce the perfectly fabricated graded porous structure was not yet fully found, the potential of the densification process for the production of the graded structure was well confirmed. The optimized fabrication method of the graded porous structure is on-going, and as further work, selective drug loading to the graded structure will be investigated for the functional release behavior of the scaffold.
The advantages of the proposed approach in this study include 1) better mechanical compatibility with biological tissues with good strength and 2) prolonged bioactivity for better biological performance. However, one of the major disadvantages is the reduced pore size that cannot promote bone ingrowth through the pore network of metallic scaffolds for a better bone-implant interface. To resolve this issue, graded pore structures have been proposed, in which the porous and dense parts coexist; thus, the porous parts allow bone ingrowth, while the dense parts provide mechanical stability and prolonged bioactivity. Therefore, functionally graded Ti implants through various structural designs will be fabricated and tested, particularly, focusing on the improved capability of bone integration. Moreover, another limitation should be the fabrication of implants with complicated geometry. In order to obtain a complex-shaped implant (e.g., femoral cone augment), the additional machining process is required after densification, imposing two major drawbacks on the final product: inefficient and uneconomical material usage because the significant volume of porous Ti block is often removed during the process, and potential contamination and loss of coated biomolecules during the machining process. Improvement on the fabrication process of the porous Ti scaffolds with complex geometry is ongoing. The densified porous metal scaffolds can be applied to various orthopedic applications, e.g., artificial disc replacement, replacing either bulk or porous metallic implants, and acting as a load support as well as a drug carrier.

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%, C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;</td></tr><tr><td>KD-4</td><td>Croda</td><td>&amp;shy;</td><td>Hypermer, polymeric dispersant</td></tr><tr><td>Phosphate Buffer Solution (PBS)</td><td>Welgene</td><td>ML 008-01</td><td>&amp;shy;</td></tr><tr><td>Green Fluorescent Protein (GFP)</td><td>Genoss Co.</td><td>-</td><td>&gt;98% purity, 1 mg/ml</td></tr><tr><td>Ball mill oven</td><td>SAMHENUG ENERGY</td><td>SH-BDO150</td><td>&amp;shy;</td></tr><tr><td>Freeze dryer</td><td>Ilshin Lab.</td><td>PVTFD50A</td><td>&amp;shy;</td></tr><tr><td>Cold isostatic pressing (CIP) machine</td><td>SONGWON SYSTEMS</td><td>CIP 42260</td><td>&amp;shy;</td></tr><tr><td>Vaccum furnace</td><td>JEONG MIN INDUSTRIAL</td><td>JM-HP20</td><td>&amp;shy;</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&amp;amp;S</td><td>AJP-200</td><td>&amp;shy;</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 camphene pores are surrounded by the Ti powder phase (Figure 1C). However, the incorrect rotation of the containers often results in inhomogeneous distribution of Ti and camphene phases in the green body, causing distortion or cracking of the porous scaffold following heat treatment. The optimal condition of the rotation speed was found to be 30 rpm, which was able to produce a homogeneous green body in most cases. Before proceeding with heat treatment, the extensive growth of camphene is confirmed by observing the cross-section of the Ti-camphene green body as shown in Figure 1C. If the camphene phase is discontinuous with a significant size distribution of pores, the temperature and time of the dynamic freeze casting needs to be reset. Typically, the camphene phase of the Ti-camphene green bodies was found to be well-developed after 12 hr dynamic freeze casting, where the camphene phase became continuous since large spherical pores were in contact with each other. The size, morphology and connectivity of pores in porous Ti were further evaluated using micro-CT analysis after heat treatment.
After sintering at 1,300 &#176;C, the porous Ti blocks are cut into multiple cylindrical-shaped samples by electrical discharge machining (Figure 2A). The obtained cylindrical specimens did not show cracks or defects. Representative micro-CT images of the porous Ti scaffolds fabricated by conventional (top) and dynamic freeze casting (bottom) are shown in Figure 2B. The pore structure of the Ti samples from the conventional freeze casting shows directional pore alignment with irregularly shaped pores because of the dendritic growth of camphene during freezing. On the other hand, the sample from dynamic freeze casting exhibits almost spherical pores with random pore distribution. Moreover, the higher resolution microscopic images of the porous Ti scaffolds with various porosities (Initial Porosity (I.P.) = 50, 60, and 70 vol%) clearly show spherical pores randomly distributed within the Ti network (Figure 2C). The pore size of the porous Ti scaffolds decreased as the volume of camphene decreased.
Subsequently, the fabricated porous Ti scaffolds are coated with biomolecules and densified within the mold by varying the applied strain as shown in Figure 3A. For the visualization of the bioactive coating layer on the Ti samples, green fluorescent protein (GFP) was used in this study. The applied strain (&#949;zz), which corresponds to the pressure (Pzz), is found to vary the degree of densification as shown in Figure 3B. The pore shape becomes flattened as the degree of densification increases and, as a result, at the highest densification, pores almost disappear because neighboring pores are in contact with each other. However, from our previous study, we confirmed that the pore channels of the densified samples are still open, with almost the same surface area as that of the porous Ti of the same porosity.25 To evaluate the densified samples with different starting porosities, the z-height should vary depending on the initial porosity in order for the densified sample to have the same final porosity. Table 2 also provides the predicted applied strains to obtain the targeted final porosity (F.P) of the densified porous scaffolds with different initial porosities. For example, to produce the densified porous specimens with F.P. = 5%, the porous scaffold with I.P. = 70% requires a strain of approximately 0.7, while the scaffold with I.P. = 50% needs approximately 0.5. Therefore, the initial heights of the porous scaffolds were carefully calculated according to the initial porosity in order to obtain samples with the same final height after densification. As shown in Figure 3C, four specimens with varying porosities from I.P. = 40% to 70% show different initial heights before densification and then at the end, with almost identical heights of 2 mm.
GFP was used to visualize the coating layer on porous (I.P. = 70%) and densified porous Ti (I.P. = 70%, F.P. = 7%) samples compared to commercial dense Ti as shown in Figure 4A. All three samples clearly display the coated surface morphology corresponding to their microstructures. The fully dense Ti surface is completely covered with a green coating layer, whereas porous and densified porous samples have green colored Ti struts with clear pores. Using these three coated samples shown in Figure 4A, the release behavior was observed (Figure 4B). The amount of released GFP from each sample was expressed as the mean &#177; standard deviation (n=3) and was tracked up to one month by measuring the intensity of fluorescence. Both dense and porous Ti were found to have fast GFP release behavior with initial bursting effect, with most released within one week. However, densified porous Ti shows continuous release up to one month, clearly showing GFP on the surface even after one month (CLSM images of Figure 4B).
The densification process can also be applied to the fabrication of functionally graded porous Ti scaffolds as introduced in Figure 5. The two potential design schematics of gradient structures were chosen, of which the inner and outer layers of a cylindrical scaffold have different porosities. For the structure with a denser core shown in Figure 5A, the outer part of the Ti scaffold was shortened by mechanical machining as shown in Figure 5B. After selective densification of the higher inner part, the gradient structure was obtained. The detailed structural information of Figures 5Bb and 5E measured by micro CT is provided in Table 3. The micro CT image of Figure 5C clearly shows the inner and outer parts of the scaffold with different porosities (inner: F.P. = ~60%, outer: F.P. = ~70%). Alternatively, a structure with denser outer layer can be produced by changing the height difference between the inner and outer parts (Figure 5D). The porous Ti with the higher outer and lower inner parts results in a denser outer part after densification (Figure 5E), in which the porosity of the outer part was lowered to ~45%, with the inner part having the preserved initial porosity (I.P. = 70%) as indicated in Figure 5F.
<img alt="Figure 1" src="/files/ftp_upload/53279/53279fig1.jpg" /> 
Figure 1. Fabrication of Ti-camphene green body by dynamic freeze casting. (A) Schematic illustration of the dynamic freeze casing process to obtain solidified Ti-camphene green body before heat treatment (Adapted with permission from Elsevier, Jung et al., 2013). (B) Optical image of a representative Ti-camphene green body after the completion of the dynamic freeze casting process. (C) Cross-sectional image of the Ti-camphene green body in which the solid camphene phase is randomly distributed within the continuous Ti powder phase. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53279/53279fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53279/53279fig2.jpg" /> 
Figure 2. Porous Ti scaffolds with various initial porosities after heat treatment. (A) Optical images of a fully sintered porous Ti block before and after machining and an obtained cylindrical porous Ti scaffold from machining. (B) Sectional micro-CT images of the porous Ti scaffolds fabricated by conventional freeze casting (top) and dynamic freeze casting (bottom). Yellow arrows on the top image of Figure 2B indicate the pore alignment in the radial direction. (c) Cross-sectional images of the porous Ti scaffolds fabricated by dynamic freeze casting with the initial porosity (I.P.) of 70% (top), 60% (middle) and 50% (bottom) where insets are the optical images of the corresponding porous Ti scaffolds (Adapted with permission from Elsevier, Jung et al., 2013). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53279/53279fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53279/53279fig3.jpg" /> 
Figure 3. Dip-coating and densification of porous Ti scaffolds. (A) Schematic illustration of the fabrication process of a densified porous metallic scaffold (Ti) coated with biomolecules (e.g., GFP) (Adapted with permission from Elsevier, Jung et al., 2015). (B) Cross-section images of the densified porous Ti scaffolds (I.P. = 70%) at the applied strain (&#949;zz) = 0, 0.53, 0.63, 0.68, resulting in the final porosity (F.P. = 70, 33, 19, 7%). (C) Optical images of the porous Ti specimens (I.P. = 70, 60, 50 and 40%) with various z-heights (top) and their corresponding densified specimens (F.P. = 7%) with the identical z-height of 2 mm after densification (bottom). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53279/53279fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53279/53279fig4.jpg" /> 
Figure 4. In vitro release behavior of GFP-loaded densified porous Ti scaffolds. (A) Typical CLSM images of GFP loaded on the surface of dense, porous and densified Ti scaffolds. (B) Cumulative amounts of GFP released from dense, porous and densified Ti scaffolds up to 29 days (n = 3) with the CLSM images of those three samples after immersion in the PBS for 24 days (scale bar = 200 &#181;m). Standard deviation (SD) is used for the descriptive error bar of each data point. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53279/53279fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53279/53279fig5.jpg" /> 
Figure 5. Fabrication of functionally graded porous metal scaffolds. (A) Schematic of a graded porous scaffold design with a denser inner part. (B) Graded porous Ti scaffold with the denser inner part fabricated through densification. (C) 2-D reconstructed micro-CT image of graded porous Ti scaffold with the denser inner part. (D) Schematic of a scaffold design with gradient porosity with the denser outer part. (E) Graded porous Ti scaffold with the denser outer part fabricated through densification in which the scaffold possesses a porous internal core surrounded by the densified outer layer. (F) 2-D reconstructed micro-CT image of graded porous Ti scaffold with the denser outer part. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53279/53279fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td rowspan="2">Target sample</td>
			<td colspan="3">Ti-camphene slurry&#160;</td>
			<td colspan="2">Coating solution</td>
		</tr>
		<tr>
			<td>Ti powder (g)</td>
			<td>Camphene (g)</td>
			<td>KD-4 (g)</td>
			<td>GFP (ml)</td>
			<td>PBS (ml)</td>
		</tr>
		<tr>
			<td>Ti scaffold with I.P. = 40%</td>
			<td>204.3</td>
			<td>90</td>
			<td>0.294</td>
			<td rowspan="4">1</td>
			<td rowspan="4">9</td>
		</tr>
		<tr>
			<td>Ti scaffold with I.P. = 50%</td>
			<td>171.4</td>
			<td>97</td>
			<td>0.268</td>
		</tr>
		<tr>
			<td>Ti scaffold with I.P. = 60%</td>
			<td>136.5</td>
			<td>103</td>
			<td>0.239</td>
		</tr>
		<tr>
			<td>Ti scaffold with I.P. = 70%</td>
			<td>100</td>
			<td>110</td>
			<td>0.21</td>
		</tr>
	</tbody>
</table>
Table 1. Detailed information of Ti-camphene slurry and coating solution for the fabrication of target porous Ti scaffolds (I.P. = 40, 50, 60, 70%) coated with GFP. (I.P. stands for initial porosity).
<table>
	<tbody>
		<tr>
			<td rowspan="2">Initial porosity (%)</td>
			<td colspan="7">Final porosity (%)</td>
		</tr>
		<tr>
			<td>60</td>
			<td>50</td>
			<td>40</td>
			<td>30</td>
			<td>20</td>
			<td>10</td>
			<td>5</td>
		</tr>
		<tr>
			<td>50</td>
			<td></td>
			<td></td>
			<td>0.17</td>
			<td>0.29</td>
			<td>0.38</td>
			<td>0.44</td>
			<td>0.47</td>
		</tr>
		<tr>
			<td>60</td>
			<td></td>
			<td>0.20</td>
			<td>0.33</td>
			<td>0.43</td>
			<td>0.50</td>
			<td>0.56</td>
			<td>0.58</td>
		</tr>
		<tr>
			<td>70</td>
			<td>0.25</td>
			<td>0.40</td>
			<td>0.50</td>
			<td>0.57</td>
			<td>0.63</td>
			<td>0.67</td>
			<td>0.68</td>
		</tr>
	</tbody>
</table>
Table 2. Predicted applied strain (&#949;zz) of porous scaffolds (I.P. = 50, 60, 70%) in terms of the targeted final porosity (F.P.) using the equation, F.P. = 1 - (1- I.P.)/(1- &#949;zz).
<table>
	<tbody>
		<tr>
			<td colspan="2" rowspan="2">Specimen</td>
			<td colspan="3">Before densification</td>
			<td colspan="3">After densification</td>
		</tr>
		<tr>
			<td>Height (mm)</td>
			<td>Porosity (%)</td>
			<td>Pore size (&#956;m)</td>
			<td>Height (mm)</td>
			<td>Porosity (%)</td>
			<td>Pore size (&#956;m)</td>
		</tr>
		<tr>
			<td rowspan="2">Graded scaffold of Fig. 5b</td>
			<td>Inner part</td>
			<td>18 &#177; 1</td>
			<td rowspan="4">70 &#177; 1</td>
			<td rowspan="4">370 &#177; 100</td>
			<td rowspan="2">13 &#177; 1</td>
			<td>57 &#177; 5</td>
			<td>285 &#177; 100</td>
		</tr>
		<tr>
			<td>Outer part</td>
			<td>14 &#177; 1</td>
			<td>70 &#177; 5</td>
			<td>365 &#177; 110</td>
		</tr>
		<tr>
			<td rowspan="2">Graded scaffold of Fig. 5e</td>
			<td>Inner part</td>
			<td>14 &#177; 2</td>
			<td rowspan="2">12 &#177; 1</td>
			<td>70 &#177; 8</td>
			<td>315 &#177; 110</td>
		</tr>
		<tr>
			<td>Outer part</td>
			<td>18 &#177; 1</td>
			<td>45 &#177; 8</td>
			<td>230 &#177; 80</td>
		</tr>
	</tbody>
</table>
Table 3. Structural information of the inner and outer parts of graded porous scaffolds (Figure 5B and Figure 5E) before and after densification in terms of the z-height, porosity and average pore size measured by micro-CT.
<table>
	<tbody>
		<tr>
			<td rowspan="2">Initial porosity of porous Ti (%)</td>
			<td colspan="2">Before densification</td>
			<td colspan="2">After densification (F.P.= 5%)</td>
		</tr>
		<tr>
			<td>Stiffness (GPa)</td>
			<td>Yield Strength (MPa)</td>
			<td>Stiffness (GPa)</td>
			<td>Yield Strength (MPa)</td>
		</tr>
		<tr>
			<td>50</td>
			<td>19</td>
			<td>143</td>
			<td>44</td>
			<td>&#62; 370</td>
		</tr>
		<tr>
			<td>60</td>
			<td>13</td>
			<td>130</td>
			<td>42</td>
			<td>&#62; 370</td>
		</tr>
		<tr>
			<td>70</td>
			<td>5</td>
			<td>58</td>
			<td>35</td>
			<td>&#62; 370</td>
		</tr>
	</tbody>
</table>
Table 4. Stiffness and yield strength of porous Ti scaffolds (I.P. = 50, 60, 70%) before and after densification (Adapted with permission from Elsevier, Jung et al., 2015).

Watch this Scientific Journal Video about Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications at JoVE.com

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 ...

fabrication-mechanically-tunable-bioactive-metal-scaffolds-for

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Unit 1

Transformations of Stress and Strain

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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

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

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, ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

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.

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 ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

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Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Novel Process for 3D Printing Decellularized Matrices