This work was financially supported by INSTM.

1. Scaffold Fabrication
<ol>
	<li>Grind NaCl in a laboratory blender for 20 min and dry it on a heater at 100 &#176;C.</li>
	<li>Put the dried NaCl (45 g at time) in a sieving machine for 30 minutes at the highest available frequency without occurring in resonance. Collect six salt fractions, ranging from 500 &#181;m to 1000 &#181;m (M 500); from 300 &#181;m to 500 &#181;m (M 300); from 100 &#181;m to 200 &#181;m (M 100); from 90 &#181;m to 100 (M 90); from 45 &#181;m to 65 &#181;m (M 45) and finally M 10 with salt particles size smaller than 45 &#181;m as schematized in Figure 1.</li>
	<li>Vacuum dry overnight all the materials in order to avoid hydrolytic scission during processing. For each material, select the temperature in order to maximize the degree of drying without overcoming - in the case of polymers - the glass transition. Therefore, choose T = 90 &#176;C for PLA and T = 25 &#176;C for PEG, T = 105 &#176;C for NaCl.</li>
	<li>Feed PLA, PEG and NaCl, with a weight percentage composition of 20/5/75 respectively, to a batch mixer operating at T = 190 &#176;C and n = 60 rpm and then process them until achieving a constant value of torque, usually after about 10 minutes. Thereafter, rapidly collect the resulting material.</li>
	<li>Prepare the mono-layers by using a laboratory press operating at 210 &#176;C, put the blends in appropriate cylindrical molds with a diameter of 10 mm and a height of 3 mm and keep them for 60 sec at ambient pressure and 3 minutes at 180 bar. Thereafter, cool the blends at room temperature, maintaining the pressure of 180 bar.</li>
	<li>Assembly of Three-layers via compression-molding
	<ol>
		<li>Prepare each single layer in the same way as that described in (1.5) but using different molds, i.e. having a diameter of 10 mm and a height of 1 mm. Finally, obtain 6 disks having a diameter of 10 mm and a height of 1 mm, containing six different particle sizes: M 500, M 300, M 100,</li>
		<li>For assembling three-layer scaffold A (TLS A), pile up M 500, M 300 and M 100 inside the cylindrical mold and compression-mold them in a laboratory press operating at 210 &#176;C for 60 sec at ambient pressure and 3 minutes at 180 bar and then cooled at room temperature, maintaining the pressure of 180 bar. 
		NOTE: Prepare the TSL B by stacking on each other M 90, M 45 and M 10 in the same molds and conduct the compression-molding operation by following the same procedure as that used for TLS A.</li>
	</ol>
	</li>
	<li>Remove the disks from the cylindrical molds and put them in a boiling demineralized water bath, without stirring. 3 hr after, remove the resulting porous structures from the bath and let them dry for 12 hr at room temperature in a chemical hood.</li>
</ol>
2. Morphological Analysis
<ol>
	<li>Evaluate the morphologies of the scaffolds by scanning electron microscopy.
	<ol>
		<li>Break down the samples under liquid nitrogen and then attach the samples on an aluminum stub using an adhesive carbon tape. Finally, sputter-coate with gold for 90 s under argon atmosphere before imaging in order to avoid electrostatic discharge during the test.</li>
	</ol>
	</li>
</ol>
3. Scaffold Pore Size
<ol>
	<li>Elaborate the images obtained by SEM analysis with an image processing software able to recognize the pore size distribution of the scaffold. 
	NOTE: In this work the pore size distribution analysis was carried out using a MATLAB-based software previously described33</li>
</ol>
4. Porosity
<ol>
	<li>Weigh the samples before leaching and evaluate the theoretical porosity according to the following expression: 
	<img alt="Equation 1" src="/files/ftp_upload/54595/54595eq1.jpg" /> 
	NOTE: mNaCl, mPEG and mPLA, are respectively the theoretical mass of NaCl, PEG, and PLA, by assuming homogeneous blends. The densities (&#961;) of NaCl, PEG and PLA are respectively 2,16 g/cm3, 1,12 g/cm3 e 1,24 g/cm3.</li>
	<li>Weigh the samples after leaching and drying in order to assess the apparent density of the samples (&#961;scaffold), and then evaluate the real porosity as the reciprocal of the ratio between the apparent density of the scaffold and the density of non-porous PLA by using expression (2). 
	NOTE: It expresses the ratio between the empty volume of the scaffold and the full volume of the scaffold (empty + full). 
	<img alt="Equation 2" src="/files/ftp_upload/54595/54595eq2.jpg" /></li>
</ol>
5. Mechanical Properties
<ol>
	<li>Test the samples under compressive mode using a tensile machine, equipped with a 1 kN load cell. Set a constant strain rate of 1 mm min-1.</li>
	<li>In order to investigate the mechanical performance of the samples in physiological environment, equip the dynamometer with a bath containing (PBS), (pH = 7.4) at 37 &#176;C and perform the test with the same setup as that described at point 5.1.
	<ol>
		<li>Before each measurement in wet environment, soak the samples with PBS in a vacuum flask for 5 min in order to let PBS fill all the pores. Thereafter, allow the scaffolds to remain in PBS at 37 &#176;C for 15 min to reach the set-point temperature.</li>
	</ol>
	</li>
	<li>Determine the interfacial adhesion strength (IAS) between the layers of TL A and TL B by using a custom-designed interfacial strength test equipment, connected to a Mechanical Testing Machine following a method described in literature32,34.
	<ol>
		<li>Fix the scaffold on the rig and ensure its correct alignment with the machine&#39;s load cell and base platen. Attach the scaffold samples to aluminum test stubs by using a high viscosity adhesive and place them into the equipment for testing.</li>
		<li>For the wet condition test, hydrate the samples in PBS for 1 hr prior to testing. Use a 1 kN load cell, under a tensile load applied at a strain rate of 1 mm min-1. 
		NOTE: Take into account that failure may occur either at the ultimate tensile strength of one of the layers or because of delamination at the interfaces. Evaluate the IAS as the maximum strength of the stress-strain curve.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The first critical step is the optimization of sieving efficiency. The high control of the NaCl particle size is fundamental for preparing scaffold with desired pore size distribution. Another critical step is avoiding the fracture of the thin PLA monolayers during the sample extraction from the mold. The image processing analysis might not be representative of the whole device.
During tensile tests, the sample can tear away from the equipment.
Prior to sieving step, ascertain that the salt have been well dried in order to get a higher control of the NaCl particle size. This issue is important especially for the smallest salt particle size, due to its extremely high hygroscopicity. Prior to compression-molding step, apply a thin Teflon spray-coating onto the molds in order to facilitate the sample removal from the molds. The image processing analysis must be carried out by considering different images taken from different regions of the scaffold to be sure that they are representative of the whole device. Finally, prior to tensile tests (especially those carried out in wet environment), carefully verify the adhesive to sample adhesion.
The principal limitation of the technique lies in the impossibility to obtain a continuous gradient of the pore size. Indeed, the method herein described allows achieving a discrete gradient of pore size, since it is based on the assembling of different layers. In most cases, a well-defined multilayer scaffold can be preferred to a continuously graded one but not ever. This limitation may be partially exceeded by increasing the number of layers, which in turns would result in thinner layers, obviously more difficult to handle.
Differently from many other production technologies, the strategy here adopted can be considered eco-friendly, since it does not require any toxic solvent potentially dangerous for environment and for living cells and tissues. Furthermore, particulate leaching provides a high control of both pore size and porosity by tuning respectively the size and the amount of the NaCl blended with the PLA.
Future advances of this technique rely on the possibility to gather layers presenting other chemo-physical differences. For instance, one can assemble different biopolymers or reinforce each layer with different nanoparticles, such as hydroxyapatite28, nanocellulose27, graphene35 or its derivatives9,36,37 in order to provide even further functionalities38. In fact, this method ensures a high control thus allowing to easily tune each region of multilayer scaffold. This challenge play a crucial role in the ITE, since the presence of multi-phasic and/or anisotropic bio-structures that gradually change from one tissue to another are typical features of interface tissues, such as ligament-to-bone, tendon-to-bone and cartilage-to-bone.

The interest in biodegradable polymers has grown in importance during the last years both in academia and in the industry, due to the rising concerns regarding plastic waste and the reduction in using non-renewable sources1-7. In particular, biocompatible and biodegradable synthetic polymers are widespread in several biomedical application fields, such as drug controlled release8,9, absorbable suture threads8,10, bioprocess intensification11 and tissue engineering12.
Tissue engineering focuses on the development of devices capable to restore and maintain normal function in diseased or injured tissues. Most of the native tissues are composed by different types of cells and extracellular matrices (ECMs) in specific spatial hierarchies. For example, articular cartilage (AC) consists of different zones with varying types and orientations of collagen fibers and collagen-binding proteins. Moreover, cartilage and bone show significantly different hierarchical structures. In this context, the preparation of multilayer scaffolds with engineered properties in each layer could allow replacing heterogeneous tissues by taking into accounts all the local microenvironments of these complex systems13,14.
Based on the cellular/biological and/or physical-chemical characteristics of the scaffolds, the main strategies adopted by the tissue engineering can be divided into monophasic, biphasic, and triphasic. Biphasic and triphasic approaches (BTA) use two or three different pores architectures, materials, or fillers to prepare multilayered functional devices. Furthermore, a single material can be used to achieve biphasic or triphasic devices, as long as it is possible to create a gradient in its physical properties12.
Cell migration plays a key-role in the morphogenesis, inflammation, wound healing and tumor metastasis. Cell movement is encouraged by the presence of a gradient of chemical-physical properties from the surface to the core of the device. Therefore, biomaterials fulfilling the above discussed requirements can be helpful in studying cell migration. In addition to chemical gradients that trigger cells migration (chemotaxis), mechanical properties of cells culture substrate can also lead to cell movement (mechanotaxis)15.
The multilayer structure can also provide the tunable release of specific drugs incorporated within the polymer matrix, by changing the specific area of the layer or the amount of loaded drugs. 
Over the past decade, in order to develop scaffolds possessing a discrete or continuous gradient of morphological properties, such as porosity or pore size, several approaches have been presented16-31. The most recent papers focused on the preparation of BTA by adopting: particle leaching 12,28,32, gas foaming technique16, electrospinning17-19, layer by layer casting technique20, rapid prototyping21,22, thermally induced phase separation (TIPS)22, centrifugation freeze drying24,25, triply periodic minimal surfaces (TPMS)26, freeze casting27-30.
Within the frame of this work, we present a fast and simple route to achieve PLA-based three-layer porous scaffolds (TLS), by combining melt mixing, compression molding and salt leaching. Differently from most of the technologies commonly used for scaffold production, the strategy herein adopted can be considered fully eco-friendly, since it does not require any toxic solvent potentially dangerous for environment and for living cells and tissues32. The basic processing-structure-property relationships established in this study by analyzing both morphological features and mechanical behavior of fabricated devices provide guidance to future advances in designing multifunctional graded scaffolds with specific target properties.

<table><tbody><tr><td>Poly(lactic acid)</td><td>NatureWorks</td><td>PLA 2002D</td><td></td></tr><tr><td>Poly(ethylene glycol)</td><td>Sigma</td><td>83797-1KG-F</td><td></td></tr><tr><td>Sodium Cloride</td><td>Sigma</td><td>793566-5KG-D</td><td></td></tr><tr><td>Phosfate Buffer Solution</td><td>Sigma</td><td>P5368-10PAK</td><td></td></tr><tr><td>Laboratory Mixer</td><td>Brabender</td><td>PLE 330 - Plasticorder</td><td></td></tr><tr><td>Laboratory Press</td><td>Carver</td><td></td><td></td></tr><tr><td>Scanning Electron Microscopy</td><td>Phenom-world</td><td>ProX</td><td></td></tr><tr><td>Universal Testing Machine</td><td>Instron</td><td>3365 (UK)</td><td></td></tr><tr><td>BioPuls Bath</td><td>Instron, Norwood</td><td></td><td></td></tr><tr><td>Sieving Machine</td><td>Endecotts</td><td>E.V.F.1.</td><td></td></tr><tr><td>Vacuum Oven</td><td>ISCO</td><td>NSV9035</td><td></td></tr><tr><td>Precision Balance</td><td>Sartorius</td><td>AX224</td><td></td></tr></tbody></table>

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.

The influence of NaCl particle size on the pore architecture of the scaffolds was assessed qualitatively and quantitatively by investigating the morphology of the samples and calculating the pore size distribution by image analysis, respectively. Figure 2a-f shows SEM micrographs of mono-layered scaffolds resulting from salt-leaching of materials containing different NaCl particle sizes.
More in detail, M 500 (Figure 2a) showed pores with an average diameter of 500 &#181;m, probably due to the breakage of salt particles with diameter higher than 500 &#181;m during the melt mixing. As clearly visible from the same figure, pore architecture is characterized by a low number of irregular pores, poorly interconnected, surrounded by walls of about 10 &#181;m. Figure 2b reports the morphology of M 300. In this case, the pores were found to exhibit an average diameter within the same range of the salt particles filled during melt mixing (300-500 &#181;m), thus confirming that no particle breakage occurred within melt mixing process. Pore walls were found to be thinner (about 5 &#181;m) than those observed in M 500 layer. M 100, (Figure 2c), shows a bimodal porous structure, characterized by a heterogeneous network composed by large pores (100-200 &#181;m) surrounded by smaller ones. This pore architecture provides a better interconnectivity and an increased pore density for volume unit, although determining a drastic thinning of walls thickness. The morphology of M 90, provided in Figure 2d, shows roughly cubic pores, homogeneously distributed throughout the polymer matrix, due to the smaller salt size range (90-100 &#181;m) used in this case. Micropores, due to PEG solvation were present as micro-tunnels inside the walls that, in fact, appear very rough. SEM micrograph of M 45, shown in Figure 2e, displays a high density of pores, which diameters range from 45 &#181;m to 65 &#181;m. M 10 (Figure 2f) displayed the highest density of pores per volume unit, an average pore size approximately equal to 20 &#181;m, with a high degree of interconnection and very thin (&#60;1 &#181;m) walls. 
Figure 3a, a&#39; ,a&#34; displays a cross-section of TLS A, after the leaching process, at different magnifications. In Figure 3a it is possible to clearly identify the three layers, each one characterized by different average pore size, while the panels a&#39; and a&#34; refer to M 100-M 300 and M 300-M 500 interface regions, respectively. As clearly visible, the whole device do not present any internal cleavages nor matrix discontinuity between the different layers. Analogously, TLS B and related interfaces are reported in Figure 3b, b&#39;, b&#34;. The images revealed a morphology similar to that of TLS A. Indeed, three layers with pores of different sizes can be easily recognized (panel b), whereas both M 10-M 45 (panel b&#39;) and M 45-M 90 (panel b&#34;) interfacial regions exhibited no flaking nor discontinuity. As expected, each single layer retains the same pore architecture after assembly and leaching steps.
Table 1 reports the compressive elastic moduli of the materials, measured in air (dry) and in PBS (wet) environment. This property was found to follow a monotonic increase with the mean pore size. The elastic moduli of the final devices are mainly determined by their respective weaker layer (i.e. M 100 for TLS A and M 10 for TLS B) for both the TLSs investigated. Table 2 reports IAS for TLS A and TLS B in dry and wet environment. No interlayer delamination phenomena were observed, since the failure always occurred in the middle of the weakest layers of TLS A (M 100) and TLS B (M 10). TLS A displayed the best IAS performance.
<img alt="Figure 1" src="/files/ftp_upload/54595/54595fig1.jpg" /> 
Figure 1: Schematics of NaCl granulometry. Granulometry of sieved NaCl particles and corresponding sample codes.
<img alt="Figure 2" src="/files/ftp_upload/54595/54595fig2.jpg" /> 
Figure 2: Morphology of monolayer scaffolds. SEM micrographs of monolayer scaffolds characterized by different pore size distributions: M 500 (a), Scale bar&#160; = 400 &#181;m; M 300 (b), Scale bar&#160; = 400 &#181;m; M 100 (c), Scale bar&#160; = 400 &#181;m; M 90 (d),Scale bar&#160; = 200 &#181;m; M 45 (e), Scale bar&#160; = 400 &#181;m and M 10 (f), Scale bar&#160; = 100 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54595/54595fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54595/54595fig3.jpg" /> 
Figure 3: Morphology of three-layer scaffolds. (a, a&#39;, a&#39;&#39;) SEM micrographs of three-layered scaffold type A (TLS A): (a) entire cross section, Scale bar&#160; = 500 &#181;m; (a&#39;) M 100-M 300 interface, Scale bar&#160; = 250 &#181;m; (a&#39;&#39;) M 300-M 500 interface, Scale bar&#160; = 250 &#181;m. (b, b&#39;, b&#39;&#39;) SEM micrographs of three-layered scaffold type B (TLS B): (b) entire cross section, scale bar 500 &#181;m; (b&#39;) M 10-M 45 interface, Scale bar&#160; = 100 &#181;m; (b&#39;&#39;) M 45-M 90 interface, Scale bar&#160; = 100 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54595/54595fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample code</td>
			<td>Dry - E (MPa)</td>
			<td>Wet &#8211; E (MPa)</td>
		</tr>
		<tr>
			<td>M 500</td>
			<td>40.33 &#177; 6.04</td>
			<td>33.23 &#177; 4.96</td>
		</tr>
		<tr>
			<td>M 300</td>
			<td>37.62 &#177; 6.89</td>
			<td>31.42 &#177; 5.83</td>
		</tr>
		<tr>
			<td>M 100</td>
			<td>32.12 &#177; 5.11</td>
			<td>28.03 &#177; 4.04</td>
		</tr>
		<tr>
			<td>M 90</td>
			<td>30.87 &#177; 4.93</td>
			<td>26.91 &#177; 3.79</td>
		</tr>
		<tr>
			<td>M 45</td>
			<td>25.36 &#177; 5.82</td>
			<td>22.83 &#177; 5.01</td>
		</tr>
		<tr>
			<td>M 10</td>
			<td>21.76 &#177; 3.91</td>
			<td>19.87 &#177; 3.93</td>
		</tr>
		<tr>
			<td>TL A</td>
			<td>33.08 &#177; 5.21</td>
			<td>29.55 &#177; 4.09</td>
		</tr>
		<tr>
			<td>TL B</td>
			<td>22.31 &#177; 5.46</td>
			<td>20.54 &#177; 3.87</td>
		</tr>
	</tbody>
</table>
Table 1: Compressive mechanical results. Compressive Young modulus of mono and three-layered scaffolds with different pore sizes in wet and dry environment. Values are given as means &#177; SD.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample code</td>
			<td>Dry - IAS (kPa)</td>
			<td>Wet &#8211; IAS (kPa)</td>
		</tr>
		<tr>
			<td>TL A</td>
			<td>350.8 &#177; 51.2</td>
			<td>299.6 &#177; 35.1</td>
		</tr>
		<tr>
			<td>TL B</td>
			<td>262.3 &#177; 62.2</td>
			<td>220.5 &#177; 31.3</td>
		</tr>
	</tbody>
</table>
Table 2: Scaffold interfacial adhesion strength. Interfacial adhesion strength test results for TL A and TL B in dry and wet condition. Values are given as means &#177; SD.

Watch this Scientific Journal Video about A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size at JoVE.com

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.

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 ...

a-facile-eco-friendly-route-to-fabricate-poly-lactic-acid-scaffolds

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.6K Views.  University of Palermo. 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.

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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

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 ...

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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.

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

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

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

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

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

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

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Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Postproduction Processing of Electrospun Fibres for Tissue Engineering

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

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

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures