Name: a facile and eco-friendly route to fabricate poly(lactic acid) scaffolds with graded pore size
Uploaded: 2016-10-17T13:00:00
Description: Watch this Scientific Journal Video about A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size at JoVE.com

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 par...

<ol>
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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...

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 border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly(lactic acid)</td>
			<td style="width:149px;">NatureWorks</td>
			<td style="width:207px;">PLA 2002D</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly(ethylene glycol)</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:207px;">83797-1KG-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cloride</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:207px;">793566-5KG-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosfate Buffer Solution</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:207px;">P5368-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Mixer</td>
			<td style="width:149px;">Brabender</td>
			<td style="width:207px;">PLE 330 - Plasticorder</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Press</td>
			<td style="width:149px;">Carver</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scanning Electron Microscopy</td>
			<td style="width:149px;">Phenom-world</td>
			<td style="width:207px;">ProX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Universal Testing Machine</td>
			<td style="width:149px;">Instron</td>
			<td style="width:207px;">3365 (UK)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BioPuls Bath</td>
			<td style="width:149px;">Instron, Norwood</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sieving Machine</td>
			<td style="width:149px;">Endecotts</td>
			<td style="width:207px;">E.V.F.1.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum Oven</td>
			<td style="width:149px;">ISCO</td>
			<td style="width:207px;">NSV9035</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Precision Balance</td>
			<td style="width:149px;">Sartorius</td>
			<td style="width:207px;">AX224</td>
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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.
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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 ...

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 in specific spatial hierarchies. For example, articular cartilage consists in different zones with varying types and orientation of collagen fibers and collagen binding proteins.The main strategies adopted in tissue engineering can be divided in monophasic, biphasic, and triphasic approaches. Biphasic and triphasic approaches use two or three different pore architectures, materials, or fillers to prepare multi-layered functional devices. A single material can also be used to achieve biphasic or triphasic devices if it possible to create a gradient of one or more physical properties.In the frame of this work, a fast and simple route to achieve polylactic acid based three layered pore scaffold is presented. Differently from many other scaffold 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 in tissues. In particular, the pathway proposed combines melt mixing, compression molding, and salt leaching.Both the morphological analysis and the mechanical behavior of the structures were investigated, paying particular attention to the processing structure property's relationships of the prepared devices. Sodium chloride was ground in a laboratory grinder for 20 minutes. Thereafter, sodium chloride was dried on a heater at 100 degrees C prior to sieving in a sieving machine for 30 minutes at the highest available frequency not showing resonance.The six selected fractions ranged from 1, 000 microns to 500 microns, from 500 microns to 300 microns, from 200 microns to 100 micron, from 100 micron to 90, and from 65 to 45, the last fraction with salt particle size smaller than 45 microns. All the materials were vacuum dried overnight, in order to avoid hydrolytic scission during processing, polylactic acid at 90 degrees C, polyethylene glycol at 25 degrees C, and sodium chloride at 105 degrees C.Before melt mixing, polylactic acid, polyethylene glycol, and sodium chloride were mechanically mixed in a beaker in the proportion of 20 weight percent for the polylactic acid, five weight percent for polyethylene glycol, and 75 weight percent for sodium chloride. The materials were then fed to a batch mixer.The temperature was set to 190 degrees C, and the rotor speed was set to 60 RPM. The materials were processed until a constant value of torque was achieved, usually after about 10 minutes. The blend was then fed out and abruptly cooled in liquid nitrogen.In order to prepare each layer, the blends were compression molded in a laboratory press at 210 degrees C.The samples were molded in an appropriate cylindrical mold with diameter of 10 millimeter and a height of three millimeter. The materials were pre-heated for 60 seconds at ambient pressure and 210 degrees C.Then the pressure was raised to 180 bars and kept for three minutes. The blends were then cooled at room temperature, keeping the pressure at 180 bars.Polymer sodium chloride blends prepared using different salt particle size were put into an appropriate cylindrical mold with a diameter of 10 millimeters and a height of three millimeters. The materials were then compression molded again for five minutes. More in detail, two different three layered scaffolds were prepared.In one case, the three layered structure was obtained by assembling blends filled with sodium chloride particle size ranged from 1, 000 microns to 500 microns, from 500 microns to 300 microns, and from 200 microns to 100 microns. The other three layered scaffold was obtained by assembling blends filled with sodium chloride particle size ranged from 100 microns to 90, from 65 to 45 microns, and the last layer with salt particle size smaller than 45 microns. The blends were then removed from the cylindrical mold, weighed, and put in boiling de-mineralized water for three hours, without stirring.The resulting structures were then left to dry for 12 hours at room temperature under fume hood. Furthermore, in order to evaluate connectivity and water uptake the samples were weighed before and after drying. The theoretical porosity for each sample was evaluated according to the following expressions.The composition of the mixture was designed in order to obtain scaffolds with a theoretical porosity of 70.8%The porosity calculated by question two is very close to the theoretical one for all the scaffold types, which makes it reasonable to conclude that all the porogen agents were extracted from the devices. The morphologies of the scaffolds were evaluated by scanning electron microscopy. The samples were broken under liquid nitrogen.Then, the specimens were attached onto an aluminum stub using an adhesive carbon tape. Before compressive tests in physiological environment, samples were filled by phosphate buffer saline solution by using a vacuum flask. This method permits the solution to fill all the void volume of the scaffold.Finally, tensile tests were performed in order to evaluate the interfacial adhesion strength occurring between the layers. Scanning electron microscopy analysis confirmed the strong influence of the sodium chloride particle size on the porous architecture of the scaffold. Figures one a, f show images obtained by scanning electron microscopy of scaffolds achieved by leaching materials filled with different salt granulometry.The measurement of pore size revealed the scaffold average pore size strongly controlled by the sodium chloride size filled into the polymer matrix. In particular, the scaffold obtained from the blend containing salt sieved in the range 1, 000 to 500 microns showed pores with an average diameter of 500 microns, probably due to the breakage of salt particles with diameter higher than 500 micron during melt mixing. As clearly visible in the same figure, pore architecture is characterized by a low number of irregular pores, poorly interconnected, and surrounded by walls of about 10 microns.Figure one b shows the morphology of the scaffold obtained by using salt in the range 500 to 300 microns. In this case, the pores were found to exhibit an average diameter within the same range of the salt particles filled during melt mixing, thus confirming that no particle breakage occurred within melt mixing process. Walls around pores were found to be thinner, about five microns, than the previous one.The scaffold obtained by using salt in the range 200 to 100 microns showed a bimodal porous structure characterized by a heterogeneous network composed by large pores 100 to 200 microns, surrounded by smaller ones. This pore architecture enhances the interconnection between pores and their amount per unit of volume, although determines a drastic thinning of wall thickness. The morphology of the scaffold obtained by using salt in the range 100, 90 microns showed roughly cubic pores, homogeneously distributed throughout the polymer matrix.Micro-pores, due to the polyethylene glycol solvation, were found inside the walls that in fact appear very rough. The scanning electron microscopy micrograph of the scaffold obtained by using salt in the range 45, 65 microns displayed a big amount of pores per unit of volume with the diameters ranging from 45 to 765 microns. Finally, the scaffold obtained by using salt smaller than 45 microns displayed the highest amount of pores per unit of volume.The pore architecture showed an average pore size of about 20 microns, with a high degree of interconnection and very thin walls with thickness lower than one micron. The morphology of the assembled scaffolds were characterized by three well distinct layers showing three different average pore size. As expected, each layer kept the same pore architecture after assembly and leaching steps.As clearly visible, the whole devices do not present any internal cleavages, nor matrix discontinuity among the different layers. During the compressive tests it was observed a decrease of the elastic modulus of the samples while decreasing their respective average pore size. Testing in wet conditions caused the decrease of the elastic moduli of about 10%Anyhow, the overall mechanical behavior of the devices did not change.The three layered devices presented an elastic modulus comparable with that of their respective weakest layer. The test highlighted that the fracture occurred about in the middle part of the weakest layer of the three layered devices, thus, not observing any delamination process. The collected data confirmed that the interfacial adhesion strength was higher than 10 size strength of the weakest layer, thus corroborating what observed by scanning electron microscopy analysis.Concluding, in this work polylactic acid based degradable scaffolds with a discrete pore size gradient has been developed by combining melt mixing, compression molding, and selective leaching. The devices present I interconnected pore structure with a porosity of about 70%and I predictability of pore size by controlling the granulometry of sodium chloride in the melt mixing step as confirmed by scanning electron microscopy analysis. Morphological analysis also showed that the three layer scaffolds are characterized by well fixed layers with different pore size.Interfacial adhesion strength tests revealed I layer adhesion without delamination.

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