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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision Balance</td>
			<td style="width:149px;">Sartorius</td>
			<td style="width:207px;">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.
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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 ...

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