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>
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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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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 ...

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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Research

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Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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 ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

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 ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

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 ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...