The authors acknowledge funding from the Australian Research Council and UNSW Australia via the Discovery Research program (DP210100094).

1. Preparation of 3D printing program and 3D printer
<ol>
	<li>Design the digital model for 3D printing following the steps below.
	<ol>
		<li>Open a computer-assisted design program (see Table of Materials).</li>
		<li>In the x-y plane, create a rectangle centered on the origin having dimensions of 80 mm x 40 mm, then extrude along the positive z-axis for 1.5 mm to make a solid rectangular prism, called the base object.</li>
		<li>Above the base object, i.e., at z = 1.5 mm, draw the desired surface patterns (in this case, two yin-yang symbols) on the surface of the rectangular prism.</li>
		<li>Extrude the surface patterns in selected regions 0.05 mm along the positive z-axis to create a slightly raised pattern relative to the base object.</li>
		<li>Export the 3D model to provide a stereolithography file with .STL file extension. 
		NOTE: In this work, dog-bone-shaped specimens were designed27. For other desired models to be printed, follow steps 1.1.1-1.1.5.</li>
		<li>Open a 3D printer slicing program (see Table of Materials) to enable single-layer settings.</li>
		<li>Open the converted .STL files from the computer hard drive by clicking on File &#62; Open then navigating to the saved .STL file.</li>
		<li>Arrange the 3D models on the build platform using the &#34;Model Rotate&#34; and &#34;Model Move&#34; buttons to fit at least 1 mm between all objects on the build stage.</li>
		<li>By entering text in the entry field boxes in the right-hand panel, change the parameters as mentioned in Table 1.</li>
		<li>Click on the blue Slice button in the bottom left-hand corner and save it as a slice file with an extension of. PWS or other 3D printer readable sliced file.</li>
		<li>Click on the Preview button once the pop-up menu appears and navigate through the sliced layers using the scroll bar on the right-hand side. Take careful note of the layer numbers for the last base layer (layer 29 in this case) and the surface pattern layer (30 in this case). 
		NOTE: The first printed layer is &#34;layer 0&#34; not &#34;layer 1&#34;.</li>
		<li>In the right-hand panel, select Single-layer settings, then expand the drop-down menu.</li>
		<li>Change the &#34;Exposure Time (s)&#34; for only the surface layer (layer 30) to 180 s, leaving all other layer exposure times as the default value.</li>
		<li>Click on Save button in the top left corner to save the sliced file to a USB.</li>
	</ol>
	</li>
	<li>Prepare the 3D printer.
	<ol>
		<li>Insert the USB containing the sliced file into the 3D printer (see Table of Materials).</li>
		<li>Before 3D printing, level the build stage and calibrate the z-axis position to the z = 0 by following the specific 3D printer method (manual or automatic calibration following the 3D printer manual).</li>
		<li>Inspect the film of the 3D printer vat to ensure a smooth and clean surface free of defects.</li>
		<li>If the vat film appears damaged, replace it according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
2. Preparation of resins
NOTE: Resins are categorized as &#34;Bulk Resin&#34; for the resin used to 3D print the original material (base substrate) and &#34;Surface Resin&#34; for the solution used to perform the surface functionalization (surface pattern).
<ol>
	<li>Prepare the Bulk Resin.
	<ol>
		<li>For preparing the bulk resin, weigh 0.36 g of 2-(n-butylthiocarbonothioylthio) propanoic acid (BTPA) into a clean 50 mL amber vial.</li>
		<li>Add 13.63 mL of poly (ethylene glycol) diacrylate average Mn 250 (PEGDA) to the amber vial using a micropipette.</li>
		<li>Add 14.94 mL of N, N-dimethylacrylamide (DMAm) to the amber vial using a micropipette.</li>
		<li>In a separate 20 mL clean glass vial covered with aluminum foil, add 0.53 g of diphenyl (2,4,6-trimethyl benzoyl) phosphine oxide (TPO).</li>
		<li>Using a micropipette, add 10 mL of DMAm to the 20 mL glass vial containing the TPO and seal the vial using the cap.</li>
		<li>Thoroughly homogenize the solution of TPO and DMAm by mixing using a vortex mixer for 10 s and then using a standard laboratory sonic bath (~40 kHz) to sonicate the mixture for 1 min at room temperature (Figure 1C, left).</li>
		<li>Using a glass pipette and rubber pipette bulb, transfer the solution from the 20 mL glass vial to the 50 mL amber vial and seal the vial with a cap and moldable plastic film.</li>
		<li>Gently shake the 50 mL amber vial and then place the vial in a sonic bath for 2 min at room temperature to ensure the mixture is homogeneous (Figure 1C, second from left).</li>
		<li>Place the sealed amber vial filled with the bulk resin in a fume hood for later use.</li>
	</ol>
	</li>
	<li>Prepare the Surface Resin.
	<ol>
		<li>For preparing the surface resin, weigh 0.50 g of TPO into a clean 50 mL amber vial.</li>
		<li>Using a micropipette, add 3.56 mL of DMAm and 11.98 mL of N, N-dimethylformamide (DMF) to the 50 mL amber vial and seal the vial with a cap moldable plastic film.</li>
		<li>Gently shake the sealed amber vial and sonicate for 1 min at room temperature using a standard laboratory sonic bath (~40 kHz).</li>
		<li>To a clean 20 mL vial covered with foil, add 0.29 g 1-pyrenemethyl methacrylate (PyMMA).</li>
		<li>Add 10 mL of DMF to the 20 mL vial and seal the vial with a cap using a micropipette.</li>
		<li>Gently shake the 20 mL glass vial and sonicate in increments of 1 min at room temperature using a standard laboratory sonic bath, visually inspecting between cycles until the PyMMA appears to be dissolved entirely (Figure 1C, third and fourth from left).</li>
		<li>Using a glass pipette and rubber pipette bulb, transfer the solution from the 20 mL glass vial to the 50 mL amber vial.</li>
		<li>Gently shake the 50 mL amber vial and then place the vial in a sonic bath for 2 min at room temperature to ensure the mixture is homogeneous (Figure 1C, right and second from right).</li>
		<li>Place the sealed amber vial filled with the bulk resin in a fume hood for later use. 
		&#8203;CAUTION: Some chemicals used in this protocol may cause severe skin and eye irritation and other toxicity to humans and the environment. Ensure safety protocols are followed in line with the safety data sheet and local regulations.</li>
	</ol>
	</li>
</ol>
3. 3D printing and surface functionalization
<ol>
	<li>Perform 3D printing of the base substrate following the steps below.
	<ol>
		<li>Pour the previously prepared bulk resin (step 2.1) into the 3D printer vat (see Table of Materials), ensuring that the solution completely covers the bottom film in the vat without any air bubbles or other inhomogeneities, and then close the 3D printer case.</li>
		<li>Navigate the USB using the 3D printer screen and select the sliced model file by clicking on the triangle Play button to begin the 3D printing process.</li>
		<li>By watching the 3D printer screen, take careful note of the number of layers printed, and pause the printing program by pressing the two vertical lines Pause button during 3D printing of the last layer of the base substrate (layer 29 in this case).</li>
		<li>Remove the entire build stage and gently rinse the build stage and printed material with undenatured 100% ethanol from a wash bottle for 10 s to remove residual bulk resin from the 3D printed material and the build stage.</li>
		<li>Using compressed air, gently dry the 3D printed material and build stage to remove residual ethanol and then reinsert the build stage into the 3D printer.</li>
		<li>Remove the vat from the 3D printer and pour the remaining bulk resin into an amber vial. Store the vial in a cool dark place.</li>
		<li>Using undenatured 100% ethanol from a wash bottle, carefully rinse the vat to remove any residual bulk resin.</li>
		<li>Dry the vat using a stream of compressed air to remove any residual ethanol and reinsert the vat into the 3D printer.</li>
	</ol>
	</li>
	<li>Perform surface functionalization.
	<ol>
		<li>Pour the previously prepared surface resin (step 2.2) into the 3D printer vat, ensuring that the solution completely covers the bottom film without any air bubbles or other inhomogeneities, and then close the 3D printer case.</li>
		<li>Resume the 3D printing program by clicking on the triangle Play button to allow the predetermined surface patterning to occur.</li>
		<li>Once the printing program has been completed, remove the build stage from the 3D printer and wash for 10 s with undenatured 100% ethanol using a wash bottle to remove residual surface resin from the 3D printed material and the build stage.</li>
		<li>Using compressed air (flow rate, 30 L/min), gently dry the 3D printed material and build stage to remove residual ethanol.</li>
		<li>While still attached to the build stage, post-cure the material by inverting the entire build stage and placing it under 405 nm light for 15 min.</li>
		<li>Gently remove the surface-functionalized 3D printed material from the build stage using a thin metal plate or paint scraper.</li>
		<li>Without further adjustments, analyze the material&#39;s mechanical and surface properties.</li>
	</ol>
	</li>
</ol>
4. Analysis of 3D printed samples
<ol>
	<li>Perform the fluorescence analysis.
	<ol>
		<li>Place the 3D printed, surface-functionalized material under a 312 nm UV gas discharge lamp (see Table of Materials) in a dark place, ensuring the surface-functionalized layer is facing up.</li>
		<li>Turn the lamp on to continuously irradiate the surface layer with 312 nm light and observe the fluorescent pattern. Take photographs if required. 
		NOTE: This is a visual inspection step; time cannot be specified. Irradiation is continuous while observation is occurring.</li>
		<li>Place the 3D printed, surface-functionalized material into a Fluorescence imager. Using the provided software, capture digital fluorescence images of the top and bottom surfaces using the Trans-UV (302 nm) gas discharge source (see Table of Materials).</li>
	</ol>
	</li>
	<li>Perform the tensile property analysis.
	<ol>
		<li>Measure the gauge with and thickness of the dog-bone specimens (in millimeters).</li>
		<li>Place the dog-bone-shaped specimens between the grips of a tensile testing machine, ensuring the 3D printed material is equally placed at a distance specified by the standards document, in this case, 50.3 mm.</li>
		<li>Set the tensile test program; in this case, the lift speed was set to 1.1 mm/min, the number of samples was set at 10 per second.</li>
		<li>Start the program to acquire force (N) vs. travel (mm) data.</li>
		<li>Once the sample gets prepared, stop the machine, and save the data as column-separated data with a .CSV file extension.</li>
		<li>Convert the force (N) data to stress (MPa) by dividing each point of the force column by the gauge area (mm2, obtained by multiplying the gauge width by the gauge thickness).</li>
		<li>Convert the travel data to strain (%) by diving the travel data by the gauge length (50.3 mm) at every point and multiplying each result by 100.</li>
		<li>Calculate toughness (MJ/m3) using the trapezoidal rule to calculate the area under the stress-strain curve.</li>
		<li>Calculate Young&#39;s modulus (MPa) by taking the gradient of the stress (MPa) vs. strain (%) curve in the elastic region, in this work from 1%-2% elongation27.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

The present protocol demonstrates a process for 3D printing of polymer materials with independently tunable bulk and interfacial properties. The procedure is performed via a two-step method by 3D printing the base substrate and subsequently modifying the surface layer of the 3D printed object using a different functional resin but using the same 3D printing hardware. While the 3D printers used in this work are designed to print crosslinked materials in a layer-by-layer fashion, the surface functionalization can ...

Additive manufacturing and 3D printing have revolutionized material manufacturing by providing more efficient and facile routes for the fabrication of geometrically complex materials1. Apart from the enhanced design freedoms in 3D printing, these technologies produce less waste than traditional subtractive manufacturing processes via the judicious use of precursor materials in a layer-by-layer manufacturing process. Since the 1980s, a wide range of different 3D printing techniques has been developed to fabricate polymeric, metal, and ceramic components1. The most commonly employed methods include extrusion-based 3D printing such as fused filament fabrication and direct ink writing techniques2, sintering techniques such as selective laser sintering3, as well as resin-based photoinduced 3D printing techniques such as laser and projection-based stereolithography and masked digital light processing techniques4. Among the many 3D printing techniques in existence today, photoinduced 3D printing techniques provide some advantages compared to other methods, including higher resolution and faster printing speeds, as well as the ability to perform solidification of the liquid resin at room temperature, which opens the possibility to advanced biomaterial 3D printing4,5,6,7,8,9.
While these advantages have allowed the widespread adoption of 3D printing in many fields, the limited ability to independently tailor the 3D printed material properties restricts future applications10. In particular, the inability to easily tailor the bulk mechanical properties independently of the interfacial properties limits applications such as implants, which require finely tailored biocompatible surfaces and often vastly differing bulk properties, as well as antifouling and antibacterial surfaces, sensor materials, and other smart materials11,12,13. Researchers have proposed surface modification of 3D printed materials to overcome these issues to provide more independently tailorable bulk and interfacial properties10,14,15.
Recently, our group developed a photoinduced 3D printing process that exploits reversible addition-fragmentation chain transfer (RAFT) polymerization to mediate network polymer synthesis15,16. RAFT polymerization is a type of reversible deactivation radical polymerization that provides a high degree of control over the polymerization process and allows for the production of macromolecular materials with finely tuned molecular weights and topologies, and broad chemical scope17,18,19. Notably, the thiocarbonylthio compounds, or RAFT agents, used during RAFT polymerization are retained after polymerization. They can thus be reactivated to modify further the chemical and physical properties of the macromolecular material. Thus, after 3D printing, these dormant RAFT agents on the surfaces of the 3D printed material can be reactivated in the presence of functional monomers to provide tailored material surfaces20,21,22,23,24,25,26. The secondary surface polymerization dictates the interfacial material properties and can be performed in a spatially controlled fashion via photochemical initiation.
The present protocol describes a method for 3D printing polymeric materials via a photoinduced RAFT polymerization process and the subsequent in situ surface modification to modulate the interfacial properties independently of the bulk material mechanical properties. Compared to previous 3D printing and surface modification approaches, the current protocol does not require deoxygenation or other stringent conditions and is thus highly accessible for non-specialists. Furthermore, the use of 3D printing hardware to perform both the initial material fabrication and the surface post-functionalization provides spatial control over the material properties and can be performed without the tedious alignment of several different photomasks to make complex patterns.

<table><tbody><tr><td>1-pyrenemethyl methacrylate</td><td>Sigma-Aldrich</td><td>765120</td><td></td></tr><tr><td>2-(&lt;em&gt;n&lt;/em&gt;-butylthiocarbonothioylthio) propanoic acid</td><td>Boron Molecular</td><td>BM1640</td><td></td></tr><tr><td>3D Printer</td><td>Photon</td><td>Mono S</td><td>light intensity at digital mask surface = 0.81 mW cm-2</td></tr><tr><td>3D Printing Slicing Software</td><td>Photon</td><td>Photon Workshop V2.1.19</td><td></td></tr><tr><td>40 kHz Ultrasonic Bath</td><td>Thermoline</td><td>UB-410</td><td></td></tr><tr><td>Compressed Air</td><td>Coregas</td><td>230142</td><td>Tank operating at 130 kPa</td></tr><tr><td>Computer Assisted Design Program</td><td>SpaceClaim</td><td>SpaceClaim Design Manager V19.1</td><td></td></tr><tr><td>Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide</td><td>Sigma-Aldrich</td><td>415952</td><td></td></tr><tr><td>Ethanol Undenatured 100% AR</td><td>ChemSupply</td><td>EL043-2.5L-P</td><td></td></tr><tr><td>Ethanol Wash bottle</td><td>Rowe Scientific</td><td>AZLWGF541P</td><td></td></tr><tr><td>Fluorescence Imager</td><td>Bio-Rad</td><td>Gel Doc XR+</td><td>Uses a 302 nm gas discharge lamp as emission source</td></tr><tr><td>Light intensity power meter</td><td>Newport</td><td>843-R</td><td></td></tr><tr><td>Mechanical Tester</td><td>Mark&amp;ndash;10</td><td>ESM303</td><td>1 kN force gauge M5&amp;ndash;200</td></tr><tr><td>Moldable plastic film</td><td>Parafilm</td><td>PM992</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;-dimethlacrylamide</td><td>Sigma-Aldrich</td><td>274135</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;,&lt;em&gt;N&lt;/em&gt;-Dimethylformamide HPLC</td><td>ChemSupply</td><td>LC1051-G4L</td><td></td></tr><tr><td>Poly(ethylene glycol) diacrylate average Mn 250</td><td>Sigma-Aldrich</td><td>475629</td><td></td></tr><tr><td>Post Cure Lamp</td><td>Leoway</td><td>&amp;lrm;B0869BY79P</td><td>60 W 405 nm</td></tr><tr><td>Standards document</td><td>ASTM</td><td>ASTM Standard D638-14</td><td></td></tr><tr><td>Tensile testing machine</td><td>Mark-10</td><td></td><td></td></tr><tr><td>UV Light</td><td>Fisher Scientific</td><td>11-982-30</td><td>6 W Spectroline E-Series, Gas discharge lamp</td></tr><tr><td>Vortex Mixer IKA Vortex 3</td><td>LabTek</td><td>3340000I</td><td></td></tr></tbody></table>

3d printing and in situ surface modification via type i photoinitiated reversible addition-fragmentation chain transfer polymerization

3D printing provides facile access to geometrically complex materials. However, these materials have intrinsically linked bulk and interfacial properties dependent on the chemical composition of the resin. In the current work, 3D printed materials are post-functionalized using the 3D printer hardware via a secondary surface-initiated polymerization process, thus providing independent control over the bulk and interfacial material properties. This process begins with preparing liquid resins, which contain a monofunctional monomer, a crosslinking multifunctional monomer, a photochemically labile species that enables initiation of polymerization, and critically, a thiocarbonylthio compound which facilitates reversible addition-fragmentation chain transfer (RAFT) polymerization. The thiocarbonylthio compound, known commonly as a RAFT agent, mediates the chain growth polymerization process and provides polymeric materials with more homogeneous network structures. The liquid resin is cured in a layer-by-layer fashion using a commercially available digital light processing 3D printer to give three-dimensional materials having spatially controlled geometries. The initial resin is removed and replaced with a new mixture containing functional monomers and photoinitiating species. The 3D printed material is then exposed to light from the 3D printer in the presence of the new functional monomer mixture. This allows photoinduced surface-initiated polymerization to occur from the latent RAFT agent groups on the surface of the 3D printed material. Given the chemical flexibility of both resins, this process allows a wide range of 3D printed materials to be produced with tailorable bulk and interfacial properties.

The general procedure for 3D printing and surface functionalization is shown in Figure 1. In this protocol, a network polymer is initially synthesized via a photoinduced RAFT polymerization process15, using a 3D printer to fabricate an object in a layer-by-layer process (Figure 1A). The bulk resin used to form the polymer network contains a photolabile initiating species (TPO), which generates radicals upon exposure to 405 nm lig...

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3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

The present protocol describes the digital light processing-based 3D printing of polymeric materials using type I photoinitiated reversible addition-fragmentation chain transfer polymerization and the subsequent in situ material post-functionalization via surface-mediated polymerization. Photoinduced 3D printing provides materials with independently tailored and spatially controlled bulk and interfacial properties.

3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

3D printing provides facile access to geometrically complex materials. However, these materials have intrinsically linked bulk and interfacial properties ...

3d-printing-situ-surface-modification-via-type-i-photoinitiated

Research

JoVE Journal

Chemistry

3.7K Views.  University of New South Wales. Met dit protocol kunnen de bulk- en interfaciale eigenschappen van 3D-geprinte materialen onafhankelijk van elkaar worden afgestemd. Dit geeft meer flexibiliteit bij het ontwerpen en fabriceren van complexe 3D-geprinte materialen. Deze techniek vereist geen strenge reactieomstandigheden en kan worden uitgevoerd met behulp van in de handel verkrijgbare apparatuur.Hierdoor maakt deze techniek het aanzienlijk eenvoudiger om complexe 3D-geprinte materialen te fabriceren. Bereid om te begin...

Video: 3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

Micro-masonry for 3D Additive Micromanufacturing

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to a different substrate by utilizing elastomeric stamps. Transfer printing enables the integration of heterogeneous materials to fabricate unexampled structures or functional systems that are found in recent advanced devices such as flexible and stretchable solar cells and LED arrays. While transfer printing exhibits unique features in material assembly capability, the use of adhesive layers or the surface modification such as deposition of self-assembled monolayer (SAM) on substrates for enhancing printing processes hinders its wide adaptation in microassembly of microelectromechanical system (MEMS) structures and devices. To overcome this shortcoming, we developed an advanced mode of transfer printing which deterministically assembles individual microscale objects solely through controlling surface contact area without any surface alteration. The absence of an adhesive layer or other modification and the subsequent material bonding processes ensure not only mechanical bonding, but also thermal and electrical connection between assembled materials, which further opens various applications in adaptation in building unusual MEMS devices.

This paper introduces a 3D additive micromanufacturing strategy (termed &#8216;micro-masonry&#8217;) for the flexible fabrication of microelectromechanical system (MEMS) structures and devices. This approach involves transfer printing-based assembly of micro/nanoscale materials in conjunction with rapid thermal annealing-enabled material bonding techniques.

Transfer printing is a method to transfer solid micro/nanoscale materials (herein called &#8216;inks&#8217;) from a substrate where they are generated to ...

Engineering

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

A Soft Tooling Process Chain for Injection Molding of a 3D Component with Micro Pillars

The purpose of this paper is to present the method of a soft tooling process chain employing Additive Manufacturing (AM) for fabrication of injection molding inserts with micro surface features. The Soft Tooling inserts are manufactured by Digital Light Processing (vat photo polymerization) using a photopolymer that can withstand relatively high temperaturea. The part manufactured here has four tines with an angle of 60&#176;. Micro pillars (&#216;200 &#181;m, aspect ratio of 1) are arranged on the surfaces by two rows. Polyethylene (PE) injection molding with the soft tooling inserts is used to fabricate the final parts. This method demonstrates that it is feasible to obtain injection-molded parts with microstructures on complex geometry by additive manufactured inserts. The machining time and cost is reduced significantly compared to conventional tooling processes based on computer numerical control (CNC) machining. The dimensions of the micro features are influenced by the applied additive manufacturing process. The lifetime of the inserts determines that this process is more suitable for pilot production. The precision of the inserts production is limited by the additive manufacturing process as well.

A protocol for fabricating injection molding inserts for complex geometry with micro features on surfaces employing Additive Manufacturing (AM) is presented.

The purpose of this paper is to present the method of a soft tooling process chain employing Additive Manufacturing (AM) for fabrication of injection ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Stereolithographic 3D Printing with Renewable Acrylates

The accessibility of cost-competitive renewable materials and their application in additive manufacturing is essential for an efficient biobased economy. We demonstrate the rapid prototyping of sustainable resins using a stereolithographic 3D printer. Resin formulation takes place by straightforward mixing of biobased acrylate monomers and oligomers with a photoinitiatior and optical absorber. Resin viscosity is controlled by the monomer to oligomer ratio and is determined as a function of shear rate by a rheometer with parallel plate geometry. A stereolithographic apparatus charged with the biobased resins is employed to produce complex shaped prototypes with high accuracy. The products require a post-treatment, including alcohol rinsing and UV irradiation, to ensure complete curing. The high feature resolution and excellent surface finishing of the prototypes is revealed by scanning electron microscopy.

A protocol for additive manufacturing with renewable photopolymer resins on a stereolithography apparatus is presented.

The accessibility of cost-competitive renewable materials and their application in additive manufacturing is essential for an efficient biobased economy. ...

Triplet Fusion Upconversion Nanocapsule Synthesis

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has significant implications for producing high energy light beyond a material's surface. However, the deployment of UC materials has been stymied due to poor material solubility, high concentration requirements, and oxygen sensitivity, ultimately resulting in reduced light output. Toward this end, nanoencapsulation has been a popular motif to circumvent these challenges, but durability has remained elusive in organic solvents. Recently, a nanoencapsulation technique was engineered to tackle each of these challenges, whereupon an oleic acid nanodroplet containing upconversion materials was encapsulated with a silica shell. Ultimately, these nanocapsules (NCs) were durable enough to enable triplet fusion upconversion-facilitated volumetric three-dimensional (3D) printing. By encapsulating upconversion materials with silica and dispersing them in a 3D printing resin, photopatterning beyond the surface of the printing vat was made possible. Here, video protocols for the synthesis of upconversion NCs are presented for both small-scale and large-scale batches. The outlined protocols serve as a starting point for adapting this encapsulation scheme to multiple upconversion schemes for use in volumetric 3D printing applications.

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has ...

Visualizing Polymer Dynamics for Optimizing 3D Printing Quality

Real-Time Imaging of Bonding in 3D-Printed Layers

In recent times, 3D printing technology has revolutionized our ability to design and produce products, but optimizing the print quality can be challenging. The process of extrusion 3D printing involves pressuring molten material through a thin nozzle and depositing it onto previously extruded material. This method relies on bonding between the consecutive layers to create a strong and visually appealing final product. This is no easy task, as many parameters, such as the nozzle temperature, layer thickness, and printing speed, must be fine-tuned to achieve optimal results. In this study, a method for visualizing the polymer dynamics during extrusion is presented, giving insight into the layer bonding process. Using laser speckle imaging, the plastic flow and fusion can be resolved non-invasively, internally, and with high spatiotemporal resolution. This measurement, which is easy to perform, provides an in-depth understanding of the underlying mechanics influencing the final print quality. This methodology was tested with a range of cooling fan speeds, and the results showed increased polymer motion with lower fan speeds and, thus, explained the poor printing quality when the cooling fan was turned off. These findings show that this methodology allows for optimizing the printing settings and understanding the material behavior. This information can be used for the development and testing of novel printing materials or advanced slicing procedures. With this approach, a deeper understanding of extrusion can be built to take 3D printing to the next level.

Author Spotlight: Real-Time Imaging of Bonding in 3D-Printed Layers  

With a non-invasive and real-time technique, nanoscopic polymer motion inside a polymer filament is imaged during 3D printing. Fine-tuning this motion is crucial for producing constructs with optimal performance and appearance. This method reaches the core of plastic layer fusion, thus offering insights into optimal printing conditions and material design criteria.

In recent times, 3D printing technology has revolutionized our ability to design and produce products, but optimizing the print quality can be ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization

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