We thank George Giudice and Steven Eliason for donating human dermal fibroblasts and Ramesh Raghupathy for help with the strain tracking algorithm.&#160;Support for this work was provided by a U.S. Department of Education Graduate Assistance in Areas of National Need Fellowship (GAANN P200A120071).

1. Stencil Preparation
Prepare a stencil on Parafilm to layout the location of each explant, following the desired geometry (Figures 1A and 1B). Space each explant approximately 1-2 mm apart. This distance corresponds to an ideal spacing for generating fiber alignment between explants. Attach the stencil underneath the area of the coverglass where the sample will be prepared with tape.
2. Sterilization
Thoroughly clean all components of the bioreactor using 70% ethanol and sterilize for 2-3 hr under UV light prior to experimentation. If an alternative vessel is being used in lieu of a bioreactor then proper sterilization techniques should be used. See step 4.12 for comments on using a glass bottom Petri dish.
3. Fibrin Gel Preparation
After the components of the bioreactor have been sterilized, cast a thin layer of fibrin gel on the surface of the coverglass. Details for preparing fibrinogen and thrombin stock solutions for making 6.6 mg/ml&#160;fibrin gels are described in Sander et al.13&#160;A similar protocol by Ye et al.19 for making 3.3 mg/ml&#160;fibrin gels can also be viewed on the JoVE website.
<ol>
	<li>Prepare a solution of fluorescent microbeads in DMEM at a concentration of 10 million beads/ml. The beads will be used to help track gel displacements. To achieve this concentration, combine 0.017 ml&#160;of microbead stock solution and 0.149 ml&#160;of DMEM into a microcentrifuge tube.</li>
	<li>Sonicate this suspension for 10 min&#160;to disperse the beads and homogenize the solution.</li>
	<li>Fibrin Solution &#8212; In a 15 ml c-tube, mix 0.22 ml of fibrinogen stock solution with 0.44 ml of 20 mM HEPES buffer. Add the 0.1667 ml of DMEM with microbeads created in step 3.1.</li>
	<li>Thrombin Solution &#8212; In a separate 15 ml c-tube, mix together 0.0328 ml of thrombin stock solution, 0.131 ml of 20 mM HEPES buffer, and 0.00246 ml of 2 M CaCl2.</li>
	<li>Carefully mix the thrombin solution (step 3.4) with the fibrinogen solution (step 3.3) by pipetting up and down 5-10x until the solution is evenly distributed. Avoid introducing bubbles as much as possible. To reduce the amount of bubbles produced, be careful not to fully discharge the pipette while mixing.</li>
	<li>The addition of thrombin will cause the solution to gel quickly (~30 sec). Pipette the mixed solution onto the coverglass as soon as possible. Allow the gel to polymerize at RT.</li>
	<li>Seal up the bioreactor, insert the heating blocks, and connect the thermocouples to the temperature controller. Incubate the gel at 37 &#176;C for 15-30 min.</li>
</ol>
4. Cell Explant Preparation
<ol>
	<li>Remove medium from the T-75 flask containing the human dermal fibroblast cells.</li>
	<li>Carefully rinse the surface with approximately 5 ml&#160;of phosphate buffered saline (PBS) to remove serum proteins. Add 1 ml of trypsin-EDTA and incubate for 3 min, or until cells have lifted.</li>
	<li>After the cells have been lifted, spin the suspension down in a centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the pellet in a volume of DMEM that will allow a final concentration of 20 million cells/ml.</li>
	<li>While the cells are spinning down in the centrifuge disconnect the bioreactor from the heating blocks and the thermocouples. Transfer the bioreactor to a biosafety cabinet and carefully remove the lid following asceptic techniques.</li>
	<li>Create explants by pipetting 0.3 &#181;l of the cell suspension onto the polymerized fibrin gel, following the pattern on the stencil. Each explant should contain approximately 6,000 cells. Make sure that low volume micropipette tips are used (0.1-10 &#181;l).</li>
	<li>Allow cells to settle and attach to the fibrin matrix for 1 hr&#160;at 37&#160;&#176;C.</li>
	<li>With the bioreactor still open, add approximately 5 ml&#160;of DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1% amphotericin B, and 10 mg/ml&#160;aprotinin directly into the bioreactor chamber. DMEM is bicarbonate buffered and requires 5% CO2&#160;to maintain a neutral pH. Since the bioreactor is not supplied with CO2, condition the medium in an incubator with 5% CO2&#160;for 2-3 hr&#160;before use. Aprotinin is a serine protease inhibitor that is widely used to reduce the rate of fibrin degradation20.</li>
	<li>Reseal the bioreactor. Use a syringe to deliver an additional 5 ml&#160;of CO2&#160;conditioned medium via the barbed fitting on the inlet port. Dispense the medium slowly and make sure the entire volume of the bioreactor chamber is filled. Carefully remove bubbles that form in the bioreactor.</li>
	<li>Supply fresh, 5% CO2 conditioned medium to the bioreactor throughout the experiment in order to maintain pH, supply nutrients, and remove waste products. Setup a syringe pump with a 10 ml&#160;or 30 ml&#160;syringe filled with 5% CO2&#160;conditioned medium. Connect the syringe directly to the inlet port on the bioreactor lid with Luer-lock fitted sterile tubing. Remove the male fitting and attach the tubing to the barbed fitting on the bioreactor (see Figure&#160;1C).</li>
	<li>Set the perfusion rate to 0.01 ml/min. Connect modified pieces of tubing to both outlet ports and place the ends into a 100 ml&#160;beaker to collect waste.</li>
	<li>Use a lab jack to set the heights of the inlet and outlet feeds so that a pressure differential does not develop in the bioreactor (consult Figure&#160;1D for reference).</li>
	<li>If a bioreactor is not available, prepare samples in 35 mm glass bottom Petri dishes with glass tops. Use one with a coverslip size that is optimized for the specific set of objectives to be used. Samples that are prepared in Petri dishes should be maintained in an incubator at 37 &#176;C and 5% CO2. 
	Note: Polystyrene depolarizes light and will interfere with DIC imaging, so glass tops should be used if DIC imaging will be conducted. Phase contrast is a suitable alternative imaging modality. Transferring dishes between the incubator and microscope will make image registration difficult. If images are not properly registered then the strain calculated in Section&#160;6 will not be accurate.</li>
</ol>
5. Time-lapse Imaging
<ol>
	<li>Once the sample has been prepared, reseal the bioreactor, reconnect the heating blocks and the thermocouples, and set the bioreactor temperature to 37 &#176;C.</li>
	<li>Set the bioreactor onto the microscope mounted motorized stage. Position the 20X&#160;DIC objective under the view-port. A lower magnification objective is acceptable, particularly if a precision motorized stage is not available. Ensure that the polarizer, analyzer, and prism are all in place. Alternatively, samples can also be imaged using phase contrast.</li>
	<li>Open the imaging software.</li>
	<li>Focus the objective on the area between the three explants.</li>
	<li>Save the coordinates (x, y, and z) of this location in the imaging software.</li>
	<li>In order to image the entire area between and around the explants select the option to acquire a large image and specify the size of the area. This will allow the acquisition of multiple images around the specified area and create a tiled image that represents a larger region of the sample.</li>
	<li>Set the exposure time and light intensity to the lowest values possible to avoid cell death caused by phototoxicity, while still providing sufficient resolution to discriminate between cells, microbeads, and fibrin fibers.</li>
</ol>
6. Strain Tracking
For details and instructions on the strain tracking software (Figure&#160;2) used see Raghupathy et al.21&#160;The algorithm is a custom MATLAB code that can be downloaded from <a href="http://www.license.umn.edu/default.aspx">http://www.license.umn.edu/default.aspx</a>. Note that DIC images often have enough texture for strain tracking. The microbeads are included to serve as a check on the calculated strain fields. If strain tracking will be done it is critical that the images obtained are taken at the exact same location so that the images are registered. Unregistered images will produce spurious strains.

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No conflicts of interest declared.

This protocol was developed for the purpose of observing and quantifying the mechanics involved in cell-mediated ECM remodeling. Such processes underlie a number of biological phenomena and have important implications for engineering tissues2,22, reducing scar1,23, and understanding pathological tissue remodeling12,24. The use of time-lapse DIC microscopy allows one to resolve and quantify the displacement and alignment of fibrin fibers that occurs as a result of cell traction forces. The...

An important tool for studying cell-matrix interactions is the cell populated collagen gel1,2. The gel provides a 3D environment that is closer to the in vivo character of the tissue and better suited for understanding cell behavior than is offered by traditional 2D cultures3. Early studies in which fibroblasts were homogenously distributed within a collagen gel found that the cells rapidly consolidate the collagen fibers and compact the gel4,5. The contractile fibroblasts in free floating gels then transition into a quiescent state soon after the gel has fully reached compaction1,6,7. The fibroblasts in gels that are constrained at the boundaries remain in an active, synthetic state8&#160;and they generate fiber alignment in a manner dependent on gel geometry and external constraints5,9. Differences in cell activity appear to be a result of the internal tension (or lack thereof) that develops as the cells exert traction forces via integrins on the collagen fibers in the gel.
A variant of this technique involves placing fibroblast explants (i.e.&#160;clumps of cells) a distance apart within a collagen gel and observing cell-matrix interactions and the gradual development of fiber alignment between the explants (sometimes called ligament-like straps)10-12. The primary advantage of the explant system is that it allows one to arrange the cells into simple geometric patterns, which makes it easier to visualize and probe the mechanisms underlying cell-driven fiber realignment. These alignment patterns &#8212; which are dependent primarily on the interplay between cell traction forces, cell spatial distribution, gel geometry, and the mechanical constraints on the gel &#8212; are important to understand because they play a central role in global tissue organization, mechanical function, and local mechanical environment13.
In the field of tissue engineering, one strategy for producing mechanically functional, engineered-tissues involves controlling the fiber alignment pattern that develops from cell compaction so that the engineered tissue possesses fiber alignment that mimics that of the native tissue14,15. Such alignment is believed necessary for engineered tissues to replicate the complex mechanical behavior of native tissues. A modification of this strategy is to replace the collagen gel with a fibrin gel16. The fibrin gel develops a similar alignment pattern as a collagen gel during compaction. Over time the fibrin is degraded and replaced with cell-synthesized ECM that follows the initial fibrin fiber alignment pattern. The resulting engineered construct has significantly improved mechanical properties compared to collagen gel derived constructs17.
The alignment process and subsequent remodeling events in fibrin gels proceed in a complicated and poorly understood manner. To better characterize these interactions and their effect on cell behavior and ECM remodeling, we have developed a procedure that is based on the explant method. In this method, fibroblast explants are positioned on a fibrin gel in different geometric patterns. The gels are maintained in an environmentally-controlled, microscope-mounted bioreactor18, and the process of compaction and fiber realignment is monitored with time-lapse differential interference contrast (DIC) microscopy. Displacement fields are quantified with custom algorithms. The data obtained from these experiments have wide ranging implications for a number of processes, including optimizing tissue engineering strategies, improving wound healing, and treating pathological tissue remodeling.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Tissue remodeling is a complex process that is driven in part by reciprocal physical interactions between cells and the surrounding matrix. The cells reorganize the surrounding fibers and generate tension in the fiber network. The alignment of fibers and mechanical environment in turn controls cell behavior, so that both cells and matrix globally reorganize to produce remodeled tissue. In this experiment, the cells of the explants, initially round in morphology, began to extend into the gel and adhere to the fibrin fiber...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

5.9K vistas.  University of Iowa. La microscopia del lapso de tiempo y las técnicas de proceso de imagen fueron utilizadas para observar y para analizar la compactación fibroblasto-mediada del gel y el realineamiento de la fibra de la fibrina en un biorreactor ambientalmente controlado durante un período de 48 horas.

Video: Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...