This work was supported by the Orthopaedic Scientific Research Foundation, NIH 5R01 AR068133, NIH TERC 5P41EB027062, and NIGMS R01 692 GM083925 (Funder ID: 10.13039/100000057).

The authors have nothing to disclose.

A dynamic mechanical environment exists within the joint as cartilage is subjected to compressive, tensile, and shear forces, and hydrostatic and osmotic pressures44,45. Although cartilage is the main load-bearing tissue of the joint, the synovium also undergoes frictional interactions with the cartilage surface and with itself in regions where the tissue folds. The physical interactions between cartilage and synovium are likely responsible for transferring cells...

Osteoarthritis (OA) is a debilitating, degenerative joint disease that affects more than 32 million American adults, with a healthcare and socio-economic cost of over $16.5 billion1. The disease has classically been characterized by the degradation of articular cartilage and subchondral bone; however, changes to the synovium have recently garnered appreciation as synovitis has been linked to OA symptoms and progression2,3,4. In primary (idiopathic) OA, normal &#39;wear and tear&#39; associated with aging inhibits cartilage&#39;s ability to sustain its load-bearing and lubrication functions. The stresses generated by prolonged sliding contact of articular cartilage layers or sliding contact of cartilage against implant materials have been shown to facilitate delamination wear through subsurface fatigue failure5,6. As a dynamic mechanical environment exists within the joint7,8, the frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device has been designed to replicate the motion of the joint with tight control over compressive and frictional loading5,6,9,10,11,12,13.
The present protocol describes a friction testing device that delivers reciprocal, translating motion and compressive load to contacting surfaces of living tissue explants. The computer-controlled device permits user control of the duration of each test, applied load, range of motion of the translation stage, and translation speed. The device is modular, allowing for testing of various counterfaces, such as tissue-on-tissue (cartilage-on-cartilage and synovium-on-cartilage) and tissue-on-glass. In addition to the functional measurements obtained by the tester, tissue and lubricating bath components can be assessed before and after testing to evaluate the biological changes imparted by a given experimental regimen.
Studies of cartilage tribology have been performed for decades, and several techniques have been developed to measure friction coefficients between cartilage and glass and cartilage on cartilage14,15. The different approaches are motivated by the joint and/or the lubrication mechanism of interest. There is often a tradeoff between the control of experimental variables and the recapitulation of physiologic parameters. Pendulum-style devices utilize intact joints as the fulcrum of a simple pendulum where one joint surface translates freely over the second surface14,16,17,18. Instead of using intact joints, friction measurements may be obtained by sliding cartilage explants over desired surfaces14,19,20,21,22,23,24,25. Reported friction coefficients of articular cartilage have varied over a wide range (from 0.002 to 0.5) depending on the operating conditions14,26. Devices have been created to replicate rotary motion23,27,28. Gleghorn et al.26 developed a multi-well custom tribometer to observe cartilage lubrication profiles using Stribeck curve analysis, and a linear oscillatory sliding motion was applied between cartilage against a flat glass counterface.
This device aims to isolate frictional responses and explore the mechanobiology of living tissues under various loading conditions. The device employs a simplified test set-up simulating joint articulation through compressive sliding, which can approximate both rolling and sliding motion with the understanding that the resistance in pure rolling motion is negligible relative to the measured friction coefficient of articular cartilage29. Originally built to study the effects of interstitial fluid pressurization on the frictional response of articular cartilage9, the tester has since been used to explore topics such as frictional effects of removing the superficial zone of cartilage10, lubricating effects of synovial fluid11, cartilage wear hypotheses5,6,30, and synovium-on-tissue friction measurements13. The friction-testing bioreactor can conduct friction experiments under sterile conditions, providing a novel mechanism to explore how frictional forces affect the mechanobiological responses of living cartilage and synovium. This design can be used as a biomimetic bioreactor to study the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.
This study presents a configuration for synovium-on-cartilage friction testing over a range of contact stresses and in different lubricating baths. The articulating surface area of most joints is, to a great extent, synovial tissue31. While synovium-on-cartilage sliding does not occur at primary load-bearing surfaces, the frictional interactions between the two tissues may still have important implications for tissue level repair and cell mechanotransduction. It has previously been shown that fibroblast-like synoviocytes (FLS) residing on the intimal layer of the synovium are mechanosensitive, responding to fluid-induced shear stress32. It has also been demonstrated that stretch33,34 and fluid-induced shear stress35 modulate FLS lubricant production. As such, direct sliding contact between synovium and cartilage may provide another mechanical stimulus to resident cells in the synovium.
Only a few reports on synovium friction coefficients have been published31,36. Estell et al.13 sought to expand on the previous characterization by utilizing biologically relevant counterfaces. With the friction testing device&#39;s ability to test living tissues, it is possible to mimic physiologic tissue interactions during joint articulation to elucidate the role of contact shear stress on synoviocyte function and its contribution to the crosstalk between synovium and cartilage. The latter has been implicated in mediating synovial joint inflammation in arthritis and post-injury. Due to the physical proximity of cartilage to synovium and synovial fluid, which contain synoviocytes that exhibit multipotent capacity, including chondrogenesis, it is postulated that synoviocytes play a role in cartilage homeostasis and repair by engrafting to the articular surface. In this context, physical contact and reciprocal shearing of cartilage-synovium and synovium-synovium may increase the accessibility of synoviocytes to regions of cartilage damage37,38,39,40. Studies utilizing synovium-on-cartilage configurations will not only provide insights into joint gross tissue mechanics and tribology, but they may also lead to new strategies for maintaining joint health.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds Group Holdings</td>
			<td>Reynolds Wrap</td>
			<td>Sterile tissue harvest</td>
		</tr>
		<tr>
			<td>Aluminum-framed acrylic enclosure</td>
			<td>Custom made</td>
			<td></td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Autoclavable instant sealing sterilization pouches</td>
			<td>Fisherbrand</td>
			<td>01-812-54</td>
			<td>Sterilization of tools</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Buxton</td>
			<td></td>
			<td>Sterilization of tools</td>
		</tr>
		<tr>
			<td>Beaker (250 mL)</td>
			<td>Pyrex Vista</td>
			<td>70000</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Betadine (Povidone Iodine Prep Solution)</td>
			<td>Medline Industries, LP</td>
			<td>MDS093906</td>
			<td>Sterile tissue harvest</td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Labconco</td>
			<td>Purifier Logic+ Class II, Type A2 BSC</td>
			<td>Sterile tissue harvest</td>
		</tr>
		<tr>
			<td>Biospy punch</td>
			<td>Steritool Inc.</td>
			<td>50162</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Box cutter</td>
			<td>American Safety Razor Company</td>
			<td>94-120-71</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Circular acrylic-sillicone post (synovium)</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Culture media</td>
			<td>Custom made</td>
			<td></td>
			<td>DMEM (Cat No. 11-965-118; Gibco) supplemented with 50 &#956;g/mL L-proline (Cat. No. P5607; Sigma), 100 &#956;g/mL sodium pyruvate (Cat. No. S8636; Sigma), 1% ITS (Cat. No. 354350; Corning), and 1% antibiotic&#8211;antimycotic (Cat. No. 15-240-062, Gibco)</td>
		</tr>
		<tr>
			<td>Cyanoacrylate (Loctite 420 Clear)</td>
			<td>Henkel</td>
			<td>135455</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Dead weights</td>
			<td>OHAUS</td>
			<td></td>
			<td>Normal load</td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc.</td>
			<td>2701</td>
			<td>Dilute to 70 %</td>
		</tr>
		<tr>
			<td>Fixed base</td>
			<td>ThorLabs, Inc.</td>
			<td>SB1T</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Forceps (synovium harvest)</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Forceps (synovium mounting)</td>
			<td>Excelta</td>
			<td>3C-S-PI</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Horizontal linear encoder (for translating stage)</td>
			<td>RSF Electronics, Inc.</td>
			<td>MSA 670.63</td>
			<td>Friction tester component; system resolution of 1 &#181;m</td>
		</tr>
		<tr>
			<td>Hot glue gun and glue</td>
			<td>FPC Corporation</td>
			<td>Surebonder Pro 4000A</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments Corporation</td>
			<td>LabVIEW&#160; 2010</td>
			<td>Friction testing program</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>JR3 Inc.</td>
			<td>20E12A-M25B</td>
			<td>Friction tester component; 0.0019 lbs resolution in x&#38;y, 0.0038 lbs resolution in z</td>
		</tr>
		<tr>
			<td>Loading platen</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>O-ring</td>
			<td>Parker</td>
			<td>S1138AS568-009</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Petri dish (60 mm)</td>
			<td>Falcon</td>
			<td>351007</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>PivotLok Work Positioner (tibia holder)</td>
			<td>Industry Depot, Pivot Lok</td>
			<td>PL325</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Removable base</td>
			<td>ThorLabs, Inc.</td>
			<td>SB1B</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Ring stand</td>
			<td></td>
			<td></td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Havel&#39;s Inc.</td>
			<td>FSC22</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>FEATHER Safety Razor Co., Ltd.</td>
			<td>No. 4</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Screwdriver</td>
			<td>Wera</td>
			<td>3334</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Stage</td>
			<td>JMAR</td>
			<td></td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Stepper motor</td>
			<td>Oriental Motor Co., Ltd.</td>
			<td>PK266-03B</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Suction tool</td>
			<td>Virtual Industries, Inc.</td>
			<td>PEN-VAC Vacuum Pen</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Support rod</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-09</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Synovial fluid (bovine)</td>
			<td>Animal Technologies, Inc.</td>
			<td></td>
			<td>Friction testing bath</td>
		</tr>
		<tr>
			<td>Testing bath</td>
			<td>Custom made</td>
			<td></td>
			<td>Phosphate-Buffered Saline (PBS) with protease inhibitors: 0.04% isothiazolone-base biocide (Proclin 950 Cat. No. 46878-U; Sigma) and 0.1% protease inhibitor - 0.05 M ethylenediaminetetraacetic acid, EDTA (Cat. No. 0369; Sigma)</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>Sterile culture</td>
		</tr>
		<tr>
			<td>Vertical linear encoder (for loading stage)</td>
			<td>Renishaw</td>
			<td>T1031-30A</td>
			<td>Friction tester component; 20 nm resolution</td>
		</tr>
		<tr>
			<td>Voice coil actuator</td>
			<td>H2W Technologies</td>
			<td>NCC20-15-027-1RC</td>
			<td>Friction tester component</td>
		</tr>
	</tbody>
</table>

a friction testing-bioreactor device for study of synovial joint biomechanics, mechanobiology, and physical regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

A synovium-on-cartilage configuration was used to friction test juvenile bovine explants. The synovium was mounted on a 10 mm diameter acrylic loading platen such that the intimal layer would be in contact with the underlying cartilage. A tibial strip was used as the cartilage counterface (Figure 6A). Tibial strips were cut with a depth of approximately 1.4 mm and a size of 10 mm x 30 mm. The samples were tested for 1 h at 37 &#176;C in a phosphate-buffered saline (PBS) bath or a bovine syno...

Watch this Scientific Journal Video about A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation at JoVE.com

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

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Friction

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2.7K Views.  Columbia University. The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces. 

Video: A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

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 ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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 ...

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.

Neural-machine interfaces (NMI) have been developed to identify the user's locomotion mode. These NMIs are potentially useful for neural control of powered artificial legs, but have not been fully demonstrated. This paper presented (1) our designed engineering platform for easy implementation and development of neural control for powered lower limb prostheses and (2) an experimental setup and protocol in a laboratory environment to evaluate neurally-controlled artificial legs on patients with lower limb amputations safely and efficiently.

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...