This work was supported by the National Science Foundation under Grant Nos. (CBET 1060990 and EBMS 1700299). Also, this material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (2018250692). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

1. Chip mask preparation
NOTE: Steps 1.1 - 1.3 only need to be performed once upon initial receipt of the chip mask. They ensure the mask does not bow during use. The design of the microfluidic masks was previously described11,14. Masks were designed in-house and commercially fabricated using high resolution stereolithography (Figure 1A).
<ol>
	<li>Cover the top surface of the mask with plastic sheeting to protect this surface from adhesive. Secure the chip mask to an equally sized acrylic sheet using spray adhesive. Clamp the pieces together overnight to allow the adhesive to fully cure. After the adhesive is dry, remove the plastic sheeting from the top of the mask.</li>
	<li>Attach the bottom of the acrylic sheet to a 3D printed leveling box (Supplementary Figure 1, Supplementary Files 1-4) using double-sided tape. Press firmly to ensure a tight bond.</li>
	<li>Seal any small openings near the detachable wall of the leveling box with a waterproof sealant. Allow the sealant to cure for 24 h.</li>
	<li>Clean the surface of the mask with 70% ethanol (EtOH). Place the leveling box with the desired chip mask in the oven. Use a digital protractor to ensure the top of the mask is level. Adjust the leveling screws if necessary.</li>
</ol>
2. PDMS fabrication
NOTE: A shallow-well (1 mm) chip design is used for functional activity (formation and resorption) assays, and a deep-well (10 mm) chip design is used for mechanical loading studies. The bottom of the deep-well is formed by attaching a separate thin PDMS membrane (Figure 1B).
<ol>
	<li>Lid layer (Layer 1)
	<ol>
		<li>Combine 63 g of PDMS prepolymer and 6.3 g of curing agent (10:1 ratio) in a plastic cup. Mix thoroughly with a disposable cell spatula and degas in a vacuum desiccator for 30 min.</li>
		<li>Slowly pour mixture into the prepared leveling box. Let the PDMS sit for 30 min and then bake at 45 &#176;C for 18 h.</li>
		<li>Loosen the edges of the PDMS with a tapered laboratory spatula and remove the polymer sheet from leveling box.</li>
		<li>Cut individual lids to size (70 mm x 34 mm) using a scalpel and a 3D printed template.</li>
		<li>Punch access holes through each lid with a biopsy punch (1 mm diameter).</li>
		<li>Surface clean the lids with packaging tape. 
		NOTE: If the lids are not being used immediately, wrap each in packaging tape and store at room temperature (RT).</li>
	</ol>
	</li>
	<li>Well and microchannel layer (Layer 2)
	<ol>
		<li>Combine the PDMS prepolymer and curing agent (10:1 ratio) in a plastic cup. The shallow-well design requires 43 g of prepolymer and 4.3 g of curing agent, and the deep-well design requires 227 g of prepolymer and 22.7 g of curing agent. Mix the polymer vigorously and degas for 30 min. 
		NOTE: This assumes a mask dimension of 152.4 mm x 152.4 mm.</li>
		<li>Slowly pour mixture over appropriate pre-leveled mask. Let the PDMS sit for 30 min and then bake at 45 &#176;C for 18 h.</li>
		<li>Loosen the edges of the PDMS with a tapered laboratory spatula and carefully peel the polymer away from the mask. Use a scalpel and 3D printed template to cut out individual chips. 
		NOTE: For loading studies it is important that the chip dimensions (70 x 34 mm) are precise and that the well is located in the center of the chip.</li>
		<li>Surface clean the PDMS with packaging tape. 
		NOTE: If the chips are not being used immediately, wrap each chip in packaging tape and store at RT.</li>
	</ol>
	</li>
	<li>Thin PDMS membrane (Layer 3) 
	NOTE: This layer is only used for the deep-well design.
	<ol>
		<li>Add 12.7 g of PDMS prepolymer and 1.3 g of curing agent (10:1 ratio) to a plastic cup. Mix vigorously and degas for 30 min.</li>
		<li>Slowly pour polymer into prepared leveling box and thoroughly scrape plastic cup to remove as much PDMS as possible.</li>
		<li>Use cell spatula to spread PDMS over entire surface. 
		NOTE: If the polymer is not manually spread out, surface tension will be sufficient to prevent the polymer from forming a uniform sheet.</li>
		<li>Let the PDMS sit for 30 min and then bake at 45 &#176;C for 18 h.</li>
		<li>Loosen the edges of the PDMS with a tapered spatula and carefully remove the PDMS sheet from the leveling box. Cut individual membranes that match the dimensions of layer 2.</li>
		<li>Measure the membrane thickness at the center of the membrane using calipers. Discard any membranes that are outside of the desired thickness (0.5 mm &#177; 0.1 mm).</li>
		<li>Carefully clean membranes with packaging tape and place on a piece of paraffin film. 
		NOTE: If the membranes are not being used immediately, cover the top of the membrane with packaging tape and store at RT.</li>
	</ol>
	</li>
</ol>
3. Functional activity substrates
NOTE: Polystyrene discs and bone wafers must be attached to the bottom of wells that will be used for osteoblast and osteoclast cultures, respectively.
<ol>
	<li>Polystyrene discs (Figure 1C)
	<ol>
		<li>Place masking tape on the back side of a tissue culture treated polystyrene coverslip. Cut circular discs from the coverslip using a sharpened cork-borer (5.4 mm diameter). Submerge the discs in 70% EtOH and leave overnight.</li>
		<li>Gently scrub the top surface of the disc with a cotton tipped applicator soaked in 70% EtOH. Ensure that the outer edge of the disc is thoroughly cleaned.</li>
		<li>Using two pairs of forceps, hold the disc and remove the masking tape backing. Place the disc treated side down and clean the back side with a cotton tipped applicator.</li>
		<li>Dip the wooden end of a cotton tipped applicator into a degassed mixture of uncured PDMS and place a small amount of the polymer on the bottom of the desired well. 
		NOTE: Remaining uncured PDMS can be stored at -20 &#176;C.</li>
		<li>Place the polystyrene disc, treated side up, into the well and gently press down on the disc with a cotton swab. Ensure that no uncured PDMS comes in contact with the treated side of the disc. 
		NOTE: If PDMS does come in contact with the treated surface of the disc, remove the disc from the well and repeat steps 3.1.4 and 3.1.5 with a new disc.</li>
		<li>Let the chip sit on a level surface for 30 min and then bake at 65 &#176;C for 4 h.</li>
	</ol>
	</li>
	<li>Bone wafers
	<ol>
		<li>Use forceps to place a bone wafer (6 mm diameter, 0.4 mm thick) on the bottom of a 100 mm dish. Hold the wafer steady with the forceps and gently etch an &#39;X&#39; on the back of the wafer with a scalpel. 
		NOTE: During the imaging process, the &#39;X&#39; is used to distinguish between the back of the wafer and the surface on which cells were seeded.</li>
		<li>Use the wooden end of a cotton tipped applicator to add a small amount of uncured PDMS to the bottom of the desired well. Place the bone wafer, marked side down, into the well and use a cotton tipped applicator to press the wafer down.</li>
		<li>Let the chip sit on a level surface for 30 min and then bake at 65 &#176;C for 4 h.</li>
	</ol>
	</li>
</ol>
4. Chip assembly and sterilization
<ol>
	<li>Activate the surfaces of layer 1 and layer 2 with a plasma cleaner for 30 s using a medium radio frequency (RF) power setting (equivalent to approximately 10.2 W).</li>
	<li>Align the access holes in layer 1 with the microchannels in layer 2 and firmly press the two layers together.</li>
	<li>For the deep-well design, repeat step 4.1 with layer 2 and layer 3. During the plasma treatment, use double-sided tape to attach the paraffin film of layer 3 to a flat surface.</li>
	<li>Use a scalpel to trim off excess material from the PDMS membrane. Carefully peel away the sheet of paraffin film from the bottom of the chip</li>
	<li>Bake the chip at 65 &#176;C for 10 min to increase bond strength between the PDMS layers.</li>
	<li>Insert angled dispensing tips (18 Gauge, 0.5 in, 90&#176;) into the access holes in the lid. Secure the dispensing tips to the lid with a two part epoxy. Use a micropipette tip to apply the epoxy around each dispensing tip.</li>
	<li>After the epoxy has fully cured, surface clean the chip with 70% EtOH and place in a biosafety cabinet. Perform all subsequent steps within the biosafety cabinet.</li>
	<li>Connect a 5 mL syringe to the dispensing tips with sterile silicone tubing (1/32&#39;&#39; ID, ~10 cm in length) and fill the entire chip with 70% EtOH for at least 30 s. For the shallow-well design, administer all liquids with a syringe pump set to a flow rate of 4 mL/h. For the deep-well design, administer all liquids by slowly dispensing the syringe by hand.</li>
	<li>Remove the EtOH from the chip and sterilize the chip with UV light overnight.</li>
	<li>Wash the chip 3 times with dH2O. Fill the chip with dH2O, remove tubing from the dispensing tips and incubate for at least 48 h at 37 &#176;C.</li>
</ol>
5. Mechanical loading device assembly
NOTE: The design and fabrication processes for the 3D printed mechanical loading device (Figure 2A-C) were previously described and all design files for printed components have been previously provided13.
<ol>
	<li>Autoclave all components of the loading device for 30 min at 121 &#176;C. 
	NOTE: To avoid warping of printed components, wrap each piece individually in foil and place on a hard flat surface during the autoclaving process. Metal hardware can be wrapped together. Complete all subsequent steps within a biosafety cabinet.</li>
	<li>Place a compression spring around the shaft of the platen and insert the platen into the central hole on the bottom of the base (Figure 2D).</li>
	<li>Attach the dial block to the bottom of the base using four self-tapping screws.</li>
	<li>Place a second compression spring around the central screw. Insert the screw into the hexagonal-shaped hole in the bottom of the dial and screw the assembly into the threaded hole in the center of the dial block.</li>
	<li>Screw four male-female standoffs into the bottom of the base.</li>
	<li>Remove slack from the device by turning the dial counterclockwise until the top of the platen is below the top of the base. Then slowly turn the dial clockwise until the top of the platen is level with the top of the base.</li>
</ol>
6. Experimentation
NOTE: Protocols for functional activity experiments were previously provided11,12.
<ol>
	<li>Loading studies (Figure 3)
	<ol>
		<li>Following step 4.9, use a 5 mL syringe to remove dH2O from the deep-well chip. Coat the bottom of the well with 200 &#181;L of 0.15 mg/mL type I collagen (CTI) in 0.02 M acetic acid for 1 h.</li>
		<li>Rinse the chip three times with Dulbecco&#39;s phosphate-buffered saline with calcium and magnesium (DPBS++).</li>
		<li>Place chip into the chip holder and seed with MLO-Y4 osteocytes at a density of 2 x 104 cells/mL in minimum essential alpha medium (MEM&#945;) supplemented with 5% calf serum, 5% fetal bovine serum, and 1% penicillin/streptomycin.</li>
		<li>Remove the tubing from the dispensing tips and place the chip into a deep-well culture dish (150 mm x 25 mm). Incubate cells at 37 &#176;C and 5% CO2 for 72 h.</li>
		<li>Attach sterile tubing to dispensing tips and use a 5 mL syringe to remove spent culture medium from the chip. Slowly dispense in fresh culture medium to refill the chip.</li>
		<li>Place the chip holder into the rectangular inset on the top of the loading device base. Feed the tubing through the slots located on the loading device lid and secure the lid to the base with four pan head screws and hex nuts. 
		NOTE: To ensure that the lid remains level, first secure two screws that are located diagonally from one another before securing the remaining two screws.</li>
		<li>Apply load to the cells by turning the dial clockwise until the desired platen displacement is reached. 
		NOTE: The device is designed so that one rotation of the dial equates to a platen displacement of 1 mm. The strain field generated on the top of the PDMS membrane was previously modeled as a function of platen displacement using finite element analysis (FEA)13.</li>
		<li>Place the loading device into an empty P1000 micropipette tip box. Incubate the cells with the applied load for 15 min. 
		NOTE: The loading time period used here serves as an example. Alternative loading times can be used.</li>
		<li>Following incubation, remove the load from the cells by turning the dial counterclockwise until the platen returns to the original starting position. Remove the lid from the device and place the chip holder into the deep-well culture plate. Incubate the cells for a 90-min post load recovery period. 
		NOTE: Again, the recovery time period used here serves as an example. Alternative recovery times can be used. For long term studies, feed cells every 72 h.</li>
		<li>Use a 5 mL syringe to remove the conditioned medium from the chip. 
		NOTE: This medium can be saved and stored at -80 &#176;C.</li>
		<li>Remove the chip from the chip holder and use a tapered spatula to break the bond between the PDMS lid and well layer. 
		NOTE: Cellular assays can now be performed following any protocol established for a cell culture plate.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article describes the foundations for fabricating a bone remodeling LOC platform for culturing osteocytes, osteoclasts, and osteoblasts. By altering the depth and size of the well within the chip, multiple configurations were developed for stimulating osteocytes with mechanical load and quantifying functional outcomes of bone remodeling (Figure 1B).
During chip assembly, optimizing the plasma oxidation protocol was critical for eliminating le...

Bone is a highly dynamic tissue that requires intricate coordination among the three major cell types: osteocytes, osteoblasts, and osteoclasts. Multicellular interactions among these cells are responsible for the bone loss that occurs during paralysis and long-term immobility and for the bone formation that occurs in response to growth and exercise. Osteocytes, the most abundant bone cell type, are highly sensitive to mechanical stimuli applied to the bone. Mechanical stimulation alters osteocyte metabolic activity and leads to an increase in key signaling molecules1,2. Through this process, known as mechanotransduction, osteocytes can directly coordinate the activities of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells). Maintaining bone homeostasis requires a tight regulation between bone formation and bone resorption rates; however, disruptions in this process can result in disease states such as osteoporosis or osteopetrosis.
The complexity of interactions between these three cell types lends itself well to investigation utilizing microfluidic and lab-on-a-chip (LOC) technologies. To that end, our lab has recently established proof of concept of a LOC platform for analyzing bone resorption and formation (functional outcomes) in the bone remodeling process. The platform can be used for the study of cellular interactions, altered loading environments, and investigational drug screening. In recent years, various microfluidic devices have been developed for investigating the molecular signaling pathways that regulate bone remodeling; however, many of these systems quantify remodeling through indirect markers that are indicative of functional activity3,4,5,6,7. An advantage of our system is that it can be used for direct quantification of functional outcomes. Bone remodeling is a long-term process. As such, direct quantification of bone resorption and formation requires a culturing system that can be maintained for a minimum of several weeks to months8,9,10,11. Thus, when developing the LOC platform, we established long-term culturing protocols necessary for formation and resorption and have maintained cells within the system for up to seven weeks11. Additionally, we incorporated appropriate culturing substrates for both cell types into the platform; osteoclasts were cultured directly on bone, and osteoblasts, which are known to be plastic adherent, were cultured on polystyrene discs. Further, we addressed issues concerning sterility, long-term cytotoxicity and chip disassembly for remodeling analysis11,12.
The LOC platform can also be used to induce osteocyte mechanotransduction through matrix deformation. A 3D printed mechanical loading device was developed to pair with the LOC and apply a static out of plane distention to stretch the cells13. To accommodate this mechanical load, the depth of the well within the LOC was increased. This small scale, simple mechanical loading device can be easily produced by labs with limited engineering experience, and we have previously shared drawings of the 3D printed components13. In the current work, we demonstrate some of the novel techniques necessary for the successful use of the LOC. Specifically, we demonstrate chip fabrication, cell seeding on functional substrates, mechanical loading and chip disassembly for remodeling quantification. We believe that the explanation of these techniques benefit from a visual format.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylic sheet</td>
			<td>Optix</td>
			<td>--</td>
			<td>3.175 mm thick</td>
		</tr>
		<tr>
			<td>Angled dispensing tips</td>
			<td>Jensen Global</td>
			<td>JG18-0.5X-90</td>
			<td>Remove plastic connector prior to use</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Robbins Instruments</td>
			<td>RBP-10</td>
			<td>1 mm diameter</td>
		</tr>
		<tr>
			<td>Bone wafers</td>
			<td>Boneslices.com</td>
			<td>0.4 mm thick</td>
			<td>Bovine cortical bone</td>
		</tr>
		<tr>
			<td>Bovine calf serum</td>
			<td>Hyclone</td>
			<td>SH30072</td>
			<td></td>
		</tr>
		<tr>
			<td>Calipers</td>
			<td>Global Industrial</td>
			<td>T9F534164</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell spatula</td>
			<td>TPP</td>
			<td>99010</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip mask</td>
			<td>ProtoLabs</td>
			<td>Custom-designed</td>
			<td>Print material: Accura SL 5530</td>
		</tr>
		<tr>
			<td>Cork borer</td>
			<td>Fisher Scientific</td>
			<td>07-865-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WCL</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish (100 mm)</td>
			<td>Corning</td>
			<td>430591</td>
			<td>Sterile, Non-tissue culture treated</td>
		</tr>
		<tr>
			<td>Culture dish (150 mm)</td>
			<td>Corning</td>
			<td>430597</td>
			<td>Sterile, Non-tissue culture treated</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>3M Company</td>
			<td>Scotch 237</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Leveling box</td>
			<td>Custom-made</td>
			<td>--</td>
			<td>3D printed</td>
		</tr>
		<tr>
			<td>Masking tape</td>
			<td>3M Company</td>
			<td>Scoth 2600</td>
			<td></td>
		</tr>
		<tr>
			<td>MC3T3-E1 preosteoblasts</td>
			<td>ATCC</td>
			<td>CRL-2593</td>
			<td>Subclone 4</td>
		</tr>
		<tr>
			<td>Mechanical loading device</td>
			<td>Custom-made</td>
			<td>--</td>
			<td>3D printed</td>
		</tr>
		<tr>
			<td>Minimum essential alpha medium</td>
			<td>Gibco</td>
			<td>12571-063</td>
			<td></td>
		</tr>
		<tr>
			<td>MLO-Y4 osteocytes</td>
			<td>--</td>
			<td>--</td>
			<td>Gift from Dr. Lynda Bonewald</td>
		</tr>
		<tr>
			<td>Packaging tape</td>
			<td>Duck Brand</td>
			<td>--</td>
			<td>Standard packaging tape</td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Bemis Parafilm</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>p4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>Expanded plasma cleaner</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane kit</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene coverslips</td>
			<td>Nunc Thermanox</td>
			<td>174942</td>
			<td>Sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Quincy Lab</td>
			<td>12-180</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 preosteoclasts</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>BD Medical</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>Saint-Gobain Tygon</td>
			<td>ABW00001</td>
			<td>ID: 1/32&#34; (0.79 mm), OD: 3/32&#34; (2.38 mm)</td>
		</tr>
		<tr>
			<td>SolidWorks software</td>
			<td>Dassault Syst&#232;mes</td>
			<td>--</td>
			<td>Used to generate 3D printed models and perform FEA</td>
		</tr>
		<tr>
			<td>Spray adhesive</td>
			<td>Loctite</td>
			<td>2323879</td>
			<td>Multi-purpose adhesive</td>
		</tr>
		<tr>
			<td>Syringe (5 ml)</td>
			<td>BD Medical</td>
			<td>309646</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-2213</td>
			<td>Pump 11 Pico Plus</td>
		</tr>
		<tr>
			<td>Tapered laboratory spatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-part expoxy</td>
			<td>Loctite</td>
			<td>1395391</td>
			<td>5 minute quick set</td>
		</tr>
		<tr>
			<td>Type I collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Rat tail collagen</td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Bel-Art</td>
			<td>F42010-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterproof sealant</td>
			<td>Gorilla</td>
			<td>8090001</td>
			<td>100% silicone sealant</td>
		</tr>
	</tbody>
</table>

a lab-on-a-chip platform for stimulating osteocyte mechanotransduction and analyzing functional outcomes of bone remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

The shallow-well configuration can be used for analyzing functional activity of osteoblasts and osteoclasts. Bone formation via osteoblasts and resorption via osteoclasts requires culturing times on the order of several weeks to months. Bone formation from MC3T3-E1 pre-osteoblasts was quantified using alizarin red and von Kossa stains11,15. At day 49, the average surface area stained with alizarin red was 10.7% &#177; 2.2% (mean &...

Watch this Scientific Journal Video about A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling at JoVE.com

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

a-lab-on-chip-platform-for-stimulating-osteocyte-mechanotransduction

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.6K Views.  The University of Akron. Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Video: A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Developmental Biology

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

The current article describes performing an intravital imaging approach to observe mechanically induced calcium signaling of embedded osteocytes in vivo in real-time in response to tissue-level mechanical loading of the mouse third metatarsal.

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, ...

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Medicine

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...