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

Summary

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).

Abstract

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.

Introduction

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 molecules^1,2. 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 activity^3,4,5,6,7. 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 months^8,9,10,11. 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 analysis^11,12.

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.

Protocol

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 described^11,14. Masks were designed in-house and commercially fabricated using high resolution stereolithography (Figure 1A).

1. 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.
2. 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.
3. Seal any small openings near the detachable wall of the leveling box with a waterproof sealant. Allow the sealant to cure for 24 h.
4. 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.

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).

1. Lid layer (Layer 1)
   1. 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.
   2. Slowly pour mixture into the prepared leveling box. Let the PDMS sit for 30 min and then bake at 45 °C for 18 h.
   3. Loosen the edges of the PDMS with a tapered laboratory spatula and remove the polymer sheet from leveling box.
   4. Cut individual lids to size (70 mm x 34 mm) using a scalpel and a 3D printed template.
   5. Punch access holes through each lid with a biopsy punch (1 mm diameter).
   6. 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).
2. Well and microchannel layer (Layer 2)
   1. 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.
   2. Slowly pour mixture over appropriate pre-leveled mask. Let the PDMS sit for 30 min and then bake at 45 °C for 18 h.
   3. 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.
   4. 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.
3. Thin PDMS membrane (Layer 3)
   NOTE: This layer is only used for the deep-well design.
   1. 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.
   2. Slowly pour polymer into prepared leveling box and thoroughly scrape plastic cup to remove as much PDMS as possible.
   3. 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.
   4. Let the PDMS sit for 30 min and then bake at 45 °C for 18 h.
   5. 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.
   6. 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 ± 0.1 mm).
   7. 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.

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.

1. Polystyrene discs (Figure 1C)
   1. 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.
   2. 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.
   3. 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.
   4. 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 °C.
   5. 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.
   6. Let the chip sit on a level surface for 30 min and then bake at 65 °C for 4 h.
2. Bone wafers
   1. 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 'X' on the back of the wafer with a scalpel.
      NOTE: During the imaging process, the 'X' is used to distinguish between the back of the wafer and the surface on which cells were seeded.
   2. 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.
   3. Let the chip sit on a level surface for 30 min and then bake at 65 °C for 4 h.

4. Chip assembly and sterilization

1. 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).
2. Align the access holes in layer 1 with the microchannels in layer 2 and firmly press the two layers together.
3. 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.
4. 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
5. Bake the chip at 65 °C for 10 min to increase bond strength between the PDMS layers.
6. Insert angled dispensing tips (18 Gauge, 0.5 in, 90°) 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.
7. 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.
8. Connect a 5 mL syringe to the dispensing tips with sterile silicone tubing (1/32'' 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.
9. Remove the EtOH from the chip and sterilize the chip with UV light overnight.
10. 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 °C.

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.

1. Autoclave all components of the loading device for 30 min at 121 °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.
2. 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).
3. Attach the dial block to the bottom of the base using four self-tapping screws.
4. 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.
5. Screw four male-female standoffs into the bottom of the base.
6. 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.

6. Experimentation

NOTE: Protocols for functional activity experiments were previously provided^11,12.

1. Loading studies (Figure 3)
   1. 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 µL of 0.15 mg/mL type I collagen (CTI) in 0.02 M acetic acid for 1 h.
   2. Rinse the chip three times with Dulbecco's phosphate-buffered saline with calcium and magnesium (DPBS++).
   3. 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 (MEMα) supplemented with 5% calf serum, 5% fetal bovine serum, and 1% penicillin/streptomycin.
   4. 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 °C and 5% CO₂ for 72 h.
   5. 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.
   6. 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.
   7. 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.
   8. 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.
   9. 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.
   10. Use a 5 mL syringe to remove the conditioned medium from the chip.
       NOTE: This medium can be saved and stored at -80 °C.
   11. 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.

Results

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 stains^11,15. At day 49, the average surface area stained with alizarin red was 10.7% ± 2.2% (mean &...

Discussion

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...

Materials

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