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

Podsumowanie

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).

Streszczenie

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.

Wprowadzenie

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 mechanot....

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Protokół

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 ....

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Wyniki

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 &.......

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Dyskusje

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.......

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Materiały

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