Fabrication of a Vertically Stacked Dentin–Pulp Complex Organ-on-a-Chip Device Using a Human Dentin Disc

Summary

To describe the fabrication and assembly of a vertically stacked dentin–pulp complex organ-on-a-chip device incorporating a human dentin disc and photolithography-fabricated microchannels, followed by dental pulp stem cell seeding and culture.

Abstract

Conventional preclinical models used in dental research, including animal models and static cell cultures, present important limitations in reproducing the physiological conditions of the dentin–pulp interface and predicting human biological responses to dental biomaterials. Organ-on-a-chip (OoC) technology provides an alternative approach by enabling the recreation of tissue-specific microenvironments within controlled microfluidic systems. This manuscript describes a step-by-step protocol for the fabrication and assembly of a dentin–pulp complex OoC device incorporating a human dentin disc within a three-layer polydimethylsiloxane (PDMS) microfluidic platform. The protocol includes dentin disc preparation, fabrication of microstructured molds using photolithography, PDMS casting and curing, plasma-assisted layer bonding, device assembly, leak testing, and dental pulp stem cell seeding under static culture conditions. Critical fabrication parameters influencing device performance are discussed, including control of PDMS layer thickness, plasma surface treatment, and integration of the dentin disc within the device to minimize leakage. Cell adhesion and viability within the device were evaluated using scanning electron microscopy and confocal live/dead staining. Representative results demonstrate successful integration of the dentin disc within the microfluidic assembly, maintenance of leak-free conditions, and adhesion of viable dental pulp stem cells to the dentin surface. This protocol provides a reproducible approach for the fabrication of a dentin–pulp complex OoC platform that may support future studies investigating cellular responses to dental biomaterials and microenvironmental stimuli.

Introduction

Animal experimentation has long been instrumental in advancing the understanding of pathological and physiological processes and has played a central role in the early stages of drug development¹. Over the course of the 20th century, human life expectancy increased substantially, and while it is not possible to precisely quantify the extent to which this improvement is attributable solely to animal research, it is widely acknowledged that many contemporary medical treatments have been developed or validated using animal models². Notable examples include the development of vaccines against poliomyelitis, measles, and hepa....

Protocol

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Universidad de La Frontera for studies involving human samples.

1. Acquisition of dentin disk

NOTE: Prepare the device design before beginning the protocol (Figure 1A–1C).

1. Collection and storage of teeth
   1. Collect healthy human molars or premolars after obtaining informed patient consent.
   2. Maintain all specimens in 0.5% chloramine solution at 4°C to prevent dentin dehydration and protein denaturation.
   <....

Results

Successful fabrication of the tooth-on-a-chip device followed the workflow illustrated in Figure 1A–1I, including dentin disc preparation (Figure 1D), fabrication of the photolithography-derived mold (Figure 1E), removal of cured PDMS layers (Figure 1F), plasma activation prior to bonding (Figure 1G), assembly of the final device (Figur.......

Discussion

This study describes a step-by-step protocol for the fabrication of a vertically stacked tooth-on-a-chip device incorporating dentin and dental pulp stem cells and demonstrates representative cell adhesion and viability within the device under static culture conditions. The protocol was developed to provide a reproducible workflow for the fabrication and assembly of a dentin–pulp complex OoC platform suitable for in vitro studies of the dentin interface. The establishment of such a workflow is particularly signific.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biological and chemical materials
Acetone	ExpertQ	SOLV 8	Silicon wafer cleaning
Alpha MEM culture medium	Cytiva	SH30265.02	Dental pulp stem cell culture medium
Chloramine (0.5%)	Merck	7080-50-4	Storage solution for dentin discs
Dental pulp stem cells	Not Applicable	Not Applicable	Primary cell culture. See Steps 3.2.1 and 3.2.2
EDTA (10%)	Winkler	N2222158E	Smear layer removal
Epoxy-based negative photoresist	Gersteltec	GM1060	Photolithography mold fabrication
Glutaraldehyde (2.5%) in 0.1 M Sorensen’s phosphate buffer	Sigma-Aldrich	210908-06	SEM fixation
Healthy human teeth (molars or premolars)	Not Applicable	Not Applicable	Human sample
Isopropanol	ExpertQ	SOLV 16	Wafer cleaning, development stopping, and PDMS cleaning
LIVE/DEAD Viability Kit	Sigma-Aldrich	451	Fluorescence viability staining.
PBS (phosphate-buffered saline)	Corning	46-013-CM	Washing and leakage testing
PDMS base and curing agent (polydimethylsiloxane)	Dow	Sylgard 184	Device fabrication
PGMEA	Sigma Aldrich	484431	Photoresist development
Trypan blue	Sigma Aldrich	T8154-100ml	Cell viability assessment
Trypsin	Corning	25-053-CI	Cell detachment
Ultrapure water	Cytiva	SH30529.01	Washing solution
Consumables and labware
19G syringe needles	Vmatic	VCB19050	Connection of device with micropump or syringe
Biopsy punch (diameter: 1 mm)	Harris Unicore	WHAWB100073	Inlet and outlet port fabrication
Cutting mat	Nicecool	Not Applicable	Surface protection during punching
Dental finishing discs (e.g., Sof-Lex)	3M	Sof-Lex	Dentin polishing
Female Luer-to-barbed adapters	Runze Fluidics	RH-M016	Tubing connection
Glass or plastic mixing stick	Duran	Not Applicable	PDMS mixing
Hypodermic syringes	Cranberry	pc-12246	Cell seeding and leakage testing
Lint-free sterile gauze	Linderson	Not Applicable	Removal of punching debris
Male Luer adapters	Runze Fluidics	RH-G016	Tubing connection
Mixing vessel	Duran	Not Applicable	PDMS preparation
Petri dish (140 mm × 20 mm)	Deltalab	2000219	Cell culture/incubation
Polytetrafluoroethylene base	CCTVal	Not Applicable	Intermediate dentin layer fabrication
Silicon wafer	Jiaozuo Commercial FineWin	Not Applicable	Mold fabrication
Silicone tube	Runze Fluidics	96421	Fluidic connection
T75 flask	Corning	353136	Dental pulp stem cell expansion
Well plate	SPL Life Sciences	30006	Confocal staining and imaging preparation
Equipment and instruments
Air dryer	METALPLAN	TITAN-040	Compressed air drying
Compressed air source	SCHULZ	CSD-18.1	Mold and surface cleaning
Confocal microscope	Olympus	Not Applicable	Model: FV1000
Direct laser lithography system	Heidelberg	uPG 101	Photoresist exposure
Environmental scanning electron microscope	Hitachi	Not Applicable	Model: Quanta 400
High-speed handpiece	Nakanishi	Not Applicable	Model: Pana-max2
Hot plate	LabTech	EH20A Plus	Wafer bake and photoresist bake
Laminar flow cabinet	BioBase	BBS8V1708313D	Device sterilization
Microfluidic pump	New Era	Not Applicable	Model: 1002X-ES. Leak testing at 10 µL/min
Microstructured mold (fabricated via photolithography)	CCTVal	Not Applicable	See Step 2.2 for mold fabrication specifications
Oven or incubator	Memmert	UN110	PDMS curing at 50°C and bond consolidation at 75°C
Vacuum Plasma Cleaning Chamber	Fari Plasma	GD-5	Wafer plasma activation
Handheld Corona Plasma	Aurora Pro Scientific	APS-CD-20AC	PDMS plasma activation
Ultrasonicator	Elma Sonic	S 10 (H)	Wafer cleaning
Vacuum chamber or desiccator (250 mm)	Winglass	Not Applicable	PDMS degassing
Vacuum pump	ROCKER	300	PDMS degassing