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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

Introduction

Joint diseases like osteoarthritis (OA) are highly prevalent and debilitating and represent a leading cause of disability worldwide1. It is estimated that in the US alone, OA affects 27 million patients and occurs in 12.1% of adults aged 60 and above2. Unfortunately, most drugs currently used to manage joint diseases are palliative, and no effective disease-modifying OA drugs (DMOADs) are available3. This unmet medical need primarily stems from the absence of an effective model for studying the disease mechanisms and developing potential DMOADs. The conventional two-dimensional (2D) cell culture d....

Protocol

The following protocol follows the ethical guidelines of the University of Pittsburgh and the human research ethics committee of the University of Pittsburgh. Information on the materials used in this study is listed in the Table of Materials.

1. Manufacturing 3D-printed bioreactors

  1. Use a computer software to design osteochondral (Figure 2A) and miniJoint bioreactors (Figure 2B) that inclu.......

Representative Results

All the tissues of the miniJoint were collected to analyze their phenotypes following 28 days of culture in the miniJoint (Figure 4A). This has been detailed in our previous publication7.

Through the use of RT-qPCR, immunostaining, and histological staining, it was confirmed that the tissue-specific phenotypes were well maintained for the individual microtissues (Figure 4). For example, the osseous component of.......

Discussion

In this article, we present a protocol for creating a knee joint-on-a-chip system, in which bone, cartilage, adipose tissue, and synovium-like tissues are formed from MSCs and co-cultured within a customized bioreactor. This multi-component, human cell-derived system with plug-and-play features represents a new tool for studying the pathogenesis of joint diseases and developing drugs.

Given that different tissues favor specific culture media, it is critical to provide the respective medium fo.......

Acknowledgements

This research was primarily supported by funding from the National Institutes of Health (UG3/UH3TR002136, UG3/UH3TR003090). In addition, we thank Dr. Paul Manner (University of Washington) for providing the human tissue samples and Dr. Jian Tan for their help in isolating the MSCs and creating the cell pool.

....

Materials

NameCompanyCatalog NumberComments
3-isobutyl-1-methylxanthineSigma -AldrichI17018-1G
6 well non-tissue culture plateCorning Falcon® Plates351146
24 well non-tissue culture plateCorning Falcon® Plates351147
30 mL syringesBD Syringe Luer Lock Cascade Health302832
Alcian blue stainEK Industries11981% w/v, pH 1.0
Advanced DMEMGibco12491-015
αMEMGibco12571-063
Antibiotic-antimycoticGibco15240-062
Biopsy punchIntegra Miltex12-460-407
BODIPY® fluorophoreMolecular Probes
Bone morphogenic protein 7 (BMP7)Peprotech
Curved forcepsFisher Brand16100110
DMEMGibco11995-065Dulbecco’s Modified Eagle Medium
DexmethasomeSigma -Aldrich02-05-2002
E-Shell 450 photopolymer inEnvisionTecRES-01-4022
Fetal Bovine SerumGemini-Bio Products900-208
GlutaMAXGibco3505-061
gelatin from bovine skinHyclone1003372809
Hank’s Balanced Salt SolutionSigma -AldrichSH30588.02
indomethacinSigma -AldrichI7378-56
Insulin-Transferrin-Selenium-Ethanolamine (ITS)Gibco51500-056
interleukin 1βPeprotech200-01B
Leur-loc connectorsCole-Parmer Instruments45508-50
L-prolineSigma -Aldrich115388-93-7
β-glycerophosphateSigma -Aldrich1003129352
Medium bagsKiYATECFC045
Methacrylic AnhydrideSigma -Aldrich102378580
Phosphate buffered SalineCorning21-040-CM
Pointed forcepsFisher Brand12000122
Silicon moldMcMaster-CarrRC00114P
Silicon o-ringsMcMaster-CarrZMCCs1X51mm x 5mm
SolidWorksDassault Systèmes SE, Vélizy-Villacoublay, France
Surgical BladesIntegra Miltex4-122
Syringe pumpLagato210P, KD ScientificZ56963110 syringe racks
T-182 tissue culture flasksFisher BrandFB012939
Tissue Culture Dish 150 mmFisher BrandFB012925
Transforming Growth Factor Beta (TGF-β3)Peprotech100-36E
TrypsinGibco25200-056
UV FlashlightKBSKB70109395 nm
Vida Desktop 3D PrinterEnvisionTec
Vitamin D3Sigma -Aldrich32222-06-31,25-dihydroxyvitamin D3

References

  1. Safiri, S., et al. Global, regional and national burden of osteoarthritis 1990-2017: A systematic analysis of the Global Burden of Disease Study 2017. Annals of the Rheumatic Diseases. 79 (6), 819-828 (2020).
  2. Lawrence, R. C., et al.

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Knee Joint on a chipMicrophysiological SystemOsteoarthritisDisease Modifying Osteoarthritis DrugsGelMAMethacrylated GelatinMesenchymal Stem Cells3D Printed BioreactorPreclinical ModelDrug Testing

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