The document discusses the development of a joint-on-chip model to study joint tissues. It proposes a stepwise approach to first engineer individual joint tissues like cartilage, bone and synovial membrane as modular organ-on-chip models. These individual models would then be combined and validated to reconstitute the human joint and study cell-cell interactions under mechanical stimulation. Key challenges include mimicking physiologically relevant mechanical forces and fluid flow, as well as including an immune component like macrophages in the synovial membrane model.

1. JOINT-ON-CHIP
PROF. DR. MARCEL KARPERIEN
DEPT. DEVELOPMENTAL BIOENGINEERING
UNIVERSITY OF TWENTE
MARCEL.KARPERIEN@UTWENTE.NL

2. dr. Marcel Karperien
Prof. in Developmental BioEngineering
TechMed Institute, University of Twente
Enschede
The Netherlands
marcel.karperien@utwente.nl
Disclosure Information
I have financial relationship(s) with:
CSO and Stockholder Hy2Care B.V.
CSO and Stockholder Orthros TR B.V.
Member of scientific advisory board and Stockholder LipoCoat B.V.
AND
My presentation does not include discussion of off-label or investigational use.

3. OARSI 2019Joint-on-Chip 3
WHAT IS AN ORGAN-ON-CHIP?
A microphysiological system with automated fluid control mimicking key
elements of human (patho)fysiology

4. Human culture models on microfluidics chips
for perfusion and 3D
OARSI 2019Joint-on-Chip 4

5.  To address scientific questions which cannot be answered with current
technologies
 To reduce animal experimentation
 To accelerate drug development
 Personalized / precission medicine
WHY DO WE NEED A JOINT-ON-CHIP?
OARSI 2019Joint-on-Chip 5

6. WHAT ARE THE MINIMAL TISSUE REQUIREMENTS
FOR A JOINT-ON-CHIP?
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7. ARTICULAR CARTILAGE
LIGAMENT
BONE
SYNOVIAL MEMBRANE Synovial fluid
Synovial fluid
Synovial fluid
Osteochondral
unit
meniscus
Tendon /
Muscle
Intra-articular
Space
(Synovial fluid)
WHAT ARE THE MINIMAL TISSUE REQUIREMENTS
FOR A JOINT ON CHIP?
OARSI 2019Joint-on-Chip 7
From: Piluso S. et al. Mimicking the Articular Joint with
In Vitro Models. Trends in Biotechnology, 2019.

8. THE UNRESOLVED BIOLOGICAL CHALLENGES
(OF ANY ORGAN-ON-CHIP)
• Appropriate organ scaling
• Creation of a universal media mimicking blood / synovial fluid
• iPSC cell sourcing / differentiation protocols
• Vascularization of tissues in particular of synovial membrane / bone
• Inclusion of immune components particular in synovial membrane
• Consideration of circadian and other cycles on cells
• Integration of nerves for assessing pain
• Appropriate mechanical actuation
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9. THE UNRESOLVED ENGINEERING CHALLENGES
(OF ANY ORGAN-ON-CHIP)
• Manufacturability, alternatives for PDMS
• Drug adsorption and binding to PDMS / biomimetic coatings
• Membrane fabrication / integration in chip
• Connection of platforms to maintain sterility and avoid bubbles / plug-and-
play breadboard
• Flow rate differences between platforms
• Physiologically relevant actuation (compression and shear strain)
• Inclusion of biosensors / non-invasive (molecular) imaging
• Creating ideal oxygenation and nutrient levels for different organs
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10. ARTICULAR CARTILAGE
LIGAMENTBONE
SYNOVIAL MEMBRANE Synovial fluid
Synovial fluid
THE START: MAKING CHOICES
Intra-articular
Space
(Synovial fluid)
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11. THE STRATEGY
1. Engineer individual joint-related functional tissues, including cartilage,
bone and synovial membrane, to be used as modular organ-on-chip
models.
2. Replicate and validate biochemical and biomechanical interactions of the
individual joint tissues on-chip, in healthy/disease settings.
3. Combine individual modules to reconstitute the human joint.
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12. SINGLE PLATFORM
- Study cells behavior during mechanical
stimulation/inflammation & combination
of the two
- Study cell response to drugs during
mechanical stimulation
- Study behavior of diseased and healthy
cells during mechanical stimulation
MULTIPLE PLATFORM
- Study cell-cell interaction between the
various tissues
- Study macrophages behavior during
mechanical stimulation
OBJECTIVES
LIGAMENT
ARTICULAR CARTILAGE
BONE
SYNOVIAL MEMBRANE
JOINT-ON-CHIP ACQUIRES STEPWISE APPROACH
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13. MIMICKING THE ARTICULATION OF CARTILAGE
femur
tibia
patella
fibula
Synovial fluid
cartilag
e
Compression &
shear stress
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14. MIMICKING ARTICULATION OF CARTILAGE ON CHIP
Blood vessels
Cartilage
Tissue
Synovial fluid
Mechanical
actuator
Bone
tibia
femur
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15. DESIGN DRAWINGS
TOP VIEW
INLETS
INLETS
INLETS
blood
Pillars / bone
Cartilage
Synovial fluid
actuation
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16. OARSI 2019Joint-on-Chip 16
SOFT LITHOGRAPHY FOR GENERATING CHIP DESIGNS

17. Chip in
PDMS
Integration of membrane Bonding of
second chip
Chip assembly
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18. 500 µm
INLETS
positive
pressure
Agarose 2% in PBS + (15 µm)
microbeads (61 µg/ml) in the
synovial fluid and cell-hydrogel
section
MECHANICAL STIMULI – COMPRESSION
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19. 500 µm
INLETS
Positive
pressure
Agarose 2% in PBS + (15 µm)
microbeads (61 µg/ml) in the
synovial fluid and cell-hydrogel
section
MECHANICAL STIMULI – COMPRESSION
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20. Static
Vacuum (-350 mbar)
Vacuumin1
chamber
Vacuumin2
chambers
500 µm
Mechanical actuation of the membrane
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21. Vacuum in a single chamber will generate a gradient in displacement (higher close
to the PDMS membrane)
By applying vacuum on multiple chambers it is possible to tune the mechanical
stimulation by combining the various displacements
Modelling of stress and strain
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22. 500 µm
Each inlet  Positive or
negative pressure
applied
INLETS
Agarose 2% in PBS + beads
(put concentration) in the
synovial fluid and cell-hydrogel
section
MECHANICAL STIMULI – WAVE FORM
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23. MECHANICALLY ACTUATED CARTILAGE-ON-CHIP
From: Petrtyl M. et al. Biomechanical Properties of
Synovial Fluid in/Between Peripheral Zones of Articular
Cartilage. Biomaterials – Physics and Chemistry, 2011.
Wave-form compression
Pressure: 200 mbar
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24. AIR PBS AGAROS
E
0 200 400 600 800 1000
0
20
40
60
80
100
120
140
160
180
membranedisplacement(µm) pressure applied (mbar)
air
collagen I
0 200 400 600 800 1000
0
50
100
150
200
250
300
membranedisplacement(µm)
pressure applied (mbar)
air
agarose 2%
0 200 400 600 800 1000
0
50
100
150
200
250
300
membranedisplacement(µm)
pressure applied (mbar)
air
PBS
500 µm
0 200 400 600 800 1000
0
50
100
150
200
250
300
350
400
membranedisplacement(µm)
pressure applied (mbar)
PDMS (10:1)
PDMS (20:1)
Membrane characterization
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25. Bead displacement as function of pressure
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26. 6 days of culture
LONG TERM CELL SURVIVAL
COMPRESSION: 800 mbar at 1 Hz for 1.5 h for 3 consecutive days
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27. Synthetic synovial fluid
Synovial fluid (horse; healthy)
HMWHA (3.5 mg/ml; 2.2MDa; in PBS)
Hyaluronic acid
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28. Synovial
membrane-on-chip
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29. SYNOVIAL MEMBRANE
The synovial membrane is a thin tissue that covers intra-articular surfaces of the joint
capsule. It serves as source of nutrients for the joints. It produces synovial fluids
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30. SYNOVIAL MEMBRANE REQUIRES AN IMMUNE COMPONENT
FLS secrete lubricin and
hyaluronic acid
• Phagocytize bacteria and debris generated from apoptotic cells
• Maintain the balance between the levels of pro-inflammatory and anti-
inflammatory cytokines in the synovial fluid
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31. Membranes – requirements
 Membranes should replicate the cellular interactions with
native matrices.
 Membranes properties, such as topography and stiffness,
can influence cell behavior (adhesion, spreading, growth).
 Mechanical deformation is important to provide mechanical
strain to cells.
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32. SILK MEMBRANES
M. McGill et al. Acta Biomaterialia 2017, 63, 76–84. Adv. Mater. 2007, 19, 2847–2850
SILK FIBROIN-BASED MEMBRANE
FDA APPROVED
IN VITRO AND IN VIVO BIOCOMPATIBILITY
ROBUST MECHANICAL PROPERTIES
RELATIVELY SLOW PROTEOLYTIC BIODEGRADATION
OARSI 2019Joint-on-Chip 32

33. Rockwood et al. Nature Protocols 2011, 6, 1612-1631.
SILK FIBROIN EXTRACTION
OARSI 2019Joint-on-Chip 33

34. Rockwood et al. Nature Protocols 2011, 6, 1612-1631.
ELECTROSPINNING OF SILK FIBROIN
After spinning After crosslinking Scale bar 8 µm; 10000 x; diameter 283 ± 59.
Silk/PEO/ photoinitiator; 0.7 mL/h; d=16 cm; 6-7 kV
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35. INTEGRATION OF MEMBRANES IN THE CHIP
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36. First design Second design
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37. First design
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38. Membrane porosity
allows passage of dye in
the system
Different sections of the
chips are isolated
(different dyes in
different sections)
Second design
Leakage control
OARSI 2019Joint-on-Chip 38

39. 1000
µm
500 µm
FluorescenceFluorescence
beadsAnchoring of
membrane
Anchoring of
membrane
Membrane stretching
500 µm 500 µm
OARSI 2019Joint-on-Chip 39

40. CROSS-SECTION
Mechanicalactuation
chamber
Mechanicalactuation
chamber
PDMS
membrane
elastic
membrane
Step 1: human synovial membrane fibroblasts are seeded in the
cell/media chamber
human synovial membrane fibroblasts
Step 2: Waiting time to allow the attachment of the fibroblasts on the
membrane
Step 3: Application of both shear stress (with flow ) and stretching (with
mechanical actuation chambers)
vacuumvacuum
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41. CELL COMPATIBILITY
OARSI 2019Joint-on-Chip 41

42. THE FUTURE
From: Piluso S. et al. Mimicking the Articular Joint with
In Vitro Models. Trends in Biotechnology, 2019.
OARSI 2019Joint-on-Chip 42

43. ACKNOWLEDGEMENTS
COLLABORATORS
UT
Séverine le Gac
Bastiën Venzac
UMCU
Jos Malda
UU
René van Weeren
Mayo Clinic
André van Wijnen
Daniël Saris
Research Center of excellence award by Dutch Arthritis Association