Watch our webinar where Professor Marc-Andre Fortin presented about the 3D printing of hydrogels and hydrated substances that have been introduced in various fields of biomedical research including regenerative medicine, cosmetic surgery, orthopedics, and medical physics. However, one of the main challenges faced by 3D printing and bioprinting is geometrical conformity. In this presentation, studies requiring hydrogel 3D printing in the fields of ophthalmology, regenerative medicine, and medical physics, were described. MRI scanning procedures were developed and optimized for these specific applications. The presentation highlighted the potential role of MRI in the development of more accurate, more precise 3D-printed hydrogel objects.

1. Clinical Field MRI as a
Measurement
Instrument for 3D
Printing and Bioprinting Marc-André Fortin Ing., Ph.D.
Professor in Biomedical Engineering
Centre de Recherche du Centre
Hospitalier Universitaire de
Québec (CR-CHU de Qc)
Université Laval, Québec, Canada

2. Marc-André Fortin, Ing. PhD
Professor in Materials Engineering, Université Laval and CR-CHU de Québec
Webinar presented for Scintica/Aspect MR, October 12, 2021
Centre de recherche du Centre hospitalier universitaire de Québec – Université Laval, axe Médecine
Régénératrice, Québec, G1L 3L5, QC, Canada; marc-andre.fortin@gmn.ulaval.ca
Clinical field MRI, 3D printing
and bioprinting
2

3. Université Laval in Québec City
• 40 000 students
• 4th oldest higher education institution in North America
• 6th in research funding in Canada
• Areas of excellence (medicine, engineering and science):
– Photonics, Optics, Lasers
– Neurosciences
– Cancer, oncology, medical physics
– Functional materials, biomaterials and regenerative
medicine

4. Fortin’s lab: Laboratoire de Biomatériaux pour l’Imagerie
Médicale (BIM)
Centre Hospitalier Universitaire de Quebec – U. Laval
• Largest Health Research center in Quebec
– 1 600 clinicians, 329 researchers, 208 associated researchers)
• 7th academic health research center in Canada (funding)
• Cancer and oncology, regenerative medicine,
neurology, immunology/infectiology, biomaterials
Biomaterials for Imaging Laboratory’s main topics:
• GOAL: Innovation in biomedical materials for enhanced
visibility in imaging through:
– A) Materials synthesis, B) Materials characterization,
C) Implementation in vivo

5. Biomaterials in Imaging (BIM) laboratory
5
Nanoparticle
synthesis
• One-pot syntheses with
biocompatible precursors
• Plasma nanoparticle synthesis
(several patents);
• 3D printing inks with
nanoparticle-based materials
Advanced Nanomaterials
Characterization
• Bio/organic materials analysis
in scanning electron
microscopy (SEM)
• Elemental analysis of
nanomaterials in transmission
electron microscopy (TEM)
• Surface analysis hybrid (nano)
materials (X-ray photoelectron
spectroscopy (XPS)
In vivo imaging
• Small-animal imaging platform: magnetic resonance
imaging (MRI), X-ray computed tomography (CT),
positron emission tomography (PET)
• Fortin et al; Advanced Healthcare Materials 2018 Aug;7(16):e1701460.
• Fortin et al; in Clinical applications of Magnetic materials, ch 8, CRC press, 2018.
Reviews:
MRI,
CT,PET

6. 2 technology platforms in the lab
Biomaterials
characterization platform
• Nanotechnology lab (DLS,
NMR, UV-Vis)
• XPS, FTIR microscopy,
SEM, AFM (in clean
room), cell culture labs,
3D printing
Small-animal imaging platform
• MRI, CT, PET, ultrasound
imaging
• Radiolabeling lab

7. Financing and Networks
U.Victoria
NSERC-CREATE
program Polymers for
drug delivery U.Alberta
(Edmonton)
U. of Toronto
U. Concordia (Montreal)
U. Laval (Quebec)

8. Outline and content
1. Introduction: contrast agents for T1-w MRI
2. MR-visible paramagnetic coatings for devices
3. Biocompatible ultra-small FeNPs “inks” for 3D
printing
4. 3D-printed contrast-enhanced biogels for eye
cancer phantoms
5. 3D-printed hydrogel implants for cervix
cancer therapy
8

9. Outline and content
• One decade of operation of ASPECT MR 1T :
chronology
9
2010 – 2013: cell tracking with Gd-based agents
2013 – 2018: Development of ultra-small FeNPs to replace Gd-based agents
2018 – : MRI contrast agents for 3D printing, bioprinting, medical physics
2010: installation
2013 – 2016: Gd-based coatings (biopsy needles)
2012
2015
2018
2020
2021

10. General knowledge on imaging modalities…
10

11. Main “whole-body” clinical imaging tools
X-ray computed
tomography (CT)
Magnetic resonance
imaging (MRI)
Positron emission
Tomography (PET)
Res.: 1-2 mm
For: Anatomical
imaging
Ex.:
-skeletal
impairment
-bone density
Res.: ≈1 mm
For: Anatomical
and functional
imaging
Ex.:
-brain, liver, fat
-neural networks
-joint imaging
-inflammation
Res.: ≈ 5 mm
For: Metabolic
and functional
Imaging
(coupled with CT
or MRI)
Ex.:
-glucose met.;
-proteins, fat;
Etc.

12. wavelength l (m)
Nuclear imaging
(PET, SPECT)
X-rays Optical imaging (fluorescence,
bioluminescence, …)
MRI ultrasound imaging
X-ray CT
 Principle: attenuation of X-rays
through high-Z elements
 Ionizing radiation → number of
repeats limited
 Anatomical imaging
 Specific applications:
 Bone density, structure,
regeneration
 Bone regeneration 12
Magnetic resonance imaging (MRI)
 Principle: stimulation of hydrogen
protons with radiofrequency
 Non-ionizing radiation → no limitation on
the number of scans
 Anatomical and functional imaging
 Specific applications:
 Soft tissue imaging (brain, liver, fat)
 Blood flow, oxygenation, water
diffusion
g-rays X-rays Radar
waves
Infra-red Microwave Radio
waves

13. Probes and contrast media for biomedical imaging
13
MRI
• Magnetic nanoparticles and
paramagnetic metal complexes
Review: Laurent S et al, Chem. Rev.
108, 6, 2064-2110 (2008)
X-ray computed
tomography (CT)
• Attenuation of X-rays by
higher-Z elements
Review: Lusic, Grinstaff et al,
Chem. Rev.201311331641-1666
Optical (fluorescent,
luminescent, opto-
acoustic)
• Fluorescent molecules
• Up-converting
luminescent particles
• Near-infrared optical
probes
• Quantum dots
• Review: Kobayashi Chem
Rev. 2010 May 12;
110(5): 2620–2640
Ultrasound imaging
• Micro/nanobubbles
• Review: Paefgen, Kiessling et al, Front. Pharmacol.,
15 Sept. 2015; Du et al, Sci Rep (2018) 8:3887
Nuclear imaging (PET,
SPECT)
• Radiolabeled
molecules;
detection of high-
energy photons
Review: Molecular imaging
with PET; Chem. Rev.108, 5,
1501-1516 (2008)
• Image source: Ghambir et al; Chem. Rev. 117, 3, 901-986 (2017)

14. Introduction: contrast agents for T1-w MRI
• Magnetization of biological
tissues in MRI: dipolar magnetic
moment (m)
– When 1H protons are submitted
to B0, a strong magnetic field,
they align in the direction of this
field
– m is parallel or antiparallel with
respect to B0
– Slightly more on the parallel
energy level.
– Generate Mz

15. Introduction: contrast agents for T1-w MRI
• T1 and T2 are intrinsic characteristics of a tissue
T1: time constant describing
the return of 63% of the spins
to equilibrium

16. Principles of MRI
• Then the RF pulse is turned-off…
The spins progressively re-align in
the direction of B0 (z) … T1
… and meanwhile the spins mutually
influence each-other, giving rise to
dephasing and extinction of the Mxy
component …. T2.
T1: time at 63%
Mz recovery T2: time at 37%
Mxy dephasing

17. Principles of MRI
• By varying TR and TE, spin echo sequences can generate T1- weighted or T2 -
weighted images
– If TR  , then 1 − e−TR/T1 1 ; If TR 0 , then 1 − e−TR/T1 = 0
– If TE 0 , then e−TE/T2  1; If TE  , then e−TE/T2 = 0
– For ideal T1 – weighting : TR  T1,
2
/
1
/
)
1
( T
TE
T
TR
H
xy e
e
M
S 



 

18. Principles of MRI
• T1 and T2 are values intrinsic to each tissue
To change the T1 and T2 of a given tissue, it is necessary to introduce a
CONTRAST AGENT

19. Principles of MRI
• Contrast agents made of gadolinium (Gd) or manganese (Mn)
chelates, as well as Fe-based nanoparticles (NPs) are
currently used in many diagnostic applications performed
under magnetic resonance imaging (MRI).
• MRI detects the signals from hydrogen (1H) protons
stimulated with radiofrequencies
– Biological tissues contain > 60% of 1H protons
“negative contrast agents”:
Fe-based
“positive contrast agents”:
Gd and Mn-based

20. Introduction: contrast agents for T1-w MRI
• T1 relaxation in MRI
– « Positive » CAs: mostly based on
paramagnetic gadolinium (7
unpaired electrons in the 4f orbital)
– Water (1H) protons bind to Gd
contrast agents and influence their
T1 (acceleration of proton
relaxation rate)
– By decreasing the T1 of H, Gd
enhance the MRI signal
2
/
1
/
)
1
( T
TE
T
TR
H
xy e
e
M
S 



 
Faucher, Fortin et al, Nanotechnology. 2011 Jul 22;22(29):295103
Faucher, Fortin et al, ACS Appl. Mater. Interfaces 2012 49, 4506-4515

21. Principles of contrast agents (CAs) in MRI
• « Negative» CAs : iron oxide nanoparticles (5 nm diam. and +)
• Hepatic diagnostics, cell tracking, molecular imaging…
T2*

22. Principles of contrast agents (CAs) in MRI
In brief, MRI signal depends on:
1H :  ; T1 ; T2
Short T1 =  signal
Long T2 =  signal
For optimal signal
enhancement, or
« positive » contrast:
•  r1
• T1/T2 (r2/r1)  1
For signal decrease
(« negative contrast» :
• T1/T2 (r2/r1)   10
2
/
1
/
)
1
( T
TE
T
TR
H
xy e
e
M
S 



 
“Positive” CA:
Gd2O3 nanoparticles;
Fortin et al, Langmuir 2011
“Negative” CA:
Fe3O4 nanoparticles;
Fortin, Oh et al,
Biomacromolecules 2014

23. Outline and content
1. Introduction: contrast agents for T1-w MRI
2. MR-visible paramagnetic coatings for
devices
3. Biocompatible ultra-small FeNPs “inks” for 3D
printing
4. 3D-printed contrast-enhanced biogels for eye
cancer phantoms
5. 3D-printed hydrogel implants for cervix
cancer therapy
23

24. MRI-visible biopsy needles
Prostate and breast cancers account for
more than 25% of all cases of cancer
• MRI is the best imaging modality to
detect small nodules (breast) and
focal points of tumor growth
(prostate)
• However: needles are not visible
enough!
• Precision is of utmost importance in
MRI-guided biopsy
– Nodes are small (mm)
– The needle must be precisely
positioned (mm)
– Strong image artifacts:
C.R. Weiss, et al., Journal of Magnetic Resonance Imaging, 2008, 27(2), 311-325
E.R. Price and E.A. Morris, Canadian Association of Radiologists Journal, 2011, 62(1), 15-21
K.M. Pondman, et al., European Urology, 2008, 54(3), 517-527.
Biopsy needle
(7 mm image
artifact)
[4] Penzkofer, T., et al.,
Cardiovasc. Intervent.
Radiol., 2010

25. What happens when a needle is inserted in a MRI voxel?
• MRI voxel (typical size)
• Filled with water (1H2O)
• Insertion of a 23G biopsy
needle
• (0.64 mm OD, 0.36 ID)
• 23% less 1H signal
• Insertion of a 23G biopsy needle
with 70% porous coating
• (1,04 mm OD, 0.36 ID)
• 61% equivalent voxel size filled
with water
• 1 mM of Gd in porous coating
• signal enhancement

26. Conditions for achieving signal enhancement in a small MRI voxel
T1-weighted MRI acquisitions (short TE, medium TR):
less magnetic susceptibility artifacts
Conditions for achieving high signal enhancement:
1. Voxel filled with water (0.2 x 0.2 x 1 mm; >
50% volume)
– A thick, dense polymer coating = NO
– Use water-rich hydrogels instead = YES
2. Contrast agent (Gd) homogeneously
distributed in the voxel
– Optimal balance: between 0.1 and 1.0 mM Gd
3. Direct contact between Gd ions and H2O
– Gd must be sequestrated in a 3D, water-filled
network to constitute functional coatings
Gd
Gd
Gd
MRI
signal
without
contrast
agent
MRI
signal
with
contrast
agent
1
mm

27. Gd-grafted onto mesoporous silica nanoparticles
27
Collaboration: Dr Freddy Kleitz, U.Laval/U.Wien
Laprise-Pelletier, Bouchoucha, Fortin et al, J Mat Chem
B; J. Mater. Chem. B, 2015,3, 748-758
Bouchoucha, Fortin, et al, Chem. Mater.2016, 28, 12,
4243-4258; Langmuir 2017, 3340, 10531-10542
H2O

28. Hydrogel coating (PEG-acrylate +
nanoparticles ) ≈ 50 – 200 microns
thick
titanium needle
(low magnetic
susceptibility;
less MRI artifacts)
Paramagnetic silica
nanoparticles
(Gd3+-labeled)
• Biocompatible
coating
• Large pore volume
for drug elution
function (silica
nanoparticles)
Preparation of TiO2/Ti surface
(phosphonate/acrylate for
hydrogel grafting)
28
Step 1
Step 2
Paramagnetic hybrid silica-PEG coatings for high MRI signal
Nanoparticle synthesis in: Bouchoucha, Fortin,
Kleitz, et al, Chem. Mater.2016, 28, 12, 4243-4258
Silencieux, Fortin et al (submitted)
Step 3

29. 29
1.6 mm diam.
1.05 mm diam.
0.51 mm diam.
T1-w MRI
T = 4h
T = 2 months
T = 4 hours

30. Toward MRI-visible, in vivo implantable 3D
bioprinted objects
MRI-visible coating materials
… used in 3D printing as MRI-
visible bioinks
General problematics of MRI
follow-up with hydrogel
scaffolds: lack of visibility
• ↑ Visibility of bioprinted
objects
• Verification of geometrical
conformity after 3D printing
• Volume and shape follow-up
post-implantation studies
Main problems of Gd-based
contrast agents: potential toxicity
(nephrogenic systemic fibrosis)
• Grobneret al; Nephrol Dial
Transplant. 2006;21(4):1104–
1108.
• No long-term implantation of
Gd-containing objects
• Necessity to use
biocompatible « positive »
MRI contrast agents:
• Ultra-small iron oxide
nanoparticles (< 6 nm diam.)
30

31. Outline and content
1. Introduction: contrast agents for T1-w MRI
2. MR-visible paramagnetic coatings for devices
3. Biocompatible ultra-small FeNPs “inks” for
3D printing
4. 3D-printed contrast-enhanced biogels for eye
cancer phantoms
5. 3D-printed hydrogel implants for cervix
cancer therapy
31

32. Ultra-small iron oxide nanoparticles (USPIONs) for
positive contrast in MRI
32
Collaboration: Dr John Oh, Concordia University, Montreal
Chan, Chevallier, Fortin, Oh et al, Biomacromolecules. 2014 Jun 9;15(6):2146-56.
Xiao, Legros, Fortin, Oh et al, ACS Appl. Nano Mater.2018, 12, 894-907

33. Ultra-small iron oxide nanoparticles (USPIONs) for
positive contrast in MRI
Fortin, Oh et al,
Biomacromolecules, 15, 6,
2146–2156, 2014

34. Ultra-small iron oxide nanoparticles (USPIONs) for
positive contrast in MRI
34
10 mm
0
25
50
75
100
125
0.00
0.50
1.00
1.50
2.00
0 h 24 h 48 h 7 d 14 d 30 d
Contrast-to-noise
ratio
(CNR)
S
x
/S
0
Time
Hydrogel+SPIONs-PEG
CNR
0 h
7 d
30 d
Hydrogel
Water
2 1
3
1. Hydrogel-SPIONs standard
2. Hydrogel standard
3. Water control
A) B)
Iron oxide nanoparticles
(USPIONs) + alginate (2% w/w)
After 30 days, USPIONs progressively diffuse
out of the hydrogels …
+
Fortin et al ACS Appl. Nano Mater. 2018, 1, 894−907
Fortin, Oh et al, Biomacromol., 15, 6, 2146–2156, 2014

35. Ultra-small iron oxide nanoparticles (USPIONs) for
positive contrast in MRI
Fortin et al ACS Appl. Nano Mater. 2018, 1, 894−907
Fortin, Oh et al, Biomacromol., 15, 6, 2146–2156, 2014
abdominal vasculature
liver
gall bladder

36. 36
Submitted
(2021)
Sugar mold printing procedure described in: Gauvin-Rossignol, Fortin, Bégin-Drolet et al; Heliyon 4 (7), e00680

37. Toward MRI-visible, in vivo implantable 3D bioprinted objects
Sugar mold printing procedure described in: Gauvin-Rossignol, Fortin, Bégin-Drolet et al; Heliyon 4 (7), e00680
Submitted
(2021)

38. Toward MRI-visible, in vivo implantable 3D bioprinted objects
Sugar mold printing procedure described in: Gauvin-Rossignol, Fortin, Bégin-Drolet et al; Heliyon 4 (7), e00680
Submitted
(2021)

39. Biocompatible MRI-visible USPIONs hydrogels
39
Published: WMIC 2021
Volume retention of
hydrogel implant (on
14 days) (from MRI)

40. Biocompatible MRI-visible USPIONs hydrogels
40
Published: WMIC 2021 Collaboration: Dr Julie Fradette, U.Laval LOEX

41. Biocompatible MRI-visible USPIONs hydrogels
41
Published: WMIC 2021
4M. Vallée, J. F. Côté and J. Fradette, Pathol. Biol., 2009, 57, 309–317.
Collaboration: Dr Julie Fradette, U.Laval LOEX

42. Follow-up of hydrogel implants by MRI
• USPIONs (FeNPs) do not cause
cell toxicity at the concentration
used for positive contrast (paper
submitted)
• USPIONs can be used as an
additive to hydrogel scaffolds
used for 3D printing of
anatomically-relevant implants
• Regenerative medicine
applications where MRI is
needed for the follow-up of soft
tissue:
– Breast reconstruction (e.g. post-
oncology)
– Facial reconstruction
– Fat tissues…
42
Source:www.3dnatives.com
Source: 3dprintingindustry.com;
MRI
Alginate +
adipose cells
+ USPIONs

43. Toward MRI-visible, in vivo implantable 3D
bioprinted objects

44. Outline and content
1. Introduction: contrast agents for T1-w MRI
2. MR-visible paramagnetic coatings for devices
3. Biocompatible ultra-small FeNPs “inks” for 3D
printing
4. 3D-printed contrast-enhanced biogels for
eye cancer phantoms
5. 3D-printed hydrogel implants for cervix
cancer therapy
44

45. Current treatment for uveal melanoma
• Brachytherapy implants (episcleral plaques) = radioactive
plaques
• Plaques are sutured on the sclera by surgery
• Removed after 2-7 days by another surgery
45
[2,3]
Eye
Episcleral
plaque
Radioactive
sources Episcleral
plaque
1. Chiu‐Tsao, S. T. et al., (2012). Medical physics, 39(10), 6161-6184.
2. Société canadienne du cancer. https://www.cancer.ca/fr-ca/cancer-information/cancer-type/retinoblastoma/treatment/radiation-therapy/?region=qc
3. Singh, A.D. et al. 2014: Springer Science & Business Media

46. Treatment planning by dose calculations in medical physics
• Radioactive source position in
the plaque must be known
before surgery→ Medical
physicists
• Dose: energy deposited in
matter by ionizing radiation per
unit mass
• The dose profile must match
with the contours of the tumor
as precisely as possible →
isodose
46
1. Schubert, L.K. and M. Miften, 2016, Springer International Publishing: Cham. p. 13-27.
2. Chiu‐Tsao, S. T. et al., (2012). Medical physics, 39(10), 6161-6184.
3. Lesperance, M. et al., Medical Physics, 2014. 41(2): p. 12.

47. Biological
phantom
Hydrogel for a phantom in eye cancer dose planning
To develop by 3D printing, an eye-shaped cell-laden hydrogel
scaffold that could be used for studying the effect of
radiotherapeutic treatments on different types of cells that are
associated with different radiosensitivity levels.
47
2. Development of a 3D printing
procedure for fabricating hydrogel
in the shape of an eye ball
3. Demonstration
of cell viability
1. Development of a
functional hydrogel
formulation
[1]
1. Branco da Cunha, C., et al., Biomaterials, 2014. 35(32)

48. Hydrogel for a phantom in eye cancer dose planning
48
Sclera Choroid
Collagen
Collagen
Proteins Fibers
Fibroblast Fibroblast
Melanocyt
e
1. Institut de l’œil, http://www.institutdeloeil.com/traitement-de-loeil/anatomie-de-loeil.html
2. DENNISTON, Alastair et al. Oxford handbook of ophthalmology. OUP Oxford, 2009.
3. Trier, K., The Sclera, in Advances in Organ Biology, J. Fischbarg, Editor. 2005, Elsevier. p. 353-373.
4. Djigo, A. D. et al., Acta Biomater. (84), 305-316, 2019.
[1]
Melanocyt
e

49. Hydrogel for a phantom in eye cancer dose planning
• Hydrogel: 3D networks of hydrophilic
polymers
• Bioprinting: Layer-by-layer production method
allowing complex geometry printing
• Criteria:
 Mechanical properties
 High cell proliferation
 Biocompatibility
49
1. Lee, K.Y. et al., Progress in Polymer Science, 2012. 37(1): p. 106-126.
2. Di Giuseppe, M., et al., Journal of the Mechanical Behavior of Biomedical Materials, 2018. 79: p. 150-157.
3. Cidonio, G., et al., Biomaterials, 2019. 209: p. 10-24.
4. Hospodiuk, M., et al., 3D printing and biofabrication, 2016: p. 1-27.

50. Hydrogel for a phantom in eye cancer dose planning
• Mechanical function: Alginate (ALG)
– Molecular weight 80-120 kDa, M/G ratio 1.56
• Cellular function: Collagen type I (COL)
– From rat tail tendons
• Printing function: Xanthan gum (XG)
– Thickener material
50
[2]
1. Ozbolat, I.T. et al., Biomaterials, 2016. 76: p. 321-343.
2. Gelinsky, M., et al., Editors. 2018, Woodhead Publishing. p. 125-136

51. Hydrogel for a phantom in eye cancer dose planning
• Optimisation of the printability with xanthan gum
• Goal: obtain a continuous filament
51
XG 1% (w/v), ALG and COL XG 1,7% (w/v), ALG and COL
Addition of xanthan gum improved printability

52. Step 2: development of a 3D printing procedure for
fabricating hydrogels in the shape of an eye ball
• Size close to that of a human eye
• In the hydrogel: diffusion of oxygen and nutrients limited
to only on few millimeters
• Support allows a better oxygen and nutrients supply to the
cells compared to a full hydrogel model
52
1. Denniston, A. and P. Murray, Oxford handbook of ophthalmology. 2009: OUP Oxford.
2. Watson, P.G. and R.D. Young, Experimental Eye Research, 2004. 78(3): p. 609-623.
3. Shanjani, Y., et al., Biofabrication, 2015. 7(4): p. 16.
Sclera
Lattice support
25 mm diameter

53. Step 2: development of a 3D printing procedure for
fabricating hydrogel in the shape of an eye ball
• Development of a
lattice support:
supports cell-containing
hydrogel
• 3D printing: provides a
thin layer of hydrogel
53

54. Step 2: development of a 3D printing procedure for
fabricating hydrogel in the shape of an eye ball
• Evaluation of accuracy of the 3D printing process → Magnetic
resonance imaging (MRI)
• Images are produced by the excitation of hydrogen atoms
• Tridimensional images of the hydrogel
• T1-weighted spin-echo sequence (TR/TE: 742 ms/10.4 ms, FOV 36
mm, 248 x 248, 30 min)
54

55. Step 2: development of a 3D printing procedure for
fabricating hydrogel in the shape of an eye ball
• Measurement of the diameter and the thickness of the hydrogel eye ball
• Diameter (26.2 ± 0.2) mm (eye diameter between 23 and 26 mm)
• Thickness of the hydrogel eye ball (1.3 ± 0.1) mm (sclera thickness at the
back 1-1.35 mm)
55
Eye ball
Eye ball
Axial view Coronal view
T1-weighted spin-echo sequence; TR/TE: 742
ms/10.4 ms, FOV 36 mm, 248 x 248, 30 min

56. Step 3: Demonstration of cell viability
• Cancer cells: cancer cell lines with different radiosensitivity levels (Mµ2 et
Mel270)
• Choiroid cells: choroid fibroblasts
• Time point:
– Day 0: cell viability after printing process
– Day 1: episcleral plaque will be added
– Day 4: episcleral plaque will be removed
56
1. Without printing 2. Printing a simple shape 3. Printing the model

57. Step 3: Cell viability assessment
• Live/dead kit test
– Green staining (calceine AM): live cells
– Red staining (ethidium homodimer-1): dead cells
• Images with epifluorescence microscope
57
Live cells image
Dead cells
image Merge image

58. Step 3: demonstration of cell viability
• Cell viability with and without printing
• Printing parameters do not affect significatively cell viability
58
Choroid fibroblasts
Cancer cell line Mel270 Cancer cell line Mµ2
0 1 4
0
20
40
60
80
100
Cell
Viability
(%)
Days
Mel270 without printing
Mel270 with printing
**
**
*
*p < 0.05
**p < 0.005
0 1 4
0
20
40
60
80
100
Cell
Viability
(%)
Days
Fibroblasts without printing
Fibroblasts with printing
*
**
**
0 1 4
0
20
40
60
80
100
Cell
Viability
(%)
Days
Mµ2 without printing
Mµ2 with printing
**
**
**
**

59. Conclusion: 3D-printed contrast-enhanced biogels
for eye cancer phantoms
• Print the complete in vitro cell-containing phantoms and
measure the cell viability at different time points
• In vitro cell-containing phantoms for testing radiotherapy
plaques
• The model could be used for a better adjustment of dose
calculation planning
59
Current
implant
1. Lesperance, M. et al., Medical Physics, 2014. 41(2): p. 12.

60. Outline and content
1. Introduction: contrast agents for T1-w MRI
2. MR-visible paramagnetic coatings for devices
3. Biocompatible ultra-small FeNPs “inks” for 3D
printing
4. 3D-printed contrast-enhanced biogels for eye
cancer phantoms
5. 3D-printed hydrogel implants for cervix
cancer therapy
60

61. Scientific objective: To develop and characterize a 3D-printed hybrid hydrogel-
nanoparticle formulation as a localized delivery system for cervical cancer therapy.
+ =
Pluronic F127
Alginate
AuNPs
(radiosensitizer)
A 3D-printed hybrid
delivery system
%
NPs
released
Time
Controlled release to the cancerous tissues
Elution/diffusion of gold nanoparticles from
hydrogels

62. Hydrogel implants for cervix cancer therapy
Hainfeld et al., Phys Med Biol. 2004, 49(18), 309-15
Beik et al., Coordination Chemistry Reviews 2019, 387, 299–324
Radiation
Nucleus
Tumor cell
e-
e-
e-
e-
e-
Photo/Auger electron
Radiosensitization effect
Stabilizing agent (e.g. PEG)
• Relative biocompatibility of Au
• High Z number element (Z = 79)
Gold nanoparticles as radiosensitizers

63. Hydrogel for Au NPs elution: PF127-alginate hydrogel
Ca2+ Ca2+
Pluronic F127 Alginate
• Thermosensitive (37 ºC)
• Poor mechanical properties
• Typical concentrations: 15% - 30% w/v
• Gelling agent for vaginal applications
• Cross-linking with divalent cations
• Improved mechanical stiffness and strength
• Typical concentrations: 1% - 5% w/v
Garg et al., Coordination Pharmaceutical Technolog 2001, 25(9), 14-24.
PEO-PPO-PEO
heating
cooling
Sol Gel
PEO PPO H2O
O
O
H
O
OH
99 67 99

64. Printability of the Pluronic F127-alginate ink
Pluronic F127 Alginate H2O
Before cross-linking with Ca2+
Low mechanical strength
37°C
1)
Extrusion-based CELLINK Bio X 3D-printer
ø 10 mm ø 3 mm, h = 3 mm
0.1 M CaCl2
Ca2+
Ca2+
Ca2+
Ca2+
After cross-linking with Ca2+
Improved mechanical strength
2)

65. In vitro degradation study of hydrogel for Au NP
release: scanning electron microscopy study
17
50 µm 50 µm 50 µm
SEM images: 3 kV, spot size 3, no coating, magnification 500 x
Day 1 Day 3 Day 5
(2.3 ± 1.0) µm (12.2 ± 3.4) µm (26.2 ± 7.5) µm
Pore size:

66. The tests were performed using a trypan blue exclusion method (n = 6)
Cytocompatibility of the PF127-alginate gel
HeLa cervical cancer cell line CRL-2616 normal vaginal cell line
> 93% viable
> 90% viable
Hydrogel implants for cervix cancer therapy

67. Biocompatibility of the PF127-alginate gel +/− AuNPs
Right flank:
gel alone/ gel + AuNPs
Left flank:
saline
Group 1,2
n = 6
• PF127 20% w/v, alginate 0.5% w/v
• AuNPs 0.1 mg/ml
• Cytokine test: IL-6, IL-10, MCP-1, IFN-γ,
TNF, IL-12p70
• Histology: H&E
• IHC: F4/80
• Balb/C mice
Day -3:
Blood sampling
12 mice
Day 0:
Administration of the
PF127-alginate gel +/−
AuNPs + MRI
12 mice
Day 3:
Blood sampling
+ MRI
12 mice
Day 10:
Blood sampling
+ MRI
12 mice
Day 14:
Blood sampling
+ MRI
+ histological
assessment
12 mice

68. MRI: in vivo gel degradation
Day 0 Day 3 Day 14
• T1-weighted 2D spin echo sequence (echo time/repetition time: 13.5/704.2 ms)
• 5 excitations
• PF127 20% w/v, alginate 0.5% w/v

69. In vivo degradation profile
**
*
• Group PF127-alginate: ~26% of initial volume
• Group PF127-alginate + AuNPs: ~20% of initial
volume

70. Immunohistochemistry: F4/80 antibody
• F4/80 is a general marker for macrophages (in green)
• Cell nuclei (in blue)
• 20 um thickness of the slide
PF127-alginate gel and surrounding skin Positive control, infected mice
• Microphages are seen on the periphery of the gel
• Mild inflammation response
Many thanks to Dr Dominique Mayrand (PhD, MBA) and Dr Julie Fradette (PhD, Professeur titulaire) for help in histological assessment
G
G
500 um 500 um
500 um
500 um

71. Printability of the Pluronic F127-alginate ink
ø 10 mm, h = 2 mm, 10 layers
ø 3 mm, h = 3 mm, 15 layers

72. 3D-printed hydrogel implants for cervix cancer
therapy
Lens-shaped gels:
T1-w. 2D SE: FOV: 40 mm; 28
slices; 0.9 mm thk; 0.1 mm gap;
dwell time 50 μs; matrix: 304 ×
304; fα 90°; echo asymmetry
50%; TE/TR: 90/3167.2 ms;
duration: 38 min). 3.5 cm
diam.coil.
Hollow tube-shaped gels: mouse
head coil.
T1-w. 2D SE: FOV: 30 mm; 30
slices; 0.6 mm slice thickness;
0.1 mm slice gap; dwell time 50
μs; matrix: 304 × 304; fα 90°;
echo asymmetry 50%; TE/TR:
85/3250.9 ms; 49 min 25 s).
(submitted)

73. Conclusion and perspectives
• Biocompatible polymers and hydrogels are often necessary in the design
of effective therapeutics
• Nanostructured materials for contrast enhancement in MRI are necessary
to design biomedical objects for diagnostic and therapeutic purposes
• Biocompatible materials with enhanced visibility in biomedical imaging
(MRI)
– Fundamentals of both imaging modalities and functional contrast agents
– Contrast agents must be used below toxicity thresholds
• Ultra-small iron oxide nanoparticles: at low concentration, as a functional
ink for 3D bioprinting (MRI visibility, geometrical conformity)
– Applications in medical physics, nanoparticle elution, etc
73

74. Research associates:
• Dr Jenny Roy
• Dr Pascale Chevallier
Students (PhD, MSc):
• Théophraste Lescot
• Mahmoud Omar
• Jean-François Sauvageau
• Mariia Kiseleva
• Sophie Lemay
• Cédric Garceau
• Lucille Samard
• Charlotte Valdenaire
• Samira Ravanbakhsh
• Zongyi Liu
Acknowledgments
Collaborators:
Dr Luc Beaulieu, Dr Claudine Bellerive, Dr Solange Landreville, Dr Julie Fradette, CHU de Québec

75. Announcement: Gold 2022 Conference in Quebec City
Chairs: MA Fortin and E.Boisselier, CR- CHU de Québec – U.Laval www.gold2022.org

76. Q&A Session
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