Journal Club Presentation - Scaffolds and Tissues

1. APPLICATION OF 3-D
BIOPRINTED MATERIALS IN
SOLVING CARDIAC PROBLEMS
JOURNAL CLUB PRESENTATION
BY
Nuruddin Bahar (2373100B)
Sri Vidhya Swaminathan ()
Nasser Alsulaiman (2339501A)

2. 3D-printed vascular networks direct therapeutic
angiogenesis in ischemia
Mirabella, T., MacArthur, J. W., Cheng, D., Ozaki, C. K., Woo, Y. J., Yang, M. T., & Chen, C. S. (2017). 3D-printed vascular
networks direct therapeutic angiogenesis in ischaemia. Nature Biomedical Engineering, 1(6), 0083.
https://doi.org/10.1038/s41551-017-0083
Paper presented by : NURUDDIN BAHAR

3. MOTIVATION
RESULT 1
RESULT 2
RESULT 3
RESULT 4
DISCUSSION
Vascular Patch
(VP) Fabrication
& Implantation
Laser Doppler
Imaging &
Analysis
Staining and
Analysis
Histological
Analysis
CDC42 Silencing
Laser Doppler
Imaging &
Analysis
Laser Doppler
Imaging &
Analysis
Geometric
Patterning of
Vascular Patch
DiI Perfusion and
Immunohistochemical
Staining
Intraventricular
pressure–volume
conductance
Transthoracic
Echocardiographic
Analysis
Staining and
Analysis
OUTLINE

4. WHY TO CONDUCT THE RESEARCH ?
• Ischaemic cardiovascular disease is the leading cause of mortality in US and worldwide –
caused by impaired blood circulation. Need to enhance perfusion.
• Pre-organizing endothelial cells into defined network architectures within small tissue-
engineered constructs can significantly enhance the speed and extent of vascularization of
the construct on implantation
TRIED & TESTED METHODS – SUCCESS & DRAWBACKS
Method Success Limitation
Artery Bypass Surgery
& Percutaneous
Revascularization
• Common medical approach • Not well-tolerated in patients
• Periprocedural complications
• Less anatomic durability
• Not applicable to microvasculature
Pro-angiogenic Therapy
(VEGF & Fibroblast Growth
Factor)
• Better results than bypass surgery
• Anatomic durability
• Rapid elimination
• Oedema
Controlled-release
Biomaterials / Expression
Vectors
• Successful in vitro • Clinically insignificant
• Slow progress
• Randomly organized
MOTIVATION

5. RESULT 1
3D-printed vascular patches functionally engraft in vivo and rescue perfusion
and function of ischaemic tissues.
• To Test : Whether organizing endothelial cells into patterned channels within
an implantable, fully degradable fibrin patch would enhance perfusion of
rodent models of ischaemia
• Model : Mouse having hind-limb ischemia
• Target : Distal ischemic limb
• Duration : 5-21 days
• Cause : Occlusion due to cauterized left femoral artery
• Effect : 50% post-operative decrease in perfusion

6. RESULT 1
Vascular Patch (VP) Fabrication & Implantation
Parallel Filaments of
3D-printed
Carbohydrate Glass
PDLGA Coating
Fibrin Bulking
PBS Removal of
Sacrificial Sugar
Endothelialisation
using HUVECs and
Shear-conditioning
Jia, Y., Qin, J., Zhi, Z., & Wang, R. K. (2011). Ultrahigh
sensitive optical microangiography reveals depth-
resolved microcirculation and its longitudinal response
to prolonged ischemic event within skeletal muscles in
mice. Journal of Biomedical Optics, 16(8), 086004.
https://doi.org/10.1117/1.3606565

7. RESULT 1
Laser Doppler Imaging & Analysis
• Groups :
VP & Sham (negative control)
• Imaging :
Laser Doppler
• VP mice perfusion levels
reach near 70% by day 3 and
95% by day 5 to perfusion
units of contralateral (non-
ischaemic) limb
STATISTICS
Mean + s.d.
N = 6 X 2 groups, *P<0.01, t-
test

8. • Ischemic distal limb
caused an
angiogenic response
in gastrocnemius
muscle of lower limb
• No. of capillaries
normalized to
number of muscle
fibres abnormally
high in sham as
compared to VP
• Increase in apoptotic
endothelial cells with
capillaries of sham
group
RESULT 1
Staining & Analysis
STATISTICS
No. of capillaries
NVP = 55, Nsham = 211
Mean ± s.e.m.
N = 3 per group, 3 gastro.
muscles per mouse
*P < 0.02, Mann–Whitney U-test

9. RESULT 2
• Direct anastomoses of VPs with host vasculature drives functional perfusion.
To observe : Vasculature at the site of implantation by using Laser Doppler
imaging and histology.

10. RESULT 2
HISTOLOGICAL ANALYSIS
• Host capillaries are present,
directly connected to the pre-
patterned endothelial channels
• Dye-labelled dextran perfused by
tail vein injection flows through
channels – host vasculature
perfusion confirmed
• 90% channels functional
• Labelling with monoclonal anti-
mouse CD31 antibody revealed
host origin of capillaries

11. RESULT 2
HISTOLOGICAL ANALYSIS (CONT..)
• Ephrin-B2 (arterial marker) forward
signalling through the EphB4 (vein
marker) receptor inhibits cell adhesion
and cell migration, whereas EphB4
reverse signalling by the transmembrane
ephrin-B2 ligand does not.
• Most endothelial cells within
microchannels expressed vein marker
EphB4 and only a few ephrinB2. =>
reverse signalling that allows cell
adhesion and migration
Hamada, et.al. (2003). Distinct roles of ephrin-B2 forward and EphB4 reverse signalling in
endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(2), 190–197.

12. RESULT 2
CDC42 SILENCING
To discard : Implanted endothelial cells
simply provide paracrine support for
host vasculature instead of
angiogenesis.
Method : Implanting constructs
containing endothelial cells with
silencing of the expression of sprout-
inducing guanosine triphosphate
hydrolase enzyme - cell division control
protein 42 (CDC42)
• Used SiRNA – SiCDC42 that lacked
CDC42 expression but showed viability
• Scrambled SiRNA (SCR) showed both
cell migration and viability
http://www.amsbio.com/trilencer27.aspx

13. RESULT 2
CDC42 SILENCING
Direct anastomoses of VPs with host
vasculature drives functional perfusion.
• Reduced integration between host
capillaries and patterned engineered
vasculature
• Fewer vessels arising from implanted VPs
• Lower anastomotic connections
between host and parent vessels (proven
by injected lectin found within channel
walls)
• Significant decrease of distal perfusion to
ischemic limbs in mice with siCDC42 VPs

14. RESULT 3
• Patterning of vascular architecture is critical to therapeutic benefit.
To test : Effect of patterning of endothelial cells against merely providing
angiocrine support.

15. RESULT 3
GEOMETRIC PATTERNING, LASER
DOPPLER IMAGING & ANALYSIS
Legend :
Par VP – channels orientated in parallel
Grid VP – channels orientated orthogonally
Sm_D VP – parallel channels (smaller diameter)
EP(endothelial patches) – HUVECs randomly
embedded into fibrin
AP(acellular patches) – patterned, not-
endothelialized

16. RESULT 3
STAINING & ANALYSIS
• EPs or APs implantation was unable to rescue
perfusion and were comparable to non-
treated shams
• Lack of anastomosis of host vessels with
implant vessels
• Decreasing channel diameter from 400 µm to
200 µm, decreased perfusion inflicting ability
• Grid VPs exhibited reduced efficacy
compared to Par VPs
• Thus, presence of only endothelial cells,
without organization into vessel-like structures
fails to establish collateral circulation AND
geometric design of vascular channels
impacts its ability to rescue perfusion

17. RESULT 4
• 3D-printed VPs rescue cardiac function in a model of myocardial infarction.
To test : the effect of VPs when applied to cardiac infarction setting
Model : Rats having myocardial infarction (MI)
Target : Athymic rats
Cause : Ligation of left anterior descending coronary artery
Duration : 4-weeks old implant assessment

18. RESULT 4
DiI Perfusion And Immunohistochemical Staining
• Groups : Healthy rats (No MI) &
sham (rats given saline ) & VPs
• Increased vascularization
throughout implant and
surrounding tissue – confirmed
by DiI perfusion post systemic
infusion
• Immunohistochemical staining
of von Willebrand factor (vWF)-
positive vessels determined
• Increase in density after VPs
applied

19. RESULT 4
Intraventricular Pressure–volume Conductance +
Transthoracic Echocardiographic Analysis
• Hearts from sham group
exhibited half the
ejection fraction and
cardiac output of no MI
• VPs resulted in partial
rescue, with values close
to no MI
• Left Ventricular Internal
Dimension (LVID) had
substantial increase
during diastole (LVIDd)
and systole (LVIDs) in
ischemic hearts which
lowered for VPs

20. DISCUSSION
• Engineered vessels became integrated with host vasculature resulting in significant
perfusion of distal ischemic tissues
• Large calibre engineered vessels may enhance blood volume distally
• Blood perfusion through disorganized endothelial cells forming branched
capillaries is biomechanically inefficient
• Geometry of the channels is a design parameter that impacts functionality of
implants
• Technique can be used as prophylaxis against progression of organ malperfusion
• Technique can be used for inoperable patients

21. CRITICAL EVALUATION
Abstract is intelligible
and accurately
describes objectives
and results obtained. It
includes all the data
presented in the paper
and material that can
be substantiated.
The motivation and aim
have been clearly
indicated in the
introduction. Background
information has been
provided adequately
where appropriate.
Methods are repeatable and
adequately referenced. Potential
problems and limitations with
methods used have not been
evaluated. Sources of drugs have
been indicated. Appropriate
statistical methods have been used.
Results : Most of the
experiments have been done
appropriately with respect to
the objective of study.
Legends to figures describe
data clearly. Appropriate
statistical analysis have been
undertaken. Scale has not
been aptly provided in a few
instances.
Insight
• Experimental design could be bettered by showing
comparison of currently used techniques or effects of anti-
MI or anti-ischemic drugs along with VP.
• How the geometric pattern affects blood perfusion and
vasculature is not explained
• It is claimed that enhanced angiogenesis is achieved by
the vascular patch implantation, but its relative
quantification has not been demonstrated with reference
to previous work

22. THANK YOU