1. health and fitness
2. disease

Research
SRN Annual Newsletter
February 2014—January 2015
Dear AOSpine members
This past year, the SRN continued to advance the field of intervertebral disc (IVD) regeneration and degeneration, and the benefits of
being a “network” continued to become apparent.
The SRN is currently comprised of 29 experts trained in various disciplines such as engineering, biochemistry, biophysics, human
medicine, epidemiology, biology, and veterinary medicine. Today, the SRN is now led by steering committee members: Barbara Chan,
Danny Chan, Lisbet Haglund, Keita Ito, Björn Meij, Tugan Muftuler, and Daisuke Sakai. These members are core members
because they hold active SRN project grants (see page 13).
While these projects are focussed on IVD regeneration and degeneration, they encompass a broad range of areas: diagnostic
imaging, genetics, cell-signalling, large and small animal models, IVD niche characterization, peptides, stem cells and differentiation,
inflammation, mechanisms of disc degeneration, disc biomechanics, drug and gene therapy (nanotherapeutics), scaffold engineering,
metabonomics, etc.
With investigators from around the world, dedicated to IVD research, the sponsored network members have published 12 manuscripts
and 23 presentations in 2014 (see detailed list on pages 20-22).
To highlight two network activities and successes:
• In May 2014, nine SRN members participated as faculty in the WFSR—World Forum for Spine Research meeting in Xi’an, China,
and a total of 15 SRN members participated in the Annual SRN meeting. Their high-level understanding and enthusiasm about
the disc provoked interesting discussions and inspired participants, including new-found Chinese colleagues (see pages 4-5).
• Three SRN Exchange awards were granted to seven SRN members. Network members are using this funding opportunity to
begin translating several of their novel techniques from small animals to human discs or to models which are more humanlike. For example, a protocol for the application of thermoreversible hydrogels in human discs was developed. More detailed
information about these exchanges can be found on page 6.
We look forward to interacting with you especially during the 2015 Annual SRN meeting and Global Spine Congress in Buenos Aires,
Argentina, and through various collaborative efforts this year.
Sincerely,
Mauro Alini
SRN Representative
2
Niccole Germscheid
Research Project Manager
Table of Contents
Section Content
3
Page
1
Making Connections
1.1 3rd Annual Meeting of the SRN—Xi’an, China
4
1.2 Involvement of SRN Members at the WFSR 2014
5
2
SRN Awards 2014
2.1 SRN Exchange Awards and Achievements
6
2.2 SRN Consortium Award
7
3
In the NEWS
8
4
SRN Meetings in 2015
9
4.1 SRN Meeting 2015
4.2 GSC—Global Spine Congress 2015
5
Funding Opportunities
5.1 3rd SRN Exchange Award
5.2 AO Start-up Grants
6
SRN Members
6.1 New SRN Members
11
6.2 New AOSpine Research Manager
11
6.3 SRN Members as of January 2015
12
7
SRN Project Overview
13
8
Research Updates from SRN Members
9
Publications in 2014
9.1 Manuscripts
20
9.2 Oral Presentations
21
9.3 Poster Presentations
22
10
Upcoming Conferences in 2015/16
23
11
Stay Connected
24
12
Contact
25
10
14-19
1. Making Connections
1.1 3rd Annual Meeting of the SRN–Xi’an, China
Several SRN members participated
in the SRN 2014 annual meeting
which took place in Xi’an, China
prior to the WFSR—World Forum for
Spine Research. Each member had
an opportunity to share and present
their project progress and their most
recent discoveries and achievements,
as well as challenges. This resulted in
a steady flow of thought-provoking
discussions. Future network strategies
and initiatives were discussed. New
ideas, connections, and collaborations
have evolved from this meeting.
This annual meeting enables global
experts within a focused and
specialized field to connect on a
high level!
The SRN members: Lisbet Haglund, Kenneth Cheung, James Iatridis, Keita Ito, Björn Meji, Stephen Ferguson,
Barbara Chan, Marianna Tryfonidou, Sibylle Grad, Jaro Karpinnen, Daisuke Sakai, Danny Chan, Yvonne Wey, Dino Samartzis,
Fackson Mwale, Benjamin Gantenbein, Niccole Germscheid
Guests: Emerson Krock, Stefan de Vries, Cora Bow, Vivian Tam, Wilson Chan, Joyce Zhang
Kenneth Cheung, Marianna Tryfonidou,
Stefan de Vries, Wilson Chan, Björn Meij
Keita Ito, Daisuke Sakai, Kenneth Cheung,
Fackson Mwale, Cora Bow, Dino Samartzis
Keita Ito, Kenneth Cheung, Niccole
Germscheid, Lisbet Haglund, Cora Bow,
Dino Samartzis
Very inspiring meeting with high-level research
discussions about the intervertebral disc. This
meeting is a platform which spurs new ideas,
draws connections, and ignites collaborations.
Wilson Chan, Danny Chan,
Marianna Tryfonidou
4
1.2 Involvement of SRN Members at the
WFSR 2014 in Xi’an, China
A very big thank you goes to the following SRN members for helping to make the WFSR 2014 such a success!
Thank you!
Kenneth Cheung
Keita Ito
Chairperson
Chairperson
Presenter
Stephen Ferguson
Lecture Title
Type of
Presentation
Biomechanics and kinematics of the cervical disc
Invited Lecture
Björn Meij
Animal models and their utility
Invited Lecture
Lisbet Haglund
Bioreactor—how good are they?
Invited Lecture
Jaro Karppinen
Is low back pain a genetic problem?
Invited Lecture
Dino Samartzis
Is disc degeneration painful?
Invited Lecture
James Iatridis
Notochordal cell derived therapies for painful disc degeneration
Invited Lecture
Are there protective genes for disc degeneration?
Invited Lecture
Schmorls nodes, disc degeneration and pain
Invited Lecture
Annulus fibrosus repair
Invited Lecture
Roles of biomaterials in cell-based therapies for disc degeneration
Invited Lecture
The LINK between degeneration and regeneration of human intervertebral discs
Invited Lecture
Pulsed Electromagnetic Field (PEMF) stimulation of intervertebral disc cells
Invited Lecture
Danny Chan
Kenneth Cheung
Sibylle Grad
Barbara Chan
Fackson Mwale
Stephen Ferguson
Dino Samartzis
Family history of low back pain is a significant predictor of pain and disability between extreme stages of lumbar
disc degeneration
Oral Presentation
Lisbet Haglund
The ability of physiological load to restore disc function
Oral Presentation
Fackson Mwale
Effect of Link N on the expression of neurotrophins and substance P release by human intervertebral disc cells
stimulated with proinflammator cytokines
Short Talk
Dino Samartzis
Facet joint tropsim and degenerative spondylolisthesis—a study from the AOSAP Research Collaboration
Short Talk
Stephen Ferguson
Simulation of spinal loading: importance of subject-specific posture and
motion patterns
Short Talk
Marianna Tryfonidou
Increased osmolarity and cell clustering preserve canine notochordal cell
phenotype in culture
Short Talk
Benjamin Gantenbein
Co-culture of notochordal cells with nucleus pulposus and annulus fibrosus
cells under normoxia and hypoxia
Short Talk
5
2. SRN Awards 2014
2.1 SRN Exchange Awards and Achievements
The AOSpine Research Commission awarded three SRN Exchange awards in 2014. The purpose of the award is to maintain an
interdisciplinary network. It provides travel funds to SRN members to facilitate collaborative research and knowledge exchange
within the network.
In April 2014:
(1) Marianna Peroglio, Research Scientist in Sibylle Grad’s laboratory at the AO Research Institute in Switzerland, visited Lisbet
Haglund’s laboratory at McGill University in Montreal, Canada.
Achievement: A protocol for the application of thermoreversible hydrogel in human discs was developed. Training was
conducted about three different analytical techniques for the characterization of proteoglycans in intervertebral disc tissue
(1. agarose gel electrophoresis, 2. sodium dodecyl sulfate/ polyacrylaminde gel electrophoresis (SDS-PAGE) followed by
immunoblotting, and 3. column chromatography).
In November 2014:
(2) Zhen Li, Research Scientist with Sibylle Grad at the AO Research Institute in Davos, Switzerland, visited Daisuke Sakai’s
laboratory at the Tokai University School of Medicine in Tokai, Japan.
Objective: to teach the technique of isolating and analyzing a subpopulation of functional AF cells performed in Japan to
Switzerland and to transfer this knowledge from mouse to sheep and/or human AF cells. This exchange will take place from
April to May 2015.
(3) Marianna Tryfonidou, Björn Meij, and Keita Ito from the Netherlands will collaborate with Danny Chan from Hong Kong
and Daisuke Sakai from Japan.
Within this award, three small exchanges have been proposed:
Characterization and isolation of canine nucleus pulposus progenitor cells (NPPC)
I. Frances Bach, PhD candidate at Utrecht University in the Netherlands, will visit Danny Chan’s laboratory at The University of Hong Kong, where he will work with PhD candidate, Joyce Zhang.
Objective: to study the Tie2, GD2, and CD24 expression of nucleus pulposus cells within healthy canine intervertebral discs of chondrodystropic and non-chondrodystrophic dogs.
II.
Frances Bach will visit Daisuke Sakai’s laboratory in Tokai, Japan.
Objective: to set up the protocol for the isolation of canine NPPC (based on their Tie2/GD2/CD24 expression) by FACS and translate it for application at the Utrecht University facilities.
The relationship between caveolin-1 KO mice with SM/J and LG/J mice
III.
Joyce Zhang will visit Utrecht University where she will work in Marianna Tryfonidou’s laboratory.
Objective: to study the caveolin-1 expression pattern and its relationship to the Tie2/GD2 expression in chondrodystrophic and non-chondrodystrophic dogs. The gene expression profile of the caveolin-1 knockout mice
will be compared to the SM/J and LG/J mouse models studied at the laboratory of Danny Chan.
Congratulations to all of the recipients!
6
2.2 SRN Consortium Award
The AOSpine Research Commission had introduced and awarded an SRN Research Consortium Award. The purpose of the award is to
build upon the SRN Exchange Award, further strengthening relationships within the SRN as well as a focused-project, cultivating longterm collaborative research activities for the future. It provides financial support for one year to a group of SRN members to facilitate
innovative collaborative research about the intervertebral disc.
Congratulations to Dino Samartzis (The University of Hong Kong), Jaro Karppinen (University of Oulu, Finland), and Tugan Muftuler
(Medical College of Wisconsin, USA) who were awarded with a Consortium Award for their project proposal entitled, “SRN ImagingPhenotypes Consortium—Multi-parametric imaging analyses of Modic changes of the lumbar spine”. From an SRN Exchange Award
received a year ago, this group has been able to evolve their research.
The main objective of their proposal is to establish an SRN imaging-phenotypes consortium to develop, test, and acquire
multi-parametric magnetic resonance imaging (MRI) in patients with modic changes and to quantitatively assess degenerative disc
status of the lumbar spine.
This group has also been successful at obtaining additional local funding support for their endeavors.
For more detailed information, refer to section 3.
Results and achievements from this award will be available once complete.
SRN Consortium across the globe
Oulu
Wisconsin
FINLAND
USA
Hong Kong
HONG KONG
7
3. In the NEWS
March 2014
During the Orthopaedic Research Society Annual 2014 Meeting, SRN members
including Fackson Mwale, Daisuke Sakai, Sibylle Grad, and James Iatridis,
organized a Research Interest Group discussion about the Spine Research Community.
MORE INFORMATION
May 2014
During the Joint Annual Meeting ISMRM-ESMRMB 2014 in Milan, Italy, Tugan L.
Muftuler was presented with the first place award in musculoskeletal MRI
research (competitive).
July 2014
Dino Samartzis with his co-investigators, Jaro Karppinen, Sai Kam Hui, Kenneth
Cheung, Tugan L. Muftuler, Keith Luk, and Nozomu Inoue, established an
international consortium with investigators from Europe, Asia, and the USA to study
modic changes of the lumbar spine. They were awarded with a prestigious General
Research Fund grant from the RGC General Research Fund in Hong Kong.
MORE INFORMATION
September 2014
The Orthopaedic Research Society awarded Mauro Alini with the 2015 Marshall R.
Urist Award.
The Marshall R. Urist, MD Award was created in 1996 and is sponsored by the Journal
of Orthopaedic Research (JOR). This prestigious award honors an investigator who has
established him/herself as a cutting-edge researcher in tissue regeneration research and
has done so with a sustained ongoing body of focused research in the area of tissue
regeneration as it relates to the musculoskeletal system.
October 2014
Fackson Mwale and James Iatridis were both invited to a National Institute of
Arthritis and Musculoskeletal Skin Disease (NIAMS) round table expert discussion about
disc degeneration and neck and back pain.
8
MORE INFORMATION
4. SRN Meetings in 2015
4.1 SRN Meeting 2015
Our next meeting will take place on Sunday, 24 May 2015 in Buenos Aires,
Argentina immediately following the Global Spine Congress 2015.
Stay tuned for more information.
4.2 GSC—Global Spine Congress 2015
May 20-23, 2015, Buenos Aires, Argentina
REGISTER NOW
9
5. Funding Opportunities
5.1 3rd SRN Exchange Award
Application 2015
This award provides travel funds to SRN members to facilitate collaborative research and
knowledge exchange within the SRN about intervertebral disc research. Exchanges may
include the transfer of personnel, knowledge, and/or material.
Funding
• The maximum amount of requested funds should not exceed CHF 8’000 per
exchange.
• The exchange period may be spread over more than one visit.
• The exchange must occur before the end of 2015.
• Funding does not include salary costs or conference/symposium visits.
Eligibility
• Applicants must include at least one member of the SRN.
• The exchange can occur with a non-SRN member.
• The proposed research conducted during the exchange must be aligned with at least
one of the SRN research projects.
Deadline: April 20, 2015
Contact Information
AOSpine International
Research Department
Clavadelerstrasse 8
7270 Davos, Switzerland
Email: [email protected]
5.2 AO Start-up Grants
This grant opportunity is designed to encourage:
• Young investigators who are within five years of the completion of his/her terminal
degree (e.g., MD, DVM, PhD, etc.) or within five years of initiation of his/her first
assistant professor position at an academic institution. Individuals currently enrolled in
residency programs or fellowships are also considered young investigators.
• Experienced researchers submitting novel high-risk projects.
10
MORE INFORMATION
Deadline: July 15, 2015
6. SRN Members
6.1 New SRN Members
We are pleased to announce and welcome two new investigators who joined the SRN in 2014.
Jean Ouellet
Lorin Benneker
McGill University
Montreal, Canada
Department of Orthopaedic Surgery,
Inselspital, Bern, Switzerland
6.2 New AOSpine Research Manager
Eberhard Denk
It’s our great pleasure to announce that Eberhard Denk started as new
AOSpine Research Manager on October 1, 2014. In addition, Eberhard is
also managing the AO Foundation R&D Platform.
Eberhard, a German citizen living in Switzerland since 2001, was working as
Management Consultant for PwC (PricewaterhouseCoopers), where he was
also involved in the R&D review for the AO Foundation.
He holds the title of Doctor of Philosophy/Doctor of Natural Sciences from
the ETH Zurich, and Master of Science in Chemistry from the Universities
of Bayreuth & Leipzig as well as a pre-diploma in Business. After working
for ETH in Zurich for almost 5 years as a PhD student and consecutively as
a Post-Doc, he moved into Management Consulting (PwC) where he was
engaged as a Manager in the Life Sciences and Pharmaceuticals industry
sector.
11
6.3 SRN Members as of January 2015
Lastname
Firstname
Institute
Department
City
Country
Alini
Mauro
AO Research Institute (ARI)
Musculoskeletal Regeneration
Davos
Switzerland
Antoniou
John
McGill University / SMBD—Jewish
General Hospital
Surgery / Division of Orthopaedic
Surgery
Montreal
Canada
Barbosa
Mario
INEB—Instituto Engenharia Biomédica;
University of Porto
Biomaterials Division
Porto
Portugal
Benneker
Lorin
Inselspital
Department of Orthopaedic Surgery
Bern
Switzerland
Boos
Norbert
Prodorso
Center of Spinal Medicine
Zurich
Switzerland
Borthakur
Ari
University of Pennslyvania School of
Medicine
Radiology
Philadelphia
USA
Chan
Danny
The University of Hong Kong
Biochemistry
Hong Kong
Hong Kong
Chan
Barbara
The University of Hong Kong
Medical Engineering; Mechanical
Engineering
Hong Kong
Hong Kong
Cheung
Kenneth
University of Hong Kong, Queen Mary
Hospital
Orthopaedics and Traumatology
Hong Kong
Hong Kong
Erwin
W. Mark
University of Toronto, Toronto Western
Hospital
Surgery—Orthopaedic and Neurological Toronto
Ferguson
Stephen
ETH Zurich
Institute for Biomechanics
Zurich
Switzerland
Gantenbeinritter
Benjamin
University of Bern
Medical Faculty / Institute for Surgical
Technology and Biomechanics
Bern
Switzerland
Grad
Sibylle
AO Research Institute (ARI)
Musculoskeletal Regeneration
Davos
Switzerland
Haglund
Lisbet
McGill University—Orthopaedic
Research Laboratory
Orthopaedics, Surgery
Montreal
Canada
Iatridis
James
Mount Sinai School of Medicine
Orthopaedics
New York
USA
Ito
Keita
Eindhoven University of Technology
Biomedical Engineering, Orthopaedic
Biomechanics Section, Biomedic
Eindhoven
Netherlands
Karppinen
Jaro
University of Oulu
Physical and Rehabilitation Medicine
Oulu
Finland
Klineberg
Eric
University of California, Davis
Orthopaedic Surgery
Sacramento
USA
Leong
Kam
Duke University
Biomedical Engineering; Surgery
Durham
USA
Meij
Björn
Utrecht University, Faculty of Veterinary
Medicine
Clinical Sciences of Companion
Animals
Utrecht
Netherlands
Muftuler
Tugan l.
Medical College of Wisconsin
Neurosurgery
Milwaukee
USA
Mwale
Fackson
McGill University
Surgery
Montreal
Canada
Ouellet
Jean
McGill University
Orthopaedic Surgery
Montreal
Canada
Potier
Esther
Eindhoven University of Technology
Biomedical Engineering
Eindhoven
Netherlands
Sakai
Daisuke
Tokai University School of Medicine
Orthopaedic Surgery
Isehara
Japan
Samartzis
Dino
University of Hong Kong
Orthopaedics and Traumatology
Pokfulam
Hong Kong
Steffen
Thomas
McGill University—Orthopaedic
Research Laboratory
Orthopaedic Surgery
Montreal
Canada
Tryfonidou
Marianna
Utrecht University, Faculty of Veterinary
Medicine
Clinical Sciences of Companion
Animals
Utrecht
Netherlands
Würtz
Karin
ETH Zurich
D-HEST
Zurich
Switzerland
12
Canada
7. SRN Project Overview
Table 1: SRN projects supported in 2014
Investigator
Center
Tugan Muftuler
Medical College of
Wisconsin
Danny Chan
The University of Hong
Kong
Daisuke Sakai
Tokai University
Keita Ito
Eindhoven University
of Technology
Project Title
Research Area
End Date
Grant Type
Project
Phase
Quantitative MRI of the intervertebral
disc degeneration
Disc Degeneration:
MRI Diagnostics
Aug 2015
(SRN ‘12—Single Center)
Execution
Discovering the genetics, understanding
progenitor cell niche and transcriptional
controls to harness the endogenous
regeneration potentials of the
intervertebral disc
Disc Regeneration:
Genetics and Endogenous
Progenitor Cells
Sept 2015
(SRN ‘12—Consortium)
Execution
Notochordal cell technology for
intervertebral disc regeneration
Disc Regeneration:
Notochordal Cells
Aug 2016
(SRN ‘11—Consortium)
Execution
Biological repair of the degenerate
intervertebral disc
Disc Regeneration:
Mechanobiology​
Sept 2016
(SRN ‘11—Single Center)
Execution
Biomaterial-assisted cell-based therapy
for disc degeneration
Disc Regeneration:
Biomaterials and Stem Cells
Oct 2016
(SRN ‘11—Consortium)
Execution
Björn Meij
Utrecht University
Lisbet Haglund
McGill University
Barbara Chan
The University of Hong
Kong
For more complete project descriptions please click:
13
FULL INFORMATION
8. Research Updates from SRN Members
Biomaterial-assisted Cell-based Therapy for Disc
Degeneration
Barbara Chan The University of Hong Kong, Kam Leong Duke University, Durham, USA
We developed a biomimetic biphasic scaffold consisting of a collagen-glycosaminoglycan co-precipitate as the nucleus pulposus-like
core, which was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes as the annulus fibrosuslike lamellae (Fig. 1). Upon mechanical test, the disc height of the biphasic scaffolds recovered in an annulus-independent manner,
suggesting the fluid replacement function of the nucleus pulposus core that mimics the unique feature of native disc. Biphasic scaffold
with 10 AF-like lamellae has the best overall mechanical performance, showing similarity with that of the native disc. The dynamic
mechanical performances of all biphasic scaffolds are similar to that of the native discs.
Figure 1
We applied microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid
cell assembly and well-controlled microenvironment for MSC spheroid culture. A molecular weight-dependent diffusion of molecules
out from the core was observed. Molecules with lower molecular weight (MW~ 400 Da) were able to diffuse across the oil layer while
larger molecules (MW~70 k Da) were trapped within the droplets. Among molecules with similar molecular weight, their oil/water
partition coefficient determines the permeability. Cells aggregated to form size-controllable (30–80 µm) spheroids in DE droplets within
150 min and could be retrieved via a droplet-releasing agent (Fig. 2). This rapid, versatile and scalable MSC assembly technology should
help advance the field of IVD tissue engineering.
Figure 2
14
The regenerative effect of notochordal
cell-conditioned medium
F.C. Bach, B.P. Meij, M.A. Tryfonidou.
Dept. of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, the Netherlands
S.A.H. de Vries, K. Ito.
Department of Biomedical Engineering, Eindhoven University of Technology, the Netherlands
A. Krouwels, L.B. Creemers, K. Ito.
Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands
Notochordal cells (NCs) secrete factors,
which can modulate the synthetic activity
of mature nucleus pulposus cells (NPCs),
making them an interesting focus for
regenerative strategies. In developing such
strategies, studies concentrate on such
factors produced from various animal
species that maintain their NCs during
life, e.g. specific canine breeds and pigs,
as opposed to humans that loose the
typical vacuolated NCs early in life. Our
first aim was to delineate whether such
secreted factors from NC-rich nucleus
pulposus (NP) tissue of different species
have a differential regenerative effect
on mildly degenerated human nucleus
pulposus cells (NPCs). Furthermore,
the stimulatory effect of NC conditioned
medium (NCCM) has so far only been
demonstrated in alginate bead and pellet
cultures. However, these methods provide
a markedly different environment from
the native NP tissue, where we ultimately
want the NC-secreted factors to have a
regenerative effect. Our second aim was
therefore to verify the stimulatory effect of
NCCM in NP tissue.
For the first aim, healthy NP tissue of
the canine (18-23 months), porcine
(3 months), and human (fetal) species
was cultured for 4 days and NCCM
was collected. Human NPC micro-
aggregates from mildly degenerated
IVDs were cultured in human, canine, or
porcine NCCM for 28 days. There were
distinct species-specific histological and
biochemical differences between healthy
NP tissue (figure 1). NCCM from all tested
species significantly increased the DNA
content and extracellular matrix production
compared with untreated human NPCs,
but porcine (3.7 ± 0.3 µg GAG/aggregate)
and canine (3.8 ± 0.3 µg GAG/aggregate)
NCCM was significantly more potent
than human NCCM (2.0 ± 0.3 µg GAG/
aggregate).
Figure 1: Cross-species differences of healthy nucleus pulposus (NP) tissue: healthy canine and porcine NPs contained
more notochordal cells with the typical vacuolated appearance than fetal human NPs.
Continued...
15
For the second aim, bovine NP tissue samples were harvested from the caudal discs of 24-month-old cows and cultured in a fiber
jacket that constrains the sample and therefore acts as an artificial annulus fibrosus. Samples were cultured in hypoxic culture conditions
in either base medium, porcine NCCM, or with addition of Link N (used as a positive control). After 4 weeks, the proteoglycan content
in the culture with porcine NCCM (10.0 ± 1.7 %ww ) had, similar to Link N (8.7 ± 1.4 %ww), increased significantly compared to day
0 (5.9 ± 1.9 %ww), and compared to the culture with base medium (5.8 ± 1.9 %ww). These findings were verified by Safranin O/
Fast Green staining (figure 2). No differences in water, DNA, and hydroxyproline content were observed between groups.
Altogether, these studies indicate that secreted factors from notochordal
NP tissues can have a regenerative effect. The matrix anabolic effect in
the NP explant culture system mimicking the native NP environment
indicates that even when sufficient matrix is present, NCCM can still
have an additional effect. In addition, this non-optimized effect can be
at least as potent as Link-N, of which the stimulatory effect on NP tissue
has been previously established in vivo.
The cross-species effect of NCCM on mildly degenerated human
NPCs indicates that distinct agents are produced by most NCs that
have a common stimulatory activity conserved across species. Given
that human NCCM was less potent than canine and porcine NCCM
generated under identical conditions, strategies based on NC-technology
employing canine or porcine animal models have a good potential for
successful translation into humans. Ongoing studies concentrate on
how NCs communicate with NPCs to achieve these effects and further
identification of the bioactive factors.
Figure 2: A Safranin O/Fast Green staining showed
a more intense red staining in the NCCM and Link N
groups, compared with Day 0 and Base medium groups,
indicating a higher proteoglycan content.
Quantitative MRI study of
intervertebral disc degeneration
Tugan Muftuler
Institution: Medical College of Wisconsin, Milwaukee, USA
We studied the structural and functional changes in degenerating intervertebral discs using a combination of new MRI techniques
and image measurement methods. We developed a normative database of healthy discs and compared the changes in degenerating
discs against this database. We observed major changes in disc morphology and physiology when the loss of disc height was beyond
1.5 standard deviations. For instance, Fig.1 shows a sharp drop in Apparent Diffusion Coefficient and Fig.2 shows a similar trend in T2
weighted MRI signal inside the nucleus pulposus when the disc height loss exceeds 1.5 standard deviations. This work was recently
published in European Spine Journal. We also demonstrated major changes in disc endplate diffusion and perfusion with increasing
degeneration. These preliminary findings were also published in European Spine Journal. In addition, our dynamic contrast enhance
MRI technique, combined with T1rho imaging method appeared to be more sensitive to degenerative endplate changes compared to
conventional T1 and T2 weighted MRI.
Figure 1. Histogram of ADC values with decreasing disc height index
16
Figure 2. Histogram of T2 weighted signal intensity values with
decreasing disc height index
Biological Repair of the Degenerate
Intervertebral Disc
Lisbet Haglund, Thomas Steffen, Jean Ouellet, and Peter Roughley
Institution: Mc Gill University, Montreal, Canada
Our group has developed organ
culture systems and bioreactors where
we can study the effect of nutrient
supply and mechanical stimulation on
disc metabolism. We demonstrated
that physiological load could restore
proteoglycan (PG) content after depletion
when loaded under sufficient nutrient
conditions. Experiments were then
performed at glucose concentration
insufficient for spontaneous proteoglycan
recovery in the loaded discs. Following PG
depletion, the content in the NP region
was about 28% lower in discs cultured
and loaded under nutrient deprivation
compared to those with a sufficient
glucose concentration. In addition to
preventing spontaneous repair, nutrient
deprivation in combination with PG
depletion and loading also consistently
resulted in cleft formation in the NP region
of the discs. The fact that we find cleft
formation under nutrient deprivation more
accurately represents degeneration seen in
human discs. We are currently evaluating if
it is possible to stimulate repair by adding
bioactive substances and stem cells in this
nutrient-deprived environment. The culture
system has also been used to evaluate the
potential for cell based therapies in loaded
human and bovine discs.
Human NP cells in HA-pNIPAN hydrogels
(HA-pNIPAM, kindly provided by Dr Eglin
at ARI) have been injected into the center
of human IVDs. The injected fluorescently
labeled cells were tracked using confocal
microscopy. The results show that it is
feasible to inject cells in a hydrogel and
culture IVDs for 14 days under dynamic
load.
Red fluorescent transplanted cells labeled with Vybrant DiI cell labeling
solution were found dispersed throughout the IVD.
17
Discovering, understanding and harnessing the repair and
regenerative potentials of the intervertebral disc
Danny Chan. The University of Hong Kong, Hong Kong, China
Daisuke Sakai. Tokai University School of Medicine, Isehara, Japan
Genetics and cell function are related key
factors influencing disc homeostasis. The
outcomes could be degeneration,
homeostasis or protection. This SRN
project focuses on genetic factors with
positive outcomes for disc maintenance
and impact on progenitor cells for repair or
regeneration of the disc. Taking advantage
of mouse genetics and the availability of
different strains of mice with varying tissue
healing potentials, we have identified poor
healer strains (C57 and SM/J) prompt for
disc degeneration in postnatal life as early
as 2-4 weeks, and good healer strains
(MRL and LG/J) that can maintain a health
disc for much of their postnatal life. We
have studied the degenerative and
protective outcomes of these mice in
relation to the different pools of cells in
the disc, from progenitors to mature
nucleus pulposus cells (1), with
differences in the maintenance of cells
with markers that are Tie2 negative and
GD2 positive (Tie2-/GD2+), that we
postulate as the key cell pool for a healthy
disc. Using histological changes in these
and other strains of mice, we have
developed a “quantitative” measure of
disc degeneration (2) that we are using
for the mapping of genes for disc
degeneration and protection, taking
advantage of the available genetic data
from the parental as well as established
recombinant inbreeds of the parental
strains. We showed that the good healer
LG/J mice indeed processes protective
genetic influences against known genetic
risk factors such as the Asporin (ASPN)
gene in transgenic mouse models that we
have generated (3).
The relevance of these genes will be
mapped to human genetic data that we
have from GWAS as well as whole exome
sequencing of a Hong Kong population
cohort for the study of intervertebral disc
degeneration. The impact on cell function
in relationship to the two key cell markers
of nucleus pulposus cell pools is being
studied concurrently with the isolation of
specific pools of NP cells that are Tie2+/
GD2+/CD24-, Tie2-/GD2+/CD24-, and
Tie2-/GD2-/CD24+, and gene expressing
profiling (micro-array and RNA
sequencing) are in progress with the aim
to identify key transcriptional and signaling
networks for cell function and
maintenance. Furthermore, these human
cells are being tested for repair potentials
in a rabbit model with disc degeneration
induced by a needle puncture with
promising preliminary findings. We also
envisage the information gained will allow
us to determine factors that can direct
differentiation or reprogramming of
somatic cells to generate appropriate
progenitors specific for disc regeneration,
providing the appropriate environment
and ready-to-go progenitor cells to mount
a regenerative process.
Tail disc of 4-week-old mice (Differences in disc integrity)
LG/J (Good healer)
SM/J (Poor healer)
1. Sakai, D., Nakamura, Y., Nakai, T., Mishima, T., Kato, S., Grad, S., Alini, M., Risbud, M. V., Chan, D., Cheah, K. S., Yamamura,
K., Masuda, K., Okano, H., Ando, K., and Mochida, J. (2012) Exhaustion of nucleus pulposus progenitor cells with ageing
and degeneration of the intervertebral disc. Nat. Commun. 3, 1264
2. Tam, V., Chan, W.C.W, Sakai, D., Cheah, K.S.E., Cheung, K.M.C. and Chan, D. (2105) A systematic histological scoring system
for the grading of mouse intervertebral disc integrity (manuscript in submission)
3. Lee, A., Yip, S. Chan, W.C.W., Sakai, D., Cheah, K.S.E. and Chan D (2015) Asporin induces intervertebral disc degeneration
in transgenic mice through activation of TGFβ signaling (manuscript in submission)
18
Activation of intervertebral disc cells by
co-culture with notochordal cells, conditioned
medium and hypoxia
Benjamin Gantenbein, Elena Calandriello, Samantha CW Chan. Tissue & Organ Mechanobiology, Institute for Surgical Technology
and Biomechanics, University of Bern, Bern, Switzerland
Benjamin Gantenbein, Karin Wuertz-Kozak. Competence Center for Applied Biotechnology and Molecular Medicine, University of
Zurich, Zurich, Switzerland
Benjamin Gantenbein, Karin Wuertz-Kozak, Lorin M Benneker. AOSpine Research Network, Duebendorf, Zurich, Switzerland
Marius JB Keel. Institute for Biomechanics, ETH Zurich, Zurich, Switzerland & Department for Orthopaedic Surgery, Inselspital, University
of Bern, Bern, Switzerland
Samantha CW Chan. Bioactive materials, EMPA, Swiss Federal Laboratories for Materials Science and Technology, St Gallen, Switzerland
Background:
Notochordal cells (NC) remain in the
focus of research for regenerative therapy
for the degenerated intervertebral
disc (IVD) due to their progenitor
status. Recent findings suggested their
regenerative action on more mature disc
cells, presumably by the secretion of
specific factors, which has been described
as notochordal cell conditioned medium
(NCCM). The aim of this study was to
determine NC culture conditions (2D/3D,
fetal calf serum, oxygen level) that lead to
significant IVD cell activation in an indirect
co-culture system under normoxia and
hypoxia (2% oxygen).
Methods:
Porcine NC was kept in 2D monolayer and
in 3D alginate bead culture to identify a
suitable culture system for these cells. To
test stimulating effects of NC, co-cultures
of NC and bovine derived coccygeal IVD
cells were conducted in a 1:1 ratio with no
direct cell contact between NC and bovine
nucleus pulposus cell (NPC) or annulus
fibrosus cells (AFC) in 3D alginate beads
under normoxia and hypoxia (2%) for 7
and 14 days. As a positive control, NPC
and AFC were stimulated with NC-derived
conditioned medium (NCCM).
Results:
We provide evidence by flow cytometry
that monolayer culture is not favorable
for NC culture with respect to maintaining
NC phenotype. In 3D alginate culture, NC
activated NPC either in indirect co-culture
or by addition of NCCM as indicated by
the gene expression ratio of aggrecan/
collagen type 2. Mass spectrometry
identified connective tissue growth factor
(CTGF, syn. CCN2) in the NCCM.
Conclusions: Our results confirm the
requirement to culture NC in 3D to best
maintain their phenotype, preferentially in
hypoxia and with the supplementation of
FCS in the culture media. Despite these
advancements, the ideal culture condition
remains to be identified.
Figure 1: Experimental set-up to test NC regenerative effects on co-culture with NPC or AF that were stimulated initially in pre-culture
with and without 10% fetal calf serum (FCS) and subsequent co-culture or exposure with conditioned medium (NCCM) from step 1.
The design also involved a comparison of normoxia (~20% oxygen) and hypoxic conditions (2% oxygen).
19
9.Publications in 2014
9.1 Manuscripts*
*Please note that some publications may not appear in the list below. These publications are derived from SRN project grants.
1. Bach FC, Willems N, Penning LC, Ito
K, Meij BP, Tryfonidou MA: Potential
regenerative treatment strategies for
intervertebral disc degeneration in dogs.
BMC Vet Res 2014; 10:3
2. Potier E, de VS, van DM, Ito K:
Potential application of notochordal
cells for intervertebral disc regeneration:
an in vitro assessment. Eur Cell Mater
2014; 28:68-80
3. Spillekom S, Smolders LA, Grinwis
GC, Arkesteijn IT, Ito K, Meij BP,
Tryfonidou MA: Increased osmolarity
and cell clustering preserve canine
notochordal cell phenotype in culture.
Tissue Eng Part C Methods 2014;
20:652-662
4. Alkhatib B, Rosenzweig D, Krock
E, Ouellet J, Weber M, Beckman L,
Roughley P, Steffen T, Haglund L.
Acute mechanical loading of the IVD:
Link to Degeneration and Pain. Eur Cell
Mater. 2014 Sep 12;28:98-110
5. Mulligan KR, Gawri R, Steffen T,
Pike GB, Jarzem P, Borthakur A,
Haglund L, Ouellet J. Axial T1ρ MRI
as a diagnostic imaging modality to
quantify proteoglycan concentration in
degenerative disc disease. Eur Spine J.
2014 Sep 19. [Epub ahead of print]
20
6. Gawri R, Moir J, Ouellet J, Beckman
L, AL Thukair R, Steffen T, Roughley
P, Haglund L. Physiological loading
can restore the proteoglycan content
in Research a model of early IVD
degeneration. PLoS One. 2014 Jul
3;9(7):e101233
10. G. Pattappa, M. Peroglio, D.
Sakai, J. Mochida, L.M. Benneker,
M. Alini, S. Grad. CCL5/Rantes is
a key chemoattractant released by
degenerative intervertebral discs in
organ culture. EuropeanCells and
Materials Vol. 27, 2014 (pages124-136)
7. Gawri R, Ouellet J, Onnerfjord P,
Alkhatib B, Heinegard D, Steffen T,
Antoniou J, Mwale F, Haglund L. Link
N is cleaved by annulus fibrosus cells
generating a product with retained
biological activity. J Orthop Res. 2014
Sep;32(9):1189-97
11. Jarman JP, Arpinar VE, Baruah
D, Klein AP, Maiman DJ, Muftuler
LT. Intervertebral disc height loss
demonstrates the threshold of
major pathological changes during
degeneration. Eur Spine J. 2014 Sep 12.
[Epub ahead of print]
8. Mwale F, Wang HT, Roughley P,
Antoniou J, Haglund L. Link N and
MSCs can induce regeneration of the
early degenerate intervertebral disc.
Tissue Eng Part A. 2014 Nov;20(2122):2942-9
12. Muftuler LT, Jarman JP, Yu HJ, Bahri
S, Gardner VO. Association Between
Intervertebral Disc Degeneration and
Endplate Perfusion Studied by DCE-MRI.
Eur Spine J. 2014 Nov 25. [Epub ahead
of print]
9. Li YY, Diao HJ, Chik TK, Chow CT, An
XM, Leung V, Cheung KM, Chan BP:
Delivering mesenchymal stem cells in
collagen microsphere carriers to rabbit
degenerative disc: reduced risk of
osteophyte formation. Tissue Eng Part A
2014;20:1379-1391
9.2 Oral Presentations 2014*
*Please note that some presentations may not appear in the list below. These publications are derived from SRN project grants.
1. Potier E, de Vries S, Tryfonidou M, Ito K.
Notochordal cells for intervertebral disc regeneration: what is
their most effective application?
WFSR 2014—World Forum for Spine Research, Xi’an,
China. May 2014.
2. Haglund, L.
Intervertebral Disc Degeneration and Pain.
Anatomy and Cell Biology Retreat, McGill University, Mont
Saint Gabriel, Quebec, Canada. May 2014.
3. Haglund, L.
Bioreactors; how good are they.
WFSR 2014—World Forum for Spine Research, Xi’an China.
May 2014.
4. Haglund, L.
The Ability of Physiological Load to Restore Disc Function.
WFSR 2014—World Forum for Spine Research, Xi’an, China.
May 2014.
Danny Chan
5. Chooi W, Chan S, Gantenbein B, Chan B.
Cellular stress response of intervertebral cells under
mechanical loading.
Molecular Chaperones & Stress Responses, Cold Spring
Harbor Laboratory, NY, USA. 2014.
6. Zhang J, Xiong S, Chan W, Tam V, Sakai D, Chan D.
Are there protective genes for Disc Degeneration?
WFSR 2014—World Forum for Spine Research, Xi’an, China.
May 2014.
7. **Muftuler LT, Jarman JP, Maiman DJ, Yu HJ, Gardner
VO. Intervertebral disc degeneration and changes in solute
transport mechanisms in disc endplates studied by DCE-MRI.
ISMRM—Annual meeting of the International Society of
Magnetic Resonance in Medicine, Milan, Italy. May 2014.
Lisbet Haglund
21
** 1st place award in the Musculoskeletal group
workshop
9.3 Poster Presentations 2014*
*Please note that some presentations may not appear in the list below. These publications are derived from SRN project grants.
1. Alkhatib BG, Rosenzweig DH, Krock E, Gawri R, Weber MH, Ouellet
JA, Haglund L.
Acute Mechanical Injury To The Human Intervertebral Disc Initiates Events
Implicated In Disc Degeneration.
ORS—Orthopedic Research Society Annual Meeting, New Orleans, USA,
March 2014.
2. Mwale F, Wang HT, Roughley P, Antoniou J, MD, Haglund L.
The effects of Link N and bone marrow-derived mesenchymal
stem cells on the regeneration of intervertebral disc.
ORS—Orthopedic Research Society Annual Meeting, New Orleans, USA,
March 2014.
3. De Vries S, Potier E, Van Doeselaar M, Meij B, Tryfonidou M, Ito K.
Stimulation of canine nucleus pulposus cells and bone marrow
derived stromal cells with notochordal cell-secreted factors.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
4. Alkhatib BG, Rosenzweig DH, Krock E, Gawri R, Beckman L,
Steffen T, Weber MH, Ouellet JA, Haglund L.
Acute Mechanical Injury To The Human Intervertebral Disc
Initiates Events Implicated In Disc Degeneration.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
5. Chik T, Chooi W, Cheng H, Choy T, Sze K, Luk K, Cheung K, Chan B.
Engineering a multi-component spinal motion segment-like
construct from mesenchymal stem cells.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
6. Chooi W, Chan S, Gantenbein B, Chan B.
Loading induced stress response in the intervertebral disc.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
7. Chan WCW, Tam V, Cheah K, Cheung K, Chan D.
A systematic and simple scoring system for histological analysis of
the mouse intervertebral disc during natural ageing, degeneration,
and regeneration.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
8. Lee KS, Lam K, Song YQ, Cheah K, Cheung K, Chan D.
In-vivo study of asporin in cartilage tissues.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
22
9. Yee A, Tam V, Cheah K, Cheung K, Chan D.
Analysis of the static protein profile of degenerated IVD indicate
increased fibrosis-related proteins.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
10. Zhang Y, Xiong C, Chan C, Sakai D, Chan D.
Roles of progenitor cells for intervertebral disc regeneration in
“healer” mice.
WFSR 2014—World Forum for Spine Research, Xi’an, China. May 2014.
11. Arpinar VE, Maiman DJ, Muftuler LT.
Pharmacokinetic analysis of DCE-MRI data from lumbar spine
reveals pathologic changes in intervertebral disc endplates and
subchondral bone.
ISMRM—Annual meeting of the International Society of Magnetic
Resonance in Medicine, Milan, Italy. May 2014.
12. Jarman JP, Maiman DJ, Muftuler LT.
Accurate Measurement of Intervertebral Disc Height Loss
Demonstrates the Threshold of Major Pathological Changes
During the Course of Degeneration.
ISMRM—Annual meeting of the International Society of Magnetic
Resonance in Medicine, Milan, Italy. May 2014.
13. Muftuler LT, Baruah D, Klein AP.
T1rho imaging demonstrates inflammatory changes in disc
endplates that were not visible in T1 or T2 weighted images.
ISMRM—Annual meeting of the International Society of Magnetic
Resonance in Medicine, Milan, Italy. May 2014.
14. Muftuler LT, Jarman JP, Yu HJ, Maiman DJ, Gardner VO.
Intervertebral disc degeneration and changes in solute transport
mechanisms in disc endplates studied by DCE-MRI.
ISMRM—Annual meeting of the International Society of Magnetic
Resonance in Medicine, Milan, Italy. May 2014.
15. Bach F, Verdonschot L, Ito K, Meij B, Tryfonidou M.
The role of caveolin-1 in IVD degeneration and regeneration.
1st Matrix Biology Europe Conference, Rotterdam, Netherlands.
June 2014.
16. De Vries S, Potier E, Van Doeselaar M, Meij B, Tryfonidou M, Ito K.
Notochordal cell-secreted factors stimulate matrix production
by canine nucleus pulposus cells and bone marrow derived
stromal cells.
ISSLS conference, Seoul, South Korea. June 2014.
10. Upcoming Conferences in 2015/16
2015
March 28 – 31, 2015
ORS—Orthopaedic Research Society Annual Meeting
Las Vegas, USA
Abstract Submission Deadline has passed.
June 17 – 20, 2015
COA Annual Meeting
Vancouver, Canada
Abstract Submission Deadline has passed.
April 25 – 28, 2015
European Calcified Tissue Society
Rotterdam, The Netherlands
Abstract Submission Deadline has passed.
July 08 – 11, 2015
IMAST—International Meeting on Advanced Spine Techniques
Kuala Lumpur, Malaysia
Abstract Submission Deadline: Feb 01, 2015.
May 20 – 23, 2015
GSC—Global Spine Congress
Buenos Aires, Argentina
Abstract Submission Deadline has passed.
September 08 – 11, 2015
TERMIS—Tissue Engineering and Regenerative Medicine International
Society—World Congress
Boston, MA, USA
Abstract Submission Deadline: March 2015.
2016
March 05 – 08, 2016
ORS—Orthopaedic Research Society Annual Meeting
Orlando, FL, USA
Abstract Submission Deadline: TBD.
April 2016
Combined Global Spine Congress and World Forum for Spine Research
Dubai, UAE
Abstract Submission Deadline: TBD
May 27 – 29, 2015
EFORT—European Federation of National Associations of Orthopaedics
and Traumatology Congress
Prague, Czech Republic
Abstract Submission Deadline has passed.
May 28 – 30, 2015
Canadian Connective Tissue Conference
Québec City, Canada
Abstract Submission Deadline: TBD.
May 30 – June 05, 2015
ISMRM—International Society for Magnetic Resonance in Medicine
Toronto, Canada
Abstract Submission Deadline has passed.
June 24 – 26, 2015
eCM XVI: Implant Infection (Main focus orthopedic and trauma related
infections)
Davos, Switzerland
Abstract Submission Deadline: mid-April 2015.
23
11. Stay Connected
The AOSpine website provides SRN members with secure access to an open Forum where only SRN
members can exchange information (comments and documents) online.
To access the forum you willl first have to log in with your AOSpine account. If you click on “myAOSpine”
on the right hand navigation window, it displays the link to “Forums”.
LOGIN PAGE
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To access the forum you willl first
have to log in with your AOSpine
account. If you click on “myAOSpine”
on the right hand navigation window,
it displays the link to “Forums”.
12. Contact
SRN Management Support Team
Niccole Germscheid
Yvonne Wey
Research Project Manager
[email protected]
Research Event Project Coordinator
[email protected]
If anyone has other inspiring publications,
ideas, stories, and/or news items about the
SRN, let us know!
25
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