Prof. dr. M.A. (Marianna) Tryfonidou

Prof. dr. H. Jakobgebouw
Yalelaan 108110
Kamer 1.076
3584 CM Utrecht

Prof. dr. M.A. (Marianna) Tryfonidou

Professor
Surgery
+31 30 253 4558

Current research focus

                                                 
My research focuses on developing treatment strategies for neck and back pain of canine and human patients in close collaboration with academia from the medical and bioengineering field. Pain in dogs and man is strongly associated with intervertebral disc (IVD) degeneration. Patients with late stage IVD degeneration that are refractory to pain medication can only be surgically treated. Surgery aims at stabilizing the spinal segment in order to relieve pain. This is primarily done both in dogs and humans by removing the IVD and fusing the two vertebrae by means of bone regeneration. These surgical treatment strategy demand long term recovery, does not lead to full recovery, and does not support a biological and hence functional repair of the diseased spine. In order to prevent the need for heavy demanding surgery, new treatment strategies concentrate on regenerating the degenerated IVD at earlier stages of the disease (Figure 1).

I address the challenges within this field by employing unique spontaneous diseased canine models, the use of cutting edge biomolecular techniques and strong implementation of the 3Rs. In this respect, the dog serves as a preclinical model for translation towards the “first in man” studies, while it serves also the veterinary patient. As a veterinary orthopedic specialist working in a referral animal university clinic I encounter frequently canine patients with neurologic deficits due to disc disease. The canine species is considered to be a suitable model to study the process of IVD degeneration: like humans, dogs suffer from spontaneous IVD degeneration, and the degenerative process involves similar macroscopic, histopathological, and biochemical changes as humans.

I have developed a preclinical platform employing the Beagle dog as an experimental animal with spontaneous IVD degeneration (Figure 2). Randomized block design strengthen the power of the studies (Reduction) and longitudinal follow up of the animals is done by modern imaging techniques (Magnetic Resonance Imaging and Computer Tomography) and objective gait analysis (force plate). From each IVD, post mortem analysis is performed by histology, biomolecular and biochemical analysis (Refinement). In collaboration with the specialists of the Neurology and Neurosurgery Center of the university clinic I have set up and maintain a biobank that enables valuable in vitro fundamental and translational studies (Replacement). The biobank contains mesenchymal stromal cells (MSCs), joint cartilage, nucleus pulposus, and annulus pulposus cells from degenerated and healthy IVDs derived from canine patients and canines that have been euthanized in unrelated experiments. Through close collaboration with the dept. of Orthopedics of the University Medical Center Utrecht, I also have access to human tissues and cells to complete the translation process towards a clinical application.

Completed Projects
Project
A novel biomimetic artificial intervertebral disc (BioAID) 01.01.2019 to 31.12.2022
General project description

We believe that fusion and current disc prostheses have failed to meet their promise because they do not restore the natural behavior of the disc, which is only one part of the complex structure in our spines. The natural disc is a complex structure that deforms, absorbs and stores energy, working together with other spinal elements. Recently, we created a novel prosthesis (BioAID) mimicking the structure and deformation properties of the natural disc, i.e. a biomimetic disc. The novelty of this biomimetic disc lies in the fact that it is composed of a hydrogel core restricted by a stiff fiber jacket, creating overall mechanical properties which are more similar to the natural intervertebral disc. Although the principal and performance of the design was demonstrated, biomimetic integration as well as in vivo proof-of-concept remain necessary for acceptance of this technology and availability of a truly biomimetic artificial intervertebral disc to alleviate back/neck pain.

The aim of the project is to demonstrate that a completely biomimetic artificial disc can be produced, become incorporated into adjacent vertebrae and return proper function to the spine. To this end, a 2nd generation prototype biomimetic artificial disc has been machine manufactured using current technology and designed according to medical device standards. The main objective of the project is to demonstrate proof of concept of primary stability and induction of biomimetic integration of the 2nd generation artificial disc with adjacent vertebrae. A secondary outcome will be the integrity of the hydrogel core within the biomimetic prosthesis.

Role
Project Leader & Supervisor
Individual project description

We contribute to the project with a preclinical translational study employing the canine model to demonstrate proof of concept and primarily feasibility of the BioAID prosthesis. For this purpose, cervical disc replacement will be conducted on one location of the caudal cervical spine, which is commonly affected in clinical disease and easily accessible. In order to determine the onset time and location of osteogenesis, a set of three different fluorochromes will be used as a labeling method within the period of (2-10 weeks). Follow up upon recovery will be conducted with periodic neurologic examinations to monitor clinical outcome and radiographic imaging. The potential efficacy of this novel BioAID will be studied by obtaining plain radiograph and/or dynamic imaging with the aid of computed tomography (CT) scan images in a longitudinal manner. Extensive post-mortem analysis includes: contact radiography of plastic-embedded thick tissue sections and micro-CT scan analysis to evaluate the osseointegration between vertebral body and BioAID. Furthermore, part of the harvested implanting site (vertebra+BioAID), will be fixed and thereafter subjected to histopathological evaluation via analysing the polymethylmethacrylate (i.e. evaluation of osseointegration and following the fluorochromes for osteogenesis) and decalcified paraffin (collagen infiltration, immunohistochemistry) sections.

Funding
NWO grant NWO TTW Open
External project members
  • dr. A. Abdelgawad (Maastricht University); dr. S. Ghazanfari (Maastricht University); PhD candidate Celien A.M. Jacobs (TU Eindhoven); Prof. S. Jockenhövel (Aachen University); dr. H. Smelt (DSM)
Project
P15-23 William Hunter Revisited: Activating intrinsic cartilage repair to restore joint homeostasis 01.03.2017 to 31.03.2022
General project description

In his famous textbook “On the diseases of articulating surfaces” published in the year 1743, William Hunter wrote “If we consult the standard Chirurgical Writers from Hippocrates down to the present Age, we shall find, that an ulcerated Cartilage is universally allowed to be a very troublesome Disease; that it admits of a Cure with more Difficulty than carious Bone; and that, when destroyed, it is not recovered”. Up until now this sentence still dictates the vision of orthopedic surgeons how to deal with joint disorders like osteoarthritis and cartilage trauma.


Recent scientific developments have challenged the view of William Hunter. First, distraction of the joint for just 6 weeks is sufficient to stimulate complete regrowth of an eroded articular cartilage surface in animal models and human patients. Second, resident stem cell populations have been identified in joint tissue such as cartilage, the bone marrow compartment below the subchondral bone plate, the synovial membrane and freely floating in the synovial fluid. Evidence shows that these cells can migrate to cartilage defects and can be activated to induce a regenerative response. Indeed, most likely by exploiting the regenerative potential of these stem or progenitor cell populations, it was shown in preliminary experiments that focal cartilage defects can be repaired by a biomaterial approach only in orthotopic focal cartilage defect equine and rabbit models. In these animals, chondral defects were filled with an injectable in situ gelating hydrogel that activates locally resident stem or progenitor cells. Indeed in rabbits, after 10 weeks defects were completely repaired in a cell-free approach. In the equine model, histological examination demonstrated the ingrowth of cells in the hydrogel two weeks after the arthroscopically guided implantation of the hydrogel in the chondral defect. A substantial part of these cells showed typical chondrocyte features. Taken together multiple lines of evidence demonstrates that articular cartilage can repair itself once the proper microenvironment is offered which unleashes its regenerative potential.


These exciting new developments open up a complete new avenue for the treatment of focal cartilage defects and osteoarthritis. Primary osteoarthritis and secondary osteoarthritis as a consequence of cartilage trauma earlier in life are among the most prevalent diseases in the Western World for which no effective cure is available. This proposal aims at bringing together leading Dutch groups on osteoarthritis research, which are well known for their pioneering work on the potential of intrinsic cartilage repair, with Dutch SMEs active in developing innovative treatments for joint regeneration. The program is furthermore supported by the Dutch Arthritis Foundation.
Within this proposal we intend to develop multidisciplinary projects along two intrinsically related themes:


Theme 1 is focusing on studies to explore the cellular basis of intrinsic cartilage repair and the role of biomechanics: This could be either stem cell populations residing in the joint but might also be chondrocyte subpopulations or alternative cell sources. Once identified we will aim at two strategies that can exploit these cell populations: i) development of bioactive scaffolds that in a cell-free manner can induce regeneration of lost cartilage tissue by activating these resident stem or progenitor cell populations; ii) controlled release strategies that release bioactive components that activate the regenerative potential of these cells.


Theme 2 is focusing on studies to explore the molecular basis of joint homeostasis by assessing the composition of synovial fluid in opposing conditions (catabolic versus anabolic conditions). This may lead to new diagnostics but also to new drugs / biologicals that can restore joint homeostasis.


Both themes are intrinsically related. For example joint distraction changes the catabolic osteoarthritic milieu in the joint into an anabolic regenerative milieu. Likewise, cell attracting hydrogels that fill up cartilage defects induce a regenerative response while in control animals similar defects result in cartilage catabolism. These opposing experimental conditions thus provide excellent models to study the fundamentals of joint homeostasis that could lead to new biomarkers of disease as well as new therapeutic interventions.

Role
Co-promotor & Project Leader
Individual project description

Our groups plays a key role in Project 3 of the William Hunter revisited consortium. This is a joint project between the dept. of Rheumatology, UMC-Utrecht and the dept. of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine. Preliminary animal experiments have been conducted and funded by both institutions and demonstrate that joint distraction is initially characterized by a mix of both anabolic and catabolic transcriptional profiles at the cartilage and subchondral bone level followed by an anabolic phase ultimately leading to cartilage repair in OA. 

The current project focusses on a better understanding of the underlying mechanisms orchestrating a reparative response initiated by distraction. For this purpose, OA is induced in the canine stifle joint according to the ‘Groove model’. Thereafter, distraction of the knee joint is applied. Using this protocol, the dog model recapitulates the clinical benefit of knee distraction.  The following key objectives will be addressed:

  1. Delineate the early and intermediate transcriptional response that mediates intrinsic cartilage repair initiated by joint distraction.
  2. Validate the regenerative effects of this transcriptional response in joint cartilage at long term follow up.
  3. Validate the identified targets in synovial fluid of OA human patients and OA canine joints who underwent knee joint distraction (collaborative with project 1 and 4 of this consortium).
  4. In a parallel project to the "William Hunter revisited"consortium our group focuses on the epigenetic mechanisms activated by temporary joint distraction.
Funding
NWO grant Applied and Engineering Sciences
External project members
  • dr. H.B.J. (Marcel) Karperien
  • Prof. dr. G.J. van Osch
  • Prof. dr. H. Weinans
  • Prof. dr. F. Lafeber
  • dr. P. van der Kraan
  • dr P. Emans
  • dr T. Welting
  • Dutch Arthritis Foundation
  • Hy2Care B.V.
  • InGell B.V.
  • Crystal Therapeutics B.V.
  • QVQ B.V.
  • Percuros B.V.
Project
Establishing a common demoninator for disc progenitor cells 01.01.2015 to 30.12.2018
General project description

A major focus within this project is to establish a common denominator for the various models that the four centers are working on, humans, dogs, and mice. The partners will perform biologic and genetic analyses that are at the forefront of a systematic biology in the understanding of cellular contribution to disc degeneration and the most appropriate exogenous cells for disc repair or regeneration, and more importantly, activation of endogenous signals, for better IVD maintenance or repair. Specifically for the canine species, we will explore the possibility of employing allogeneic disc progenitor cells to achieve biologic repair.

Role
Project Leader
Funding
Utrecht University
Project members UU
External project members
  • prof dr Chan (Hong Kong University)
  • dr Sakai (Tokai University Japan)
Project
ArIADNE: Controlled release of Anti-Inflammatory Agents for prolonged inhibition of inflammation, pain and DegeNeration in degenerative joint diseasE 01.09.2014 to 31.12.2018
General project description

In Western societies, osteoarthritis (OA) and chronic low back pain caused by intervertebral disc degeneration are the musculoskeletal diseases with most impact, resulting in decreased productivity and high health costs. Anti-Inflammatory Agents (AIA) are effective but have systemic side effects or limited duration of local activity. The ArIADNE consortium will develop slow release systems of anti-inflammatory drugs for the long term inhibition of inflammation, thus enhancing regeneration. Furthermore, in order to fine-tune these systems for clinical application, the local side effects of high local and prolonged doses of AIAs will be studied, as well as the in vivo release kinetics of AIA in relation to the implantation site and biomaterial degradation . Alongside, ArIADNE will study the role of inflammation in regeneration and kartogenin and caveolin-1 in particular as possible new regenerative compounds.

Role
Co-promotor & Project Leader
Funding
External funding Life Sciences Health sector: Impulse
External project members
  • dr LB Creemers (UMC Utrecht)
  • I Jansen (UMC Utrecht)
  • Nina Voike (DSM)
  • George Mihov (DSM)
  • Jens Thies (DSM)
  • Ingrid Lether (Dutch Arthritis Foundation)
Project
Mimicking developmental biology to regenerate the intervertebral disc 01.01.2012 to 30.12.2017
General project description

Low back pain is a widespread disorder, with tremendous socioeconomical impact. While this condition is multifactorial, intervertebral disc degeneration is one of its main causes. Actual treatments are only symptomatic (to alleviate pain) and new therapies are needed. Disc degeneration is characterized by the inability of the resident cells to keep the disc tissue intact due to a change of their phenotype and their decreasing number. Thus, delivery of exogenous cells may help. The disc originally contains two distinct cell populations: the nucleus pulposus cells (NPCs, chondrocyte-like cells) and the notochordal cells (NCs). Because NCs do not exist in human adults, regenerative therapies have only focused on using chondrocyte-like cells, e.g. adult mesenchymal stem cells (MSCs). However, their transplantation has been of limited success as disc degeneration was only inhibited and not reversed. We believe that this failure is in part due to inability of the transplanted cells to produce enough/appropriate disc matrix to restore tissue properties.

Using an innovative approach, we propose to mimic the natural cell combination of a developing disc, i.e. NCs and NPCs/MSCs, when the largest amount of matrix is produced. Different approaches will be tested for their efficacy using a succession of model systems moving from higher volume:cost ratio systems to more realistic but lower volume:costs systems. Another novelty of our approach will be to use the canine model in which disc degeneration is a frequent spontaneous spinal disease and whose NCs, NPCs and MSCs are readily available.

First, canine NCs, NPCs and MSCs (used to complement the decreasing disc cell population) will be combined in different co-culture systems to compare the ability of various cell combinations to stimulate the production of an appropriate disc matrix. We will also determine if the NCs directly contribute to matrix production or regulate other cells through biochemical signals. The most successful concepts will be further developed pre-clinically in experimental dogs with early stage spontaneous disc degeneration. Finally as proof of principle, a clinical study will be performed in client-owned canine patients with disc disease as the basis for a trial to be set up in human patients. In parallel with this sequential approach, the applicability of our findings to human cells will be demonstrated and if the NC regulatory effects are mediated through soluble factors, these will be identified for further exploitation. This project will (1) provide new and important insights on the role of NCs in disc regeneration and (2) may provide a new biological therapy to treat one of the most common disorders in modern society.

Role
Project Leader
Funding
External funding Financed by the AO Spine Research Network
External project members
  • Prof. dr. Keita Ito (TU/e)
  • dr. Laura Creemers (UMCU)
  • Ing. Stefan de Vries (TU/e)
  • Ing. Irene Arkensteijn (TU/e)
  • dr. Esther Potier (TU/e)
Project
Fundamental studies on endochondral bone formation: focus on keeping up the pace of bone regeneration 01.09.2011 to 31.08.2013
General project description

Despite numerous advances in the treatment of the diseased / damaged musculo-skeletal system, the field of clinical orthopedics is still challenged by the treatment of large bone defects. One major problem of bone Regenerative Medicine (RM) is that osteoblasts are metabolically highly demanding. Due to the lag-time of vascularization, hypoxia and nutrient deficiency occur within the construct and result into decreased proliferation of the seeded osteoblasts and into reduction of the matrix (quality and quantity) produced by the osteoblasts.

Contrary, chondrocytes are specialized in surviving without direct blood supply, proliferate optimally and produce better matrix (quality and quantity) under hypoxic conditions. This physiologic aspect can bethe solution to the problem of bone RM. Chondrocyte-based bone formation may conquer the strenuous conditions within large bone defects and complicated fracture healing. Therefore, novel successful strategies in bone RM are based on the understanding of the mechanisms by which the extensive network of local signaling pathways facilitates the cross-talk of chondrocytes, osteoblasts, and osteoclasts during endochondral bone formation.

Key objective of this proposal is to study local networks that define an accelerated pace of endochondral bone formation. This proposal studies the orderly occurring endochondral bone formation at the growth plate level. Dogs represent an excellent model for studying this process. They reveal large growth disparity between breeds, and hence distinct physiological differences in the rate of endochondral bone formation. The distal growth plate of the ulna from growing large and small breed dogs is studied with a two-colour microarray experiment. The data-set is validated for its biological significance by 

  1. studying the osteogenic potential of the selected novel pathways in the chondrocytic lineage of canine and human MSCs through endochondral bone formation.
  2. comparing the protein expression pattern in the canine and human growth plate
  3. studying the expression pattern of the potent target or its ligand, that is crucial in determining a higher pace of endochondral bone formation, in fracture healing of critical bone defects.

The obtained knowledge can be further employed in translational RM to develop rational-driven therapies for amending endochondral bone formation during fracture healing or in cell-seeded scaffolds employed in large bone defects in human and veterinary patients.

Role
Project Leader
Funding
External funding AO foundation
Project members UU
External project members
  • dr. Laura Creemers
  • Prof. dr. Wouter Dhert
Project
New Early Therapies for Intervertebral Disc Diseases. Drug Delivery and Augmentation through Smart Polymeric Biomaterials 01.01.2009 to 30.06.2014
General project description

Do not remove…

The spinal column has a heavy task. It protects our spinal cord and its nerves, carries our body weight, supports the head and absorbs the shocks resulting from physical exercise. No wonder that lower back pain is a very common problem. Two out of three Dutch people will be confronted with this problem during their lifetime. Some people have chronic lower back pain. An important cause of chronic back pain is wear and tear of the intervertebral disc, the soft disc between the vertebrae that together form the spinal column. This degeneration of the intervertebral disc develops due to a previously incurred hernia or gradual wear and tear. Current treatments for lower back pain require the replacement of afflicted vertebrae with bone from elsewhere in the body, or replacement by intervertebral disc prostheses. In both cases, the entire intervertebral disc is removed and only the symptoms are suppressed. These operations also carry risks, such as further damage to the adjacent intervertebral disc, and persistent pain at the sites where the bone was removed.

... but repair

The purpose of this study is to determine how the function of the degenerated intervertebral disc can be repaired instead of removed. If the degeneration of a vertebral disc is not far advanced, a treatment could consist of injectable, biodegradable materials that release drugs over a longer period of time, thereby stimulating the self-recovery of the intervertebral disc. The materials for the incorporation and release of these compounds may be watery gels, very small spheres (microparticles) or a combination of these two. Next to this, methods are being developed to repair the outside of the intervertebral disc to ensure the injected materials will not diffuse out of the disc. These methods are based on a biodegradable plaster.

A unique aspect of this project is the investigation into the ethical aspects of these new self-repair methods and the moral guidelines for implementing these techniques in patients.

Role
Co-promotor & Project Leader & Researcher
Individual project description
  1. Project Researcher of Work Package 4 (Animal studies); 09/2009-08/2013.
  2. Coordinator and project researcher of Work Package 7  (Animal studies employing hydrogels with OP-1) ; 09/2011-09/2013.
  3. Coordinator and project researcher of Work Package 9  (Animal studies employing hydrogels with CXB, dose response study) Completion Call ; 01/2013-12/2013.
Funding
External funding Funded by BMM which is financed by the Dutch government (50%), academia (25%) and industry (25%)
External project members
  • dr LB Creemers
  • prof dr K Ito
  • prof dr T Smit
  • InGell BV.
  • DSM
  • TNO