Intervertebral Disc Tissue Engineering Hôpital Orthopédique de la Suisse Romande (CHUV) Unit of Orthopedic Cell Therapy (UNIL/CHUV) Laboratory of Biomechanical Orthopedics (EPFL) Laboratory of Composite and Polymer Technology (EPFL)
2006 Activity Report
Table of contents
Intervertebral Disc Tissue Engineering……………..............…………3 Staff……………………………………………………………………………4 Collaborations……………………………………………………………….4 Projects report………………………………………………………………5 1. 2. 3. 4.
Composite hydrogels for the replacement of the nucleus pulposus.……..……5 In vitro characterization of fetal disc and cartilage cells……………....………6 Intervertebral disc biomechanics................................................…......………..7 Spine biomechanics......................……………………………………………..8
Publications 2006…………….…………………..………...……………....9 Acknowledgements.……………………………………..…………………9 Contacts………..…………………………………………………………...10
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Intervertebral Disc Tissue Engineering Following the philosophy developed for the bone tissue engineering project, in 2006 we have initiated a new field of research in intervertebral disc tissue engineering. The team composed of engineers, biologists, and surgeons works in close collaboration to develop new solutions in the treatment of disc degeneration. These solutions will be based on the development of a new injectable gel and the use of spinal fetal cells. The biomechanical aspects of the natural intervertebral disc will be taken into account in these developments. The same partners as for the bone tissue engineering project will work together and the addition of Dr. Constantin Schizas, specialist spinal surgeon will force the new solution to be clinically relevant.
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Staff During the year 2006, the following people were directly involved in the project “Intervertebral Disc Tissue Engineering”:
Lab directors: • • •
Prof. Pierre-François Leyvraz (HOSR/CHUV) Prof. Jan-Anders Månson (LTC/EPFL) Prof. Dominique Pioletti (LBO/EPFL)
Group leaders • • • •
Dr. Lee Laurent-Applegate (UTC/UNIL) Dr. Pierre-Etienne Bourban (LTC/EPFL) Dr. C. Plummer (LTC/EPFL) Dr. Constantin Schizas (HOSR)
Laboratory assistants • •
Sandra Jaccoud (LRO/EPFL) Corinne Scaletta (LMF/UNIL)
PhD students • • •
Ana Borges (LTC /EPFL) Aurélie Quintin (LBO/EPFL) Arne Vogel (LBO/EPFL)
MS students • •
dsssd (LTC/EPFL) Martin Aeberhard (LBO/EPFL)
Collaborations The following laboratories collaborate for the project Intervertebral Disc Tissue Engineering”: • IIT Madras (Prof A. Jayakrishna) • AO Research Institute (Prof. Keita Ito)
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Composite hydrogels for the replacement of the nucleus pulposus Collaborator: A. Borges Supervisors: P.-E. Bourban, D. Pioletti, C. Plummer
Description Back pain due to intervertebral disc (IVD) degeneration is a major health problem and costs several billion $ every year. Degeneration always starts in the nucleus pulposus (NP), a gel-like structure containing over 70% of water.
NP AF Figure 1: The normal and degenerate lumbar IVD. The figure shows a normal IVD (left). The AF lamellae surrounding the softer NP are clearly visible. In the highly degenerate disc (right), the nucleus is desiccated and the annulus is disorganized. Urban, J.P.G. and S. Roberts, Degeneration of the intervertebral disc. Arthritis Research and Therapy, 2003. 5(3): p. 120-130
The strategy to alleviate pain is nowadays to replace the NP by a synthetic hydrogel. Several hydrogels are being studied but none gained universal acceptance. The goal of the research project proposed here is to design a new type of hydrogel, an injectable, in situ UV-curing composite hydrogel that will have better mechanical properties than neat hydrogels and that will restore the mechanical functions of the IVD. In vitro and in vivo studies will be carried out to evaluate the potential of the composite hydrogel as a nuclear implant.
Results obtained in 2006 A literature review of the state-of-the art of hydrogels for nucleus pulposus replacement was carried out during the first months. Then, the synthesis of the monomers that will crosslink to form the hydrogel has been performed. The monomers were partially characterized by FTIR and H NMR.
Perspective for 2007 The monomers will be completely characterized by a MALDI-TOF analysis. Neat and composite hydrogels will be synthesized, characterized (polymerization parameters, mechanical properties) and compared. Biocompatibility tests will be carried out on hydrogel samples.
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In vitro characterization of fetal disc and cartilage cells Collaborator: A. Quintin Supervisors: D. Pioletti, L. Applegate, C. Schizas
Description The overall project consists in evaluating the capacity of fetal disc and cartilage cells to regenerate intervertebral discs. In a first step, fetal disc and cartilage cells will be characterized in vitro by their proliferation capacity, consistency in in vitro passaging and matrix synthesis capacity.
Results obtained in 2006 Fetal disc and cartilage cells proliferated faster than adult disc cells. Proliferation rate of fetal cells is stable from passage 4 to 8 with a tendency for fetal cartilage cells to proliferate faster than disc ones. Fetal disc and cartilage cells were positive for HLA-A, -B and –C and were negative for HLA-DP, -DQ and –DR. Consistency in fetal disc and cartilage cell culture was assessed by flow cytometry. The first results showed that disc and cartilage cells were stable from passage 4 to 11 notably regarding the expression of fibrau, galectin-3 and HIF-1. Fetal disc and cartilage were encapsulated in alginate beads and cultured for 28 days. DNA content of the alginate beads seemed to decrease during the first 5 days of culture and then became stabilized. Both type of cells produced matrix up to 28 days. Fetal cartilage cells seemed to produce more matrix than fetal disc cells.
Perspective for 2007 Primary results of the in vitro characterization will be confirmed. Then the matrix synthetized by fetal disc and cartilage cells in alginate beads will be characterized regarding its aggrecan and type II collagen content.
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Intervertebral disc biomechanics Collaborator: A. Vogel Supervisors: D. Pioletti, C. Schizas
Description The intervertebral disc (IVD) is a dissipative joint allowing great mobility of the spine. Throughout its life, the tissue is subjected to important biological and biochemical changes leading to a very different mechanical behavior. Particularly, viscoelasticity of the disc dramatically decreases within the 3 first decades after birth. This period of life is also known to be subjected to the first signs of degeneration. This observation motivates the investigation of viscosity changes in relation with biological tissues as the intervertebral disc.
Results obtained in 2006 Our findings on the viscoelastic properties of the annulus fibrosus (AF) showed that, at high strain rate, viscoelastic effects are more pronounced in compression than in traction.
Figure 2: Force/displacement curves of porcine annulus fibrosus obtained at different elongation rate.
The mechanical bearing is very different in both cases as in compression, the load is mainly supported by the proteoglycans rather than by the collagen fibers which are recruited mainly in traction. A viscoelastic constitutive law has been developed to describe the AF behavior. The law was based on pseudo-potentials and allow us to take into account large deformations.
Perspective for 2007 The developed viscoelastic law will be implemented in a numerical code (ABAQUS) and anisotropy will be considered. Mechanical tests will also be performed on nucleus pulposus.
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Spine biomechanics Collaborator: A. Vogel, M. Aeberhard Supervisors: D. Pioletti, C. Schizas
Description In the framework of spine traumatology, burst fractures are severe and are often caused by car accidents or by falls. The vertebra breaks under the traumatic compression and pieces of it are shattered in all directions. This type of fracture is often treated by spinal fusion using a posterior system with rods and pedicular screws. By decompressing the traumatic region, the fracture is allowed to heal and to restore to a physiological geometry. However, a progressive kyphosis (flexion of the spine) is clinically observed arising from the rotation of the pedicular screws in the vertebra. The correction is therefore lost and proper healing compromised. The project consisted in analyzing and in optimizing the stress around the pedicular screws under compressive loading of a posterior spinal fusion system.
Results obtained in 2006 The outcome of this study is mostly qualitative. It has been demonstrated that the geometry of the screw can be optimized to reduce stress concentration. The influence of osteoporosis and of different elastic-density relation of the trabecular bone was also assessed and understanding of the instability mechanism was gained.
Figure 3: von Mises stress distribution with 200N load vertically applied on the screw.
In parallel, this model was also developed to quantify mechanical aspects in the development of an artificial NP implant. Constitutive modeling reviewing and numerical modeling specification was also partly assessed in this preliminary model.
Perspective for 2007 One original design of a pedicular screw can potentially be used but requires further developments for feasibility assessment. Speculation on the mechanism of the stability loss should also be confirmed by numerical and/or experimental investigations.
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Publications 2006 Manuscripts •
Quintin A, Hirt-Buri N, Scaletta C, Schizas C, Pioletti DP, Applegate LA. Consistency of fetal cell banks for research and clinical use, Cell Trans, in press, 2007.
Abstracts •
Quintin A, Scaletta C, Jaccoud S, Pioletti DP, Schizas C, Applegate LA. Preliminary characterization of cells isolated from fetal spine intervertebral disc tissue engineering. Annual Meeting of the International Society for the Study of Lumbar Spine, 2006.
Acknowledgements The studies are partially supported by grants from: •
AO Foundation (#04-S33);
•
Hôpital Orthopédique de la Suisse Romande;
•
LTC/EPFL
•
LBO/EPFL
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•
Contacts for the project“Intervertebral Disc Tissue Engineering”
General “Intervertebral Disc Tissue Engineering” project supervision Dominique P. Pioletti, PhD EPFL STI IBME LBO Station 15 1015 Lausanne Switzerland Tel: +41 21 693 83 41 Fax: +41 21 693 86 60 E-mail:
[email protected] Fetal cell project supervision Lee Laurent-Applegate, PhD Unit of Orthopedic Cell Therapy Pavillon 3 CHUV 1011 Lausanne Switzerland Tel: +41 21 314 35 10 Fax: +41 21 314 31 69 E-mail:
[email protected] Scaffold project supervision Pierre-Etienne Bourban, PhD EPFL STI IMX LTC Station 12 1015 Lausanne Switzerland Tel: 41 21 693 58 06 Fax: 41 21 693 58 80 E-mail:
[email protected] Medical project supervision Constantin Schizas, MD Hôpital Orthopédique de la Suisse Romande Av. Pierre Decker 4 1005 Lausanne Switzerland Tel: 41 21 545 06 30 Fax: 41 21 310 34 27 E-mail:
[email protected]
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