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WORKSHOP
Cell and Tissue Engineering for
Annulus Fibrosus Repair:
AO Foundation Collaborative
Research Project
Organizers:
Sibylle Grad, PhD
James C. Latridis, PhD
Speakers:
Daisuke Sakai, MD, PhD
Stephen J. Ferguson, PhD
Abhay Pandit, PhD
2 ORS 2014 Annual Meeting
March 15-18, 2014 New Orleans, Louisiana
WORKSHOP
Cell and Tissue Engineering for Annulus Fibrosus Repair: AO Foundation Collaborative Research Project Organizers: Sibylle Grad, PhD, AO Research Institute Davos, Davos, Switzerland
James C. Iatridis, PhD, Dept. of Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA [email protected]
SIGNIFICANCE AND PURPOSE
Disc herniation is a major clinical problem and the most prominent pathological condition requiring spinal surgery. Though surgical discectomy provides favorable results in the majority of cases, there are conditions (e.g., large disc protrusions with minimal disc degeneration and adolescent disc protrusions) where alternative treatment solutions involving annulus fibrosus closure and repair are desirable. Regardless of the surgical approach, lumbar spine surgery results are often unsatisfactory, and the economic burden to society is enormous. Novel strategies towards annular healing and biological restoration have potential to improve surgical outcomes in patients with contained disc herniation but otherwise minor degenerative changes.
This workshop will give insight into an interdisciplinary consortium effort aimed to develop tissue-engineered implants to stimulate annulus fibrosus repair. The general concept is to develop 3D materials with targeted biomechanical properties and activate them with a specific stem cell population and/or by the tailored release of active molecules. The ultimate goal is to progress towards an intra-operative procedure which would prevent re-herniation of repaired annulus fibrosus tissue and provide sustained pain relief for patients. In the past considerable advances have been made in the field of intervertebral disc regeneration and repair; nevertheless, comprehensive interdisciplinary approaches remain rare. To address this we assembled an interdisciplinary, international team of experts (Figure 1, Table 1) who are working collaboratively towards a clinical solution. In this workshop we provide a summary of the achievements during the first three years of this AO Foundation sponsored international consortium.
Ref.: Guterl CC, See EY, Blanquer SB, Pandit A, Ferguson SJ, Benneker LM, Grijpma DW, Sakai D, Eglin D, Alini M, Iatridis JC, Grad S. Challenges and strategies in the repair of ruptured annulus fibrosus. Eur Cell Mater 25:1-21, 2013.
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Figure 1. Consortium partners of the Collaborative Research Program "Annulus Fibrosus Repair" funded by the AO Foundation.
Table 1. Consortium members and their expertise
Daisuke Sakai, MD, PhD, Department of Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan
Clinical input; stem/progenitor cells; in vivo models
Stephen J. Ferguson, PhD, Institute for Biomechanics, ETH Zürich, Zürich, Switzerland
Spine/Disc biomechanics; instrumentation
Lorin M. Benneker, MD, Department of Orthopaedic Surgery, Inselspital, University of Bern, Bern, Switzerland
Clinical input; instrumentation; surgery
Abhay Pandit, PhD, Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland
Functional biomaterials; delivery systems
James Iatridis, PhD, Department of Orthopaedics, Mount Sinai School of Medicine, New York, NY, USA
Spine/Disc biomechanics; adhesive materials; organ culture
Dirk Grijpma, PhD, Department of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands
Structured biomaterials; scaffolds
David Eglin, PhD, AO Research Institute Davos, Davos, Switzerland
Functional biomaterials; closure membrane
Sibylle Grad, PhD, Mauro Alini, PhD, AO Research Institute Davos, Davos, Switzerland
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Parametric Simulation to Define Biomechanical Targets for Annulus Fibrosus Repair Benedikt Helgason1, Lorin M. Benneker2, Stephen J. Ferguson1
1Institute for Biomechanics, ETH Zurich, Zurich, Switzerland
2Department of Orthopedic Surgery, Inselspital, University of Bern, Bern Switzerland
The direct repair of lesions of the annulus fibrosus (AF) is currently limited to recent investigational studies of the use of suturing methods, membrane barriers or injectable sealants. The optimal biomechanical properties for such a repair remain an open question. Our aim was to establish the functional requirements for next-generation annulus repair methods through iterative, parametric simulation.
A parameterized finite element model of an L4-L5 spinal segment was developed to characterize the window of mechanical response that a candidate annulus repair material has to fall within, in order to restore not only the range of motion after focal annulus repair to a healthy state, but to also withstand the local strains acting on the repair site. Vertebral body geometry and ligament attachment points were derived from literature. Ligaments were defined as multi-linear elastic structures, facet joint surfaces were modeled as frictionless, elastic cartilage layers, with the angulation of the facet planes in the three anatomical directions derived from literature. The width, height, depth and lordosis of the intervertebral disc were parametrically varied. A non-planar, concave endplate curvature was defined. With a script, the internal annulus layers and nucleus pulposus geometry were defined for each model. The material model for the annulus was based on a continuum mechanical model with multi-direction fiber reinforcement; the strain energy function for the annulus material includes separate ground substance and fiber contributions, with the fiber contribution taking into account the alteration in fiber direction in alternating layers. Model validation was based on comparison of the intact model response (kinematic) to published literature values for in vitro testing.
For investigating the influence of disc morphology on the mechanical response of the IVD, nine FE-model geometries were created (see Figure 1, left). This was done by varying the disc size in the transverse plane within the range for lumbar vertebra geometries reported by Panjabi et al. (Spine, 1992. 17(3): p. 299-306). The disc height and lordotic angle were also varied within the range of values reported by Abuzayed et al. (Surg Radiol Anat, 2010. 32(1): p. 75-85). All models were subjected to four different load cases, generated by loading the vertebrae with a 7.5 Nm moment about the anatomical axes (flexion, extension, lateral bending and torsion respectively) in addition to an axial compressive follower load of 1000 N. Principal stresses and principal strains were sampled for all annulus volume elements over the full range of model geometries and loading cases to assemble representative ranges defining appropriate candidate implant material properties.
The principal stresses vs. principal strains for all annulus elements, for all load steps and all load cases, for the nine different vertebra models analyzed, were combined and are illustrated in Figure 1, right. We found that the disc geometry had a large influence on the mechanical response of the disc. Our results indicated that a small-thick disc configuration produced the most severe response in the annulus tissue. The maximum absolute principal tensile (7.85 MPa) and compressive (6.07 MPa) stress values were found in a model simulating a small vertebra-thick disc IVD geometry. Corresponding values for a FE model simulating a large vertebra-thin disc IVD geometry were 0.66 MPa and 1.60 MPa respectively. We suggest a window of stress strain response (Figure 1, right) for a candidate implant or annulus repair material for IVDs. This window is relatively broad but provides a domain for narrowing the search for such materials.
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Figure 1. Left: A Parameterized FE model of a L4-L5 spinal segment incorporating rigid bony elements and variable geometry for the disc, ligaments and facets. Loads were applied at the reference point located in the centroid of the superior vertebra of the motion segment. Right: Principal stresses vs. principal strains for all annulus elements for all load steps and all load cases for the nine different segmental geometries simulated.
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Effect of the pore characteristics of scaffolds built by stereolithography on mechanical properties and human annulus fibrosus cells behavior
S.B.G. Blanquera, S. Haimia, A.A. Poota and D.W. Grijpmaa,b
a Dept. of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands;
b University of Groningen and University Medical Centre Groningen, Dept. of Biomedical Engineering, Groningen,
The Netherlands. [email protected]
Introduction
Disk degeneration can lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). The current surgical repair strategies are suboptimal, and a tissue engineering repair strategy seems to be a better alternative. However, regeneration of the AF is challenging due to its complex and non-homogeneous structure. In the engineering of AF tissue, the biomaterial-cell constructs should ideally resemble the native structural and mechanical properties.1 Therefore we aim to regenerate AF tissue using resorbable scaffolds that take into
account the complexity, flexibility and elasticity of the native AF tissue.
Stereolithography (SL) can generate 3D objects with specific predetermined geometries, porosities, structure and morphologies.2 Using rubber-like poly(trimethylene carbonate) (PTMC) macromers, complex porous structures
with gyroid pore architecture were prepared, and used to culture human annulus fibrosus cells (HAFCs). Results
Different gyroid PTMC scaffolds with porosities close to 70% and pores sizes of 400, 500 and 600 µm were prepared and used to investigate the effect of the pore size on mechanical properties and on the adhesion/proliferation of HAFCs cells.
Table 1 shows that the compression modulus of the scaffolds increases with decreasing pore size. For the lowest pore sizes, the compression is close to the confined compression modulus of human AF tissue which ranges from 0.4 to 0.7 MPa.3
Table 1. Compression modulus of PTMC scaffolds.
Pore size (µm) 400 500 600
Ec Modulus (MPa) 0.31±0.09 0.23±0.05 0.21±0.03
The effect of pore size on the adhesion and proliferation of HAFCs was also investigated using gyroid scaffolds with of pore sizes. Each cubic scaffold (5x5x5 mm3) was seeded with 10x104 cells in 50 µl of medium. After 1 and
14 days, the HAFCs distribution was visualized using methylene blue staining. The viability of the cells in the scaffolds was evaluated using the Live/Dead assay. Figure 1A shows favorable adhesion of the cells in a scaffold and an excellent distribution of the cells, exactly following the shape of the pores. After 14 days (Figure 1B) the pores were almost completely filled with HAFCs forming tissue-like structures.
Figure 1. Stereomicroscopic images of methylene blue stained HAFCs in scaffolds after 1 (A) and 14 days (B) of culturing.
The DNA content of the cell-seeded scaffolds with equal porosity was determined after 1 and 14 days of culturing. The values presented in Table 2 show favorable adhesion into the scaffold after one day. DNA content at 14 days
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indicates good proliferation in all cases. The amount of DNA (and thus cell numbers) tends to increase with increasing pore size of the scaffold.
Table 2. Relative DNA contents of scaffolds after 1 and 14 days of culturing
Pore sizes (µm) 400 500 600
Relative DNA content after 1 day 1.0±0.2 1.5±0.7 2.2±0.3 Relative DNA content after 14 days 3.2±0.2 4.1±0.1 4.3±0.2 Conclusions
The PTMC scaffolds prepared by SL show favorable mechanical properties, comparable with those of native AF tissue. They proved adequate substrates for HAFCs cells.
References
1. Nerurkar, N. L., et al. (2009). Nat Mater 8(12):986-992.
2. Melchels, F. P. , et al. (2009. Biomaterials 30(23-24): 3801-3809. 3. D. Périé. et al. (2005) Journal of Biomechanics 38 :2164–2171.
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Annulus fibrosus cell characterization for efficient tissue engineering Daisuke Sakai MD, PhD
Tokai University, Kanagawa, Japan [email protected]
Recent stem cell research reports the presence of a stem/progenitor cell system as key in the maintenance of normal homeostasis and self-renewal in various organs. A decrease in number and/or altered function in stem/progenitor cells of the intervertebral disc relates to ageing and degeneration1. In order to facilitate a fully
functional annulus fibrosus (AF) repair using tissue engineering techniques, selection of the appropriate cell type is a critical issue. To date, despite some evidence of cells that demonstrate partial characteristics of a stem/progenitor cell3,4, no specific AF cell population has been reported to demonstrate functional properties of AF
stem/progenitor cells.
In this workshop, we will discuss several AF cells that were analyzed for the expression of various surface markers by flow-cytometry techniques, in order to define specific markers relating to clonogenicity, self-renewal and multipotency of AF cells in transgenic (TG) mice (P0-Cre Floxed EGFP). The use of P0-Cre Floxed EGF enables separation of NP cells more easily, as notochord derived cells in this TG mice express EGFP. We sorted EGFP- cells from the AF region and used them for various assays. Gene expression analyses for expression of defined markers for functional AF cells may identify candidate cell populations for AF stem/progenitor cells that may contribute to normal homeostasis and repair of the AF. Finally, integration of functional cell populations with ideal biomaterials developed through the AO consortium will be tested. This information is expected to provide insight into cellular and biochemical reactions during disc degeneration and to be utilized in the development of new treatment options.
References:
1. Sakai D, Nakamura Y, Nakai T, Mishima T, Kato S, Grad S, Alini M, Risbud MV, Chan D, Cheah KS, Yamamura K, Masuda K, Okano H, Ando K, Mochida J. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun. 2012;3:1264.
2. Risbud MV, Guttapalli A, Tsai TT, Lee JY, Danielson KG, Vaccaro AR, Albert TJ, Gazit Z, Gazit D, Shapiro IM. Evidence for skeletal progenitor cells in the degenerate human intervertebral disc. Spine 2007;32:2537-44.
3. Feng G, Yang X, Shang H, Marks IW, Shen FH, Katz A, Arlet V, Laurencin CT, Li X. Multipotential differentiation of human anulus fibrosus cells: an in vitro study. J Bone Joint Surg Am. 2010;92:675-85.
9 Functionalization Strategies: Designing for the IVD Abhay Pandit
Network of Excellence for Functional Biomaterials, National University of Ireland, Galway
Biomaterials are no longer considered innate structures and using functionalization strategies to modulate a desired response whether it be a host or implant responsive is an important focus in current research paradigms. Using functionalization strategies such as enzymatic and dendrimeric linkers, we have been able to link biomolecules to different structural moieties.
The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of functionalized scaffolds. Recent design efforts have utilized a bottom-up approach toward both understanding and engineering supramolecular protein assemblies.
We have synthesized a wide variety of functionalization systems but have also contributed broadly to the physical characterization and the development of applications of these dendritic macromolecules. Studies involving rheological, thermal, optical, and other methods have revealed that these polymers have unique properties that diverge widely from the established patterns of conventional macro-molecules. These properties, and the unique ability to tailor the polymers at the molecular level, have led to explorations of the use of these systems in a host of innovative applications.
These include functionalization of nanoparticles with biomolecules that include designed peptide motifs, growth factors and a multitude of gene vector systems. Structural moieties have taken a variety of different forms such as nanofibers and nanoparticulate. Functionalization on a microscale and macroscale has also been successfully attempted. Such strategies with examples from in vitro and in vivo studies will be illustrated. Development of complex geometrical structures and quantification of these geometries that have aided these investigations will be exemplified.
