Theodoor H. Smit

Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed.

Engineering alginate for intervertebral disc repair

Alginate is frequently studied as a scaffold for intervertebral disc (IVD) repair, since it closely mimics mechanical and cell-adhesive properties of the nucleus pulposus (NP) of the IVD. The aim of this study was to assess the relation between alginate concentration and scaffold stiffness and find preparation conditions where the viscoelastic behaviour mimics that of the NP. In addition, we measured the effect of variations in scaffold stiffness on the expression of extracellular matrix molecules specific to the NP (proteoglycans and collagen) by native NP cells. We prepared sample discs of different concentrations of alginate (1%-6%) by two different methods, diffusion and in situ gelation. The stiffness increased with increasing alginate concentration, while the loss tangent (dissipative behaviour) remained constant. The diffusion samples were ten-fold stiffer than samples prepared by in situ gelation. Sample discs prepared from 2% alginate by diffusion closely matched the stiffness and loss tangent of the NP. The stiffness of all samples declined upon prolonged incubation in medium, especially for samples prepared by diffusion. The biosynthetic phenotype of native cells isolated from NPs was preserved in alginate matrices up to 4 weeks of culturing. Gene expression levels of extracellular matrix components were insensitive to alginate concentration and corresponding matrix stiffness, likely due to the poor adhesiveness of the cells to alginate. In conclusion, alginate can mimic the viscoelastic properties of the NP and preserve the biosynthetic phenotype of NP cells but certain limitations like long-term stability still have to be addressed.

Quantifying intervertebral disc mechanics: a new definition of the neutral zone

BackgroundThe neutral zone (NZ) is the range over which a spinal motion segment (SMS) moves with minimal resistance. Clear as this may seem, the various methods to quantify NZ described in the literature depend on rather arbitrary criteria. Here we present a stricter, more objective definition.MethodsTo mathematically represent load-deflection of a SMS, the asymmetric curve was fitted by a summed sigmoid function. The first derivative of this curve represents the SMS compliance and the region with the highest compliance (minimal stiffness) is the NZ. To determine the boundaries of this region, the inflection points of compliance can be used as unique points. These are defined by the maximum and the minimum in the second derivative of the fitted curve, respectively. The merits of the model were investigated experimentally: eight porcine lumbar SMSs were bent in flexion-extension, before and after seven hours of axial compression.ResultsThe summed sigmoid function provided an excellent fit to the measured data (r2 > 0.976). The NZ by the new definition was on average 2.4 (range 0.82-7.4) times the NZ as determined by the more commonly used angulation difference at zero loading. Interestingly, NZ consistently and significantly decreased after seven hours of axial compression when determined by the new definition. On the other hand, NZ increased when defined as angulation difference, probably reflecting the increase of hysteresis. The methods thus address different aspects of the load-deflection curve.ConclusionsA strict mathematical definition of the NZ is proposed, based on the compliance of the SMS. This operational definition is objective, conceptually correct, and does not depend on arbitrarily chosen criteria.

The Use of Poly(L-lactide-co-caprolactone) as a Scaffold for Adipose Stem Cells in Bone Tissue Engineering: Application in a Spinal Fusion Model

Since the early 1990s, tissue engineering has been heralded as a strategy that may solve problems associated with bone grafting procedures. The original concept of growing bone in the laboratory, however, has proven illusive due to biological, logistic, and regulatory problems. Fat-derived stem cells and synthetic polymers open new, more practicable routes for bone tissue engineering. In this paper, we highlight the potential of poly(L-lactide-co-caprolactone) (PLCL) to serve as a radiolucent scaffold in bone tissue engineering. It appears that PLCL quickly and preferentially binds adipose stem cells (ASCs), which proliferate rapidly and eventually differentiate into the osteogenic phenotype. An in vivo spinal fusion study in a goat model provides a preclinical proof-of-concept for a one-step surgical procedure with ASCs in bone tissue engineering.

A Biodegradable Glue for Annulus Closure: Evaluation of Strength and Endurance

Study Design. A biodegradable glue was biomechanically tested for annulus closure using nondegenerated goat intervertebral discs. Ultimate strength and endurance tests were performed using native and punctured discs as positive and negative controls, respectively. Objective. The aim of this study was to investigate the feasibility and biomechanical properties of a biodegradable glue for annulus closure. Summary of Background Data. There is an unmet clinical need for annulus closure techniques. Isocyanate-terminated tissue glues show potential because they adhere to annulus tissue, have an elastic modulus similar to the annulus, and show limited cytotoxicity to human annulus fibrosus cells. Methods. Three biomechanical tests were performed divided in 2 parts: part 1: ultimate strength tests comparing native, punctured (2.4-mm needle), and glued caprine intervertebral discs (n = 11 per group); part 2: 10 discs per group were subjected to a 10-day ex vivo endurance test of 864,000 load cycles, followed by ultimate strength tests. Outcome parameters include the restoration of strength after puncture, reduction of herniation in the endurance test, and conservation of glue strength after endurance testing. Results. Part 1: The glue partially restored subsidence to failure and yield strength/ultimate strength ratio of intervertebral discs. Part 2: During endurance testing, 40% of punctured discs failed compared with none of the glued discs. Endurance testing did not affect glue strength, and pooling of ultimate strength tests showed that the glue restored ultimate strength, work to failure, and yield strength/ultimate strength to 79%, 75%, and 119% of native values, respectively. Conclusion. A biodegradable isocyanate-terminated glue increases the force at which nucleus protrusion occurs, and it limits herniations during endurance or ultimate strength tests. Biomechanical tests in a bioreactor provide a low-cost assessment for annulus repair strategies; however, the clinical efficacy needs to be further addressed using long-term in vivo studies. Level of Evidence: N/A

Biomechanical evaluation of a novel nucleus pulposus prosthesis in canine cadaveric spines.

Partial disc replacement is a new surgical technique aimed at restoring functionality to degenerated intervertebral discs (IVDs). The aim of the present study was to assess biomechanically the behaviour of a novel nucleus pulposus prosthesis (NPP) in situ and its ability to restore functionality to the canine IVD after nuclectomy alone or after combined dorsal laminectomy and nuclectomy. Nine canine T13-L5 specimens (L2L3 group) and 10 L5-Cd1 specimens (LS group) were tested biomechanically in the native state, after nuclectomy (L2L3 group) or after combined dorsal laminectomy and nuclectomy (LS group), and after insertion of the NPP. Range of motion (ROM), neutral zone (NZ), and neutral zone stiffness (NZS) were determined in flexion/extension, lateral bending, and axial rotation. Nuclectomy alone and combined dorsal laminectomy and nuclectomy caused significant instability in all motion directions. Implantation of the NPP resulted in significant restoration of the parameters (ROM, NZ, and NZS) towards the native state; however, fragmentation/herniation of the NPP occurred in 47% of the cases. In conclusion, the NPP has the ability to improve functionality of the nuclectomized canine IVD. The high rate of NPP failure requires modifications directed at the integrity of the NPP and its confinement to the nuclear cavity.

Long-term creep behavior of the intervertebral disk: comparison between bioreactor data and numerical results

The loaded disk culture system is an intervertebral disk (IVD)-oriented bioreactor developed by the VU Medical Center (VUmc, Amsterdam, The Netherlands), which has the capacity of maintaining up to 12 IVDs in culture, for approximately 3 weeks after extraction. Using this system, eight goat IVDs were provided with the essential nutrients and submitted to compression tests without losing their biomechanical and physiological properties, for 22 days. Based on previous reports (Paul et al., 2012, 2013; Detiger et al., 2013), four of these IVDs were kept in physiological condition (control) and the other four were previously injected with chondroitinase ABC (CABC), in order to promote degenerative disk disease (DDD). The loading profile intercalated 16 h of activity loading with 8 h of loading recovery to express the standard circadian variations. The displacement behavior of these eight IVDs along the first 2 days of the experiment was numerically reproduced, using an IVD osmo-poro-hyper-viscoelastic and fiber-reinforced finite element (FE) model. The simulations were run on a custom FE solver (Castro et al., 2014). The analysis of the experimental results allowed concluding that the effect of the CABC injection was only significant in two of the four IVDs. The four control IVDs showed no signs of degeneration, as expected. In what concerns to the numerical simulations, the IVD FE model was able to reproduce the generic behavior of the two groups of goat IVDs (control and injected). However, some discrepancies were still noticed on the comparison between the injected IVDs and the numerical simulations, namely on the recovery periods. This may be justified by the complexity of the pathways for DDD, associated with the multiplicity of physiological responses to each direct or indirect stimulus. Nevertheless, one could conclude that ligaments, muscles, and IVD covering membranes could be added to the FE model, in order to improve its accuracy and properly describe the recovery periods.

The poro-elastic behaviour of the intervertebral disc: A new perspective on diurnal fluid flow

Diurnal disc height changes, due to fluid in- and outflow, are in equilibrium while daytime spinal loading is twice as long as night time rest. A direction-dependent permeability of the endplates, favouring inflow over outflow, reportedly explains this; however, fluid flow through the annulus fibrosus should be considered. This study investigates the fluid flow of entire intervertebral discs. Caprine discs were preloaded in saline for 24h under four levels of static load. Under sustained load, we modulated the disc׳s swelling pressure by exchanging saline for demineralised water (inflow) and back to saline (outflow), both for 24h. We measured disc height creep and used stretched exponential models to determine time-constants. During inflow disc height increased in relation to applied load, and during outflow disc height decreased to preload levels. When comparing in- and outflow phases, there was no difference in creep, and time-constants were similar indicating no direction-dependent resistance to fluid flow in the entire intervertebral disc. Results provoked a new hypothesis for diurnal fluid flow: in vitro time-constants for loading are shorter than for unloading and in vivo daytime loading is twice as long as night time unloading, i.e. in diurnal loading the intervertebral disc is closer to loading equilibrium than to unloading equilibrium. Per definition, fluid flow is slower close to equilibrium than far from equilibrium; therefore, as diurnal loading occurs closer to loading equilibrium, fluid inflow during night time unloading can balance fluid outflow during daytime loading, despite a longer time-constant.

Mechanical Loading 1

The problem is approached with Biot’s theory of poro-elasticity, and calculated with the finite element method. The tunnelling osteon was modelled axisymmetrically as a cilindrical gap with a spherical end, and a diameter of 200mm. The bone matrix was described as an isotropic material with a fully saturated lacunar–canalicular porosity of 5.0%. The Young’s modulus (15.8GPa) and Poisson’s ratio (0.33) of the drained bone material, as well as the bulk modulus of the solid matrix (Ks=18.7GPa) were derived from literature using experimental testing data and equations from the theory of poro-elasticity 2 . The bulk modulus of the bone fluid Kf=2.3GPa (water). The resistance of the bone matrix against fluid flow is expressed by the hydraulic permeability k estimated at 1.2 � 10 � 7 mm 4 /Ns. The force applied to the model was a typical loading curve recorded from a person walking at 4 km/h 3 . The maximum deformation of the bone matrix during the walking cycle (t=0.275s) was 1500mstrain (0.15%). The fluid flows at the osteonic tunnel wall and inside the bone matrix were calculated in 40 increments of 0.025 s (total step time 1.0s).

Minimally Invasive Micro-Indentation: mapping tissue mechanics at the tip of an 18G needle

Experiments regarding the mechanical properties of soft tissues mostly rely on data collected on specimens that are extracted from their native environment. During the extraction and in the time period between the extraction and the completion of the measurements, however, the specimen may undergo structural changes which could generate unwanted artifacts. To further investigate the role of mechanics in physiology and possibly use it in clinical practices, it is thus of paramount importance to develop instruments that could measure the viscoelastic response of a tissue without necessarily excising it. Tantalized by this opportunity, we have designed a minimally invasive micro-indenter that is able to probe the mechanical response of soft tissues, in situ, via an 18G needle. Here, we discuss its working principle and validate its usability by mapping the viscoelastic properties of a complex, confined sample, namely, the nucleus pulposus of the intervertebral disc. Our findings show that the mechanical properties of a biological tissue in its local environment may be indeed different than those that one would measure after excision, and thus confirm that, to better understand the role of mechanics in life sciences, one should always perform minimally invasive measurements like those that we have here introduced.