Effects of dynamic loading on solute transport through the human cartilage endplate

Nutrient and metabolite transport through the cartilage endplate (CEP) is important for maintaining proper disc nutrition, but the mechanisms of solute transport remain unclear. One unresolved issue is the role of dynamic loading. In comparison to static loading, dynamic loading is thought to enhance transport by increasing convection. However, the CEP has a high resistance to fluid flow, which could limit solute convection. Here we measure solute transport through site-matched cadaveric human lumbar CEP tissues under static vs. dynamic loading, and we determine how the degree of transport enhancement from dynamic loading depends on CEP porosity and solute size. We found that dynamic loading significantly increased small and large solute transport through the CEP: on average, dynamic loading increased the transport of sodium fluorescein (376 Da) by a factor of 1.85 ± 0.64 and the transport of a large dextran (4000 Da) by a factor of 4.97 ± 3.05. Importantly, CEP porosity (0.65 ± 0.07; range: 0.47–0.76) strongly influenced the degree of transport enhancement. Specifically, for both solutes, transport enhancement was greater for CEPs with low porosity than for CEPs with high porosity. This is because the CEPs with low porosity were susceptible to larger improvements in fluid flow under dynamic loading. The CEP becomes less porous and less hydrated with aging and as disc degeneration progresses. Together, these findings suggest that as those changes occur, dynamic loading has a greater effect on solute transport through the CEP compared to static loading, and thus may play a larger role in disc nutrition.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Exploiting notochord cells for stem cell-based regeneration of the intervertebral disc

The nucleus pulposus is an avascular and aneural tissue that has significant influence on the homeostasis and overall function of the intervertebral disc. The nucleus pulposus is comprised of a heterogeneous population of cells including large notochord cells and smaller chondrocyte-like cells. Loss of notochord cells has been correlated with the pathogenesis of disc degeneration and consequently, it has been hypothesized that regeneration of the disc could be mediated by notochord cells. Attempts to grow and expand notochord cells in vitro have thus far been limited by cell availability and ineffective culturing methodologies. As a result, co-culturing techniques have been developed in order to exploit notochord-derived signals for the differentiation of proliferative mesenchymal stem cells. A recent study by Korecki et al. has demonstrated that notochord cell conditioned medium has the ability to differentiate mesenchymal stem cells toward a nucleus pulposus-like fate, producing high levels of glycosaminoglycans and type III collagen. These findings suggest that growth factors and other soluble proteins may be able to stimulate endogenous IVD tissue maintenance in vivo. While this study advances our understanding of intervertebral disc cell-cell interactions, limitations remain in our ability to determine the phenotype of terminally differentiated cells within the nucleus pulposus (ie mature notochord cells) and therefore assess the relevance of differentiated mesenchymal stem cells for disc regeneration. In order for the field to progress, elucidation of the notochord phenotype remains of utmost importance.     

An extended biphasic model for charged hydrated tissues with application to the intervertebral disc

Finite element models for hydrated soft biological tissue are numerous but often exhibit certain essential deficiencies concerning the reproduction of relevant mechanical and electro-chemical responses. As a matter of fact, singlephasic models can never predict the interstitial fluid flow or related effects like osmosis. Quite a few models have more than one constituent, but are often restricted to the small-strain domain, are not capable of capturing the intrinsic viscoelasticity of the solid skeleton, or do not account for a collagen fibre reinforcement. It is the goal of this contribution to overcome these drawbacks and to present a thermodynamically consistent model, which is formulated in a very general way in order to reproduce the behaviour of almost any charged hydrated tissue. Herein, the Theory of Porous Media (TPM) is applied in combination with polyconvex Ogden-type material laws describing the anisotropic and intrinsically viscoelastic behaviour of the solid matrix on the basis of a generalised Maxwell model. Moreover, other features like the deformation-dependent permeability, the possibility to include inhomogeneities like varying fibre alignment and behaviour, or osmotic effects based on the simplifying assumption of Lanir are also included. Finally, the human intervertebral disc is chosen as a representative for complex soft biological tissue behaviour. In this regard, two numerical examples will be presented with focus on the viscoelastic and osmotic capacity of the model.     

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.     

A combined numerical and experimental technique for estimation of the forces and moments in the lumbar intervertebral disc

Evaluation of the loads on lumbar intervertebral discs (IVD) is critically important since it is closely related to spine biomechanics, pathology and prosthesis design. Non-invasive estimation of the loads in the discs remains a challenge. In this study, we proposed a new technique to estimate in vivo loads in the IVD using a subject-specific finite element (FE) model of the disc and the kinematics of the disc endplates as input boundary conditions. The technique was validated by comparing the forces and moments in the discs calculated from the FE analyses to the in vitro experiment measurements of three corresponding lumbar discs. The results showed that the forces and moments could be estimated within an average error of 20%. Therefore, this technique can be a promising tool for non-invasive estimation of the loads in the discs and may be extended to be used on living subjects.     

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.     

Preliminary results of a new approach for three-dimensional reconstruction of Dynamic AngioThermography (DATG) images based on the inversion of heat equation

Dynamic AngioThermography (DATG) is a contact-plate technique capable of producing a digital representation of breast vascularity. The inception and growth of a tumor are associated with neoangenesis, which may result in a demonstrable alteration in the regional blood flow, while in normal health conditions the vascularity remains unchanged throughout life. DATG, if included in the clinical evaluation for breast cancer, could potentially improve the accuracy of the diagnosis of this disease.Conventional DATG is limited, however, in that it is a projection (i.e. two-dimensional) imaging technique that does not provide any information on the depth and its effect on the pattern of the perfusion revealed by this technique. In fact, the blood pattern is detected by projecting temperature signals on the plate, thus acquiring a digital two-dimensional image.In this article we propose a new approach for extracting information on depth through the inversion of the Fourier heat equation. The idea is to extract the information along the third axis while acquiring and analyzing the temporal sequence during the process of image formation. The method implemented has been tested on a dedicated “electric phantom” and in one in vivo experiment.In spite of the limits of these preliminary tests, the experimental results have shown that this method makes it possible to obtain a 3D representation of the vascularity. Although it appears to be promising, further validation and characterization of our technique are required.