Neil A. Duncan

Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison

The nuclei pulposi of the intervertebral discs (IVDs) contain a mixed population of cell types at various stages of maturation. This tissue is formed either by or with the help of cells from the embryonic notochord, which appear to be replaced during development by a population of chondrocyte‐like cells of uncertain origin. However, this transition occurs at widely varying times, depending upon the species – or even breed – of the animal being examined. There is considerable debate among spine researchers as to whether the presence of these residual notochordal cells has a significant impact upon IVD degeneration models, and thus which models may best represent the human condition. The present study examines several different species commonly used in lumbar spine investigations to explore the variability of notochordal cells in the IVD.

Material properties of the human calcaneal fat pad in compression: experiment and theory.

The structural behaviour of the human heel pad has been studied extensively due to its ability to absorb shock, protect against excessive local stress, and reduce plantar pressures. However, the material properties of the tissue have not been adequately measured. These must be known in order to perform a finite element analysis of the effect of factors such as foot geometry and shoe/surface construction on heel pad function. Therefore, the purposes of this study were to (a). measure the viscoelastic behaviour of the fat pad in compression, and (b). to determine an appropriate constitutive equation to model the tissue. A series of unconfined compression tests were performed on 8mm diameter cylinders of fat pad tissue, consisting of quasi-static, 175, 350 mm/s and stress-relaxation tests to approximately 50% deformation. The tissue exhibited nonlinear, viscoelastic behaviour. No significant difference was found in the quasi-static behaviour between samples from different locations and orientations in the heel. The stress-relaxation tests were used to determine the time constant (tau(1)=0.5s), the 175 mm/s test to determine the relaxation coefficient (g(1)=28), and the 350 mm/s compression test to determine the material constants (C(100)=C(010)=0.01, C(200)=C(020)=0.1 Pa) of a single-phase, hyperelastic, linear viscoelastic strain energy function (r(2)=0.98).

Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc

The three‐dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and inner annulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlike cells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the discs periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regions of the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site‐specific changes in tissue architecture, biochemistry and mechanical properties during degeneration, injury and healing.

The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc

Cells from the nucleus pulposus of young (< 2 years) and old (> 5 years) non‐chondrodystrophoid dogs were studied using routine histology, confocal laser scanning microscopy and transmission electron microscopy. The architecture of cell structures – from the tissue scale down to subcellular scale – was reported. Clusters of notochordal cells were observed in young nuclei pulposi, ranging from 10 to 426 cells each. These clusters resisted mechanical disruption and showed evidence of cell–cell signalling via gap junctions. Cells (30–40 µm in diameter) within the clusters had a physaliferous appearance, containing numerous large inclusions which ranged from 1 to 20 µm in diameter. The inclusions were surrounded by a dense actin cortex but were not contained by a lipid bilayer. The contents of the inclusions were determined not to be predominantly carbohydrate or neutral lipid as assessed by histochemical staining, but the exact composition of the contents remained uncertain. There were striking differences in the cell architecture of young vs. old nuclei pulposi, with a loss of both cell clusters and physaliferous cells during ageing. These observations demonstrate unique cell structures, which may influence our understanding of the differences between notochordal and chondrocytic cells in the nucleus pulposus. Such differences could have substantial impact upon how we think about development, degeneration and repair of the intervertebral disc.

ISSLS prize winner: Collagen fibril sliding governs cell mechanics in the anulus fibrosus: an in situ confocal microscopy study of bovine discs.

Study Design. In situ investigation of collagen and cell mechanics in bovine caudal discs using novel techniques of confocal microscopy. Objective. To measure simultaneously the in situ intercellular and collagen matrix mechanics in the inner and outer anulus fibrosus of the intervertebral disc subjected to flexion. Summary of Background Data. Mechanobiology studies, both in vivo and in vitro, clearly demonstrate that mechanical factors can influence the metabolic activity of disc cells, altering the expression of key extracellular matrix molecules. Essential to elucidating the mechanotransduction mechanisms is a detailed understanding of the in situ mechanical environment of disc cells in response to whole-body mechanical loads. Methods. Confocal microscopy was used to simultaneously track and capture in situ images of fluorescently labeled cells and matrix during an applied flexion. The position of the nuclear centroids was calculated before and after applied flexion to quantify the in situ intercellular mechanics of both lamellar and interlamellar cells. The deflection patterns of lines photobleached into the extracellular matrix were used to quantify collagen fibril sliding and collagen fibril strains in situ. Results. The extracellular matrix was observed to deflect nonuniformly due to the relative sliding of the collagen fibrils. Intercellular displacements within the lamellar layers were also nonuniform, both along a cell row andbetween adjacent rows. Within a cell row, the intercellular displacements were small (<1%). Conclusions. The in situ cell mechanics of anular cells was found to be strongly influenced by collagen fibril sliding in the extracellular matrix and could not be inferred directly from applied tissue loads.

In situ intercellular mechanics of the bovine outer annulus fibrosus subjected to biaxial strains

In situ intercellular strains in the outer annulus fibrosus of bovine caudal discs were determined under two states of biaxial strain. Confocal microscopy was used to track and capture images of fluorescently labelled nuclei at applied Lagrangian strains in the axial direction (E(A)(S)) of 0%, 7.5% and 15% while the circumferential direction (E(C)(S)) was constrained to either 0% or -2.5%. The position of the nuclear centroids were calculated in each image and used to investigate the in situ intercellular mechanics of both lamellar and interlamellar cells. The intercellular Lagrangian strains measured in situ were non-uniform and did not correspond with the biaxial Lagrangian strains applied to the tissue. A row-oriented analysis of intercellular unit displacements within the lamellar layers found that the magnitudes of unit displacements between cells along a row (delta;(II)) were small (|delta;(IIavg)|=1.6% at E(C)(S)=0%, E(A)(S)=15%; |delta;(IIavg)|=3.0% at E(C)(S)=-2.5%, E(A)(S)=15%) with negative unit displacements occurring greater than one-third of the time. Evidence of interlamellar shear and increased intercellular Lagrangian strains among the cells within the interlamellar septa suggested that their in situ mechanical environment may be more complex. The in situ intercellular strains of annular cells were strongly dependent upon the local structure and behaviour of the extracellular matrix and did not correspond with applied tissue strains. This knowledge has immediate relevance for in vitro investigations of disc mechanobiology, and will also provide a base to investigate the mechanical implications of disc degeneration at the cellular level.

The Functional Significance of Cell Clusters in the Notochordal Nucleus Pulposus : Survival and Signaling in the Canine Intervertebral Disc

Study Design. Cell viability was assessed in relation to cell clustering, and mechanisms of cell–cell signaling in the clusters were investigated. Objectives. To explore the functional role of cell clustering in the notochordal nucleus pulposus. Summary of Background Data. The intervertebral disc of some species contains residual cells from the embryonic notochord. These cells form large three-dimensional clusters in the young, healthy disc but are replaced by chondrocyte-like cells during aging and degeneration. Methods. Forty nucleus pulposi of adult dog lumbar intervertebral discs were isolated, and were left untreated, mechanically disrupted through a syringe, or enzymatically digested. The presence of functional gap junctions was determined by the fluorescence recovery after photobleaching method. Cell viability was also assessed over 20 days in vitro. Results. The cell clusters were interconnected via functional gap junctions. Mechanical disruption of the tissue had little effect on long-term cell viability, but enzymatic disruption of the tissue had a substantial negative impact on cell survival. Conclusions. These results demonstrate that the notochordal cells in adult dog nucleus pulposi are able to communicate via cytoplasmic signals and that such communications may influence the functionality of these cells in the young disc.

A voltage‐dependent K+ current contributes to membrane potential of acutely isolated canine articular chondrocytes

The electrophysiological properties of acutely isolated canine articular chondrocytes have been characterized using patch‐clamp methods. The ‘steady‐state’ current–voltage relationship (I–V) of single chondrocytes over the range of potentials from −100 to +40 mV was highly non‐linear, showing strong outward rectification positive to the zero‐current potential. Currents activated at membrane potentials negative to −50 mV were time independent, and the I–V from −100 to −60 mV was linear, corresponding to an apparent input resistance of 9.3 ± 1.4 GΩ (n= 23). The outwardly rectifying current was sensitive to the K+ channel blocking ion tetraethylammonium (TEA), which had a 50% blocking concentration of 0.66 mm (at +50 mV). The ‘TEA‐sensitive’ component of the outwardly rectifying current had time‐ and membrane potential‐dependent properties, activated near −45 mV and was half‐activated at −25 mV. The reversal potential of the ‘TEA‐sensitive’ current with external K+ concentration of 5 mm and internal concentration of 145 mm, was −84 mV, indicating that the current was primarily carried by K+ ions. The resting membrane potential of isolated chondrocytes (−38.1 ± 1.4 mV; n= 19) was depolarized by 14.8 ± 0.9 mV by 25 mm TEA, which completely blocked the K+ current of these cells. These data suggest that this voltage‐sensitive K+ channel has an important role in regulating the membrane potential of canine articular chondrocytes.

Strain transfer in the annulus fibrosus under applied flexion

A detailed understanding of the anatomical and mechanical environment in the intervertebral disc at the scale of the cell is necessary for the design of tissue engineering repair strategies and to elucidate the role of mechanical factors in pathology. The objective of this study was to measure and compare the macroscale to microscale strains in the outer annulus fibrosus in various cellular regions of intact discs over a range of applied flexion. Macroscale strains were measured on the annulus fibrosus surface, and contrasted to in situ microscale strains using novel confocal microscopy techniques for dual labeling of the cell and the extracellular matrix. Fiber oriented surface strains were significantly higher than in situ fiber strains, which implies a mechanism of load redistribution that minimizes strain along the fibers. Non-uniformity of the strains and matrix distortion occurred immediately and most interestingly varied little with increase in flexion (3-16 degrees), suggesting that inter-fiber shear is important in the initial stages of strain redistribution. Fiber oriented intercellular strains were significantly larger and compressive compared to in situ strains in other regions of the extracellular matrix indicating that the mechanical environment in this region may be unique. Further examination of the structural morphology in this pericellular region is needed to fully understand the pathway of strain transfer from the tissue to the cell. This study provides new knowledge on the complex in situ micro-mechanical environment of the annulus fibrosus that is essential to understanding the mechanobiological behavior of this tissue.

In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.