➤ Tissue engineering is a rapidly evolving discipline that seeks to repair, replace, or regenerate specific tissues or organs by translating fundamental knowledge in physics, chemistry, and biology into practical and effective materials, devices, systems, and clinical strategies.➤ Stem cells and pro

Engineering principles of clinical cell-based tissue engineering.

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration.

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The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is an often investigated pathophysiological condition because of its implication in causing low back pain. As human material for such studies is difficult to obtain because of ethical and government regulatory restriction, animal tissue, organs and in vivo models have often been used for this purpose. However, there are many differences in cell population, tissue composition, disc and spine anatomy, development, physiology and mechanical properties, between animal species and human. Both naturally occurring and induced degenerative changes may differ significantly from those seen in humans. This paper reviews the many animal models developed for the study of IVD degeneration aetiopathogenesis and treatments thereof. In particular, the limitations and relevance of these models to the human condition are examined, and some general consensus guidelines are presented. Although animal models are invaluable to increase our understanding of disc biology, because of the differences between species, care must be taken when used to study human disc degeneration and much more effort is needed to facilitate research on human disc material.

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Are animal models useful for studying human disc disorders/degeneration?

Injectable alginate hydrogels for cell delivery in tissue engineering

The pericellular matrix (PCM) is a narrow tissue region surrounding chondrocytes in articular cartilage, which together with the enclosed cell(s) has been termed the "chondron." While the function of this region is not fully understood, it is hypothesized to have important biological and biomechanical functions. In this article, we review a number of studies that have investigated the structure, composition, mechanical properties, and biomechanical role of the chondrocyte PCM. This region has been shown to be rich in proteoglycans (e.g., aggrecan, hyaluronan, and decorin), collagen (types II, VI, and IX), and fibronectin, but is defined primarily by the presence of type VI collagen as compared to the extracellular matrix (ECM). Direct measures of PCM properties via micropipette aspiration of isolated chondrons have shown that the PCM has distinct mechanical properties as compared to the cell or ECM. A number of theoretical and experimental studies suggest that the PCM plays an important role in regulating the microenvironment of the chondrocyte. Parametric studies of cell-matrix interactions suggest that the presence of the PCM significantly affects the micromechanical environment of the chondrocyte in a zone-dependent manner. These findings provide support for a potential biomechanical function of the chondrocyte PCM, and furthermore, suggest that changes in the PCM and ECM properties that occur with osteoarthritis may significantly alter the stress-strain and fluid environments of the chondrocytes. An improved understanding of the structure and function of the PCM may provide new insights into the mechanisms that regulate chondrocyte physiology in health and disease.

The Pericellular Matrix as a Transducer of Biomechanical and Biochemical Signals in Articular Cartilage

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Study Design. The mRNA levels of aggrecan and collagen were quantified in intervertebral disc cells cultured under three conditions: primary alginate culture, monolayer culture, and re-encapsulation in alginate after monolayer culture. Objectives. To compare the phenotype of intervertebrai disc cells under different culture conditions and to investigate the reversibility of cell phenotype after reencapsulation in alginate after monolayer culture. Summary of Background Data. The intervertebral disc contains heterogeneous populations of cells that vary with anatomic region. These cells possess significant differences in phenotype that can be preserved in vitro, although the effect of culture conditions on the phenotype of these cells is poorly understood. Methods. The intervertebral disc cells of 4-5-month-old pigs were isolated enzymaticaily from three anatomic zones: anulus fibrosus (AF), transition zone (TZ), and nucleus puiposus (NP). Gene expression levels of aggrecan and collagen Types 1 and II were measured using a quantitative reverse transcriptase-polymerase chain reaction. Results, Gene expression levels of anulus fibrosus and transition zone cells were shifted in monolayer compared with alginate, although the shift was partially reversed when re-encapsulated in alginate. However, NP cells appeared to be insensitive to culture conditions. Furthermore, characteristic patterns of gene expression among AF, TZ, and NP cells in primary alginate culture did not exist in monolayer culture, but they were also observed after re-encapsulation in alginate. Conclusion. The findings of this study suggest that anulus fibrosus and transition zone cells undergo a reversible shift in phenotype when cultured in monolayer compared with alginate. These differences suggest that the culture system exerts a strong influence on cell phenotype and may play a role in the response of these cells to biophysical and biochemical stimuli in vitro.

Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture.

Study design A combined experimental and theoretical biomechanical study to quantify the mechanical properties of living cells of the porcine intervertebral disc. Objectives To quantify zonal variations in the mechanical properties and morphology of cells isolated from the intervertebral disc. Summary of background data Cellular response to mechanical stimuli is influenced by the mechanical properties of cells and of the extracellular matrix. Significant zonal variations in intervertebral disc matrix properties have been reported. No information is currently available on the corresponding regional variations in the mechanical properties of intervertebral disc cells, despite evidence of significant differences in cellular phenotype and biologic response to loading. Methods The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of cells isolated from the anulus fibrosus, transition zone, and nucleus pulposus. Results Intervertebral disc cells exhibited viscoelastic solid behaviors. Highly significant differences were observed in the morphology, cytoskeletal arrangement, and biomechanical properties of the nucleus pulposus cells as compared with anulus fibrosus or transition zone cells. Cells of the nucleus pulposus were approximately three times stiffer and significantly more viscous than cells of the anulus fibrosus or transition zone. Conclusions The findings of this study provide new evidence for the existence of two biomechanically distinct cell populations in the intervertebral disc. These differences in mechanical behavior may be related to observed differences in the cytoskeletal architecture between these cells, and may further play an important role in the development, maintenance, and degeneration of the intervertebral disc.

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations.

Cellular response to mechanical loading varies between the anatomic zones of the intervertebral disc. This difference may be related to differences in the structure and mechanics of both cells and extracellular matrix, which are expected to cause differences in the physical stimuli (such as pressure, stress, and strain) in the cellular micromechanical environment. In this study, a finite element model was developed that was capable of describing the cell micromechanical environment in the intervertebral disc. The model was capable of describing a number of important mechanical phenomena: flow-dependent viscoelasticity using the biphasic theory for soft tissues; finite deformation effects using a hyperelastic constitutive law for the solid phase; and material anisotropy by including a fiber-reinforced continuum law in the hyperelastic strain energy function. To construct accurate finite element meshes, the in situ geometry of IVD cells were measured experimentally using laser scanning confocal microscopy and three-dimensional reconstruction techniques. The model predicted that the cellular micromechanical environment varies dramatically between the anatomic zones, with larger cellular strains predicted in the anisotropic anulus fibrosus and transition zone compared to the isotropic nucleus pulposus. These results suggest that deformation related stimuli may dominate for anulus fibrosus and transition zone cells, while hydrostatic pressurization may dominate in the nucleus pulposus. Furthermore, the model predicted that micromechanical environment is strongly influenced by cell geometry, suggesting that the geometry of IVD cells in situ may be an adaptation to reduce cellular strains during tissue loading.

Anthony E. Baer

Papers

Collagen gene expression and mechanical properties of intervertebral disc cell–alginate cultures

Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture.

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations.

The micromechanical environment of intervertebral disc cells determined by a finite deformation, anisotropic, and biphasic finite element model.

Matrix protein gene expression in intervertebral disc cells subjected to altered osmolarity.