Mauro Alini

The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration.

Very little is known about the turnover of extracellular matrix in the human intervertebral disc. We measured concentrations of specific molecules reflecting matrix synthesis and degradation in predetermined regions of 121 human lumbar intervertebral discs and correlated them with ageing and Thompson grade of degeneration. Synthesis in intervertebral discs, measured by immunoassay of the content of a putative aggrecan biosynthesis marker (846) and the content of types I and II procollagen markers, is highest in the neonatal and 2-5-yr age groups. The contents of these epitopes/molecules progressively diminished with increasing age. However, in the oldest age group (60-80 yr) and in highly degenerated discs, the type I procollagen epitope level increased significantly. The percentage of denatured type II collagen, assessed by the presence of an epitope that is exposed with cleavage of type II collagen, increased twofold from the neonatal discs to the young 2-5-yr age group. Thereafter, the percentage progressively decreased with increasing age; however, it increased significantly in the oldest group and in highly degenerate discs. We identified three matrix turnover phases. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of type II collagen. Phase II (maturation and ageing) is distinguished by a progressive drop in synthetic activity and a progressive reduction in denaturation of type 11 collagen. Phase III (degeneration and fibrotic) is illustrated by evidence for a lack of increased synthesis of aggrecan and type II procollagen, but also by an increase in collagen type II denaturation and type I procollagen synthesis, both dependent on age and grade of tissue degeneration.

Are animal models useful for studying human disc disorders / 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.

Compression-induced changes in intervertebral disc properties in a rat tail model.

STUDY DESIGN An Ilizarov-type apparatus was applied to the tails of rats to assess the influence of immobilization, chronically applied compression, and sham intervention on intervertebral discs of mature rats. OBJECTIVES To test the hypothesis that chronically applied compressive forces and immobilization cause changes in the biomechanical behavior and biochemical composition of rat tail intervertebral discs. SUMMARY OF BACKGROUND DATA Mechanical factors are associated with degenerative disc disease and low back pain, yet there have been few controlled studies in which the effects of compressive forces on the structure and function of the disc have been isolated. METHODS The tails of 16 Sprague-Dawley rats were instrumented with an Ilizarov-type apparatus. Animals were separated into sham, immobilization, and compression groups based on the mechanical conditions imposed. In vivo biomechanical measurements of disc thickness, angular laxity, and axial and angular compliance were made at 14-day intervals during the course of the 56-day experiment, after which discs were harvested for measurement of water, proteoglycan, and collagen contents. RESULTS Application of pins and rings alone (sham group) resulted in relatively small changes of in vivo biomechanical behavior. Immobilization resulted in decreased disc thickness, axial compliance, and angular laxity. Chronically applied compression had effects similar to those of immobilization alone but induced those changes earlier and in larger magnitudes. Application of external compressive forces also caused an increase in proteoglycan content of the intervertebral discs. CONCLUSIONS The well-controlled loading environment applied to the discs in this model provides a means of isolating the influence of joint-loading conditions on the response of the intervertebral disc. Results indicate that chronically applied compressive forces, in the absence of any disease process, caused changes in mechanical properties and composition of tail discs. These changes have similarities and differences in comparison with human spinal disc degeneration.

The use of biodegradable polyurethane scaffolds for cartilage tissue engineering: potential and limitations.

The aim of the present study was to evaluate the capability of novel biodegradable polyurethane scaffolds to support attachment, growth and maintenance of differentiated chondrocytes in vitro for up to 42 days. After an initial decrease, although not significant, the DNA content of the constructs remained constant over the culture time. A progressive increase in glycosaminoglycans and collagen was observed during the culture period. However, a significant release of matrix molecules into the culture medium was also noticeable. At the transcriptional level, a decrease in aggrecan and procollagen II mRNA expression was noticeable, whereas procollagen I expression was increased. To conclude, the present data demonstrate that biodegradable polyurethane porous scaffolds seeded with articular chondrocytes support cell attachment and the production of extracellular matrix proteins. The limitations of the system are the diffusion of large amounts of matrix molecules into the culture medium and the dedifferentiation of the chondrocytes with prolonged time in culture. However, due to the favourable mechanical properties of the polyurethane scaffold, stimulation of chondrocytes by mechanical loading can be considered in order to improve the formation of a functional cartilage-like extracellular matrix.

The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix.

Study Design. The use of a cell-seeded biomatrix for tissue engineering of the intervertebral disc. Objective. To evaluate the ability of a biomatrix to support the viability of intervertebral disc cells and to accumulate the extracellular matrix that they produce. Summary of Background Data. Intervertebral disc degeneration is a common occurrence during adult life that has adverse economic consequences on the health care system. Current surgical treatments are aimed at removing or replacing the degenerate tissue, which can alter the biomechanics of the spine and result in degeneration at adjacent disc levels. The ideal treatment of the degenerate disc would involve biologic repair, and tissue-engineering techniques offer a means to achieve this goal. Methods. Scaffolds of type I collagen and hyaluronan were seeded with bovine nucleus pulposus or anulus fibrosus cells and maintained in culture for up to 60 days in the presence of fetal calf serum or a variety of growth factors to try to generate a tissue whose properties could mimic those of the nucleus pulposus with respect to proteoglycan content. Results. During the culture period, various proteoglycans (aggrecan, decorin, biglycan, fibromodulin, and lumican) and collagens (types I and II) accumulated in the scaffold. Proteoglycan accumulation in the scaffold was greatest under conditions in which transforming growth factor-&bgr;1 was present, but under all conditions, more proteoglycan was lost into the culture medium than retained in the scaffold. Both the nucleus and anulus cells behaved in a similar manner with respect to their ability to synthesize matrix macromolecules and have them retained in the scaffold. By day 60 of culture, the proteoglycan content of the scaffolds never exceeded 10% of that present in the mature nucleus pulposus, although this figure could have been considerably increased if most of the proteoglycan being synthesized could have been retained. Furthermore, proteoglycan retention was not uniform within the scaffold, but increased near its periphery. Conclusions. This work demonstrates that although it is possible to maintain functional disc cells in a biomatrix, it will be necessary to optimize proteoglycan synthesis and retention if any resulting tissue is to be of value in the biologic repair of the degenerate disc. The ability of the anulus cells to replicate the matrix production of the nucleus cells, at least in the collagen/hyaluronan scaffold, suggests that repair may not be limited to the availability of authentic nucleus cells.

2004 Young Investigator Award Winner: vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration.

Study Design. Intervertebral disc degeneration was evaluated by morphologic appearance, magnetic resonance imaging, and by biochemical matrix composition. Caliber and distribution of openings of the adjacent vertebral osseous endplates were measured. Objectives. Correlation between occlusion of vertebral endplate openings and intervertebral disc degeneration was quantified. Summary of Background Data. Calcifications of vertebral endplates with disease and age have suggested insufficient nutrition as a mechanism for intervertebral disc degeneration. It has been proposed that occlusion of endplate openings, which contain vascular sources for the disc, may limit the transport of nutrients, leading to disc degeneration. Methods. Fresh magnetic resonance images from 39 human lumbar discs were scored. Sectioned discs with endplates were morphologically graded. Samples of nuclear and anular regions were evaluated for proteoglycan and collagen contents. Backlight microscopic images of 4 endplate regions were obtained, and caliber and distribution of endplate openings for each disc were measured. Analysis of variance regression models were used to assess correlation between endplate openings and disc degeneration. Results. The decrease in opening density significantly correlated to morphologic degeneration grade, best for openings with 20 to 50 ìm equivalent diameter and in the nuclear region. Although the density of 20 to 50 ìm openings also significantly indirectly correlated to age, it was not as strong as the correlation to degeneration grade. Opening density was also significantly correlated to proteoglycan content in all regions. However, all other biochemical parameters as well as the T2 intensity score showed only weak or no correlation to opening density. Conclusions. A high indirect correlation between the density of openings in the osseous endplate (particularly of the size of the capillary buds) and the morphologicdegeneration grade of the disc support the hypothesis that occlusion of these openings may deprive the cells of nutrients, leading to insufficient maintenance of the extracellular matrix and disc degeneration.

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Effects of immobilization and dynamic compression on intervertebral disc cell gene expression in vivo

Study Design. An in vivo analysis of the intervertebral disc’s cellular response to dynamic compression and immobilization was performed using a rat-tail model. Objective. To assess the effects of immobilization and short-term dynamic compression on intervertebral disc cell expression of anabolic and catabolic genes. Summary of Background Data. Static compressive loads applied in vivo alter the composition of the disc matrix and cell viability in a dose-dependent manner. The effects of in vivo dynamic compression, which is a more physiologic load, and reported risk factor for low back pain have not been investigated. Methods. An Ilizarov-type device was implanted on the rat tail and used to determine the effects from 72 hours of immobilization (n = 6), 2 hours of dynamic compression (1 MPa/0.2 Hz) (n = 8), and the coupled effect of immobilization followed by compression (n = 8). Real-time reverse transcription-polymerase chain reaction was used to measure changes in anabolic and catabolic gene levels relative to both internal control subjects and a sham-operated group (n = 7). Results. Immobilization and dynamic compression affect anabolic and catabolic genes, with an overall downregulation of types 1 and 2 collagen and upregulation of aggrecanase, collagenase, and stromelysin in the anulus. The effects of immobilization and compression appear to be additive for collagen types 1 and 2 in the anulus, but not in the nucleus, and not for catabolic genes. Conclusions. Short-duration dynamic compression and immobilization alter gene expression in the rat disc. In studying the response of the disc to loading, it is necessary to look at both anabolic and catabolic pathways, and to consider strain history.

Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc

Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.

Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic.

Mesenchymal stem cells (MSCs) are increasingly being used in tissue engineering and cell‐based therapies in all fields ranging from orthopedic to cardiovascular medicine. Despite years of research and numerous clinical trials, MSC therapies are still very much in development and not considered mainstream treatments. The majority of approaches rely on an in vitro cell expansion phase in monolayer to produce large cell numbers prior to implantation. It is clear from the literature that this in vitro expansion phase causes dramatic changes in MSC phenotype which has very significant implications for the development of effective therapies. Previous reviews have sought to better characterize these cells in their native and in vitro environments, described known stem cell interactions within the bone marrow, and discussed the use of innovative culture systems aiming to model the bone marrow stem cell niche. The purpose of this review is to provide an update on our knowledge of MSCs in their native environment, focusing on bone marrow‐derived MSCs. We provide a detailed description of the differences between naive cells and those that have been cultured in vitro and examine the effect of isolation and culture parameters on these phenotypic changes. We explore the concept of “one step” MSC therapy and discuss the potential cellular and clinical benefits. Finally, we describe recent work attempting to model the MSC bone marrow niche, with focus on both basic research and clinical applications and consider the challenges associated with these new generation culture systems. Stem Cells 2014;32:1713–1723