Coming together is a beginning: the making of an intervertebral disc.

The intervertebral disc (IVD) is a complex fibrocartilaginous structure located between the vertebral bodies that allows for movement and acts as a shock absorber in our spine for daily activities. It is composed of three components: the nucleus pulposus (NP), annulus fibrosus, and cartilaginous endplate. The characteristics of these cells are different, as they produce specific extracellular matrix (ECM) for tissue function and the niche in supporting the differentiation status of the cells in the IVD. Furthermore, cell heterogeneities exist in each compartment. The cells and the supporting ECM change as we age, leading to degenerative outcomes that often lead to pathological symptoms such as back pain and sciatica. There are speculations as to the potential of cell therapy or the use of tissue engineering as treatments. However, the nature of the cells present in the IVD that support tissue function is not clear. This review looks at the origin of cells in the making of an IVD, from the earliest stages of embryogenesis in the formation of the notochord, and its role as a signaling center, guiding the formation of spine, and in its journey to become the NP at the center of the IVD. While our current understanding of the molecular signatures of IVD cells is still limited, the field is moving fast and the potential is enormous as we begin to understand the progenitor and differentiated cells present, their molecular signatures, and signals that we could harness in directing the appropriate in vitro and in vivo cellular responses in our quest to regain or maintain a healthy IVD as we age.

Hypertrophic chondrocytes at the junction of musculoskeletal structures.

Hypertrophic chondrocytes are found at unique locations at the junction of skeletal tissues, cartilage growth plate, articular cartilage, enthesis and intervertebral discs. Their role in the skeleton is best understood in the process of endochondral ossification in development and bone fracture healing. Chondrocyte hypertrophy occurs in degenerative conditions such as osteoarthritis. Thus, the role of hypertrophic chondrocytes in skeletal biology and pathology is context dependent. This review will focus on hypertrophic chondrocytes in endochondral ossification, in which they exist in a transient state, but acting as a central regulator of differentiation, mineralization, vascularization and conversion to bone. The amazing journey of a chondrocyte from being entrapped in the extracellular matrix environment to becoming proliferative then hypertrophic will be discussed. Recent studies on the dynamic changes and plasticity of hypertrophic chondrocytes have provided new insights into how we view these cells, not as terminally differentiated but as cells that can dedifferentiate to more progenitor-like cells in a transition to osteoblasts and adipocytes, as well as a source of skeletal stem and progenitor cells residing in the bone marrow. This will provide a foundation for studies of hypertrophic chondrocytes at other skeletal sites in development, tissue maintenance, pathology and therapy.

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followed by comparisons with human. Leveraging on a dataset of over 2,700 proteins from 31 proteomic profiles, we identified key molecular and cellular differences between disc compartments and spine levels, but not gender. The nucleus pulposus (NP) and annulus fibrosus (AF) compartments differ the most, both in matrisome and cellularity contents. Differences in the matrisome are consistent with the fibrous nature required for tensile strength in the AF and hydration property in the NP. Novel findings for the NP cells included an enrichment in cell junction proteins for cell-cell communication (Cdh2, Dsp and Gja1) and osmoregulation (Slc12a2 and Wnk1). In NP cells, we detected heterogeneity of vacuolar organelles; where about half have potential lysosomal function (Vamp3, Copb2, Lamp1/2, Lamtor1), some contain lipid droplets and others with undefined contents. The AF is enriched in proteins for the oxidative stress responses (Sod3 and Clu). Interestingly, mitochondrial proteins are elevated in the lumbar than tail IVDs that may reflect differences in metabolic requirement. Relative to the human, cellular and structural information are conserved for the AF. Even though the NP is more divergent between mouse and human, there are similarities at the level of cell biology. Further, common cross-species markers were identified for both NP (KRT8/19, CD109) and AF (COL12A1). Overall, mouse is a relevant model to study IVD biology, and an understanding of the limitation will facilitate research planning and data interpretation, maximizing the translation of research findings to human IVDs.

Development of a standardized histopathology scoring system using machine learning algorithms for intervertebral disc degeneration in the mouse model-An ORS spine section initiative.

Mice have been increasingly used as preclinical model to elucidate mechanisms and test therapeutics for treating intervertebral disc degeneration (IDD). Several intervertebral disc (IVD) histological scoring systems have been proposed, but none exists that reliably quantitate mouse disc pathologies. Here, we report a new robust quantitative mouse IVD histopathological scoring system developed by building consensus from the spine community analyses of previous scoring systems and features noted on different mouse models of IDD. The new scoring system analyzes 14 key histopathological features from nucleus pulposus (NP), annulus fibrosus (AF), endplate (EP), and AF/NP/EP interface regions. Each feature is categorized and scored; hence, the weight for quantifying the disc histopathology is equally distributed and not driven by only a few features. We tested the new histopathological scoring criteria using images of lumbar and coccygeal discs from different IDD models of both sexes, including genetic, needle-punctured, static compressive models, and natural aging mice spanning neonatal to old age stages. Moreover, disc sections from common histological preparation techniques and stains including H&E, SafraninO/Fast green, and FAST were analyzed to enable better cross-study comparisons. Fleiss's multi-rater agreement test shows significant agreement by both experienced and novice multiple raters for all 14 features on several mouse models and sections prepared using various histological techniques. The sensitivity and specificity of the new scoring system was validated using artificial intelligence and supervised and unsupervised machine learning algorithms, including artificial neural networks, k-means clustering, and principal component analysis. Finally, we applied the new scoring system on established disc degeneration models and demonstrated high sensitivity and specificity of histopathological scoring changes. Overall, the new histopathological scoring system offers the ability to quantify histological changes in mouse models of disc degeneration and regeneration with high sensitivity and specificity.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

Matrix remodeling during intervertebral disc growth and degeneration detected by multichromatic FAST staining.

Various imaging techniques have been used to assess degeneration of the intervertebral disc, including many histological methods, but cartilage-oriented histological stains do not clearly show the comparatively complex structures of the disc. In addition, there is no integrated method to assess efficiently both the compartmental organization and matrix composition in disc samples. In this study, a novel histological method, termed FAST staining, has been developed to investigate disc growth and degeneration by sequential staining with fast green, Alcian blue, Safranin-O, and tartrazine to generate multichromatic histological profiles (FAST profiles). This identifies the major compartments of the vertebra-disc region, including the cartilaginous endplate and multiple zones of the annulus fibrosus, by specific FAST profile patterns. A disc degeneration model in rabbit established using a previously described puncture method showed gradual but profound alteration of the FAST profile during disc degeneration, supporting continual alteration of glycosaminoglycan. Changes of the FAST profile pattern in the nucleus pulposus and annulus fibrosus of the postnatal mouse spine suggested matrix remodeling activity during the growth of intervertebral discs. In summary, we developed an effective staining method capable of defining intervertebral disc compartments in detail and showing matrix remodeling events within the disc. The FAST staining method may be used to develop a histopathological grading system to evaluate disc degeneration or malformation.

Histological and reference system for the analysis of mouse intervertebral disc.

A new scoring system based on histo-morphology of mouse intervertebral disc (IVD) was established to assess changes in different mouse models of IVD degeneration and repair. IVDs from mouse strains of different ages, transgenic mice, or models of artificially induced IVD degeneration were assessed. Morphological features consistently observed in normal, and early/later stages of degeneration were categorized into a scoring system focused on nucleus pulposus (NP) and annulus fibrosus (AF) changes. "Normal NP" exhibited a highly cellularized cell mass that decreased with natural ageing and in disc degeneration. "Normal AF" consisted of distinct concentric lamellar structures, which was disrupted in severe degeneration. NP/AF clefts indicated more severe changes. Consistent scores were obtained between experienced and new users. Altogether, our scoring system effectively differentiated IVD changes in various strains of wild-type and genetically modified mice and in induced models of IVD degeneration, and is applicable from the post-natal stage to the aged mouse. This scoring tool and reference resource addresses a pressing need in the field for studying IVD changes and cross-study comparisons in mice, and facilitates a means to normalize mouse IVD assessment between different laboratories. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:233-243, 2018.

Early onset of disc degeneration in SM/J mice is associated with changes in ion transport systems and fibrotic events.

Intervertebral disc degeneration (IDD) causes back pain and sciatica, affecting quality of life and resulting in high economic/social burden. The etiology of IDD is not well understood. Along with aging and environmental factors, genetic factors also influence the onset, progression and severity of IDD. Genetic studies of risk factors for IDD using human cohorts are limited by small sample size and low statistical power. Animal models amenable to genetic and functional studies of IDD provide desirable alternatives. Despite differences in size and cellular content as compared to human intervertebral discs (IVDs), the mouse is a powerful model for genetics and assessment of cellular changes relevant to human biology. Here, we provide evidence for early onset disc degeneration in SM/J relative to LG/J mice with poor and good tissue healing capacity respectively. In the first few months of life, LG/J mice maintain a relatively constant pool of notochordal-like cells in the nucleus pulposus (NP) of the IVD. In contrast, chondrogenic events are observed in SM/J mice beginning as early as one-week-old, with progressive fibrotic changes. Further, the extracellular matrix changes in the NP are consistent with IVD degeneration. Leveraging on the genomic data of two parental and two recombinant inbred lines, we assessed the genetic contribution to the NP changes and identified processes linked to the regulation of ion transport systems. Significantly, "transport" system is also in the top three gene ontology (GO) terms from a comparative proteomic analysis of the mouse NP. These findings support the potential of the SM/J, LG/J and their recombinant inbred lines for future genetic and biological analysis in mice and validation of candidate genes and biological relevance in human cohort studies. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD008784.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

Structure and biology of the intervertebral disk in health and disease.

The intervertebral disks along the spine provide motion and protection against mechanical loading. The 3 structural components, nucleus pulposus, annulus fibrosus, and cartilage endplate, function as a synergistic unit, though each has its own role. The cells within each of these components have distinct origins in development and morphology, producing specific extracellular matrix proteins that are organized into unique architectures fit for intervertebral disk function. This article focuses on various aspects of intervertebral disk biology and disruptions that could lead to diseases such as intervertebral disk degeneration.

A splice-site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas.

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation.