Comparative transcriptomic profiling of myxomatous mitral valve disease in the cavalier King Charles spaniel.

BACKGROUND: Almost all elderly dogs develop myxomatous mitral valve disease by the end of their life, but the cavalier King Charles spaniel (CKCS) has a heightened susceptibility, frequently resulting in death at a young age and suggesting that there is a genetic component to the condition in this breed. Transcriptional profiling can reveal the impact of genetic variation through differences in gene expression levels. The aim of this study was to determine whether expression patterns were different in mitral valves showing myxomatous degeneration from CKCS dogs compared to valves from non-CKCS dogs. RESULTS: Gene expression patterns in three groups of canine valves resulted in distinct separation of normal valves, diseased valves from CKCS and diseased valves from other breeds; the latter were more similar to the normal valves than were the valves from CKCS. Gene expression patterns in diseased valves from CKCS dogs were quite different from those in the valves from other dogs, both affected and normal. Patterns in all diseased valves (from CKCS and other breeds) were also somewhat different from normal non-diseased samples. Analysis of differentially expressed genes showed enrichment in GO terms relating to cardiac development and function and to calcium signalling canonical pathway in the genes down-regulated in the diseased valves from CKCS, compared to normal valves and to diseased valves from other breeds. F2 (prothrombin) (CKCS diseased valves compared to normal) and MEF2C pathway activation (CKCS diseased valves compared to non-CKCS diseased valves) had the strongest association with the gene changes. A large number of genes that were differentially expressed in the CKCS diseased valves compared with normal valves and diseased valves from other breeds were associated with cardiomyocytes including CASQ2, TNNI3 and RYR2. CONCLUSION: Transcriptomic profiling identified gene expression changes in CKCS diseased valves that were not present in age and disease severity-matched non-CKCS valves. These genes are associated with cardiomyocytes, coagulation and extra-cellular matrix remodelling. Identification of genes that vary in the CKCS will allow exploration of genetic variation to understand the aetiology of the disease in this breed, and ultimately development of breeding strategies to eliminate this disease from the breed.

The Expression of PHOSPHO1, nSMase2 and TNAP is Coordinately Regulated by Continuous PTH Exposure in Mineralising Osteoblast Cultures.

Sustained exposure to high levels of parathyroid hormone (PTH), as observed in hyperparathyroidism, is catabolic to bone. The increase in the RANKL/OPG ratio in response to continuous PTH, resulting in increased osteoclastogenesis, is well established. However, the effects of prolonged PTH exposure on key regulators of skeletal mineralisation have yet to be investigated. This study sought to examine the temporal expression of PHOSPHO1, TNAP and nSMase2 in mineralising osteoblast-like cell cultures and to investigate the effects of continuous PTH exposure on the expression of these enzymes in vitro. PHOSPHO1, nSMase2 and TNAP expression in cultured MC3T3-C14 cells significantly increased from day 0 to day 10. PTH induced a rapid downregulation of Phospho1 and Smpd3 gene expression in MC3T3-C14 cells and cultured hemi-calvariae. Alpl was differentially regulated by PTH, displaying upregulation in cultured MC3T3-C14 cells and downregulation in hemi-calvariae. PTH was also able to abolish the stimulatory effects of bone morphogenic protein 2 (BMP-2) on Smpd3 and Phospho1 expression. The effects of PTH on Phospho1 expression were mimicked with the cAMP agonist forskolin and blocked by the PKA inhibitor PKI (5-24), highlighting a role for the cAMP/PKA pathway in this regulation. The potent down-regulation of Phospho1 and Smpd3 in osteoblasts in response to continuous PTH may provide a novel explanation for the catabolic effects on the skeleton of such an exposure. Furthermore, our findings support the hypothesis that PHOSPHO1, nSMase2 and TNAP function cooperatively in the initiation of skeletal mineralisation.

Large animal models of cardiovascular disease.

The human cardiovascular system is a complex arrangement of specialized structures with distinct functions. The molecular landscape, including the genome, transcriptome and proteome, is pivotal to the biological complexity of both normal and abnormal mammalian processes. Despite our advancing knowledge and understanding of cardiovascular disease (CVD) through the principal use of rodent models, this continues to be an increasing issue in today's world. For instance, as the ageing population increases, so does the incidence of heart valve dysfunction. This may be because of changes in molecular composition and structure of the extracellular matrix, or from the pathological process of vascular calcification in which bone-formation related factors cause ectopic mineralization. However, significant differences between mice and men exist in terms of cardiovascular anatomy, physiology and pathology. In contrast, large animal models can show considerably greater similarity to humans. Furthermore, precise and efficient genome editing techniques enable the generation of tailored models for translational research. These novel systems provide a huge potential for large animal models to investigate the regulatory factors and molecular pathways that contribute to CVD in vivo. In turn, this will help bridge the gap between basic science and clinical applications by facilitating the refinement of therapies for cardiovascular disease.

miRNA-221 and miRNA-222 synergistically function to promote vascular calcification.

Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans-differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR-microarray analysis revealed the significant down-regulation of a range of miRs following nine days in culture, including miR-199b, miR-29a, miR-221, miR-222 and miR-31 (p < 0.05). Subsequent studies investigated the specific role of the miR-221/222 family in VSMC calcification. Real-time quantitative polymerase chain reaction data confirmed the down-regulation of miR-221 (32.4%; p < 0.01) and miR-222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR-221 and miR-222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two-fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR-221 and miR-222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit-1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels.

Metformin ameliorates valve interstitial cell calcification by promoting autophagic flux.

Calcific aortic valve disease (CAVD) is the most common heart disease of the developed world. It has previously been established that metformin administration reduces arterial calcification via autophagy; however, whether metformin directly regulates CAVD has yet to be elucidated. In the present study we investigated whether metformin alleviates valvular calcification through the autophagy-mediated recycling of Runx2. Calcification was reduced in rat valve interstitial cells (RVICs) by metformin treatment (0.5-1.5 mM) (P < 0.01), with a marked decrease in Runx2 protein expression compared to control cells (P < 0.05). Additionally, upregulated expression of Atg3 and Atg7 (key proteins required for autophagosome formation), was observed following metformin treatment (1 mM). Blocking autophagic flux using Bafilomycin-A1 revealed colocalisation of Runx2 with LC3 puncta in metformin treated RVICs (P < 0.001). Comparable Runx2 accumulation was seen in LC3 positive autolysosomes present within cells that had been treated with both metformin and hydroxychloroquine in combination (P < 0.001). Mechanistic studies employing three-way co-immunoprecipitation with Runx2, p62 and LC3 suggested that Runx2 binds to LC3-II upon metformin treatment in VICs. Together these studies suggest that the utilisation of metformin may represent a novel strategy for the treatment of CAVD.

Increased bone mass, altered trabecular architecture and modified growth plate organization in the growing skeleton of SOCS2 deficient mice.

Suppressor of cytokine signalling-2 (SOCS2) negatively regulates the signal transduction of several cytokines. Socs2(-/-) mice show increased longitudinal skeletal growth associated with deregulated GH/IGF-1 signalling. The present study examined the role of SOCS2 in endochondral ossification and trabecular and cortical bone formation, and investigated whether pro-inflammatory cytokines associated with pediatric chronic inflammatory disorders mediate their effects through SOCS2. Seven-week-old Socs2(-/-) mice were heavier (27%; P < 0.001) and longer (6%; P < 0.001) than wild-type mice. Socs2(-/-) tibiae were longer (8%; P < 0.001) and broader (18%; P < 0.001) than that of wild-type mice, and the Socs2(-/-) mice had wider growth plates (24%; P < 0.001) with wider proliferative and hypertrophic zones (10% (P < 0.05) and 14% (P < 0.001) respectively). Socs2(-/-) mice showed increased total cross-sectional bone area (16%: P < 0.001), coupled to increased total tissue area (17%; P < 0.05) compared to tibia from wild-type mice. Socs2(-/-) mice showed increased percent bone volume (101%; P < 0.001), trabecular number (82%; P < 0.001) and trabecular thickness (11%; P < 0.001), with associated decreases in trabecular separation (19%; P < 0.001). TNFalpha exposure to growth plate chondrocytes for 48 h increased SOCS2 protein expression. Growth of metatarsals from 1-day-old Socs2(-/-) and Socs2(+/+) mice, as well as expression of Aggrecan, Collagen Type II and Collagen Type X, were inhibited by TNFalpha, with no effect of genotype. Our data indicate that physiological levels of SOCS2 negatively regulate bone formation and endochondral growth. Our results further suggest that pro-inflammatory cytokines mediate their inhibitory effects on longitudinal bone growth through a mechanism that is independent of SOCS2.

Inflammatory cytokines and the GH/IGF-I axis: novel actions on bone growth.

Longitudinal bone growth is a tightly regulated process that relies on complex synchronized mechanisms at the growth plate. Chronic paediatric inflammatory diseases are well accepted to lead to growth retardation and this is likely due to raised inflammatory cytokine levels and reduced growth hormone (GH)/insulin-like growth factor-1 (IGF-I) signalling. The precise cellular mechanisms responsible for this inhibition are unclear and therefore in this article, we will review the potential interactions between inflammatory cytokines and the GH/IGF-I axis in the regulation of bone growth. In particular, we will emphasis the potential contribution of the suppressors of cytokine signalling (SOCS) proteins, and in particular SOCS2, in mediating this process.

Inflammatory cytokines in juvenile idiopathic arthritis: effects on physical growth and the insulin-like-growth factor axis.

OBJECTIVE: To investigate the relationship between markers of inflammation with physical growth and systemic markers of GH secretion in JIA. DESIGN: This is a cross sectional prospective study of patients with JIA recruited during therapeutic arthrocentesis of 17 children with JIA (F,10): 8 oligoarticular (OJIA) and 9 polyarticular (PJIA). RESULTS: Median adjusted height (AHt) SDS was -0.3 (-2.2 to 1.6). Serum ALS SDS (median -1.3, range -2.7 to -0.6) was reduced compared with serum IGFBP-3 SDS (median -0.5, range -7.7 to 2.3) and IGF-1 SDS (median -0.2, range -0.5 to 0.5). Log serum IL5 (95% CI -3.25, -0.81) and log serum IL15 (95% CI -9.58, -4.10) were independent factors associated with AHt SDS. Inflammatory cytokines individually showed no association with IGF-1, IGFBP-1, -2, -3 and ALS. CONCLUSION: Children with JIA and mild degree of growth retardation show decreased ALS and IGFBP-3. Cytokines did not show an association to systemic markers of GH secretion. However, this study reports the novel, preliminary association between serum levels of IL5 and IL15 and the extent of short stature.

A comparison of breast muscle characteristics in three broiler great-grandparent lines.

Genetic selection of broiler chickens has led to a gross overdevelopment of the broiler breast muscle pectoralis major. This may have resulted in increased myopathy and detrimental effects on meat quality. The present study examined 3 commercial great-grandparent lines (lines A, B, and C). Lines A and B are female lines, and line C is a male line. The mean BW of line C (2.7 kg) was significantly greater than those of lines A and B (both 2.3 kg). However, the mean breast yield of both lines B and C (8.9 and 8.7%, respectively) was significantly greater than that of line A (6.9%). Line B therefore matched the meat yield of line A while maintaining a high reproductive capacity. The mean breast fillet weight of line A (169 g) was significantly lower than lines B (207 g) and C (235 g). No differences were observed between lines in either mean fiber size or amount of connective tissue. Therefore, additional fibers must provide the additional weight in the breast fillet of lines B and C, compared with A. Plasma creatine kinase activity, a commonly used marker of muscle damage, was significantly higher in line A (1368 IU/L) than in lines B (995 IU/L) and C (982 IU/L). However, qualitative evaluations of muscle pathology revealed no differences among lines. Selection for increased embryonic muscle fiber number, rather than for increased radial fiber growth, could improve growth potential and may also alleviate muscle damage.

Myxomatous Degeneration of the Canine Mitral Valve: From Gross Changes to Molecular Events.

Myxomatous mitral valve disease (MMVD) is the single most common acquired heart disease of the dog, but is also of emerging importance in human medicine, with some features of the disease shared between both species. There has been increased understanding of this disease in recent years, with most research aiming to elucidate the cellular and molecular events of disease pathogenesis. For gross and histological changes, much of our understanding is based on historical studies and there has been no comprehensive reappraisal of the pathology of MMVD. This paper reviews the gross, histological, ultrastructural, cellular and molecular changes in canine MMVD.

The restricted potential for recovery of growth plate chondrogenesis and longitudinal bone growth following exposure to pro-inflammatory cytokines.

Childhood chronic inflammatory disease can be associated with transient and permanent growth retardation. This study examined the potential for spontaneous growth recovery following pro-inflammatory cytokine exposure. Murine ATDC5 chondrogenic cells and postnatal metatarsals were exposed to interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNFalpha), and their growth and proliferative capacity were determined following recovery. TNFalpha and IL-1beta reduced chondrocyte proliferation and aggrecan and collagen types II and X expression at minimum concentrations of 10 ng/ml and 0.1 ng/ml respectively. TNFalpha but not IL-1beta exposure led to increased caspase-3 activity and altered cellular morphology, consistent with reduced viability. Cytokine exposure particularly inhibited proteoglycan synthesis. This effect was dose and duration dependent. Compared with the control, IL-1beta and TNFalpha led to a 71% and 45% reduction in metatarsal growth after 8 days of exposure respectively (P < 0.05). An additive effect of IL-1beta combined with TNFalpha was observed (110% decrease; P < 0.05). Metatarsals exposed to IL-1beta or TNFalpha individually for a 2-day period, and allowed to recover spontaneously in the absence of cytokines for a further 6 days, showed normal growth trajectories. In combination, growth was 59% lower (P < 0.01) compared with control metatarsals at the end of the recovery period. Exposure to the combination for 4 days followed by a 4-day recovery period resulted in 87% decrement compared with controls (P < 0.05). IL-6 did not alter any parameter studied. IL-1beta and TNFalpha exert diverse inhibitory effects on ATDC5 chondrocyte dynamics and metatarsal growth. The extent of recovery following cytokine exposure depends on the duration of exposure, and may be incomplete following longer periods of exposure.

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

Cytokine actions in growth disorders associated with pediatric chronic inflammatory diseases (review).

Growth disorders are commonly observed in children suffering from chronic inflammatory diseases such as Juvenile Idiopathic Arthritis (JIA) and Inflammatory Bowel Disease (IBD). These disorders range from general growth retardation to local acceleration of growth in the affected limb and are associated with the increased production of pro-inflammatory cytokines. In this article, we review how cytokines influence child growth by exerting a local effect at the level of the growth plate, and through systemic effects throughout the whole body.

Characterisation of matrix vesicles in skeletal and soft tissue mineralisation.

The importance of matrix vesicles (MVs) has been repeatedly highlighted in the formation of cartilage, bone, and dentin since their discovery in 1967. These nano-vesicular structures, which are found in the extracellular matrix, are believed to be one of the sites of mineral nucleation that occurs in the organic matrix of the skeletal tissues. In the more recent years, there have been numerous reports on the observation of MV-like particles in calcified vascular tissues that could be playing a similar role. Therefore, here, we review the characteristics MVs possess that enable them to participate in mineral deposition. Additionally, we outline the content of skeletal tissue- and soft tissue-derived MVs, and discuss their key mineralisation mediators that could be targeted for future therapeutic use.

Endocrine role of bone: recent and emerging perspectives beyond osteocalcin.

Recent developments in endocrinology, made possible by the combination of mouse genetics, integrative physiology and clinical observations have resulted in rapid and unanticipated advances in the field of skeletal biology. Indeed, the skeleton, classically viewed as a structural scaffold necessary for mobility, and regulator of calcium-phosphorus homoeostasis and maintenance of the haematopoietic niche has now been identified as an important regulator of male fertility and whole-body glucose metabolism, in addition to the classical insulin target tissues. These seminal findings confirm bone to be a true endocrine organ. This review is intended to detail the key events commencing from the elucidation of osteocalcin (OC) in bone metabolism to identification of new and emerging candidates that may regulate energy metabolism independently of OC.

Direct stimulation of bone mass by increased GH signalling in the osteoblasts of Socs2-/- mice.

The suppressor of cytokine signalling (Socs2(-/-))-knockout mouse is characterised by an overgrowth phenotype due to enhanced GH signalling. The objective of this study was to define the Socs2(-/-) bone phenotype and determine whether GH promotes bone mass via IGF1-dependent mechanisms. Despite no elevation in systemic IGF1 levels, increased body weight in 4-week-old Socs2(-/-) mice following GH treatment was associated with increased cortical bone area (Ct.Ar) (P<0.01). Furthermore, detailed bone analysis of male and female juvenile and adult Socs2(-/-) mice revealed an altered cortical and trabecular phenotype consistent with the known anabolic effects of GH. Indeed, male Socs2(-/-) mice had increased Ct.Ar (P<0.05) and thickness associated with increased strength. Despite this, there was no elevation in hepatic Igf1 expression, suggesting that the anabolic bone phenotype was the result of increased local GH action. Mechanistic studies showed that in osteoblasts and bone of Socs2(-/-) mice, STAT5 phosphorylation was significantly increased in response to GH. Conversely, overexpression of SOCS2 decreased GH-induced STAT5 signalling. Although an increase in Igf1 expression was observed in Socs2(-/-) osteoblasts following GH, it was not evident in vivo. Igf1 expression levels were not elevated in response to GH in 4-week-old mice and no alterations in expression was observed in bone samples of 6-week-old Socs2(-/-) mice. These studies emphasise the critical role of SOCS2 in controlling the local GH anabolic bone effects. We provide compelling evidence implicating SOCS2 in the regulation of GH osteoblast signalling and ultimately bone accrual, which maybe via mechanisms that are independent of IGF1 production in vivo.

A new method for the quantification of aortic calcification by three-dimensional micro-computed tomography.

To gain a better understanding of the mechanisms that underpin aortic calcification, rodent models have been previously utilised. Regions of calcium and phosphate deposition are commonly visualised using labor-intensive two-dimensional histomorphometric techniques. In this study, we developed a novel micro-computed tomography (µCT) imaging protocol to quantify calcification in vascular tissues using high resolution three-dimensional (3D) reconstructions of aortae derived from the well-established Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 knockout (Enpp1-/-) mouse model of medial aortic calcification. A dual-contrast method was employed for specimen preparation and the application of corn oil as a submersion medium for the samples during scanning, which allowed the definition and quantification of soft tissue. 3D µCT was utilised to produce reconstructions of calcified and non-calcified aortae. A highly accurate quantification of a standardized region of calcium deposition was undertaken on these reconstructions. An excellent correlation between images obtained from µCT and those obtained with Alizarin red staining, of whole aortae for calcium deposition, was observed. This imaging protocol provides a powerful tool for studying the development of aortic calcification and potential therapeutic approaches for clinical intervention.

New insights into NPP1 function: lessons from clinical and animal studies.

The recent elucidation of rare human genetic disorders resulting from mutations in ectonucleotide pyrophosphotase/phosphodiesterase (ENPP1), also known as plasma cell membrane glycoprotein 1 (PC-1), has highlighted the vital importance of this molecule in human health and disease. Generalised arterial calcification in infants (GACI), a frequently lethal disease, has been reported in recessive inactivating mutations in ENPP1. Recent findings have also linked hypophosphataemia to a lack of NPP1 function. A number of human genetic studies have indicated that NPP1 is a vital regulator that influences a wide range of tissues through various signalling pathways and when disrupted can lead to significant pathology. The function of Enpp1 has been widely studied in rodent models, where both the mutant tiptoe walking (ttw/ttw) mouse and genetically engineered Enpp1(-/-) mice show significant alterations in skeletal and soft tissue mineralisation, calcium/phosphate balance and glucose homeostasis. These models therefore provide important tools with which to study the potential mechanisms underpinning the human diseases associated with altered NPP1. This review will focus on the recent advances in our current knowledge of the actions of NPP1 in relation to bone disease, cardiovascular pathologies and diabetes. A fuller understanding of the mechanisms through which NPP1 exerts its pathological effects may stimulate the development of novel therapeutic strategies for patients at risk from the devastating clinical outcomes associated with disrupted NPP1 function.

MEPE is a novel regulator of growth plate cartilage mineralization.

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and µCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide.

Chondrogenic ATDC5 cells: an optimised model for rapid and physiological matrix mineralisation.

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM ß-glycerophosphate (ßGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with ßGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM ßGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.