Jeroen Leijten

Gremlin 1, frizzled-related protein, and dkk-1 are key regulators of human articular cartilage homeostasis

OBJECTIVE The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. METHODS Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. RESULTS Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. CONCLUSION Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate

Significance Multipotent cells, such as mesenchymal stromal cells (MSCs), have the capacity to differentiate into cartilage-forming cells. Chondrocytes derived from MSCs obtain an epiphyseal cartilage-like phenotype, which turns into bone upon implantation via endochondral ossification. Here, we report that the chondrogenic fate of MSCs can be metabolically programmed by low oxygen tension to acquire an articular chondrocyte-like phenotype via mechanisms that resemble natural development. Our study identifies metabolic programming of stem cells by oxygen tension as a powerful tool to control cell fate, which may have broad applications for the way in which stem cells are now prepared for clinical use. Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of oxygen, with lower oxygen tension in the region destined to become articular cartilage and higher oxygen tension in transient hypertrophic cartilage. Here, we prove that metabolic programming of MSCs by oxygen tension directs chondrogenesis into either permanent or transient hyaline cartilage. Human MSCs chondrogenically differentiated in vitro under hypoxia (2.5% O2) produced more hyaline cartilage, which expressed typical articular cartilage biomarkers, including established inhibitors of hypertrophic differentiation. In contrast, normoxia (21% O2) prevented the expression of these inhibitors and was associated with increased hypertrophic differentiation. Interestingly, gene network analysis revealed that oxygen tension resulted in metabolic programming of the MSCs directing chondrogenesis into articular- or epiphyseal cartilage-like tissue. This differentiation program resembled the embryological development of these distinct types of hyaline cartilage. Remarkably, the distinct cartilage phenotypes were preserved upon implantation in mice. Hypoxia-preconditioned implants remained cartilaginous, whereas normoxia-preconditioned implants readily underwent calcification, vascular invasion, and subsequent endochondral ossification. In conclusion, metabolic programming of MSCs by oxygen tension provides a simple yet effective mechanism by which to direct the chondrogenic differentiation program into either permanent articular-like cartilage or hypertrophic cartilage that is destined to become endochondral bone.

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

Fibroblast growth factor-1 is a mesenchymal stromal cell-secreted factor stimulating proliferation of osteoarthritic chondrocytes in co-culture

Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis patients were co-cultured with human bone marrow-derived MSCs (hMSCs) in pellets. Genome-wide mRNA expression analysis and quantitative polymerase chain reactions (qPCR) were used to identify soluble factors that were specifically induced in co-cultures. Immunofluorescent staining combined with cell tracking and enzyme-linked immunosorbent assay (ELISA) were performed to validate up-regulation at the protein level and to identify the cellular origin of the increased proteins. Chemical blockers and neutralizing antibodies were used to elucidate the role of the identified candidate genes in co-cultures. A number of candidate factors were differentially regulated in co-cultures at the mRNA level. Of these, fibroblast growth factor-1 (FGF-1) mRNA and protein expression were markedly increased in co-cultures predominantly due to up-regulated expression in MSCs. Blocking of FGF signaling in co-culture pellets by specific FGF receptor inhibitors or FGF-1 neutralizing antibodies completely blocked hPCs proliferation. We demonstrate that MSCs increase FGF-1 secretion on co-culture with hPCs, which, in turn, is responsible for increased hPCs proliferation in pellet co-cultures.

Cardiovascular Organ-on-a-Chip Platforms for Drug Discovery and Development

Cardiovascular diseases are prevalent worldwide and are the most frequent causes of death in the United States. Although spending in drug discovery/development has increased, the amount of drug approvals has seen a progressive decline. Particularly, adverse side effects to the heart and general vasculature have become common causes for preclinical project closures, and preclinical models do not fully recapitulate human in vivo dynamics. Recently, organs-on-a-chip technologies have been proposed to mimic the dynamic conditions of the cardiovascular system-in particular, heart and general vasculature. These systems pay particular attention to mimicking structural organization, shear stress, transmural pressure, mechanical stretching, and electrical stimulation. Heart- and vasculature-on-a-chip platforms have been successfully generated to study a variety of physiological phenomena, model diseases, and probe the effects of drugs. Here, we review and discuss recent breakthroughs in the development of cardiovascular organs-on-a-chip platforms, and their current and future applications in the area of drug discovery and development.

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs

Bioprinting is the most convenient microfabrication method to create biomimetic three-dimensional (3D) cardiac tissue constructs, which can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, we developed a gold nanorod (GNR) incorporated gelatin methacryloyl (GelMA)-based bioink for printing 3D functional cardiac tissue constructs. The GNR concentration was adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentration of GNR, the nanocomposite bioink had a low viscosity, similar to pristine inks, which allowed for the easy integration of cells at high densities. As a result, rapid deposition of cell-laden fibers at a high resolution was possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells showed improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridged the electrically resistant pore walls of polymers, improved the cell-to-cell coupling, and promoted synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.

Hypoxia Inhibits Hypertrophic Differentiation and Endochondral Ossification in Explanted Tibiae

Purpose Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself. Materials and Methods Fetal mouse tibiae (E17.5) explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively). Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion. Results The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation. Conclusions Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

Abstract. Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular “fingerprint” of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward’s clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis-associated factors

IntroductionOsteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1), frizzled-related protein (FRZB) and dickkopf 1 homolog (Xenopus laevis) (DKK1) as articular cartilage’s natural brakes of hypertrophic differentiation. In this study, we investigated whether factors implicated in osteoarthritis or regulation of chondrocyte hypertrophy influence GREM1, FRZB and DKK1 expression levels.MethodsGREM1, FRZB and DKK1 mRNA levels were studied in articular cartilage from healthy preadolescents and healthy adults as well as in preserved and degrading osteoarthritic cartilage from the same osteoarthritic joint by quantitative PCR. Subsequently, we exposed human articular chondrocytes to WNT, BMP, IL-1β, Indian hedgehog, parathyroid hormone-related peptide, mechanical loading, different medium tonicities or distinct oxygen levels and investigated GREM1, FRZB and DKK1 expression levels using a time-course analysis.ResultsGREM1, FRZB and DKK1 mRNA expression were strongly decreased in osteoarthritis. Moreover, this downregulation is stronger in degrading cartilage compared with macroscopically preserved cartilage from the same osteoarthritic joint. WNT, BMP, IL-1β signaling and mechanical loading regulated GREM1, FRZB and DKK1 mRNA levels. Indian hedgehog, parathyroid hormone-related peptide and tonicity influenced the mRNA levels of at least one antagonist, while oxygen levels did not demonstrate any statistically significant effect. Interestingly, BMP and WNT signaling upregulated the expression of each other’s antagonists.ConclusionsTogether, the current study demonstrates an inverse correlation between osteoarthritis and GREM1, FRZB and DKK1 gene expression in cartilage and provides insight into the underlying transcriptional regulation. Furthermore, we show that BMP and WNT signaling are linked in a negative feedback loop, which might prove essential in articular cartilage homeostasis by balancing BMP and WNT activity.