O-Phenanthroline as modulator of the hypoxic and catabolic response in cartilage tissue-engineering models.

Hypoxia has been shown to be important for maintaining cartilage homeostasis as well as for inducing chondrogenic differentiation. Ensuring low oxygen levels during in vitro culture is difficult, therefore we assessed the chondro-inductive capabilities of the hypoxia-mimicking agent O-phenanthroline, which is also known as a non-specific matrix metalloproteinase (MMP) inhibitor. We found that O-phenanthroline reduced the expression of MMP3 and MMP13 mRNA levels during chondrogenic differentiation of human chondrocytes (hChs), as well as after TNFα/IL-1ß exposure in an explant model. Interestingly, O-phenanthroline significantly inhibited matrix degradation in a TNFα/IL-1ß-dependent model of cartilage degeneration when compared to control and natural hypoxia (2.5% O2 ). O-Phenanthroline had limited ability to improve the chondrogenic differentiation or matrix deposition in the chondrogenic pellet model. Additionally, O-phenanthroline alleviated MMP-induced cartilage degradation without affecting chondrogenesis in the explant culture. The data presented in this study indicate that the inhibitory effect of O-phenanthroline on MMP expression is dominant over the hypoxia-mimicking effect. Copyright © 2014 John Wiley & Sons, Ltd.

MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors.

The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Since the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. In this study, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty hMSC donors were initially tested for marker expression and differentiation potential. In particular, the chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight showed low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a 7-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low-performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have a prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration.

Differentiation of mesenchymal stem cells under hypoxia and normoxia: lipid profiles revealed by time-of-flight secondary ion mass spectrometry and multivariate analysis.

Mesenchymal stem cells (MSC) have the ability to self-renew and differentiate into multiple cell types valuable for clinical treatment of rheumatic pathologies. To study the chondrogenic potential of MSC and identify the conditions that recreate the native cartilage environment, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) for label-free detection of cell-type- and environmental-condition-specific molecular profiles. We observed that coculture of human MSC and chondrocytes under standard culture conditions leads to improved extracellular matrix (ECM) deposition. In marked contrast, this effect was lost under low oxygen tension. This improved extracellular matrix deposition was associated with a significant decrease in lipids and in particular cholesterol under low oxygen tension as revealed by TOF-SIMS coupled to principal component analysis and discriminant analysis. We furthermore demonstrate that the higher cholesterol levels under normoxia might regulate fibroblast growth factor 1 (FGF-1) gene expression which was previously implemented in increased ECM production in the cocultures. In conclusion, our study shows an unexpected role of lipids as orchestrators of chondrogenesis in response to oxygen tension which is, at least in part, mediated through FGF-1.

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

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.

Mesenchymal stromal/stem cell-or chondrocyte-seeded microcarriers as building blocks for cartilage tissue engineering.

In this study we have tested the use of mesenchymal stromal/stem cell (MSC)- or chondrocyte (hch)-laden microcarriers as building blocks for engineered cartilage tissue. MSCs and hchs expanded on microcarriers were used in chondrogenic coculture and compared with monoculture of MSCs or hchs. The use of cell-laden microcarriers as building blocks for cartilage tissue engineering led to a compact tissue formation with significant volume increase compared to the biomaterial-free approach. After 28 days of differentiation culture, formation of cartilaginous matrix in cocultures and chondrocyte monoculture approaches was observed. Coculture resulted in beneficial glycosaminoglycan deposition compared with monoculture of MSCs or chondrocytes attached to microcarriers. Further, the microcarrier-adhered coculture displayed increased levels of the differentiation marker ACAN and reduced levels of the dedifferentiation marker COL1A1. To our knowledge, this is the first article that successfully combines an innovative combination of cell expansion on microcarriers and the direct use of MSC- or hch-cell-laden microcarriers as building blocks in cartilage tissue engineering.

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

INTRODUCTION: Osteoarthritis 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. METHODS: GREM1, 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-1ß, 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. RESULTS: GREM1, 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-1ß 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. CONCLUSIONS: Together, 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.

The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells.

Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering.

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.

Cell sources for articular cartilage repair strategies: shifting from monocultures to cocultures.

The repair of articular cartilage is challenging due to the sparse native cell population combined with the avascular and aneural nature of the tissue. In recent years, cartilage tissue engineering has shown great promise. As with all tissue engineering strategies, the possible therapeutic outcome is intimately linked with the used combination of cells, growth factors, and biomaterials. However, the optimal combination has remained a controversial topic and no consensus has been reached. In consequence, much effort has been dedicated, to further design, investigate, and optimize cartilage repair strategies. Specifically, various research groups have performed intensive investigations attempting to identify the single most optimal cell source for articular cartilage repair strategies. However, recent findings indicate that not the heavily investigated monocell source, but the less studied combinations of cell sources in coculture might be more attractive for cartilage repair strategies. This review will give a comprehensive overview on the cell sources that have been investigated for articular cartilage repair strategies. In particular, the advantages and disadvantages of investigated cell sources are comprehensively discussed with emphasis on the potential of cocultures in which benefits are combined, while the disadvantages of single-cell sources for cartilage repair are mitigated.

Cartilage tissue engineering.

Cartilage tissue engineering is the art aimed at repairing defects in the articular cartilage which covers the bony ends in the joints. Since its introduction in the early 1990s of the past century, cartilage tissue engineering using ACI has been used in thousands of patients to repair articular cartilage defects. This review focuses on emerging strategies to improve cartilage repair by incorporating fundamental knowledge of developmental and cell biology in the design of optimized strategies for cell delivery at the defect site and to locally stimulate cartilage repair responses.

Trophic effects of mesenchymal stem cells increase chondrocyte proliferation and matrix formation.

Previous studies showed that coculture of primary chondrocytes (PCs) with various sources of multipotent cells results in a higher relative amount of cartilage matrix formation than cultures containing only chondrocytes. The aim of this study was to investigate the mechanism underlying this observation. We used coculture pellet models of human mesenchymal stem cells (hMSCs) and human PCs or bovine PCs (bPCs) and studied the fate and the contribution to cartilage formation of the individual cell populations during coculture. Enhanced cartilage matrix deposition was confirmed by histology and quantification of total glycosaminoglycan deposition. Species-specific quantitative polymerase chain reaction demonstrated that cartilage matrix gene expression was mainly from bovine origin when bPCs were used. Short tandem repeat analysis and species-specific quantitative polymerase chain reaction analysis of genomic DNA demonstrated the near-complete loss of MSCs in coculture pellets after 4 weeks of culture. In coculture pellets of immortalized MSCs and bPCs, chondrocyte proliferation was increased, which was partly mimicked using conditioned medium, and simultaneously preferential apoptosis of immortalized MSCs was induced. Taken together, our data clearly demonstrate that in pellet cocultures of MSCs and PCs, the former cells disappear over time. Increased cartilage formation in these cocultures is mainly due to a trophic role of the MSCs in stimulating chondrocyte proliferation and matrix deposition by chondrocytes rather than MSCs actively undergoing chondrogenic differentiation.

Strategies for zonal cartilage repair using hydrogels.

Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrogel-based tissue-engineering strategies have recently been developed to form constructs with biomimetic zonal variations to improve cartilage repair. Modular hydrogel systems allow for systematic control over hydrogel properties, and advanced fabrication techniques allow for control over construct organization. These technologies have great potential to address many unanswered questions involved in prescribing zonal properties to tissue-engineered constructs for cartilage repair.