Search

Hypertrophic differentiation occurs during in vitro chondrogenesis of mesenchymal stem cells (MSCs), decreasing the quality of the cartilage construct. Previously we identified WNT pathway antagonists Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in blocking hypertrophic differentiation of human MSCs (hMSCs). In this study, we investigated the role of endogenously expressed DKK1 and FRZB in chondrogenesis of hMSC and chondrocyte redifferentiation and in preventing cell hypertrophy using three relevant human cell based systems, isolated hMSCs, isolated primary human chondrocytes (hChs), and cocultures of hMSCs with hChs for which we specifically designed neutralizing nano-antibodies. We selected and tested variable domain of single chain heavy chain only antibodies (VHH) for their ability to neutralize the function of DKK1 or FRZB. In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan and collagen type II staining were significantly reduced in monocultured hMSCs and monocultured chondrocytes. Furthermore, in cocultures, cells in pellets showed hypertrophic differentiation. In conclusion, endogenous expression of the WNT antagonists DKK1 and FRZB is necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are indispensable for the initial steps of chondrogenic differentiation of hMSCs, second they are necessary for chondrocyte redifferentiation, and finally in preventing hypertrophic differentiation of articular chondrocytes., Competing Interests: Author Disclosure Statement No competing financial interests exist.

Angiogenesis and neovascularization are fundamental for the success of clinically relevant-sized tissue-engineered (TE) constructs. The next generation of TE constructs relies on providing instructive materials combined with the delivery of angiogenic growth factors and cells to avoid tissue ischemia. However, the majority of materials and cell types screened so far show limited clinical relevance, either due to insufficient number of cells or due to the use of animal-derived matrixes. Here, we investigated whether endothelial-like cells derived from mesenchymal stromal cells (EL-MSCs) can be used for vascular TE in combination with injectable dextran-hyaluronic acid (Dex-g-HA) hydrogels. These hydrogels can be easily modified, as demonstrated by the incorporation of vascular endothelial growth factor (VEGF). We examined in vitro the reciprocal influences between cells and matrix. Dex-g-HA enabled higher EL-MSC metabolic rates associated with optimal cell sprouting in vitro compared to human umbilical vein endothelial cells. In vivo evaluation demonstrated the absence of an acute inflammatory response, and EL-MSCs incorporated within Dex-g-HA formed a functional vascular network integrated with the host vascular system. This work demonstrates that Dex-g-HA is an efficient delivery method of VEGF to induce angiogenesis. Additionally, functional neovascularization can be achieved in vitro and in vivo by the combination of Dex-g-HA with EL-MSC.

In cartilage, tissue engineering bioreactors can create a controlled environment to study chondrocyte behavior under mechanical stimulation or produce chondrogenic grafts of clinically relevant size. Here we present a novel bioreactor that combines mechanical stimulation with a two compartment system through which nutrients can be supplied solely by diffusion from opposite sides of a tissue-engineered construct. This design is based on the hypothesis that creating gradients of nutrients, growth factors, and growth factor antagonists can aid in the generation of zonal tissue-engineered cartilage. Computational modeling predicted that the design facilitates the creation of a biologically relevant glucose gradient. This was confirmed by quantitative glucose measurements in cartilage explants. In this system, it is not only possible to create gradients of nutrients, but also of anabolic or catabolic factors. Therefore, the bioreactor design allows control over nutrient supply and mechanical stimulation useful for in vitro generation of cartilage constructs that can be used for the resurfacing of articulated joints or as a model for studying osteoarthritis disease progression.

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-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., 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-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., 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.

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain μl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

Objective: When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms., Methods: RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated., Results: In vitro expanded human chondrocytes showed progressive changes in gene expression. Strikingly, an overall decrease in total gene expression was detected, which was both gradual and cumulative. DNA methylation was in part responsible for the expression downregulation of a number of genes. Genes involved in many pathways such as the extracellular-signal-regulated kinase (ERK) and Bone morphogenetic protein (BMP) pathways exhibited significant changes in expression. Inhibition of ERK pathway did not show dramatic effects in counteracting dedifferentiation process. BMP-2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture., Conclusion: Our study not only improves our knowledge of the intricate signaling network regulating maintenance of chondrocyte phenotype, but also contributes to improved chondrocyte expansion and chondrogenic performance for cell therapy., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2012 by the American College of Rheumatology.)

Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline 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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.