A. Cremers

Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures.

OBJECTIVE Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. DESIGN Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. RESULTS 3D cultures specifically expressed chondrogenic mRNAs [collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. CONCLUSIONS For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.

Autologous engineering of cartilage

Treatment of full-thickness damage to hyaline cartilage is hampered by the limited availability of autologous healthy cartilage and the lengthy, cost-prohibitive cell isolation and expansion steps associated with autologous cartilage implantation (ACI). Here we report a strategy for de novo engineering of ectopic autologous cartilage (EAC) within the subperiosteal space (in vivo bioreactor), through the mere introduction of a biocompatible gel that might promote hypoxia-mediated chondrogenesis, thereby effectively overcoming the aforementioned limitations. The EAC is obtained within 3 wk post injection of the gel, and can be press-fit into an osteochondral defect where it undergoes remodeling with good lateral and subchondral integration. The implanted EAC showed no calcification even after 9 mo and attained an average O’Driscoll score of 11 (versus 4 for controls). An “on demand” autologous source of autologous cartilage with remodeling capacity is expected to significantly impact the clinical options in repair of trauma to articular cartilage.

Activation of NF-κB/p65 Facilitates Early Chondrogenic Differentiation during Endochondral Ossification

Background NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling. Methodology/Principal Findings The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. Conclusions/Significance The presented data demonstrate that NF-κB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Methods Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). Results The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. Conclusion We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types

BackgroundImmediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells.MethodsTo further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach.ResultsWe show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1β than the NP progenitor cells and aspects of this response were controlled by EGR1.ConclusionsOur current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells’ epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.

Indomethacin induces differential effects on in vitro endochondral ossification depending on the chondrocyte's differentiation stage

Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossifications are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossification on a molecular level; therefore, we aimed to determine whether indomethacin might influence heterotopic ossification via impairing the chondrogenic phase of endochondral ossification. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a decrease in gene‐ and protein expression of chondrogenic and hypertrophic markers (measured by RT‐qPCR and immunoblotting) as well as decreased glycosamino‐glycan content (by alcian blue histochemistry) was observed. Even when hypertrophic differentiation was provoked, the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre‐differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onward, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocytes differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rational behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications.

MicroRNA Profiling in Cartilage Ageing

Osteoarthritis (OA) is the most common age-related joint disorder in man. MicroRNAs (miRNA), a class of small noncoding RNAs, are potential therapeutic targets for regulating molecular mechanisms in both disease and ageing. Whilst there is an increasing amount of research on the roles of miRNAs in ageing, there has been scant research on age-related changes in miRNA in a cartilage. We undertook a microarray study on young and old human cartilages. Findings were validated in an independent cohort. Contrasts between these samples identified twenty differentially expressed miRNAs in a cartilage from old donors, derived from an OA environment which clustered based on OA severity. We identified a number of recognised and novel miRNAs changing in cartilage ageing and OA including miR-126: a potential new candidate with a role in OA pathogenesis. These analyses represent important candidates that have the potential as cartilage ageing and OA biomarkers and therapeutic targets.

The Role of Prostaglandins and COX-Enzymes in Chondrogenic Differentiation of ATDC5 Progenitor Cells

Objectives NSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation. Methods ATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses. Results Inhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD2 and PGE2 followed the COX-2 expression pattern, whereas PGF2α and TXA2 levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD2 and PGF2α in the COX-1 inhibited condition. Addition of PGE2 and PGF2α resulted in increased expression of chondrogenic markers, whereas TXA2 increased expression of hypertrophic markers. Conclusions Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production.

168 AN EARLY INFLAMMATORY RESPONSE DETERMINES THE ONSET OF CHONDROGENESIS

chondrocyte cultures. Genes already associated with hypertrophic cartilage or OA (ALPL, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two donor groups. The expression of 661 genes was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished, and only 184 genes were differentially regulated. Conclusions: All in all, our data confirm already known data on many characteristic features of native OA cartilage, but we have also identified new candidate genes that are differentially expressed during OA. For the development of new OA cartilage treatment strategies, such a deeper insight into phenotypical alterations occurring in OA is important. Only a few genes were differentially expressed between OA and ND chondrocytes in hyaff-11 culture. So, the risk of generating hypertrophic cartilage does not seem to be increased for OA chondrocytes. Importantly, our findings suggest that the chondrogenic capacity is not significantly affected by OA, and OA chondrocytes fulfill the requirements for ACT.

175 DECREASED CHONDROCYTE HYPERTROPHIC DIFFERENTIATION BY INHIBITION OF CYCLOOXYGENASE-2

INTRODUCTION: Chondrocyte hypertrophic differentiation is an essential process during endochondral bone formation, however it hampers the application of many cartilage regenerative techniques and may play a role at the onset of osteoarthritis (1). Heterotopic bone formation after orthopaedic surgery is suppressed by non-steroidal anti-inflammatory drugs (NSAIDs) and recent studies point to an essential role of Cyclooxygenase-2 (COX-2) in osteocytes during endochondral ossification (2). COX-2 and its metabolite PGE2 are thought to regulate the function of bone morphogenic protein-2 (BMP-2) and vice versa, which might provide an explanation for the role of COX-2 during endochondral ossification (3). It is largely unknown how and in which phase NSAIDs affect endochondral bone formation. In this study, we aim to determine the involvement of COX-2 in chondrocyte hypertrophy and provide an explanation for the suppressive effect of NSAIDs on heterotopic ossification and fracture healing. .