M.M. Caron

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.

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.

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

Osmolarity determines the in vitro chondrogenic differentiation capacity of progenitor cells via nuclear factor of activated T-cells 5

INTRODUCTION Previous studies have shown that human articular chondrocytes in vitro are osmolarity-responsive and increase matrix synthesis under cartilage-specific physiological osmolarity. The effects of increased osmolarity on chondrogenesis of progenitor cells in vitro are largely unknown. We therefore aimed to elucidate whether hyperosmolarity facilitates their chondrogenic differentiation and whether Nfat5 is involved. MATERIALS AND METHODS ATDC5 cells and human bone marrow stem cells (hBMSCs) were differentiated in the chondrogenic lineage in control and increased osmolarity conditions. Chondrogenic outcome was measured by gene- and protein expression analysis. RNAi was used to determine the role of Nfat5 in chondrogenic differentiation under normal and increased osmolarity. RESULTS Increasing the osmolarity of differentiation medium with 100mOsm resulted in significantly increased chondrogenic marker expression (Col2a1, Col10a1, Acan, Sox9, Runx2 and GAGs) during chondrogenic differentiation of the two chondroprogenitors, ATDC5 and hBMSCs. Nfat5 knockdown under both control and increased osmolarity affected chondrogenic differentiation and suppressed the osmolarity-induced chondrogenic induction. Knockdown of Nfat5 in early differentiation significantly decreased early Sox9 expression, whereas knockdown of Sox9 in early differentiation did not affect early Nfat5 expression. CONCLUSIONS Increasing the osmolarity of chondrogenic culture media by 100mOsm significantly increased chondrogenic gene expression during the course of chondrogenic differentiation of progenitor cells. Nfat5 may be involved in regulating chondrogenic differentiation of these cells under both normal and increased osmolarities and might regulate chondrogenic differentiation through influencing early Sox9 expression.

The Immediate Early Gene Product EGR1 and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis

Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.

BAPX-1/NKX-3.2 Acts as a Chondrocyte Hypertrophy Molecular Switch in Osteoarthritis

Osteoarthritis (OA) development involves a shift of the articular chondrocyte phenotype toward hypertrophic differentiation via still poorly characterized mechanisms. The purpose of this study was to test our hypothesis that the function of BAPX‐1/NKX‐3.2 is impaired in OA chondrocytes and leads directly to loss of hypertrophic protection of the articular chondrocyte, which is central in the changing chondrocyte phenotype that drives OA.

Endochondral ossification in a case of progressive osseous heteroplasia in a young female child

Progressive osseous heteroplasia (POH) (OMIM 166350) is a rare autosomal dominant condition, characterized by heterotopic ossification of the skin, subcutaneous fat, and deep connective tissue. This condition is distinct from Albright’s hereditary osteodystrophy or McCune Albright syndrome (OMIM 103580) and fibrodysplasia ossificans progressiva (OMIM 135100). We present an unusual presentation of POH in a 7-year-old female child. The clinical features included a painful swelling on the left foot, with mechanical complaints. There was no congenital hallux valgus. Family anamnesis was positive in the father. There were subcutaneous ossifications of his left upper arm, right-sided thorax, and lateral side of the right ankle. The father did not allow any radiographs or further examinations. Radiographic examination of the patient revealed ossified subcutaneous plaques on the left foot, lumbar spine, and left scapulae. Additional blood samples were analyzed, revealing no pseudohypoparathyroidism. Sequence analysis of the gene associated with POH, the GNAS1 gene, revealed the heterozygote mutation c.565_568del, previously found in Albright’s hereditary osteodystrophy. Histopathological examination of the subcutaneous ossification showed presence of chondrocyte clusters, a feature usually found in fibrodysplasia ossificans progressiva. The combination of the clinical features, the absence of pseudohypoparathyroidism, histology revealing chondrocyte clusters, and the specific GNAS mutation in this patient makes this a truly unusual presentation of POH. The findings in the described case might denote subdivisions of POH. The condition is associated with progressive superficial to deep ossification, progressive restriction of range of motion, and recurrence if excised. We hope to inform pediatricians and orthopedic surgeons to create more awareness of this disorder so that unnecessary treatments can be avoided and proper counseling offered.

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.

Expression of RMRP RNA is regulated in chondrocyte hypertrophy and determines chondrogenic differentiation

Mutations in the RMRP-gene, encoding the lncRNA component of the RNase MRP complex, are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in RMRP RNA lead to skeletal dysplasia. Since chondrogenic differentiation of the growth plate is required for development of long bones, we hypothesized that RMRP RNA plays a pivotal role in chondrogenic differentiation. Expression of Rmrp RNA and RNase MRP protein subunits was detected in the murine growth plate and during the course of chondrogenic differentiation of ATDC5 cultures, where Rmrp RNA expression was found to be correlated with chondrocyte hypertrophy. Genetic interference with Rmrp RNA expression in ATDC5 cultures caused a deregulation of chondrogenic differentiation, with a prominent impact on hypertrophy and changes in pre-rRNA processing and rRNA levels. Promoter reporter studies showed that Rmrp RNA expression responds to chondrogenic morphogens. Chondrogenic trans-differentiation of cartilage-hair hypoplasia fibroblasts was impaired with a pronounced impact on hypertrophic differentiation. Together, our data show that RMRP RNA expression is regulated during different stages of chondrogenic differentiation and indicate that RMRP RNA may play a pivotal role in chondrocyte hypertrophy, with potential consequences for CHH pathobiology.

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.