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.

BMP-2 and BMP-7 display opposing actions on the chondrogenic outcome of differentiating progenitor cells: BMP-2 acts a specific inducer of chondrocyte hypertrophy, while BMP-7 appears to increase or maintain chondrogenic potential and prevent chondrocyte hypertrophy. Our results pave the way for an application-dependent differential use of BMP-2 or BMP-7.

BackgroundNF-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 FindingsThe 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/SignificanceThe 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.

Skeletogenesis and bone fracture healing involve endochondral ossification, a process during which cartilaginous primordia are gradually replaced by bone tissue. In line with a role for cyclooxygenase-2 (COX-2) in the endochondral ossifi cation process, non-steroidal anti-inflammatory drugs (NSAIDs) were reported to negatively affect bone fracture healing due to impaired osteogenesis. However, a role for COX-2 activity in the chondrogenic phase of endochondral ossifi cation has not been addressed before. We show that COX-2 activity fulfi ls an important regulatory function in chondrocyte hypertrophic differentiation. Our data reveal essential cross-talk between COX-2 and bone morphogenic protein-2 (BMP-2) during chondrocyte hypertrophic differentiation. BMP-2 mediated chondrocyte hypertrophy is associated with increased COX-2 expression and pharmacological inhibition of COX-2 activity by NSAIDs (e.g., Celecoxib) decreases hypertrophic differentiation in various chondrogenic models in vitro and in vivo, while leaving early chondrogenic development unaltered. Our fi ndings demonstrate that COX-2 activity is a novel factor partaking in chondrocyte hypertrophy in the context of endochondral ossifi cation and these observations provide a novel etiological perspective on the adverse effects of NSAIDs on bone fracture healing and have important implications for the use of NSAIDs during endochondral skeletal development.

Osteoarthritis (OA) is an extremely prevalent age-related condition. The economic and societal burden due to the cost of symptomatic treatment, inability to work, joint replacement, and rehabilitation is huge and increasing. Currently, there are no effective medical therapies that delay or reverse the pathological manifestations of OA. Current treatment options are, without exception, focused on slowing down progression of the disease to postpone total joint replacement surgery for as long as possible and keeping the associated pain and joint immobility manageable. Alterations in the articular cartilage chondrocyte phenotype might be fundamental in the pathological mechanisms of OA development. In many ways, the changing chondrocyte phenotype in osteoarthritic cartilage resembles the process of endochondral ossification as seen, for instance, in developing growth plates. However, the relative contribution of endochondral ossification to the changing chondrocyte phenotype in the development and progression of OA remains poorly described. In this review, we will discuss the current knowledge regarding the cartilage endochondral phenotypic changes occurring during OA development and progression, as well as the molecular and environmental effectors driving these changes. Understanding how these molecular mechanisms determine the chondrocyte cell fate in OA will be essential in enabling cartilage regenerative approaches in future treatments of OA.

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.

Objective. 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.Methods. Human articular chondrocytes (HACs; from healthy and OA donors) and SW-1353 chondrocytic cells were exposed to bone morphogenetic protein 7 (BMP-7), interleukin-1b (IL-1b), tumor necrosis factor, or OA synovial fluid (SF; 20% [volume/volume]). Loss-offunction and gain-of-function experiments for BAPX-1/ NKX-3.2 were performed. Mouse experimental models of OA were used, and (immuno)histochemistry of tissue sections was performed. Gene and protein expression of BAPX-1/NKX-3.2 and chondrogenic, hypertrophic, and OA-related mediators were determined by real-time quantitative polymerase chain reaction analysis and immunoblotting. In addition, alkaline phosphatase (AP) activity and prostaglandin E 2 levels were measured.Results. BAPX-1/NKX-3.2 expression correlated negatively with expression of chondrocyte hypertrophic markers (RUNX-2, COL10A1, AP), cartilage-degrading enzymes (matrix metalloproteinase 13, ADAMTS-5), and mediators of inflammation (cyclooxygenase 2, IL-6) in healthy and OA chondrocytes, as well as in OA induced chondrocytes. BAPX-1/NKX-3.2 positivity was diminished in articular chondrocytes in the knee joints of mice with experimental OA. Knockdown of BAPX-1/NKX-3.2 in HACs did not influence the expression of SOX9, COL2A1, or aggrecan, but led to an acute hypertrophic shift in the HAC phenotype. Overexpression of BAPX-1/NKX-3.2 decreased hypertrophic gene expression in HACs. Furthermore, the hypertrophic OA chondrocyte phenotype could be counteracted by overexpression of BAPX-1/NKX-3.2 and by BMP-7 in a BAPX-1/NKX-3.2 dependent manner.Conclusion. Our findings indicate that BAPX-1/ NKX-3.2 is a molecular switch that is involved in controlling the hypertrophic phenotype of the postdevelopmental (OA) articular chondrocyte.Osteoarthritis (OA) is the most common degenerative joint disorder worldwide and presents with degradation of the articular cartilage, leading to loss of joint mobility and function, and is accompanied by chronic pain (1). From a biochemical perspective, OA is characterized by uncontrolled synthesis of mediators of inflammation (e.g., interleukins, prostaglandins) and extracellular matrixdegrading enzymes, such as aggrecanases (ADAMTS) and matrix metalloproteinases (MMPs), resulting in the active breakdown of the cartilage tissue (2).Changes in the articular cartilage and chondrocytes that characterize OA resemble the cellular developmental process that drives skeletal development by endochondral ossification (3-5). Many of the cartilagedegrading enzymes that are secreted by hypertrophic chondrocytes in the growth plate are also centra...

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