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

Abstract Introduction: Chondrocytes experience a hypertonic environment compared to plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to non-physiological 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. Methods: Human 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 (TonEBP/NFAT5) in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference. Results: Moderately 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’ (non-osteoarthritic) 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. Conclusions: Physiological 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

The establishment, maintenance, and adaptation of high- and low-impact chronic pain: a framework for biopsychosocial pain research

We present a framework for the study of states of chronic pain and transitions between those states. We capture in the framework the dynamic nature of pain: people live with pain that changes over time. First, we offer definitions of both acute and chronic pain and explore the contextual considerations related to the common use of this temporal dichotomy. Second, we promote the importance of incorporating the impact pain has on a person's life. Finally, we discuss the challenges and opportunities inherent in implementing this common approach. Our goal is to produce a framework for the study of the development, maintenance, and resolution of chronic pain. Whether a single brief event or a constant feature of life, pain interrupts to prioritise protection, interferes with activity, reduces quality of life, and can alter identity.44 Protection is achieved by escape from harm, avoidance of perceived danger, withdrawal for respite and repair, and communication of incapacity and environmental risk; longer-term protection is achieved by learning the cues for pain and injury.53 From this perspective, pain is most usefully considered a need state, fundamentally a motivational drive to protect.49 This approach centres our attention on the consequences of pain for the person in their context, on its duration and its impact

Management of the stiff elbow: a literature review

• The elbow is prone to stiffness due to its unique anatomy and profound capsular reaction to inflammation. The resulting movement impairment may significantly interfere with a patient’s activities of daily living. • Trauma (including surgery for trauma), posttraumatic arthritis, and heterotopic ossification (HO) are the most common causes of elbow stiffness. • In stiffness caused by soft tissue contractures, initial conservative treatment with physiotherapy (PT) and splinting is advised. In cases in which osseous deformities limit range of motion (e.g. malunion, osseous impingement, or HO), early surgical intervention is recommended. • Open and arthroscopic arthrolysis are the primary surgical options. Arthroscopic arthrolysis has a lower complication and revision rate but has narrower indications. • Early active mobilization using PT after surgery is recommended in postoperative rehabilitation and may be complemented by splinting or continuous passive motion therapy. Most results are gained within the first few months but can continue to improve until 12 months

Calcineurin inhibitors promote chondrogenic marker expression of dedifferentiated human adult chondrocytes via stimulation of TGFss1 production

In-vitro chondrocyte expansion is required for several cell-based approaches for the repair of chondral lesions. During expansion, loss of chondrogenic phenotype takes place (dedifferentiation). The objective of this study was to investigate calcineurin as a potential target to improve chondrocyte phenotype for cartilage repair purposes. Calcineurin activity in human articular chondrocytes was significantly increased during dedifferentiation and decreased during redifferentiation in vitro. Inhibition of calcineurin activity by FK506 increased the expression of chondrogenic markers collagen type 2, aggrecan and SOX9 in culture expanded cells. Addition of FK506 increased endogenous Transforming Growth Factor (TGF) beta1 expression on both mRNA and protein level. The effect of FK506 on chondrogenic markers was abolished by addition of anti-TGFbeta1 antibody, indicating that the endogenous TGFbeta1 was necessary to increase chondrogenic marker expression. We also showed that chondrocyte redifferentiation by TGFbeta requires calcium influx and does not depend on changes in calcineurin activity. In conclusion, inhibition of calcineurin activity by FK506 increases the expression of chondrogenic markers via endogenous TGFbeta1 production in human articular chondrocytes. Calcineurin inhibitors might be an alternative for the application of (recombinant) TGFbeta, to promote chondrocyte phenotype for cell-based cartilage repair procedures

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies