In vivo use of PRP, but also of PPP to a certain extent, in tendon injuries might accelerate the catabolic demarcation of traumatically injured tendon matrices and promote angiogenesis and formation of a fibrovascular callus. Whether this will also be beneficial for degenerative tendinopathies remains to be elucidated.

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.

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Objective. The physiologic interstitial tonicity of healthy articular cartilage (350-480 mOsm) is lowered to 280-350 mOsm in osteoarthritis (OA). This results in loss of tissue prestress, altered compressive behavior, and, thus, inferior tissue properties. This study was undertaken to determine whether physiologic tonicity in combination with the inhibition of calcineurin (Cn) activity by FK-506 has synergistic effects on human articular chondrocytes and explants in vitro.Methods. OA chondrocytes and explants and non-OA chondrocytes were cultured in cytokine-free medium of 280 mOsm or 380 mOsm with or without Cn inhibition by FK-506. Chondrogenic, hypertrophic, and catabolic marker expression was evaluated at the messenger RNA (mRNA), protein, and activity levels.Results. Compared to OA chondrocytes cultured at 280 mOsm, those cultured at 380 mOsm had increased expression of mRNA for chondrogenic markers (e.g., ϳ13 fold for COL2; P < 0.001), and decreased COL1 expression (ϳ0.5 fold, P < 0.01). Inhibiting Cn activity under physiologic tonicity further enhanced the expression of anabolic markers at the mRNA level (ϳ50 fold for COL2; P < 0.001, ϳ2 fold for AGC1; P < 0.001, and ϳ3.5 fold for SOX9; P < 0.001) and at the protein level (ϳ6 fold for type II collagen; P < 0.001). Cn inhibition suppressed relevant collagenases as well as hypertropic and mineralization markers at the mRNA and activity levels. Expression of aggrecanase 1 and aggrecanase 2 was not influenced by tonicity or FK-506 alone, but the combination suppressed both, by ϳ50% (P < 0.05) and ϳ40% (P < 0.001), respectively. Generally, similar anabolic and antihypertrophic effects were observed in ex vivo cartilage explant cultures and non-OA chondrocytes.Conclusion. Our findings indicate that Cn at physiologic tonicity exerts a superior effect compared to physiologic tonicity or FK-506 alone, increasing anabolic markers while suppressing hypertrophic and catabolic markers. Our data may aid in the development of improved cell-based chondral repair and OA treatment strategies.Articular cartilage has unique properties related to the structure and composition of its extracellular matrix (ECM), which is mainly composed of proteoglycans (PGs), such as aggrecan, entangled in a network of collagen fibers, such as type II collagen, and large amounts of water (1). Water and inorganic salts represent the bulk mass portion, while collagens, PGs, and other glycoproteins and proteins constitute only 20% of the wet weight of the tissue. Collagens represent 50-60% of the dry weight of cartilage, while PGs, the second largest solid-phase portion, account for 5-10%.In normal cartilage there is a balance between matrix synthesis and degradation. Depletion of aggrecan from articular cartilage by matrix metalloproteinase (MMP) and aggrecanase (ADAMTS) action is an essential early pathophysiologic event in osteoarthritis (OA) (2-4). This decrease in PG content can result in reduced viscoelastic properties and increased deformation of

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 (Cn) as a potential target to improve chondrocyte phenotype for cartilage repair purposes. Cn activity in human articular chondrocytes was significantly increased during dedifferentiation and decreased during redifferentiation in vitro. Inhibition of Cn 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 2 (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 Cn activity. In conclusion, inhibition of Cn activity by FK506 increases the expression of chondrogenic markers via endogenous TGFbeta1 production in human articular chondrocytes. Cn inhibitors might be an alternative for the application of (recombinant) TGFbeta, to promote chondrocyte phenotype for cell-based cartilage repair procedures.