Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).Peer reviewedPublisher PD

A homeostatic function of CXCR2 signalling in articular cartilage

Funding This work was funded by Arthritis Research UK (grants 17859, 17971, 19654), INNOCHEM EU FP6 (grant LSHB-CT-2005-51867), MRC (MR/K013076/1) and the William Harvey Research FoundationPeer reviewedPublisher PD

Distinct mesenchymal progenitor cell subsets in the adult human synovium

Objective. To analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM. Methods. Cell populations were enzymatically released from the knee joint synovium of adult human individuals. Single cell-derived clonal populations were obtained by limiting dilution and serially passaged to determine growth rates. Phenotypic analysis was carried out by flow cytometry. Replicative senescence was assessed by the senescence-associated β-galactosidase staining. Telomere lengths were determined semiquantitatively by Southern blotting. Telomerase activity was measured using a real-time quantitative telomerase repeat amplification procedure. Culture-expanded clonal populations were subjected to in vitro differentiation assays to investigate their mesenchymal multipotency. Results. The 50 clones analysed displayed wide variations in the proliferation rates, even within the same donor sample. The time taken to reach 20 population doublings ranged from 44 to 130 days. The phenotype of the clones tested was compatible with that of mesenchymal stem cells. Mean telomere lengths ranged from 5.2 to 10.9 kb with positive linear trend with telomerase activity, but no correlation with proliferative rates or cell senescence. All clones tested were capable of chondrogenic and osteogenic differentiation, though with large variability in potency. In contrast, only 30% of the clones were adipogenic. Conclusions. We report for the first time the co-existence, within the synovium, of progenitor cell subsets with distinct mesenchymal differentiation potency. Our findings further emphasize the need for strategies to purify cell populations with the clinically desired tissue formation potential

Cell-based Approaches to Joint Surface Repair : A Research Perspective

The authors are grateful for support to their research from Arthritis Research UK (grants 19271, 19429, 19667, 20050). None of the authors received any funding related to the writing of this manuscript, and the funding bodies did not play any role in the writing of the manuscript or decision to submit the manuscript for publication.Peer reviewedPublisher PD

Stem Cells and Cartilage Development: Complexities of a Simple Tissue

Cartilage is considered to be a simple tissue that should be easy to engineer because it is avascular and contains just one cell type, the chondrocyte. Despite this apparent simplicity, regenerating cartilage in a form that can function effectively after implantation in the joint has proven difficult. This may be because we have not fully appreciated the importance of different structural regions of articular cartilage or of understanding the origins of chondrocytes and how this cell population is maintained in the normal tissue. This review considers what is known about different regions of cartilage and the types of stem cells in articulating joints and emphasizes the potential importance of regeneration of the lamina splendens at the joint surface and calcified cartilage at the junction with bone for long-term survival of regenerated tissue in vivo. Stem Cells 2010;28:1992–199

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits

Purpose: Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. Methods: A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. Results: Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. Conclusions: Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.This study was supported by the Spanish Ministry of Science and Innovation through MAT2010-21611-C03-00 project (including the FEDER financial support), by Conselleria de Educacion (Generalitat Valenciana, Spain) PROMETEO/2011/084 grant, and by CIBER-BBN en Bioingenieria, Biomateriales y Nanomedicina. The work of JLGR was partially supported by funds from the Generalitat Valenciana, ACOMP/2012/075 project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the - Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Sancho-Tello Valls, M.; Forriol, F.; Gastaldi, P.; Ruiz Sauri, A.; Martín De Llano, JJ.; Novella-Maestre, E.; Antolinos Turpín, CM.... (2015). 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Small animal models to understand pathogenesis of osteoarthritis and use of stem cell in cartilage regeneration

Osteoarthritis (OA) is one of the most common diseases, which affect the correct functionality of synovial joints and is characterized by articular cartilage degradation. Limitation in the treatment of OA is mostly due to the very limited regenerative characteristic of articular cartilage once is damaged. Small animal models are of particular importance for mechanistic analysis to understand the processes that affect cartilage degradation. Combination of joint injury techniques with the use of stem cells has been shown to be an important tool for understanding the processes of cartilage degradation and regeneration. Implementation of stem cells and small animal models are important tools to help researchers to find a solution that could ameliorate and prevent the symptoms of OA

Η πρωτεΐνη YAP στα μεσεγχυματικά βλαστικά κύτταρα

Mesenchymal stem cells (MSCs) are the most studied subtype of adult stem cells and have been derived from most postnatal organs and tissues. MSCs are defined as having the capacity to self-renew and to differentiate into both mesodermal and non-mesodermal lineages, and are immunosuppressive. For these properties, MSCs have been considered ideal candidates for regenerative medicine and have been used in several clinical trials. The difficulty, however, to preserve the potency of the cells during culture expansion and to monitor differentiation are obstacles in their use in the clinic and have emphasized the need to investigate molecular pathways underlying stem cell fate-decisions during differentiation in more detail. The Hippo pathway is a recently identified molecular pathway in Drosophila melanogaster and mammals, which controls organ size by regulating cell proliferation, apoptosis and differentiation. The pathway is composed of a core of serine/threonine kinases [Hpo/(MST1/2) and Wts/(LATS1/2)] and scaffold proteins [(Sav/Shrp)/SAV1 and Mats/MOB] that when are activated, phosphorylate and inhibit yes-associated protein (YAP) transcriptional co-activator and tumour suppressor. Inactivation of YAP in some stem and progenitor cells by the Hippo pathway is required for their differentiation. On the contrary, failure to inhibit YAP enhances proliferation and may cause oncogenic transformation.In the present study, the expression of multiple YAP variants was confirmed in human and mouse MSCs. In both human and mouse, YAP was inhibited in response to cell-contact inhibition and remained unchanged during in vitro chondrogenic differentiation. Overexpression of human (hYAP1) variant in C3H/10T1/2 MSC-like cells did not appear to affect colony formation, cell cycle distribution or cell size, but increased cell proliferation, induced cell transformation and reduced the capacity of the cells to differentiate in vitro towards the chondrogenic, adipogenic and osteogenic lineages. The effects of hYAP1 overexpression are hypothesized to be either a result of a nuclear co-factor function or indirectly via protein interactions in the cytoplasmic compartment.YAP and the Hippo pathway are possible pharmacological targets for modulation of MSC functionΤα μεσεγχυματικά βλαστοκύτταρα (MSCs) αποτελούν τον πιο μελετημένο υποτύπο ενηλίκων βλαστοκυττάρων και έχουν προέλθει από τα περισσότερα μεταγεννητικά όργανα και ιστούς. Τα MSCs χαρακτηρίζονται από την ικανότητά τους να αυτοανανεώνονται (self-renew) και να διαφοροποιούνται τόσο σε μεσοδερμικές, όσο και σε μη μεσοδερμικές γενεές και είναι ανοσοκατασταλτικά. Για αυτές τις ιδιότητες, τα MSCs έχουν θεωρηθεί ιδανικοί υποψήφιοι για την αναγεννητική ιατρική και έχουν χρησιμοποιηθεί σε αρκετές κλινικές δοκιμές. Η δυσκολία, ωστόσο, να διατηρηθεί η ισχύς των κυττάρων κατά την καλλιέργειά τους, και να παρακολουθηθεί η διαφοροποίησή τους, αποτελούν εμπόδια στη χρήση τους στην κλινική, γεγονός που κρίνει την ανάγκη να διερευνηθούν λεπτομερέστερα οι μοριακές οδοί στις οποίες βασίζεται η μοίρα των βλαστοκυττάρων κατά τη διαφοροποίηση.Το σηματοδοτικό μονοπάτι Hippo είναι ένα πρόσφατα αναγνωρισμένο μοριακό μονοπάτι στη Drosophila melanogaster και στα θηλαστικά, το οποίο ελέγχει το μέγεθος των οργάνων ρυθμίζοντας τον πολλαπλασιασμό των κυττάρων, την απόπτωση και τη διαφοροποίηση. Το μονοπάτι αποτελείται από έναν πυρήνα κινασών σερίνης/θρεονίνης [Hpo/(MST1/2) και Wts/(LATS1/2)] και πρωτεϊνών ικριώματος [(Sav/Shrp)/SAV1 και Mats/MOB] που όταν ενεργοποιούνται, φωσφορυλιώνουν και αναστέλλουν την πρωτεΐνη YAP (yes-associated protein), η οποία λειτουργεί ως μεταγραφικός συν-ενεργοποιητής και ογκοκαταστολέας. Η αδρανοποίηση του ΥΑΡ είναι απαραίτητη σε ορισμένα βλαστοκύτταρα και προγονικά κύτταρα, μέσω του μονοπατιού Hippo, για τη διαφοροποίησή τους. Αντίθετα, η αποτυχία αναστολής του ΥΑΡ ενισχύει τον κυτταρικό πολλαπλασιασμό και δύναται να προκαλέσει τον ογκογόνο μετασχηματισμό τους.Στην παρούσα μελέτη, η έκφραση πολλαπλών παραλλαγών/ισομερών του YAP επιβεβαιώθηκε σε MSCs προερχόμενα από ιστούς ανθρώπων και ποντικιών. Τόσο στον άνθρωπο όσο και στα ποντίκια, το ΥΑΡ ανεστάλη ως απόκριση στην «κυτταρική αναστολή επαφής» (cell contact inhibition) και παρέμεινε αμετάβλητο κατά τη διάρκεια της in vitro χονδρογενούς διαφοροποίησης. Η υπερέκφραση της ανθρώπινης παραλλαγής (hYAP1) σε κύτταρα της σειράς C3H/10T1/2, που μοιάζουν με MSCs, δεν φάνηκε να επηρεάζει τον σχηματισμό αποικιών, την κατανομή του κυτταρικού κύκλου ή το μέγεθος των κυττάρων, αλλά αύξησε τον κυτταρικό πολλαπλασιασμό, προκάλεσε κυτταρικό μετασχηματισμό και μείωσε την ικανότητα των κυττάρων να διαφοροποιούνται in vitro προς τις χονδρογενείς, λιπογενείς και οστεογενείς γενεές. Τα αποτελέσματα της υπερέκφρασης του hYAP1 θεωρείται ότι είναι είτε αποτέλεσμα της λειτουργίας του ως πυρηνικού συμπαράγοντα, είτε έμμεσα μέσω αλληλεπιδράσεων διαφόρων πρωτεϊνών στο κυτταρόπλασμα. Το YAP και το σηματοδοτικό μονοπάτι Hippo είναι πιθανοί φαρμακολογικοί στόχοι για τη ρύθμιση της λειτουργίας των MSCs

Sensitivity Analysis of Hydrodynamic Parameters on the Dynamic Response of the Semisubmersible Floating Offshore Wind turbine with the use of OpenFAST

The advancement of floating wind turbine technology in recent years necessitates accurate predictions of their behaviour using various simulation tools. Utilizing multi-fidelity tools like OpenFAST allows for comprehensive calculations of the entire floating wind turbine structure. However, the precision of these calculations is challenged by the uncertainties associated with different system parameters.The main goal of this research is to conduct a comprehensive exploration of various parameters, with a particular emphasis on hydrodynamic factors, and their impact on the structural and aerodynamic performance of semi-submersible floating wind turbines. To achieve this, an extensive sensitivity analysis approach is employed, utilizing the multi-fidelity model OpenFAST. The simulations are conducted on a semi-submersible floating wind turbine, specifically the one used in the OC4 experiment, featuring the 5MW NREL wind turbine. The Elementary Effects (EE) method is employed in this study to assess the significance of various factors. These factors include the hydrodynamic drag coefficients, wave height, peak wave period, wind speed, wind direction, current speed, current direction, and turbulence intensity. Their influence on the Damage Equivalent Load (DEL) of different loads acting on the floating offshore wind turbine (FOWT) is thoroughly examined. The analysis conducted in this study has unveiled several key findings. The most influential parameters identified are the current speed, primarily impacting mooring line tension, and the significant wave height, which exhibits a balanced effect across various outputs, including hydrodynamic loads, tower loads, and rotor dynamics. Additionally, turbulence intensity emerges as a significant factor, particularly concerning rotor thrust and torque.When employing the Morison equation, the order of parameter significance remains consistent with the hybrid theory (Potential + Drag). However, it is noteworthy that the added mass coefficients carry greater significance compared to the drag coefficient. Furthermore, simulations conducted using probability distributions for different locations confirm that wave height consistently ranks as the most significant parameter. However, the overall significance may vary and decrease in more severe environmental conditions.The comparison with the OC3 spar platform revealed a similar pattern when examining the influence of current speed and significant wave height. Nonetheless, the simple hydrodynamic drag coefficient used for the whole platform also displayed significance, emphasizing the sensitivity of the spar platform loads to this modelling parameter. This significance is much higher than the corresponding one of the semisubmersible platform.The research underscores it is imperative to mitigate the uncertainty associated with various parameters. This can be achieved through experimental verification and establishing correlations between modelling parameters and the specific conditions being simulated. Additionally, given that the most influential parameters are related to external conditions, it becomes crucial to simulate conditions that closely resemble the anticipated deployment location for the floating offshore wind turbine (FOWT). By doing so, the accuracy and reliability of the simulations will be enhanced to better inform FOWT design and deployment strategies.Electrical Engineering | Sustainable Energy Technolog