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Engineering in vitro microenvironments for translation of cell-based therapies for tendon repair

Periodic Reporting for period 2 - Tendon Therapy Train (Engineering in vitro microenvironments for translation of cell-based therapies for tendon repair)

Période du rapport: 2018-02-01 au 2020-01-31

Tendon Therapy Train is a network of 5 universities and 3 companies based across Europe which aimed to develop an effective tissue-engineered therapy for human and equine tendon injury. Over 20 million tendon procedures take place annually worldwide with associated healthcare expenditure at €145 billion. Current surgical repairs do not effectively restore tendon function. The consortium’s solution was to develop tendon-like cell culture environments by recruiting the cells’ natural ability to build tissues and produce tendon-like tissues in the lab from these cells. Tendon-derived cells (TDCs), adipose-derived stem cells (ADSCs), bone-marrow derived stem cells (BMSCs) and dermal fibroblasts (DFs) were investigated. By growing the cells in a lab environment that resembles tendons in the body, early stage researchers (ESRs) were able to maintain TDCs properties and to induce ADSCs, BMSCs and DFs to differentiate into tendon-like tissue cells in culture. To mimic the natural tendon environment in cell cultures, ESRs Dominika Berdecka (University of Minho), Alessandro Dei (StemCell Technologies), Andrea De Pieri (Proxy Biomedical), Dimitrios Tsiapalis, Eugenia Pugliese and Salome Guillaumin (National University of Ireland, Galway/NUIG) and Sergio Garnica Galvez, Adrian Djalali Cuevas and Andrea Rampin (University of Ioannina) experimented with culture media, adding different factors and macromolecules. Steven Vermeulen and Aysegul Dede-Eren (Maastricht University), Sofia Ribeiro (Sofradim Production), Dimitrios Tsiapalis, Eugenia Pugliese, Salome Guillaumin and Ignacio Sallent (NUIG), Andrea De Pieri, and Adrian Djalali Cuevas experimented with cell culture substrates. Dimitrios Tsiapalis, Ignacio Sallent and Salome Guillaumin subjected the cells to different levels of oxygen tension or mechanical forces. Giulia Sivelli (Royal Veterinary College, RVC) progressed the use of extracellular vesicles derived from equine cells to treat tendon inflammation. The ESRs work has advanced tendon tissue engineering; they have engineered cultures which maintain tendon cells and induce stem cells to grow as tendon-type cells. Promising implants have been validated in preclinical models by Andrea De Pieri and Ise Francois (RVC).
Dimitrios Tsiapalis, Alessandro Dei, Dominika Berdecka, Andrea Rampin and Adrian Djalali Cuevas established protocols for the isolation, characterisation and culture of cells. They experimented with culture media to encourage tissue formation. Alessandro Dei developed a tendon-specific culture medium which can culture tendon cells and induce stem cells to develop into tendon-like tissues. Adrian Djalali Cuevas conducted an in-depth study of TDC proteins which may make the characterization of tendon cells possible for the first time. Giulia Sivelli progressed knowledge on treatment strategies for tendon inflammation. Traditionally, cell culture takes place on smooth, rigid culture plastics. Sofia Ribeiro developed biodegradable films of varying stiffness and topography which direct stem cell differentiation into tendon-type cells. Ignacio Sallent produced collagen-based substrates for cell culture which maintain topographical features, imitating the cells natural environment. Eugenia Pugliese engineered intricate multi-layered collagen scaffolds for complex tendon tissues. Steven Vermeulen identified the optimal topographies for the maintenance of tendon cell culture. Andrea de Pieri used a temperature-responsive polymer as a cell scaffold which is dissolved once a tissue of implantable size is produced. This allows tissue detachment from the culture surface; a feat which is not easily achieved. Aysegul Dede-Eren developed a protocol to remove cells from tendon tissue and use the resulting scaffold as a basis for tendon tissue engineering. Dimitrios Tsiapalis and Adrian Djalali Cuevas assessed fibrous constructs that closely imitate native tendon architecture. Dimitrios Tsiapalis, Andrea Rampin and Sergio Garnica Galvez investigated the use of macromolecules in the culture media seeking to increase the rate at which tissue is laid down. They identified molecules that support the tendon-specific culture of TDCs, BMSCs and ADSCs. These molecules crowd the culture space, driving cells to deposit more extracellular matrix; the structure which supports cells in the body. They successfully increased the amount of matrix laid down, as well as the rate at which the tissue forms, overcoming two bottlenecks to tissue culture. Tissue culture using a mechanical bioreactor or oxygen tension chambers was investigated by Dimitrios Tsiapalis, Ignacio Sallent and Salome Guillaumin. The bioreactor was used to apply mechanical forces to the cultures. This is important for tendon tissues which are load-bearing and subject to significant forces. Optimal mechanical stimulation for tendon work was identified as well as optimal oxygen tension. RVC developed a preclinical study for research outputs using an equine model. For the first time, equine stem cells cultured with tendon-specific topographies were implanted in an equine patient. 3D tissue implants developed using the temperature-responsive polymer, MMC and ADSCs, were implanted at NUIG by Andrea De Pieri demonstrating superior preclinical outcomes to control groups. 15 ESRs have received interdisciplinary training and experience in all aspects of Advanced Therapy Medicinal Product (ATMP) development with input from academic, industry and clinical partners. Over 100 conference presentations and over 30 journal publications have resulted so far. Exploitation plans have been prepared for cell and cell-culture products, scaffolds and tissue implants. Andrea de Pieri has co-drafted an IDF on cell sheet engineering which is being considered for patent application. Alessandro Dei developed the serum-free media for tendon applications. Sofia Ribeiro and Steven Vermeulen received conference awards. Public engagement activity included school visits (Dominika Berdecka, Sofia Ribeiro, Andrea de Pieri, Eugenia Pugliese, Ignacio Sallent, Salome Guillaumin) science fairs and events (Alessandro Dei, Steven Vermeulen, Giulia Sivelli) newspaper articles (Andrea Rampin) and a film documentary broadcast on National Television and at the Galway Film Festival.
Tissue grafts are the gold standard in clinical practice, however, in severe injuries and degenerative conditions there is insufficient material for autograft. Allo- and xeno-grafts and biomaterials have limited therapeutic use due to inflammatory issues. Cell injections show limited cell localisation and retention. Although ATMPs can address these limitations, the prolonged culture periods required to develop an implantable device has impeded commercialisation. Outside of their optimal tissue context for prolonged periods, cells ‘drift’ and lose their functionality and therapeutic potential. ESRs have optimised culture conditions for TDCs, BMSCs and ADSCs using tendon-specific environmental cues to maintain or induce tendon-like cultures. Tendon-specific media formulations were developed with plans for market placement. Tissue implants were developed in a fraction of the time, using a fraction of the cells that traditional approaches require. Proof-of-principal has been demonstrated in a preclinical study resulting in an IDF. The possibility of better therapies for patients, both human and equine, has been communicated to public and scientific audiences.
Engineering the tenocyte in vitro niche
Community Outreach; ESRs teaching the Healing Tendons workshops at local primary school in Galway
Equine BM-MSCs and SDFT TDCs cultured on commercial tissue culture plastic and treated with 10ng/mL
Extracellular matrix-rich cell layers, bidirectionally aligned, using bioreactors
Ignacio Sallent presenting at ICORS 2019
Andrea de Pieri, Ignacio Sallent and Eugenia Pugliese work on in vitro characterization at NUIG
Sofia Riberio presenting her research at ISBPPB 2018
Eugenia Pugliese presents at TERMIS EU 2019
Andrea de Pieri demonstrates tendon anatomy to school children at a national event in Irealand
Dominika Berdecka presenting her research at EORS 2018