54 The Stem Cell Niche In Tendon And Ligament – Investigating Alterations With Ageing And Disease

Abstract

Introduction Ligament and tendon are prone to degeneration through ageing and injury and current therapies are largely ineffective. The identification of a cell population within tendon with stem cell-like characteristics (Bi, 2007) holds potential for regeneration of tendon and ligament. Tendon stem cells differentiate into tenocytes (Zhang, 2010); the predominant cell type within tendon, responsible for producing extracellular matrix (ECM). The local stem cell environment (niche) is vital for stem cell maintenance and function in many tissues, and tenascin C in particular has been shown to play an important role within stem cell niches (Garcion, 2004). Tendon and ligament are composed of fascicles and interfascicular matrix (IFM) which vary considerably in composition providing definitive niches within the tissue. This study aims to characterise ECM components of the stem cell niche in equine tendon and canine ligament, which are prone to age-related degeneration. The goal of this research is to produce an in vitro environment for stem cells which mimics the stem cell niche, for treatment of tendon and ligament disease. Methods Putative stem cells were isolated from equine superficial digital flexor tendon (SDFT) and canine anterior cruciate ligament (ACL) by low-density plating and differential adhesion to plastic and fibronectin substrates. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers CD90, CD73 and CD105, as well as qRT-PCR for stem cell and tenogenic markers. ECM components of the fibroblast and stem cell niche were analysed using radioisotope labelling. Cells were labelled with 14C-labelled amino acids to specifically label newly synthesised collagenous (proline) and non-collagenous (lysine/arginine) ECM, prior to extraction of ECM. Immuno-histochemistry and histology were conducted to analyse the structure and composition of SDFT. Results Tendon and ligament cells formed colonies after low-density plating, however only ligament cells formed colonies after differential adhesion to fibronectin. A subpopulation of tendon cells expressed CD90 in both freshly isolated cells and putative stem cells, but were CD105 and CD73 negative. Putative tendon stem cells, isolated by differential fibronectin adhesion did not exhibit increased expression of stem cell markers when compared with tenocytes. However there was a significant increase in expression of stem cell markers in putative ligament stem cells compared with ligamentocytes. Tenocytes and putative tendon stem cells (isolated by low-density plating) labelled with 14C-labelled amino acids both displayed similar labelling profiles. Histological analysis of SDFT tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to IFM (see Figure.1). Abstract 54 Figure 1 The expression of tenascin C in equine SDFT tissue Conclusion The absence of stem cell marker expression in putative stem cell populations indicates that further testing of stem cell isolation procedures is ...

TENOgenic MODULating INsider factor: systematic assessment on the functions of tenomodulin gene

“…Cumulatively the few articles providing some information on this topic are suggesting that the above process might be mediated by the adult TSPCs or perivascular stem cells or tendon intra-fascicular matrix stem cells, or even a mixture of all these cell types depending on the circumstances (Bi et al 2007, Tempfer et al 2009, Rui et al 2010, Mienaltowski et al 2013, Liu et al 2014, Alberton et al 2015, Docheva et al 2015, Lee et al 2015). Mienaltowski et al, 2013 actually proposed that in tendons there is a regional distribution of different stem/progenitor cells.…”

“…TNMD is marked in orange, transcription factors in blue and other genes in black. Based on Alberton et al 2015; Berasi et al 2011; Eloy-Trinquet et al 2009; Havis et al 2014; Huang et al 2015; Lee et al 2015; Liu et al 2014; Mienaltowski et al 2013; Pryce et al 2009; Shen et al 2013; Tempfer et al 2009. Abbreviations: Aqp1, aquaporin 1; BMP, bone morphogenetic protein; Col, collagen; COMP, cartilage oligomeric matrix protein; Egr, early growth response protein; Eya, eyes absent transcription factor; FGF, fibroblast growth factor; Htra3, HtrA serine peptidase 3; IFM, interfascicular matrix; Mkx, Mohawk; Sca-1, stem cells antigen-1; Scx, scleraxis; SMA, smooth muscle actin; TGF, transforming growth factor; Thbs, thrombospondin; Tnmd, tenomodulin.…”

Viscoelastic characteristics of the canine cranial cruciate ligament complex at slow strain rates

“…There is a large economic cost associated with managing canine CCLR, for example in the United States alone the economic cost was estimated to be at least one billion dollars in 2003 ( Wilke et al, 2005 ). Both human and canine CCL failure significantly increases the incidence of age-associated joint degeneration ( Lee et al, 2014 ; Liu et al, 2003 ; Peters et al, 2018 ) and so understanding the tissue’s fundamental material properties can assist with the prediction and effective management of ligament injuries.…”

Immortalized murine tenocyte cells – a novel and innovative tool for tendon research

“…Flow cytometry is less frequently used for tendon characterization since the markers are more established for progenitor cells (e.g. mesenchymal stem cells or tendon progenitor cells) or fibroblasts in general 29,[32][33][34][35] . However, although mesenchymal stem cells and tenocytes are different cell types, similar fibroblastic surface marker expression has previously been reported 35,36 and adds another valuable tool to define a tenogenic phenotype.…”