Knee joint ligaments provide stability to the joint by preventing excessive movement. There has been no systematic effort to study the effect of OA and ageing on the mechanical properties of the four major human knee ligaments. This study aims to collate data on the material properties of the anterior (ACL) and posterior (PCL) cruciate ligaments, medial (MCL) and lateral (LCL) collateral ligaments. Bone-ligament-bone specimens from twelve cadaveric human knee joints were extracted for this study. The cadaveric knee joints were previously collected to study ageing and OA on bone and cartilage material properties; therefore, combining our previous bone and cartilage data with the new ligament data from this study will facilitate subject-specific whole-joint modelling studies. The bone-ligament-bone specimens were tested under tensile loading to failure, determining material parameters including yield and ultimate (failure) stress and strain, secant modulus, tangent modulus, and stiffness. There were significant negative correlations between age and ACL yield stress (p = 0.03), ACL failure stress (p = 0.02), PCL secant (p = 0.02) and tangent (p = 0.02) modulus, and LCL stiffness (p = 0.046). Significant negative correlations were also found between OA grades and ACL yield stress (p = 0.02) and strain (p = 0.03), and LCL failure stress (p = 0.048). However, changes in age or OA grade did not show a statistically significant correlation with the MCL tensile parameters. Due to the small sample size, the combined effect of age and the presence of OA could not be statistically derived. This research is the first to report tensile properties of the four major human knee ligaments from a diverse demographic. When combined with our previous findings on bone and cartilage for the same twelve knee cadavers, the current ligament study supports the conceptualisation of OA as a whole-joint disease that impairs the integrity of many peri-articular tissues within the knee. The subject-specific data pool consisting of the material properties of the four major knee ligaments, subchondral and trabecular bones and articular cartilage will advance knee joint finite element models.
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 ...
Tendon fibroblasts, or tenocytes, are the main cell type in tendon and serve to synthesize and maintain collagen fibrils and other extracellular matrix proteins within the tissue. Despite the high prevalence of tendon injury, the underlying biological mechanisms of postnatal tendon growth and repair are not well understood. Insulin‐like growth factor (IGF‐1) signaling is stimulated by growth hormone (GH) and promotes the growth and development of many different tissue types. While IGF‐1 is a potent hypertrophic signaling molecule in skeletal muscle, less is known about the role of IGF‐1 in tendon biology. Therefore, the purpose of this study was to determine the importance of IGF‐1 signaling in postnatal tendon growth. Our hypothesis was that IGF‐1 signaling is required for the response of tendon to a mechanical load stimulus. To test this hypothesis, we used both in vitro studies of cultured tenocytes, and in vivo studies using genetically modified mice that allowed us to delete the IGF‐1 receptor (IGF1R) specifically in tenocytes. Treatment of tenocytes in vitro with IGF‐1 increased the expression of Mki67 transcript levels after 24 hours, and resulted in activation of the Akt and ERK1/2 pathways. When tenocyte proliferative activity was measured, cells treated with IGF‐1 displayed increased proliferation compared to controls, but inhibition of ERK prevented the IGF‐1‐mediated increase in cell proliferation and Mki67 (Ki67) expression. Inhibition of Akt signaling did not impact tenocyte proliferation or Mki67 expression. In addition, treatment of tenocytes with IGF1 increased the rate of protein synthesis and ribosomal RNA expression. Analysis of mechanically overloaded plantaris tendons in vivo revealed significantly reduced size of the neotendon matrix which forms around the original tendon in response to mechanical loads, and total tendon cross‐sectional area (CSA) at 14 days in the IGF‐1 knockdown mice compared to the controls. This was accompanied by decreased number of proliferating cells and proteins involved in cell proliferation and initiation of translation, as measured by proteomic analysis. The results of this work support IGF‐1 signaling as a fundamental component of postnatal tendon growth in response to mechanical loading, specifically through the regulation of ERK1/2‐dependent tenocyte proliferation and initiation of protein synthesis. Further studies of the growth factors and proteins involved, and the potential to evaluate IGF‐1 to promote tendon regeneration, are warranted.Support or Funding InformationNIH R01‐AR063649 and F32‐AR067086.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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