Metabolic disorders are among known risk factors for tendinopathies or spontaneous tendon ruptures. However, the underlying cellular and molecular mechanisms remain unclear. We have previously shown that human and rat tendon cells produce and secrete insulin upon glucose stimulation. Therefore, we hypothesize that nutritional glucose uptake affects tendon healing in a rat model. Unilateral full-thickness Achilles tendon defects were created in 60 female rats. Animals were randomly assigned to three groups receiving different diets for 2 weeks (high glucose diet, low glucose/high fat diet, control diet). Gait analysis was performed at three time points (n=20/group). In addition, tendon thickness, biomechanical (n=14/group), and histological and immunohistochemical analysis was conducted. Subsequently, a subtractive-suppression-hybridization (SSH) screen comparing cDNA pools (n=5) prepared from repair tissues of the high glucose and the control diet group was conducted to identify differentially expressed genes.Introduction
Materials and Methods
Tendon cells originate from yet poorly described precursor cells and develop in a particular “niche” close to vascular walls. Several factors have been described to determine this niche such as mechanical stimuli, oxygen tension, composition and structure of the extracellular matrix (ECM). Also, the vasculature is considered to play a crucial role for tendon cell development, yet evidence of how this is accomplished is lacking. In this study we therefore focussed on the endothelium of tendon vessels postulating the existence of a paracellular barrier. By electron microscopy, immunohistochemistry, and RT-PCR we investigated the presence of constituents making up such an endothelial barrier which we subsequently tested for its functionality by tracer injection. Moreover, we performed differentiation experiments into the adipogenic, chondrogenic and osteogenic lineage on tendon derived cells in the presence and absence of serum. Expression levels and activity of matrixmetalloproteinases (MMPs) were assessed by western blot and zymography.Introduction
Materials and Methods
The ability of tendons to withstand stress generally decreases with age, often resulting in increased tissue degeneration and decreased regeneration capacity. However, the underlying molecular and cellular mechanisms of tendon senescence remain poorly characterized. Therefore, the aim of the current study was to identify genes showing an age-dependent altered expression profile in tendons. A suppression-subtractive-hybridization (SSH) screen comparing cDNA libraries generated from Achilles tendons of mature-adult (3 months) and old (18 months) female C57BL/6 mice was conducted. Subsequently, the differential expression of the identified genes was validated by RT-qPCR and selected genes were then further analysed by immunohistochemistry and Western blot. To investigate age-related structural alterations in the collagenous extracellular matrix we applied SHG-microscopy and TEM. In vitro experiments with young and old tendon derived stem/progenitor cells (TDSCs) involved wounding assays, tendon-like constructs as well as collagen gel contraction assays.Introduction
Materials and Methods
