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47 Effects Of Chronic High Glucose On Primary Human Tenocytes
  1. Zuzana Kalivodova,
  2. Philippa Hulley
  1. Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, UK

Abstract

Introduction Tendon disease and injury is more prevalent in type II diabetic patients than in healthy individuals [Longo, 2009; Abate, 2013]. Tendon healing is possibly impaired, too [Ahmed, 2012; Bedi, 2010]. Chronic high blood glucose levels cause damage to cells in several ways, including increased oxidative stress [Nishikawa, 2000]. Elucidating further and targeting these mechanisms in tendon as well as developing new therapeutic strategies for diabetic patients is necessary. Therefore we investigated the cellular responses to a range of chronic glucose conditions in human tenocytes.

Methods Healthy primary human tenocytes were chronically cultured in 8 mM, 17 mM (standard high glucose) and 38 mM glucose medium. The EdU incorporation assay as well as Live stain imaging were used to evaluate the proliferative capacity of the cells. Alamar blue assay was employed to estimate the metabolic activity of cells. The amount of soluble collagen secreted by cells was assessed by Sircol dye; DMB assay was used to evaluate the levels of glycosaminoglycans (GAGs). In vitro monolayer scratch assay was performed to evaluate the healing capacity of cells. Cells were subjected to hydrogen peroxide to induce oxidative stress and then examined by Live and Dead stain and Alamar Blue assay.

Results The EdU assay revealed that tenocytes chronically cultured in different glucose conditions proliferate significantly faster in 38 mM glucose (under 5% and 20% FCS). Intriguingly the resulting total cell number was the lowest in 38 mM glucose (under all FCS conditions), whereas it was the highest in 17 mM glucose (under all FCS conditions) despite cells being seeded at equal densities at origin. This data is consistent with our Live stain data which visualises the cell layer density. Metabolic activity showed an interesting pattern: 38 mM glucose tenocytes were significantly more metabolically active than 8 mM glucose tenocytes once cell number was corrected for. The main structural component of the tendon extracellular matrix, collagen, was significantly reduced in 38 mM glucose, unlike GAGs which were stable across the entire glucose range. In monolayer healing 38 mM glucose decreased the healing rate overall and significantly reduced the ability of cells to respond to 20% FCS stimulation. The capacity of tenocytes to survive under acute oxidative stress (1 mM hydrogen peroxide) was severely compromised under 38 mM glucose while cells in 8 mM glucose were viable despite the treatment.

Discussion We have demonstrated glucose sensitivity of primary human tenocytes. High extracellular glucose compromises several aspects of tendon cellular biology, most notably collagen production and response of cells to oxidative stress. In this way normal physiological processes may be shifted into pathological status under high glucose, ultimately rendering the tissue more prone to degeneration. Uncovering the molecular mechanisms responsible will offer hope for improved therapies in the future.

References Abate et al. Rheumatology. 2013;52(4):599–608

Ahmed et al. J Appl Physiol. 2012;113(11):1784–91

Bedi et al. J Shoulder Elb Surg. 2010;19(7):978–988

Longo et al. Brit J of Sport Med. 2009;43(4):284–287

Nishikawa et al. Nature 2000;404(6779):787–790

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