Abstract
Backgroundhyaluronan biopolymer is used in dermatology but the underlying mechanism and the impact of its molecular weight have not yet been investigated in skin wound healing. The aim of our work was to study the role of HA molecular weight in the proliferative phase of wound healing and to understand how this physiological biopolymer acts to promote wound healing on a human keratinocyte in vitro model.Methodology and Findingswound healing closure was evaluated using scratch test assay, cell proliferation by counting cell with haemocytometer, expression of CD44 and ZO-1 (protein present in tight junctions specific of epithelia) using flow cytometry, and P2X7 receptor activation on living using a cytoflurometric method. Our study showed that medium hyaluronan fragment (MMW-HA, between 100 and 300 kDa) induced a significant increase in wound closure, increased ZO-1 protein expression and induced a slight activation of P2X7 receptor, contrary to high (between 1000 and 1400 kDa) and low (between 5 and 20 kDa) molecular hyaluronan fragments that had no healing effects. Basal activation of P2X7 receptor is already known to stimulate cell proliferation and this activation in our model plays a pivotal role in MMW-HA-induced wound healing. Indeed, we showed that use of BBG, a specific inhibitor of P2X7 receptor, blocked completely the beneficial effects of MMW-HA on wound healing.Conclusiontaken together, our results showed for the first time the relationship between P2X7 receptor and hyaluronan in wound healing, and that topical use of MMW-HA (fragment between 100 and 300 kDa) could represent a new therapeutic strategy to promote healing.
Highlights
Hyaluronan (HA), the major component of the extracellular matrix (ECM), is the only linear non-sulfated glycosaminoglycane composed of alternating b-1,4-glucuronic acid and b-1,3-N-acetylglucosamin [1;2]
We show that only Medium Molecular Weight Hyaluronan (MMW-HA) induced a significant wound healing compared to control and other HA (***: p,0.001 compared to culture medium, n = 3)
HA and Low Molecular Weight Hyaluronan (LMW-HA) had no effect on wound healing compared to control. % of wound area was 77.70%, 59.24%, 5.83% and 43.58% respectively for culture medium, High Molecular Weight Hyaluronan (HMW-HA), MMW-HA and LMW-HA
Summary
Hyaluronan (HA), the major component of the ECM, is the only linear non-sulfated glycosaminoglycane composed of alternating b-1,4-glucuronic acid and b-1,3-N-acetylglucosamin [1;2]. Its major function is the modulation of inflammatory cell and dermal fibroblast activities, e.g. cellular migration, proinflammatory cytokine synthesis and the phagocytosis of invading microbes [6]. Low molecular weight HA binds to TLR4 to induce inflammatory responses stimulating IL-6, TNFa and IL-1b [7]. Proliferative step is a set of complex biological responses requiring extracellular matrix (ECM) and cytoskeletal remodelling, signal cascades, and gene regulation to induce fibroblast and keratinocyte migration and proliferation. CD44 is localised in lipid raft domains which are plasma membrane domains that contain high levels of cholesterol and sphingolipids. Proteins such as receptors involved in cell signalling are enriched in lipid rafts [12]. Our laboratory showed that HA of molecular weight superior to Nomenclature High Molecular Weight Hyaluronan (HMW-HA) Medium Molecular Weight Hyaluronan (MMW-HA) Low Molecular Weight Hyaluronan (LMW-HA)
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