- مبلغ: ۸۶,۰۰۰ تومان
- مبلغ: ۹۱,۰۰۰ تومان
Nowadays there is a great interest in investigating the effect of particular hyaluronan fragments in the biomedical field and in cosmeceutical applications. Literature has reported that very low molecular weight HA (Mw < 5 kDa) has an inflammatory effect, whilst HA ranging from 15 to 250 has shown controversial effects. This work aims to give better elucidation on the correlation between the different sized HA fragments and their biological functions. In this respect, a simple and effective degradation strategy is used to obtain several HA fragments. Also, an hydrodynamic and structural characterization was performed in order to obtain samples suitable to evaluate cellular response. In particular an in vitro scratch test in time lapse experiments was used to study the effect of HA fragments, ranging from 1800 to 6 kDa on wound dermal reparation based on human keratinocytes. All high and low Mw HA used in this study allowed for faster wound closure compared to the un-treated cells, except for 6 kDa that, on the contrary, prevented repair. In addition, TGF- 1, TNF and IL-6, representative biomarkers of the inflammation phase occurring in wound healing process, were quantified by RT-PCR. A general up-regulation trend of these biomarkers was found with the HA molecular weight reduction. LHA6 kDa was the only treatment that induced a major inflammatory response (over 30 fold increase respect to control) confirming the recent literature outcomes. IL-6 protein level evaluated through ELISA assay corroborated the previous results. Furthermore, activation of key HA receptors, such as CD44, RHAMM, TLR4, with respect to hyaluronan size, was evaluated, at transcriptional level showing selective recognition by HA 1800, 1400, 500 for CD44, whilst the lower Mw fragments activated TLR-4 moderately at 50 and 15 kDa. An increase to “alarm” level was found for 6 kDa fragments. Immunofluorescence staining confirmed this data. The present research work demonstrated that the diverse pharma grade hyaluronan fragments could modulate cellular processes differently. From 1800 kDa down to 50 kDa, CD44 was the recognized receptor and pro-inflammatory biomarkers were only slightly up-regulated during wound healing in the presence of HA. Finally our outcomes showed that the lower the fragment size the higher the concern for inflammatory cytokines up-regulation; repair process impairment was highlighted only for 6 kDa chains. © 2
For the first time, a parallel comparative study has been run on the same model, using specific markers for diverse hyaluronan of pharmaceutical grade ranging from 1800 to 6 kDa in order to eventually assess specific features. All have proved to speed up wound healing except for 6 kDa HA that was also the one to increase outmostinflammatory cytokine levels. Differently from the others, HA, 6 kDa treatments could not prompt CD44 expression but activated the TLR-4 receptor. Overall, down to 100 kDa residues, all hyaluronan behaved similarly, the only differences were highlighted for 15 and 6 kDa HA. Therefore, except for the one below 6 kDa, all hyaluronan fragments studied are safe, biologically active, can support cell activation prompting repair of dermis. On the basis of the experimental results, general concern about low molecular weight hyaluronan is reasonable only when highly concentrated LHA, with average molecular weight lower than 15 kDa, is present. All the otherHAchains concur inbiochemical activationandthereforemay help in skin reparation procedures and biological remodeling.