Biological Activities Of Agarose Grafting Hyaluronan And Its Application In Skin Regeneration

Objective: In order to disclose the role of agarose-g-HA in the process of skinregeneration.Methods: Agarose was activated with epichlorohydrin, and then grafted withhyaluronan with molecular weight of5kDa, to obtain agarose grafting hyaluronancopolymer (agarose-g-HA). The molecular weight of agarose-g-HA was measured byviscosimetry; and its structures were characterized with FT-IR、EA and1H-NMR. Thecell toxicity of agarose-g-HA was evaluated by MTT assay. Cell adhesion experimentwas performed to test the biocompatibility of agarose-g-HA, and agarose-g-HApromotes the mRNA level of collagen Ⅰ and collagen Ⅲ in3T3cells and TNF-αinRAW264.7cells with Real-time PCR. In addition, the effect of agarose-g-HA on theprotein level of collagen Ⅰ and collagen Ⅲ in3T3cells was analyzed by westernblotting. Finally, the agarose-g-HA dressing was used to repair mice skin lesion andits role in the process of skin regeneration was evaluated. Then, the wound healing ofmice was checked by HE staining.Result: Results of FT-IR, EA and1~H-NMR indicated that HA was successfullygrafted into agarose chain. And the molecular weight of agarose-g-HA was about100kD. The MTT assays proved that agarose-g-HA had no cell toxicity in theconcentration range from0to2mg/mL in72h. The result of cell adhesionexperiments showed that agarose-g-HA film promotes3T3cell adhesion. Results ofReal-time PCR revealed that agarose-g-HA can affect collagen Ⅰ and collagen ⅢmRNA level in3T3cell in a concentration-dependent manner. These results werefurther confirmed by western blotting which showed a dramatical increase of collagenⅠ and collagen Ⅲ protein levels in3T3cell with raising the concentration ofagarose-g-HA. Mice skin regeneration experiments disclosed that agarose-g-HAinhibited inflammation and scar, and promoted angiogenesis as well as mice woundhealing. Finally, agarose-g-HA facilitated skin repair and regeneration by HE staininganalyses. Conclusions: agarose-g-HA played a crucial role in promoting cell adhesionthrough interacting with3T3cell, and increased the protein level of collagen Ⅰ andcollagen Ⅲ in3T3cell, which are favorable for facilitating skin repair andregeneration.