The Promotion To Wound Healing And Skin Regeneration Of Biological Hydrogels And Its Molecular Mechanism Investigation

Objective:The biological hydrogels were fabricated from hyaluronic acid(HA)with physical and chemical methods,aiming to explore the potential function of the hydrogels in wound healing and skin regeneration.Method:In this work,two kinds of hyaluronic acid(HA)-based hydrogels were fabricated: one is made from physical freezing-thawing HA solution(HA1),and the other is from chemical cross-linking of HA and polysaccharide(HA2).They were applied to repair full-thickness skin defects with New Zealand rabbits as the test animals,using powder HA and cotton dress as the references.The wound starts to heal after wounds were disinfected with iodine followed by coating with HA2,HA1,HA and cotton dress(the control),respectively.They were recorded as 4 treatments(groups),HA2,HA1,HA and the control.The healing progress was followed and tested in the duration of 56 days,and the biological repairing mechanism was explored.Result:The results of WBC detection displayed that animals in group HA2 possessed the smallest values during the first 17 days,which implied that they got the least serious inflammation.The reduction of inflammation at the first stage when the wound took place is beneficiary for the wound healing.The serious extent for animals in other groups was HA1<HA < the control,which leads to the healing efficiency of HA1>HA>the control from the wound over-viewing.H&E staining helped to show the microscopic observation of the healing progress at various time points.Since the 3rd day,there were a great number of cells with the secretion of exudate followed by the formation of scabs.The hydrogel would cling to the wounds like a bed for the newborn cells to adhere and to migrate around,which was beneficial for the tissue to form a protective skin epidermis on the top of wound.Neonatal epidermis appeared at day 7 and extended to the wound center in the following days in HA2 animals.At day 14,the cells became fewer in number and sparser in space like the normal tissue.More mature epidermis formed in HA2 than in animals of other group like HA1,HA,and the control.Meanwhile,the appendages of skin such as blood vessels and hair follicles regenerated.A large amount of blood vessels exhibited mainly in granulation tissues at initial phase and then gradually disappeared at remodeling phase.The hair follicles began to be observed at around 14 days in the regenerated tissue in both HA1 and HA2 group,with the larger regeneration part and the smaller scab area after then.Until day 56,there was only a fine X-shape scar with no hair follicles,which was considered to be in the center of the original wound.In our study,high expressions of ?-SMA were shown in HA2 and HA1 group at day 7,but at day 14 in HA and control group days.The result of RT-PCR supports quantitatively this conclusion.That is,hydrogel of HA2 improved the secretion of ?-SMA,which was beneficial to wound contraction and vessel angiogenesis.After 7 days,the amount of ?-SMA in both HA2 and HA1 animals decreased,faster than the animals of HA and the control,both of them decreased until day 14.This indicated that ?-SMA expressions in HA2 group reduced more quickly than the all other groups(HA,HA1 and control)leading to reduce angiogenesis of granulation tissues and thus to fewer scab formation.At day 7,the amount of TGF-?1 expression was higher in HA1 and HA2 than in HA and control groups.After then,they decreased.However,in HA and control groups,the amount of TGF-?1 expression were higher since day 14.This is accounted for the more serious inflammatory in the control animals than in HA,HA1 and HA2,with the least serious inflammatory in HA2.It could be inferred that HA2 decreased the secretion of TGF-?1 relatively after day 14,therefore alleviating the inflammation and relieved the scar formation.Our results showed that HA1 and HA2 group reached the highest amount of the expression of VEGF in 7 days,with higher amount in HA2 than in HA1 group;HA and control group reached the highest VEGF expression in 14 days,with higher VEGF in HA than in control.In addition,highest amount of VEGF expression in HA2 group(lowest ?Ct).This demonstrated that HA2 improved the level of VEGF,which was helpful to form more blood vessels to provide nutrition so that it could accelerate the process of wound repairing.It was positive for wound healing within a short time though the increased expression might cause the final scar formation.Conclusion:From the wound area alteration,white blood cell(WBC)measurements and H&E staining,HA2 was the most promising treatment in promoting the wound healing with least serious scar formation.Immunochemistry analyses and real-time PCR tests of the bio-factors involved in the wound healing,vascular endothelial growth factor(VEGF),alpha-smooth muscle actin(?-SMA)and transforming growth factor beta-1(TGF-?1),exhibited that HA2 enhanced VEGF and ?-SMA secretion but reduced TGF-?1 expression at early stage,which alleviated the wound inflammation,improved the skin regeneration and relieved the scar formation.