| ObjectiveHyaluronic acid(HA) has been widely used to improve the quality of the skin,fill wrinkles and improve the facial contour and so on.In this study,we study the healing process of MTZs,and in the healing process used HA.Meanwhile,we study the change of percutaneous penetration ability of HA in the process of MTZs healing.Materials and methods1.Prepared skin in vitro and selected fractional CO2 laser’s energy:We had anesthetized SD rats by intraperitoneal injection of chloral hydrate(0.003 m L·g-1)(the same below),and had preserved skin of the back of SD rats with an electric scissors and a shaving knife(the same below),had observed the effect of skin.The whole layer of skin was taken,and the changes of the anterior and posterior subcutaneous tissues were observed histologically;in addition,when we cut off the subcutaneous tissue,histological observation the depth of excised rat skin were used different profractional energy fractional CO2 laser,so that we were selected the depth of energy size in the papillary dermis.2.Orthogonal experiments were designed to optimize the fractional density(D),HA molecular weight(Mr),and the penetration time(T): The design of orthogonal experiment was produced by orthogonal design assistant II V3.1.spots density(0、6.3%、11.1%、25.0%),molecular weight of hyaluronic acid(10k Da、100k Da、1000k Da、1500k Da),transdermal time(1h、3h、5h、7h)was regarded as investigation factors.According to the orthogonal table L16(45),the 16 experimental groups were obtained.3.In vitro skin permeation test was performed with modified Franz diffusion cell.The hyaluronic acid concentration of skin tissue were measured by using Rat HA ELISA KIT.4.Selected the better combination of “D-Mr-T”: three healthy SD rats have been shaved and were anesthetized,circled with a diameter of about 1.50 cm was draw on the spinal bilateral symmetry parts,each side has 3 circles.The fractional CO2 laser,different spots densities 6.3%,11.1%,25%,irradiated the skin in the circle from head to tail,and observed and recorded the skin reaction and healing at different time points.Combined with the orthogonal test in vitro,we were chosen the optimum combination of “D-Mr-T”.5.In vivo calcein staining,HE staining histological observation and in vivo HA transdermal experiment time arrangement were as follows.A total of 18 SD rats were randomly divided into 9 groups.Marked SD rats from head to tail side,the left 3 circles of one SD rat were designed as 0h,6h,12 h,the right corresponded to 3h,9h,15 h.The left 3 circles of another SD rat were designed as 18 h,24h,the normal skin test group(only by using HA),the right corresponded to 21 h,27h,blank control group(only by using PBS).The normal control and blank control were used in the HA transdermal test in vivo.Methods 1,4 selected the better parameters of the fractional CO2 laser(the same below)in SD rats,then they were covered by sterile wet normal saline gauze.We taken the gauze after each part of the wound healing time,conducted the calcein staining,HE staining and HA transdermal in vivo,respectively.6.Calcein staining: we alternate had used of normal saline and 75% ethanol rinse for three times after calcein staining 10 min,and wiped the surface moisture and drawing materials.Then observed under the fluorescence microscope with the excitation wavelength is 490 nm and the emission wavelength is 515 nm,as well as obtaining fluorescence image.We had taken the size of 0.50cm×1.00 cm back skin tissue of SD rats,conducted HE staining: Then observed under the optical microscope and recorded image.7.HA transdermal assay in vivo: we had used hyaluronic acid transdermal delivery 4h at corresponded to moments.We separated the skin and subcutaneous dartos,and then successively proceeded homogenate,centrifugation,supernatant and filtration.The last,we detected concentration of hyaluronic acid within skin tissue,by means of Rat HA ELISA KIT.8 Statistical analysis: Excel was used to establish the database of all the data in vivo and in vitro.SPSS19.0 software were used for statistical analysis.Two independent samples were compared using T test,the comparison of multiple sample mean use one-way analysis of variance.Between the every two groups were compared by SNK-q test,and the difference was statistically significant(P<0.05).Results1.The results of skin in vitro and fractional CO2 energy: The skin have not visible residual subcutaneous tissue and skin tissue defect after excluding subcutaneous tissue,and the dermis were completely retain in vitro.The skin have clear layers of stratum corneum,epidermis and dermis under optical microscope.Fractional CO2 laser irradiated skin formed the characteristic microthermal zones(MTZs).When the other parameters are constant,with the increased of energy,the depth of MTZs are increased dermal reticular layer.We choose the energy of the dermal papilla layer was 30.0m J.2.The result of orthogonal test in vitro: The experiment combination "D-Mr-T" was HA Mr=100k Da,the better penetration time 4h,spots density 11.1% and 25% in vitro orthogonal test.The optimal parameters of fractional density depend on the skin reaction.3.The results of skin reaction in vivo: with the increased of spots density,the skin reaction aggravated and the healing time was prolonged in vivo.When the spots density was 6.3%,the wound was healed at about 15 h,and the skin appearance was close to normal after 18h;When the spots density was 11.1%,the wound had been healed about 21 h,and skin restored close to the normal appearance after 24h;When the spots density was 25.0%,SD rat skin edge contraction after irradiaton,formation of eschar.The wound contraction decreased gradually at 21 h after irradiaton.The eschar are gradually disappear at 24 h,but was still visible at 27 h.So,the better experimental combination was "11.1%-100 k Da-4h".4.The result of calcein staining by using the laser with optimum laser parameters(profractional energy was 30.0m J and spots density was 11.1%)(the same below): Calcein staining showed microchannel,which hollow did not staining but solidification zone had been staining thus strong green fluorescence.In the MTZs healing process,the fluorescent intensity gradually becomes shallow in the process of healing.MTZs has shrunk at 3 hours,the part of MTZs hollow had appeared fluorescent signals during 9-12 h,and there was no obvious hollow part at 21 h,and there had not obvious fluorescence spot at 24 h.The results showed that the micro channel was healed in 21 h,and the skin continuity was restored.5.The results of HE staining by using the laser with optimum laser parameters: The typical MTZs was observed after the operation immediately(0h).The center of tissues formed a micro-channel by evaporation and evaporation,solidification zone around it.It was observed that there was an obvious inflammatory reaction zone around the solidification zone in vivo.MTZs has been partially reduced at 3h,there was a tendency slightly to shrink and shallow.The continuity of epidermis was restored and the micro-channel was closed in 21 h.However,Thermal solidification layer were still visible at 27 hours after irradiation.6.Percutaneous penetration of HA during MTZs healing: the fractional CO2 laser with optimum laser parameters can increase the content of HA in the skin tissue on 0h、3h、6h、9h、12h、15h、18h、21h after irradiation,compared with the blank control group 1.62、1.54、1.45、1.38、1.28、1.18、1.12、1.07 folds,respectively(P<0.05),showed a decreasing trend.However,there was no difference in the concentration of HA in skin of 24 h and 27 h group compared with blank control group(P>0.05).There was no difference in HA concentration between normal control group and blank control group(P > 0.05).7.Changes of HA content in the dartos after percutaneous penetration: the fractional CO2 laser with optimum laser parameters had irradiated the SD rats,compared with blank control group,there was no difference in HA concentration on 0h、3h、6h、9h、12h、15h、18h、21h、24h、27h(P>0.05).Conclusions1.The wound caused by low profractional energy(≤30.0m J)and spots density(≤11.1%)fractional CO2 laser healed within 24 h,at the same time,the skin continuity was basically restored.2.After irradiated by fractional CO2 laser,the concentration of HA(Mr=100k Da)permeation into skin was increased immediately.With the healing time going on,the permeation of HA gradually decreases,and it is unable to percutaneous penetration when the wounds healed and the skin’s continuity was restored after 21 h.3.With the increase of the profractional energy within limits,the depth of the MTZs was deepened;With the increase of the spots density within limits,the penetration of HA was increased. |
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