| ObjectiveAt present,the research of microneedle percutaneous drug delivery technology has focused on its effect of promoting permeability,but ignores the healing process of wound caused by microneedle.This paper is aimed to sdudy the wound healing process and in-depth study of its effects on the percutaneous penetration of hyaluronic acid.Materials and methods1.Shaving the back hair of SD rat,observing the bleeding of skin.Cutting off the shaved skin and pruning the subcutaneous tissue,then observing the pruning level.Taking the full skin,the skin before and after pruning subcutaneous and the skin after microneedle acupuncture to stain with HE,observing the integrity of the epidermis,the pruning level and the depth of needling histologically.2.The design of orthogonal experiment was conducted by orthogonal design sofetware V3.1.Needle model(G),molecular weight of hyaluronic acid(Mr),transdermal time(T)was served as examine factors,each factor was divided into four levels,the needle models contain needle-free,25 G,22G,18 G.Molecular weight contain 10 kDa,100kDa,1000 kDa,1500kDa.Transdermal time contain 1h,3h,5h,7h.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.The concentration of each group minus the blank control group that with the same penetration time as examining index.Screening and optimizing the optimum combination of “G-Mr-T”.4.Three healthy rats which have been shaved were anesthetized,circle with a diameter of about 1.5cm was draw on the spinal bilateral symmetry parts,about 3 on one side,the circles on the spinal left and right were acupunctured by microneedle array of 22 G and 18 G respectively.Taking photographs,observing skin reaction and the wound healing process.5.The group of rats in vivo experiment: Eighteen healthy SD rats which have been shaved,weight about 180 g,histologic observation of wound healing,calcein dyeing and hyaluronic acid percutaneous penetration randomly selected two,six and ten rats respectively,after anesthetized,circle with a diameter of about 1.5cm was draw on the spinal bilateral symmetry parts,about 3 on each side.Time point design of every two rats are as follows,on one rat,the left 3 circles were designed as0 h,3h,6h.The right 3 circles were designed as 9h,12 h,15h.On the other one,the left 3 circles were designed as 18 h,21h,24 h.The right 3 circles were designed as normal control and blank control,After microneedle acupuncture,acupuncture sites were covered by sterile gauze,waiting for natural healing,when reachs to the appointed time,removing the ganze,then doing the following experiments.6.Calcein dyeing experiment: covering with the calcein cotton ball,dyeing 10 min in dark place,removing the calcein cotton,then using saline solution and alcohol cotton to wipe 2 times alternately.Taking full thickness skin and triming the subcutaneous tissue,then putting the skin on microscope slides and observing under the fluorescence microscope with the excitation wavelength is 490 nm and the emission wavelength is 515 nm,recording the number of spots,as well as obtaining fluorescence image.7.The histologic observation of wound healing experiment: Taking full thickness skin staine with HE,then observing histologically.8.Hyaluronic acid percutaneous penetration experiment in vivo: Using hyaluronic acid about 4h,using swab with PBS liquid to wipe excess HA of skin surface,taking full thickness skin and washing,separating the dartos from full thickness skin,then the skin and dartos were homogenated respectively.The concentration of hyaluronic acid in skin tissue and dartos were detected by using Rat HA ELISA KIT.9.The data of orthogonal experimental was analysed by using orthogonal design sofeware V3.1.The data of hyaluronic acid concentration of skin and dartos were showed as mean±SD.The statistical analysis was calculated by SPSS19.0,all experiment groups were comparied with each other comprehensively by using SNK-q test(n = 5),when p-valued less than 0.05,there were significant difference.All sample rates were comparied with each other comprehensively by using partitions of chi-square method,in order to ensure the same of type I errors a in hypothesis testing(a=0.05),it was necessary to reset the inspection level a'(a'=a/2(k-1),a=0.05,k stands for groups),and arrived at the conclution: When p-valued less than 0.00313,there were significant difference.Results1.The hair on both sides of spine had been shaved cleanly,there was no scars and big bleeding spots.The skin had a clear,hierarchical histologic structure which contain Corneous layer,granular layer and stratum spinosum,base layer and the dermis,the corneous layer is intact,the skin barrier structure is normal;Observing the skin after pruning,there was no subcutaneous tissue and hole,the skin was complete and smooth,keep the intact of dermis;The corneous layer was destructed after acupuncturing skin by microneedle array,the needling depth reachs to papillare cutis.2.In the orthogonal experimental design,the greater the needle,the smaller the molecule weight,the longer the percutaneous penetration time,the more does of percutaneous penetration.the percutaneous penetration does(ng·L-1)of “18G-10k-7h”?“18G-100k-5h”?“22G-10k-5h”?“22G-100k-7h” were 410.555,231.700,294.705,215.440,when considering that the larger the molecular weight,the slower the degradation speed and more lasting effect,the 100 kDa was choiced.When the percutaneous penetration time at 3~5h,the slope was the largest,so the time was setted at 4h.The needle type was choiced either 22 G or 18 G,and the result was depend on the healing process.3.Observing by eyes: After acupuncturing by microneedle array,the needed healing time of 22 G and 18 G needle were 15 h and 21 h at least,when time to 24 h,the skin restores its normal morphology.Therefore,the best experiment combination is “22G-100k-4h”,select 22 G microneedle to do the calcein dyeing experiment,the histologic observation of wound healing experiment and in vivo transdermal experiment of HA.4.Calcein dyeing experiment: Acupuncturing by 22 G microneedle,after a period of wound healing time about 0h,3h,6h,9h,12 h,15h,then using calcein dyeing for 10 min,observing immediately.The number of spots reduced only a little,the dyeing rate were 97.8%,98.9%,95.6%,94.4%,92.2%,91.1% respectively,compared with each other,the dyeing rate fell only a little(P>0.00313).After a period of wound healing time about 18 h,21h,24 h,The number of spots reduced steeply,the dyeing rate were 3.3%,1.1%,2.2%.and compared with 0h,the dyeing rate reduced greatly(P<0.00313).With the healing time going on,the spot's silhouette become smaller and smaller,when to 18 h,there is no spot.5.Histologic observation of wound healing experiment: Acupuncturing by 22 G microneedle,skin barrier structure was destroyed,the needling depth reach to papillare cutis.With the healing time going on,the micropores contraction and the size become smaller,to 18 h,micropores covered by a proliferous of epdermal cells,and the skin barrier structure basic recoveried.6.Hyaluronic acid percutaneous penetration experiment in vivo(the skin): The Content of hyaluronic acid in normal control Group was equal to the Content in blank control group(P>0.05).After 0h,3h,6h,9h,12 h,15h of healing time,then using hyaluronic acid.The HA content were higher than in blank control group(P<0.05).With the increase of healing time,the HA content was on the decline(P<0.01).However,after 18 h,21h,24 h of healing time,then using hyaluronic acid.The content in the skin were similar to blank control group(P>0.05).7.Hyaluronic acid percutaneous penetration experiment in vivo(the dortos): Acupuncturing by 22 G microneedle,after 0h,3h,6h,9h,12 h,15h,18 h,21h,24 h of healing time,then using hyaluronic acid.Hyaluronic acid content in the dartos were similar to blank control group(P>0.05).Conclusions1.The wound caused by microneedle array healed within 24 h,at the same time,the skin barrier function restored.2.After acupuncturing by 22 G microneedle,the does of hyaluronic acid(Mr=100kDa)permeation into skin increase.With the healing time going on,the permeation dose decreases gradually.When reaching to 18 h,wound healed and skin barrier function recoveried,the hyaluronic acid is unable to percutaneous penetration.3.Microneedle have contributed to large molecular weight of HA(from 10 kDa to 1500kDa)to transdermal absorption. |
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