The Clinical Study Of Hematoporphyrin Monomethyl Ether Photodynamic Treatment Of Port Wine Stains And The Construction Of 3D Skin Equivalent Disease Model

Part?Analysis of clinical factors affecting the effectiveness and safety of hematoporphyrin monomethyl ether photodynamic therapy of port wine stainsBACKGROUND:Port-Wine Stain(PWS)treatment is a challenge for dermatologists.In most cases,pulsed dye laser treatment cannot completely remove the skin lesions.At present,hematoporphyrin monomethyl ether photodynamic therapy(HMME-PDT)has become a promising alternative for the treatment of port wine stains.OBJECTIVE:To analyze the relationship between gender,age,skin lesion type and Fitzpatrick skin type and other clinical factors and the effectiveness and safety of HMME-PDT in port wine stains,and provide certain basis and reference for clinical treatment.METHODS:The data of 239 cases of port wine stains(PWS)treated with hematoporphyrin monomethyl ether photodynamic therapy(HMME-PDT)in our department from March 2017 to June 2020 were retrospectively analyzed.All were followed up for 6 months.All patients were pumped with HMME 5 mg/kg intravenously,the normal skin was covered,and the skin lesions were exposed to 532 nm LED green light10 minutes after drug infusion,and the irradiation power density was 80-100 m W/cm~2.Each spot(10×10cm~2)was irradiated for 20-25 minutes,according to the patient's gender,age group,skin lesion type,Fitzpatrick skin type,skin lesion location,skin size,previous treatment history,and the type of immediate postoperative response are classified.Dermatologists evaluated the clinical efficacy based on the comparison of VISIA before and after PWS treatment.The overall efficacy were evaluated in accordance with basic recovery,marked effect,improvement,and ineffectiveness.The pain score was based on the Visual Analogue Scale/Score(VAS):0 points(no pain);1-3 points(mild pain);4-6points(moderate pain);7-10 points(severe pain),and recorded adverse reactions(thick scab,thin scab,hyperpigmentation,hypopigmentation,phototoxic reaction)after PDT.The Pearson?~2 test and Fischer's exact test were used to analyzing the effects of the above factors on the efficacy,pain score,and adverse reactions.RESULTS:(1)Efficacy:50 cases of basic recovery(20.9%),111 cases of marked effect(r46.4%)improvement in 48 cases(20.1%),30 cases of ineffectiveness(12.6%),the total number of effective cases(basic recovery+marked effect+improvement)were 209 cases(87.4%);clinical factors related to good curative effect included female,pink and purple skin lesion types,Fitzpatrick skin type?,skin lesions located in the head and neck,skin lesion area?40cm~2,and pale edema and purpura appeared immediately(p<0.05).(2)Pain score:97 cases were severe(40.6%),65 cases were moderate(27.2%),50 cases were mild(20.9%),and 27 cases were painless(11.3%);clinical factors related to severe pain including males,over 5 years of age,skin lesions located on the head and neck,skin lesions area>40cm~2,and the color became darker immediately after PDT(p<0.05).(3)Adverse reaction:46 cases(19.2%)of adverse reactions occurred,193 cases(80.8%)did not occur;adverse reactions included 8 cases of thick crust,30 cases of thin crust,5 cases of hyperpigmentation,and 1 case of hypopigmentation,and 2 cases of phototoxic reaction,which returned to normal within 3-6 months after treatment;clinical factors related to the occurrence of adverse reactions included pink type and thickened nodular skin lesion types and previous treatment history(p<0.05).CONCLUSION:HMME-PDT is safe and effective in the treatment of PWS.At the same time,its effectiveness and safety are affected by a variety of clinical factors.The results of this study provide a basis for the rationalization and individualized treatment of PWS patients,which is conducive to achieving the safest experience and optimal curative effect.Part ? The relationship between dermoscopic vascular pattern and the effect of hematoporphyrin monomethyl ether photodynamic therapy on port wine stainsBACKGROUND: HMME-PDT is a novel,safe and effective treatment strategy for PWS.At the same time,its effectiveness and safety are affected by a variety of clinical factors,which provide a basis for the rationalization and individualized treatment of PWS patients.However,considering the shortcomings of the high economic cost of PDT treatment,how to accurately screen out PWS candidates that meet the HMME-PDT treatment indications has always been a clinical problem.It is very important to provide patients with non-invasive guidance and predict the effect of curative effect.However,there are few reports about the PWS vascular model of non-invasive dermoscopy to predict curative effect of HMME-PDT.OBJECTIVE: To analyze whether the vascular pattern classifications of PWS by dermoscopy can predict the efficacy of PDT.METHODS: This prospective cohort study included 163 patients with a clinical diagnosis of PWS who were treated twice with hemoporfin-mediated photodynamic therapy(HMME-PDT)at two-month intervals and followed up for 6 months.The vascular manifestations of dermoscopy with PWS were independently classified into 8 categories by 3 dermatologists.Images of the lesions were taken using VISIA,and the vascular patterns were imaged by dermoscopy by the same investigator.Images were captured before and after each treatment.The efficacy was evaluated with pre-and post-treatment VISIA images,and correlations between efficacy and vascular patterns were analyzed by four dermatologists in a blinded and independent manner,between 10 January 2019 and 11 December 2019.RESULTS: In the dermoscopy images for the whole cohort,dotted and globular vessels (25 cases,15.3%),short clubbed vessels(30 cases,18.4%),and curved vessels(21 cases,12.9%),patients with vascular patterns had the best effect;in patients with vascular patterns of pale halos surrounding brown dots(13 cases,8.0%)and arborizing vessels(16 cases,9.8%),HMME-PDT had moderate efficacy in the treatment of PWS;Patients with mixed(21 cases,12.9%),grey-whitish veil(19 cases,11.7%),and reticular patterns(18 cases,11.0%)vascular patterns were the worst or ineffective after receiving HMME-PDT(p<0.05).CONCLUSION: There is a clear correlation between the efficacy of PDT and the dermoscopy pattern in patients with PWS.Dermoscopy may therefore provide very useful clinical information prior to treatment in these cases.In addition,the vascular manifestations of PWS determined by dermoscopy help to predict response to PDT and manage patient expectations.Part ? Construction of 3D full-thickness human skin equivalent disease model and exploration of drug action mechanismBACKGROUND: Clinically,we found that dermoscopic vascular model is of significance in predicting the efficacy,so it is of great research value to construct related disease models in vitro and explore the vascular injury of PWS after treatment.At present,although animal and cell models provide research platforms for diseases,species differences and the lack of complex physiological microenvironment of human skin remain bottlenecks in the study of PWS diseases.Therefore,it is very necessary to construct an in vitro 3D model that can simulate the physiological conditions of the human body and is similar to the characteristics of PWS.OBJECTIVE: Based on skin tissue engineering,3D full-thickness skin equivalent disease model was constructed in vitro to preliminarily explore the vascular injury and mechanism of action of PWS after different drug treatments.METHODS: The normal human dermal fibroblasts(NHDFs)and human dermal microvascular endothlial cells(HDMECs)isolated from the foreskin,were mixed evenly at a ratio of 1:1 and then inoculated into the skin model scaffold.Above,after continuous cultivation for 28 days to obtain mature 3D dermal equivalent(DE),the first part of the experiment was divided into the following groups: blank control group,PWS model group.In the second part of the experiment,we inoculated normal human epidermal keratinocytes(NHEK)into artificial dermal tissue,and maintained the full-thickness skin model for gas-liquid incubation for 14 days to obtain 3D full-thickness skin.After the full-thickness skin equivalent(SE),the treatment groups were as follows: blank control group,PWS model group,low-concentration rapamycin(Rapamycin,RA)group,high-concentration RA group,and low-concentration imiquimod(Imiquimod,IM)group,high concentration IM group and DMSO group.The blank control group was still cultured with conventional culture medium,and the pathogenic factor group was cultured with conventional culture medium supplemented with VEGF-A,FGF,MMP-2,and MMP-9(both 50 ng/ml)for 7 days.After making paraffin sections,perform HE staining,observed the tissue morphology under a microscope,and used immunohistochemistry and immunofluorescence CD31 specific staining to study the p-PI3 K,p-AKT,p-ERK and p-JNK of human capillary endothelial cells expression and the formation of capillary-like structures in the 3D model.RESULTS:(1)DE(3D dermal equivalent)immunohistochemical staining results showed that the expression of p-PI3 K,p-AKT,p-ERK and p-JNK in human capillary endothelial cells of the PWS model was significantly increased compared with the blank control group(p<0.05);The results of DE CD31 immunofluorescence staining showed that the blood vessel density and diameter of the PWS model group were significantly higher than those of the blank control group(p<0.05)(2)The results of SE(full-thickness skin equivalent model)immunohistochemical staining scoring showed that the human capillary endothelial cells of the PWS model group expressed p-PI3 K,p-AKT,p-ERK and p-JNK compared with the control group significantly enhanced(p<0.05).Compared with the RA treatment group in the PWS model group,the expressions of p-PI3 K and p-AKT were reduced after the treatment of RA 10 ?g/ml and RA 20 ?g/ml(p <0.05),there was no change in the rest of the groups(p>0.05);compared with the IM treatment group in the PWS model group,the expression of p-JNK in human capillary endothelial cells decreased after IM 10 ?g/ml and IM 20 ?g/ml(p<0.05),and there was no significant difference between the other groups(p>0.05)).The results of SE(full-thickness skin equivalent model)CD31 immunofluorescence staining showed that the blood vessel density and diameter of the PWS model group were higher than those of the blank control group(p<0.05).The blood vessel density and diameter of the RA 10 ?g/ml and RA 20 ?g/ml treatment groups were significantly lower than those of the PWS model group(p<0.05),and there was no difference between the groups of different RA concentrations(p>0.05);the blood vessel density of IM 10?g/ml and IM 20?g/ml group was significantly lower than that of the PWS model group(p<0.05),but the blood vessel diameter of IM 10?g/ml was not different from that of the PWS model group(p>0.05);the blood vessel diameter of IM 20?g/ml was significantly lower than that of the PWS model group(p <0.05),and the blood vessel density and diameter of different IM concentration groups were different(p <0.05).There was no difference in blood vessel density and blood vessel diameter between the DMSO treatment group and the PWS model group(p>0.05).CONCLUSION: In this study,3D dermal and full-thickness skin equivalent models were successfully constructed in vitro,which can be used as a research platform for the pathophysiology and complex structure of PWS.The successful construction of the disease model can bring convenience for the testing of new drugs and the screening of therapeutic technologies.