Basic Research On The Anti-aging Effect Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells On The Skin And The Reconstruction Of The Auricle With Insufficient Skin In The Mastoid Are

BackgroundSkin aging is divided into endogenous aging and exogenous aging.The primary cause of exogenous aging is photoaging due to the long-term UV exposure.Human umbilical cord mesenchymal stem cells(hucMSCs)are one class of proliferative,readily available mesenchymal stem cells(MSCs)which secrete a wide range of bioactive factors.HucMSCs may play a therapeutic role through exosomes and therefore have broad prospects for clinical applications.A few studies have found that human umbilical cord mesenchymal stem cell derived exosomes(hucMSCs-exo)can improve skin photoaging,but the number of studies available is small,and the mechanisms are not clear.Microtia is a common congenital craniofacial malformation,which is often associated with different extent of auricular deformities,atresia or stenosis of the external auditory meatus,as well as middle ear abnormities.Currently,the most commonly used methods of auricular reconstruction are the two-stage Nagata method and the tissue expander method.None of the existing methods,however,can guarantee safety and efficacy for patients with extremely insufficient skin in the mastoid region.Objectives1.To study the effect and mechanism of hucMSCs-exo on skin anti-aging.2.To explore the anti-aging effects of bipolar electrical stimulation on the skin surface of the hucMSCs-exo as a noninvasive method.3.This paper presents and reviews the application of a modified tissue expander method for auricular reconstruction in patients with severe skin insufficiency in the mastoid region.Methods1.HucMSCs were isolated,amplified and cultured in vitro by enzymatic collagen assay.HucMSCs were identified by microscopy and by flow cytometry for cell surface markers.HucMSCs-exo was isolated by hypervelocity centrifugation,the morphology was observed by transmission electron microscopy,the vesicle size distribution by NTA and the surface marker protein wad tested by Western-Blot.2.The dorsal skin of Bama pigs was exposed in simulated sunlight for 8 weeks,after which the changes in appearance were observed using a skin magnifying glass,changes in skin structure and dermal collagen were seen by HE and Masson staining,and gene and protein expression of MMP-1 and collagen I in skin tissue was assessed by qRT-PCR and Western-Blot.3.There were four experimental groups in addition to the control group and the photoaging group:the high-dose hucMSCs-exo injection group,the low-dose hucMSCs-exo injection group,the hucMSCs-exo external application group and the combined hucMSCs-exo external application and electrical stimulation group.At the end of the treatment,the appearance was observed using a skin magnifying glass,changes in skin structure and dermal collagen were observed by HE and Masson staining,and gene and protein expression of MMP-1 and collagen I in skin tissue was assessed by qRT-PCR and Western-Blot.The effects of hucMSCs-exo on skin anti-aging and the comparison between the different treatment groups were evaluated.4.A retrospective review of the patients who underwent auricular reconstruction with a modified tissue expander from January 2010 to December 2019 was performed at the center.A detailed description of the surgery procedure,the analysis of the efficacy and management of complications,and the summary of the experiences were presented.Results1.A morphological observation showed that the hucMSCs of P3 generation formed like a swirl in a petri dish with uniform spindle morphology.The cell surface markers were identified by flow cytometry and positive expression of CD44,CD73,CD90,CD105,as well as negative expression of CD11b,CD19,CD34,HLA-DR,and CD45 were identified.The vesicles isolated by hypervelocity centrifugation were characterized by transmission electron microscopy and observed to have the classic circular or cup-shaped shapes of exosomes.NTA vesicle size analysis revealed that the peak diameter of the vesicles was about 120 nm.Western-Blot suggested that Alix,CD63 and CD81 were expressed in high amounts on the obtained vesicles.2.After eight-week exposure to the ultraviolet light,the photoaging group showed rough,dry skin with visible wrinkles,slight pigmentation,slight desquamation and a leathery texture,compared to the control group.Histological HE and Masson staining showed marked thickening of the epidermis,marked thinning of the dermis,as well as sparse and disordered collagen tissue in the dermis.Gene and protein expression of MMP-1 and collagen I in skin tissues were detected by qRT-PCR and Western-Blot,which showed the decreased expression of collagen I and increased expression of MMP-1.3.After eight-week exposure to the ultraviolet light and hucMSCs-exo treatments,the general observation,histological HE staining,Masson staining,and qRT-PCR and Western-Blot detection of MMP-1 and collagen I expression levels in skin tissue revealed that the hucMSCs-exo treatment group had a more fine skin appearance,less pigmentation and desquamation,decreased epidermal thickness,increased dermal thickness,and more ordered collagen tissue in the dermis.Increased expression of collagen I and decreased expression of MMP-1 were detected in skin tissues.Intergroup comparison of four hucMSCs-exo treatment groups showed that intradermal injection had the greatest anti-aging effect on skin at the same dose and concentration of hucMSCs-exo.Compared with hucMSCs-exo external application alone,hucMSCs-exo combined with electrical stimulation was more effective against skin aging.4.In this clinical study,fourty-five patients underwent the modified tissue expander method for auricular reconstruction with extremely insufficient skin in the mastoid region were followed between 6 months and 10 years.93.3%of the patients were satisfied with the outcome,4.4%were generally satisfied,and 2.2%were not satisfied with the outcome.Conclusions1.HucMSCs-exo can improve skin photoaging induced by ultraviolet light.2.Bipolar electrical stimulation,as a noninvasive method,can synergize with hucMSCs-exo on the skin surface to promote UV-induced skin photoaging,which is superior to hucMSCs-exo externally applied on the skin surface alone.3.The modified tissue expander method for auricle reconstruction is a safe and effective method for patients with extremely insufficient skin in mastoid region.