Inhibition Of Extracorporeal Shock Wave Therapy On Hypertrophic Scarring In Rabbit Ear Model

Despite rapid development of medical technology in the past decades, hypertrophic scarsremain one of the challenges need to be solved. Hypertrophic scar (HS) can have devastatingoutcomes, ranging from function impairment and body disfigurement to disturbance ofeveryday activities and quality of lives because of pain, discomfort, anxiety and depression.Studies on prevention and treatment of HS are of vital importance for patients involved toimprove their quality of lives.Currently，the mechanisms involved in the emergence and development of HS includecells, cytokines, microcirculation, immunological factors and genetics. Nowadays, variousstrategies are available for managing and preventing HS, including certain drugs appliedtopically or intravenously, surgery, laser and particle beams, compression therapy andmassage, radiotherapy, cryotherapy and so on. However，to some extent, there are someintrinsic shortcomings and disadvantages with these therapies, e.g.，the invasion of surgery,pigmentation of pulsed dye laser. In addition to invasion, intralesional injection also results inpain, pigmentation changes, blistering, ulcer and necrosis. It is reported that compressiontherapy for HS could induce severe discomfort and limitation of movement. Thedisadvantages of cryotherapy and radiotherapy are their potential to cause hypopigmentationand carcinogenesis, respectively. Explore a non-invasive, safe, efficacious and cost-effectivetreatment for HS is necessary.Extracorporeal shock wave therapy(ESWT) belongs to one kind of pulsed acoustic waveresulted from dramatic changes of pressure, with the advantages of non-invasive, safe,efficacious, easily to operate. ESWT has been originally introduced and adopted for urinarylithotripsy, and subsequently its application has extended to other areas including treatingfracture and nonunion, acute and chronic musculoskeletal disorders and soft tissue wounds.The potential mechanisms of ESWT include promote angiogenesis and increasing blood supply to the wound, inhibiting inflammation of the wound, recruiting mesenchymal stemcells and endothelial progenitor cells to the injured site and so on.There is a paucity of research on its role in treating HS. In this study, full-thickness softtissue defects were created in the ventral surface of rabbit ear. The wound healedspontaneously three weeks later and HS formed (verified by pathological examination).Rabbits were class into three groups randomly, i.e., group A（with energy flux density of0.1mJ/mm2), group B（with energy flux density of0.2mJ/mm2), and group C (control groupwithout any treatment). ESWT was applied, changes were documented and biopsies werecollected and tested on the pre-established time points. Methods in this study are as follows:1. To measure the changes of diameters, areas, volume of elevations, toughness,wrinkles, haemoglobin, melanin of HS with Antera3D system.2. To observe the histological changes of HS, such as hypertrophic index(HI),morphology and density of fibroblasts, density of microvessels, and infiltration ofinflammatory cells, with hymatoxylin-eosin staining. To observe the morphologyand distribution of collagen fibers with Masson trichrome staining.3. Cell proliferation and differentiation of HS: to detect the mRNA expression ofproliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) byreverse transcriptase polymerase chain reaction and expression in fibroblasts andmyofibroblasts by immunohistochemical staining.4. To measure the mRNA and protein expression of TGF-β1, Smad2, Smad3, Smad7with reverse transcriptase polymerase chain reaction and enzyme-linkedimmunosorbent assay, respectively.Primary results of this study:1. Morphologic changes Scars faded three weeks after the application of lower andhigher ESWT, and became flatten and soften too. However, scars in the control groupwere still elevated, red and tough. More obvious differences were observed with longertime. No significant difference was observed between two treatment groups.2. Results of Antera3D examination Wrinkles and haemoglobin of scars diminished significantly in both two ESWT groups, and these changes appeared in group A was alittle earlier. Texture of scars also improved in group A which did not observed in groupB. However, no influence of ESWT was observed on diameter, area, volume ofelevation, melanin of scars.3. Results of histopathology1) HE staining No significant difference of HI was noted between three groups afterESWT treatment. However, fibroblast density was much lower in group A and group B,which was more obvious in group A. Two to five weeks later, the dermis in group A andgroup B became thinner than group C, with less microvessels, fibroblasts and collagenfibers observed. When compared with group C, HI and fibroblast density in group A andgroup B were also declined.2) Masson trichrome staining There was no significant difference was observed in theearly stage. With time going on, the collagen fiber became less, and arranged regularly intwo ESWT groups when compared with control group. However, no significantdifference was noticed between two ESWT groups.4. Influences of ESWT on cell proliferation and differentiation of HS1) Immunochemistry examination of PCNA and α-SMA Downregulation of PCNA ingroup A appeared at an early stage, and four weeks later, this phenomenon was alsoobserved in group B. There is no significant difference between two ESWT groups onPCNA expression. Both low and high energy flux density ESWT wound decrease theexpression of α-SMA significantly, and about four weeks later, significant difference alsoobserved between these two ESWT groups.2) mRNA expression of PCNA and α-SMA No significant influence on PCNA mRNAexpression was observed in two ESWT groups when compared with control group.Expression of α-SMA mRNA decreased significantly in group A, while no differencewas noted between group B and control group.5. Influences of ESWT on TGF-β1/Smad signal pathway of HS1) mRNA expression of TGF-β1，Smad2，Smad3of HSNo significant influence of ESWT was noticed on TGF-β1mRNA expression in both ESWT groups. No significant difference was observed in Smad2mRNA expression in groupA either, however，Smad3mRNA level declined significantly in this group. On the other hand,group B up-regulated Smad2and Smad3mRNA expression distinctly.2) Determination of protein content of TGF-β1，Smad2，Smad3and Smad7Content of Smad3protein decreased significantly in group A when compared withcontrol group, but exert no influence on the content of TGF-β1, Smad2and Smad7. Nosignificant change was noted of all these proteins in group B.Conclusions:1. ESWT is non-invasive, safe and well tolerated. Wrinkles，haemoglobin and toughnesswas improved significantly in both two ESWT groups. In addition, low energy fluxdensity ESWT could also improve the texture of scar. However, no influence of ESWT ondiameter, area and melanin of scars was noted.2. Histological examination indicated that both two ESWT could alleviate the infiltrationextent of inflammatory cells, decrease the number and density of fibroblasts, reducehypertrophic index and improve collagen arrangement.3. The potential mechanism of low energy flux density ESWT in inhibiting HS maybedecreases the expression of Smad3. Moreover, low energy flux density ESWT alsodecreases the expression of α-SMA mRNA which may play a vital role in managing scarcontracture.4. High energy flux density ESWT can decrease the expression of α-SMA mRNA too, butcan increase the expression of Smad2and Smad3simultaneously. Further research of themechanism of this therapy on scar management is necessary.