Implantation Instrument on the Expression of Collagen-I and Cell Proliferation Markers in Pathological Scar

Meng WANG ,Gang ZHANG ,Wen-guang ZHANG ,Qian HUANG ,Wan-hong CHEN ,Cheng WANG

1Shenzhen Longhua People’s Hospital,Guangdong Province,518109,China;

2The Fifth Affiliated Hospital of Zunyi Medical University,Guangdong Province,519100,China;

3Affiliated Hospital of Guangdong Medical University,Guangdong Province,524001,China

ABSTRACT Objective To explore the efficacyof Scar Infusion Instrument on expression of Type I Collagen and Cell Proliferation Markers in Pathological Scars.Methods 70 cases patients with pathologic scar admitted in our hospital from Augst 2016 to July 2017 were randomly divided into study group and control group.The control group was treated with scar ointment.The treatment group was treated with scar ointment combined with scar The expression of COL-I in scar tissue was determined by enzyme-linked immunosorbent assay (ELISA).The expression of ki-67 nuclear protein was determined by immunohistochemical staining (sp).Results The efficacy of the study group was significantly better than the control group,the total efficiency of the study group was significantly higher than the control group after 6 months treatment(P＜0.05);The average COL-I and Ki67 nuclear protein expression in the single slice of the study group was significantly lower than that of the control group at the 3th months,6th months after treatment(P＜0.001).Conclusions The scarring device is more effective in the treatment of pathological scar,and its mechanism may be related to the better inhibition of COL-I and ki-67 nucleoprotein expression

KEY WORDS scarring instrumental;pathological scar;COL-I;ki-67 nucleoprotein

Scar is a kind of changes in appearance and histopathology on normal skin tissue caused by various kinds of trauma.Any tissue and organ will inevitably produce scar in the process of wound repair[1].Pathological scar is the product of pathological healing of human skin which is traumatized to the dermis.Pathological scar has the following characteristics:protruding above the injured skin tissue;higher toughness than normal skin;redder and darker than normal skin;and more obvious degree of congestion.Therefore,if the pathological scar occurs at the joint,it may cause joint dysfunction[2-3].At present,the common treatments for pathological scars are surgical treatment,local injection of corticosteroids,silicone gel membrane therapy,pressure therapy,radiotherapy,laser therapy and drug therapy,which report to have good curative effect[4-5].Ki-67 is a nucleoprotein,which is a marker of cell proliferation.Its function is closely related to mitosis and it is indispensable in cell proliferation.Ki-67 nucleoprotein reflects the degree of cell proliferation to a certain extent.Overexpression of COLlagen-I (COL-I)is widely recognized as an important cause of pathological scar.This paper prospectively analyzed the effect of scar implantation instrument on the expression of Collagen-I and cell proliferation markers in pathological scar.

MATERIALS AND METHODOLOGY

General Material

70 patients with pathological scars admitted to our hospital from July 2016 to June 2017 were selected as the research objects.35 patients in each group were divided into study group and control group.There were 19 males and 16 females in the control group,aged 23-68 years old,with an average age of 39.2±10.5 years old.19 patients were educated with junior middle school or below level,12 with senior high school and vocational education level,and 4 with diploma and above level.Scar etiology:16 cases burned,8 cases scalded,6 cases traumatized and 5 cases infected.There were 22 males and 13 females in the study group,aged 22-62 years old,with an average age of 39.7±10.9 years old.6 patients were educated with junior middle school or below level,13 with senior high school and vocational education level,and 6 with diploma and above level.Scar etiology:14 cases burned,11 cases scalded,7 cases traumatized and 3 cases infected.There was no significant difference in age,gender,education level and scar etiology between the two groups (P >0.05).

Inclusion and exclusion criteria

(1)pathological scar;(2)course of disease <3 months;(3)informed of the details of this study.Exclusion criteria:(1)excluding patients with severe heart,liver,kidney and other organ diseases,malignant tumor,malignant hematological diseases,psychiatric diseases;(2)excluding patients with cognitive dysfunction and mental diseases;(3)excluding patients with scar damage and ulcer;(4)excluding patients who rejected this study.

Therapeutic Methods

The control group used clean water to clean the skin,took appropriate amount of scar ointment (produced by Shanghai Meibao Life Technology Co.,Ltd.,approval number:Shanghai Food,Drug &Devices No.264092,2012)and applied it on the scar,gently massaged for 5-10 minutes,three times a day,for 6 months.

The study group was treated with scar implantation instrument combined with scar ointment.Scar implantation instrument:produced by Shenzhen Demai Technology Co.,Ltd.,model DM-200B.The methods of scar skin cleaning and scar ointment smearing were the same as those of the control group.The output of scar implantation instrument was 30-40khz.The mobile ultrasound probe moved in a circular or linear way at a speed between 0.5-2.0 cm/s.Pulse wave and continuous wave were used alternately.Each treatment lasted 15-20 minutes,2 times a day in the morning and in the evening,for 6 months.

Evaluation Indicators

Therapeutic evaluation refers to the objective criteria for evaluating the therapeutic effect of pathological scars formulated by Shanghai Seventh People’s Hospital[6]:(1)the thickness of scars measured by ultrasound:1 point for reduction by more than 30%,2 points for 30% -20%,3 points for 20% -10%,4 points for less than 10%,and 5 points for no change;(2)the color of scars is divided into four grades,namely,red,light red,dark purple and normal,which are scored 4-1 points respectively;(3)Scar ulceration was classified according to the ulceration area around the scar:5 points for scar ulceration 1 cm around the scar,4 points for 0.75-0.99 cm,3 points for 0.50-0.74 cm,2 points for 0.25-0.49 cm,and 1 point for <0.25 cm;(4)Scar hardness:5 points for scare not depressed under pressure,4 points for depression 1/4 of scar’s hardness,3 points for 1/2,2 points for 3/4,and 1 point for scar hardness equals to surrounding skin.(5)Peri-scar hyperemia:5 points for hyperemia >1 cm,4 points for 0.75-0.99 cm,3 points for 0.50-0.74 cm,2 points for 0.25-0.49 cm,and 1 point for hyperemia <0.25 cm.The total score is all the indicators added up:if the score after treatment decreased by 75%,the treatment is obviously effective;if the score after treatment decreased by 50%-75%,the treatment is effective;if the score after treatment decreased by 50% and below,the treatment is ineffective.Effective rate=obviously effective+effective.The evaluation was conducted before treatment,3 months after treatment and 6 months after treatment.

COL-I and ki-67 nucleoprotein expression was adopted to measure COL-I:Scar tissue was cut under aseptic environment,washed,cut,cultured and stained.The expression of COL-I was determined by ELISA.The COL-I positive substance was brown granule.It was collected and analyzed under optical microscope (OLYMPUS,CH20)system.Each slice was randomly observed in 5 visual fields,and the positive particles in the visual field were accurately selected.ki-67 nucleoprotein was measured by immunohistochemical staining (sp).Scar tissue was cut under aseptic environment.After paraffin section dewaxing and hydration,antigen was repaired by microwave for 10 minutes.50 ml peroxidase blocking solution (produced by Maixin Company in Fuzhou,Fujian Province)was added to each slice.It was rinsed three times and 50 ml Ki67-MIBI monoclonal antibody was added for overnight at 4℃ before rinsing for another 3 times and stained with hematoxylin R.Cells stained to claybank or chocolate brown under 400 times high power field (HighPowerField,Hp)were Ki67 positive cells.

Statistical Analysis

Data was processed by SPSS 17.0 statistical software.The counting data were expressed by percentage,tested byχ2,Manng-Whitely U test for grade data,(±s)for measurement data and t test was adopted;P＜0.05 means that the difference has statistical significance.

RESULTS

Comparison of Effect

After 6 months of treatment,the study group was significantly better than the control group,and the effective rate of the study group was significantly higher than that of the control group,p <0.05,Table 1.

The expression of average COL-I and Ki67 in the study group was significantly lower than that in the control group in single slice after 3 months and 6 months of treatment,p＜ 0.001,Table 2.

DISCUSSIONS

Therapeutic Effect of Scar Implantation Instrument on Pathological Scars

Scar is the inevitable outcome of wound healing.When abnormal wound healing occurs,collagenbased extracellular matrix deposits in large quantities,resulting in excessive proliferation of dermal tissue and then pathological scars.Scar treatment mostly focused on increasing collagen decomposition and reducing its synthesis.Zhang Shuiliang[7]used scar antipruritic ointment combined with elastic socks to treat pathological scar patients and after 6-16 months,the total effective rate was 94.4%.Song Haiyun[8]treated pathological scar patients with acupuncture combined with ultrasound for 2-9 courses,the effective rate was 93.9%.Gilman T H[9],a foreign scholar,has achieved remarkable improvement in pain experience,scar appearance and scar flexibility after long-term hydration therapy (moisturizing cream and dressing)for hypertrophic scars.The results of this study showed that after 6 months of treatment,the study group was significantly better than the control group,and the effective rate of the study group was significantly higher than that of the control group(P<0.05).It is suggested that the scar implantation instrument is effective in the treatment of pathological scars.The effective ingredients of scar ointment are oleic acid,linoleic acid,sesamin,cactus extract,beeswax,etc.Oleic acid and linoleic acid can inhibit the proliferation of fibroblasts,damage and destroy the structure and function of cells,which however,when used on scar skin,can effectively reduce the expression of related proteins,smooth wrinkles and repair damaged skin.Sesamin,one of the lignans,has remarkable antioxidant effect.It can inhibit the proliferation of fibroblasts in scar skin,promote the proliferation of epidermal cells by regenerating substances,promote the rearrangement of collagen fibers in scar tissue,and achieve the purpose of preventing,alleviating or eliminating scars.

Table 1:Comparison of 2 Groups at 3 months and 6 months after Treatment

Table 1:Comparison of 2 Groups at 3 months and 6 months after Treatment

Cactus extract has many pharmacological effects.Fang Quan[10]found that cactus extract can reduce the expression of COL-I in scar tissue,promote the expression of COL-III,promote the expression of MMP-1 and smooth the scar.Beeswax can relieve itching and pain of new epidermis or scars,moisturize tissues,regulate the proportion of epithelial cells to collagen cells,and change their morphology.Ultrasound introducer is the direct contact between the ultrasound microwave and the treated skin.By changing the pressure and particle of cells and tissues,the effective ingredients in scar ointment can be introduced into deep cell tissues to give full play to the efficacy of scar ointment.Ultrasound introducing has certain biological massage,hyperthermia and improvement of scar group.Tissue cell microcirculation can relieve the symptoms of scar itching,pain,dry crack,pigmentation and functional limitation.

Effect of Scar Implantation Instrument on the Expression of COL-I and Ki67 in Scar Tissues

The results showed that the average expressions of COL-I and Ki67 in the study group were significantly lower than those in the control group after 3 months and 6 months of treatment,p <0.001.These results suggest that scar implantation instrument can reduce the expression of COL-I and Ki67 in scar tissue,which may be the potential mechanism of scar implantation instrument in the treatment of pathological scars.Over-expression of COL-I is widely recognized as an important cause of pathological scars.The imbalance of collagen metabolism leads to a large amount of collagen deposition in pathological scars.Scar histopathological sections show that pathological scars contain a large amount of collagen,which is arranged in disorder.Collagen bundles overlap and thicken,showing wavy and marked“collagen nodules”.Study[11]found that the proportion of COL-I in patients with pathological scar increased significantly.Collagen is mainly produced by endothelial cells and fibroblasts,which have stronger ability to produce collagen.

Hasegawa T[12]measured related proteins in patients with pathological scars and found that collagenase inhibitor,α-globulin,fibrinolysin inhibitor-1 and COL-I were significantly increased in the scar tissues of patients with pathological scars,but the secretion of degradable collagenase was insufficient.Ki-67 is an ENA associated with proliferating cells.Its function is closely related to mitosis.The expression range of Ki-67 covers the proliferative cycles except G0phase.It first appears in the second half of the prophase of DNA synthesis (G1),increases significantly in the phase of DNA synthesis(S),and peaks at the late stage of cells.Kang Shensong[13]used immunohistochemical method to detect the expression of Ki-67 protein in pathological scar tissue and found that Ki-67 index in pathological scar tissue was significantly higher than that in non-pathological scar tissue.

Tao Maochan[14]cultured keloid fibroblasts with Quscar Decoction.The expression of transforming growth factor-β1 (TGF-β1)and Ki-67 was detected.It was found that there was a positive correlation between TGF-β1 and ki-67.The increase of TGF-β1 expression could promote fibroblast proliferation and increase collagen secretion.This leads to hypertrophic scars.Teofoli[15],a foreign scholar,used Ki-67 nuclear Proliferating Antigen Ki67-MIBI monoclonal antibody immunohistochemical staining to detect scar fibroblasts.It was found that the expression of Ki-67 protein could be used as a reliable indicator of the proliferation activity and level changes of scar fibroblasts.

The effect of scar implantation instrument on the expression of COL-I and Ki67 in scar tissue has not been found yet.The mechanism of reducing the expression of COL-I and Ki67 may be as follows:(1)Scar implantation instrument combines computerbased IF therapy technology with IF drug introducing technology and thermal and magnetic therapy function by continuous wave,and the effective ingredient of scar ointment is introduced deeper in the scar tissues.The effective ingredient of scar ointment can better inhibit the expression of COL-I and Ki67;2)Scar implantation instrument can activate collaterals and promote blood circulation by rising temperature on local scar skin,degrade collagen in the extracellular matrix of scar under good blood circulation condition,regulate TGF-β1 and CD44,and reduce the expression of COL-I and Ki67;3)Fibroblasts can repair cell degeneration,necrosis and tissue defect to some extent.The imbalance of proliferation and apoptosis of fibroblasts plays a very important role in the formation of pathological scars.In the early stage of wound healing,fibroblasts proliferate by mitosis to promote wound healing;when wound healing forms pathological scar tissues,fibroblasts have always maintained high proliferation activity,leading to the growth of pathological scars.The proliferation of fibroblasts is regulated by the body and influenced by many environmental factors.Scar implantation instrument may interfere with fibroblast activity and decrease the expression of COL-I and Ki67 in scar proliferation phase in some way.

In this study,we found that the effect of scar implantation instrument combined with Meibao ointment on pathological scars was more remarkable,while inhibiting the expression of COL-I,it also decreased the expression of ki-67,a cell proliferation marker.This may be the potential mechanism of scar implantation instrument in the treatment of pathological scars.