| BackgroundsBurns are common accidental injuries.Clinically,different treatment plans are adopted according to the depth of burns.After a burn occurs,the integrity of the skin is destroyed,and it is prone to secondary ischemia,infection,and scar formation.The purpose of burn treatment is to achieve wound re-epithelialization in the shortest time to reduce the chance of infection and the formation of scars.Deep partial-thickness burns involve all layers of the skin epidermis and part of the dermis.Scars are usually left after healing,causing psychological and physical discomfort for the patient,reducing the patient's quality of life.The scars on joints may even affect joint function.However,unlike full thickness burns,deep partial-thickness burns still have part of the dermis and skin appendages left,which can provide seed cells for the re-epithelialization of the wound.Therefore,how to promote the re-epithelialization of the wound on the basis of the remaining dermal tissue and skin appendages to accelerate wound repair is a hot research topic.Epidermal stem cells(ESCs)are the key cells in the process of re-epithelialization of wounds.Their proliferation,differentiation,migration biological behaviors play key roles in the self-renewal of skin and wound repair.When the injury occurs,ESCs can produce new sub-representative skin cells,which migrate,proliferate,and differentiate from the remaining skin appendages of the wound to the center,and finally reach to coverage the wound.Wound healing means the restoration of skin integrity.This is the key to wound repair.In the process of wound healing,ESCs not only need to proliferate in large quantities to supplement the missing epidermal cells,but also need to migrate from the original settlement site to the wound and differentiate into epidermal cells to cover the wound.Any functional changes such as proliferation,migration and differentiation will directly affect the ability of ESCs to repair wounds.At the same time,ESCs have slow periodicity,and their differentiation,proliferation and migration are closely related to their own microenvironment.In addition,changes in pathological conditions(such as pregnancy,diabetes,burns)have a negative impact on the biological activity of ESCs.Therefore,how to adjust the proliferation and migration ability of ESCs by changing the internal or external factors of ESCs to promote wound repair is the target of our research.Changing the external factors of ESCs,adjusting the microenvironment of ESCs,such as increasing the content of epidermal growth factor,can promote their proliferation and migration,thereby further promoting the re-epithelialization of the wound.Platelet-rich plasma(PRP)is plasma that contains platelets several times higher than the physiological concentration,and the platelet concentration is at least 3-5 times higher than the whole blood concentration.The high concentration of platelets in PRP can be activated to release a large number of growth factors,including PDGF,EGF,FGF,IGF,VEGF and TGF-?.These growth factors alone or in concert promote the repair and reconstruction of wounds through various mechanisms.Studies have shown that PRP can enhance the therapeutic effect of transplanted stem cells including ADSCs,BMSCs and ESCs.However,there are few studies on the effect of PRP on epidermal stem cells,and the mechanism is still unclear.Through review of previous literature,we have concluded that PRP can regulate the proliferation and migration of ESCs,but the specific concentration is still uncertain,and whether there is a concentration dependence is still unclear.Besides,burns are different from general traumas in that they have additional heat injury to the skin.Does PRP promote proliferation and migration of ESCs under such pathological damage conditions?In addition,in vivo conditions,can PRP change the microenvironment of ESCs by releasing a large number of growth factors to promote the re-epithelialization of wounds by regulating the biological behavior of ESCs?To answer these questions,the first part of this paper intends to study the effect of autologous PRP on the proliferation of human ESCs to determine the concentration under in vitro conditions,and further study the effect of PRP on the biological behavior of heat-injured ESCs,and explore its mechanism of action.The second part is to study the regulation effect of PRP on ESCs and its effect on deep partial-thickness burn wound healing in vivo.In addition,the wounds of the split-thickness skin grafts donor sites is similar to deep partial-thickness burns in the depth.Sometimes the skin graft area has healed but the donor sites have a weak healing.The situation caused distress to doctors and patients.Can PRP promote wound healing and reduce scars by regulating the proliferation of ESCs in the donor sites of partial-thickness skin grafts?In the third part of our study,PRP was clinically applied to wounds in the donor sites to evaluate its therapeutic effects and anti-scarring effects.It is hoped that our research will provide new theoretical basis and ideas for the treatment of deep partial-thickness burns and donor site wounds.Objective1.In vitro conditions,the effect of autologous PRP on the proliferation of human ESCs was evaluated,and the concentration was determined.The effect of PRP on the biological behavior of heat-injured ESCs was evaluated and its mechanism of action was explored.2.In vivo conditions,the effect of PRP on the biological behavior of ESCs was evaluated during deep partial-thickness burn wounds healing process.The mechanism of PRP action in the process was also explored3.To evaluate the treatment effect of PRP on wound healing in skin donor sites of split-thickness skin grafts in clinical trials.The effect of aspirin on the role of PRP in wound healing process was also explored.Materials and Methods1.Experimental Study of PRP in Regulating the Biological Behavior of Heat-Injured Epidermal Stem Cells(1)Cultivation and identification of ESCs.Type ? collagen adhesion method was used to culture ESCs.Flow cytometry was used to detect cell markers.(2)The effect of PRP concentration on the proliferation of ESCs.PRP was prepared by the two-step centrifugation method.CCK-8 was used to measure the effect of different volume concentrations of PRP on the proliferation of ESCs.Growth curves was mapped according to CCK-8.(3)The effect of PRP on the biological behavior of heat-injured ESCs.Heat-injured ESCs model was prepared for further research.Flow cytometry was used to detect the effect of PRP on the apoptosis of heat-injured ESCs.CCK-8 was used to evaluate the effect of PRP on the proliferation of heat-injured ESCs.The cell scratch test was used to evaluate the effect of PRP on the migration of heat-injured ESCs.Western blot was used to evaluate the expressions of proteins in the signaling pathway after PRP acted on heat-injured ESCs.2.Experimental Study of PRP in Regulating Epidermal Stem Cells to Repair Deep Partial-Thickness Burns in Pigs(1)Establishment of deep partial-thickness burn model.A temperature-controlled heat press was used to prepare deep partial-thickness burn wounds on the back of Bama pigs.PRP was prepared by the two-step centrifugation method.After injury,PRP and normal saline were applied to the wound as PRP group and control group.(2)The effect of PRP on deep partial-thickness burn wound healing in pigs.Wound healing time was compared between the two groups.H&E staining was used to analyze the thickness and structure of the wound skin after healing.(3)The effect of PRP on cell proliferation and apoptosis in deep partial-thickness burn wounds.Immunohistochemical staining was used to compare the number of Ki67(+)cells and CK19(+)cells at different time points in the two groups.TUNEL staining was used to detect cell apoptosis.Western blot was used to evaluate the expressions of AKT and pAKT in tissues.(4)The effect of PRP on blood vessel formation in deep partial-thickness burn wounds.Immunohistochemical staining was used to compare the number of ?-SMA(+)blood vessels at different time points in the two groups.(5)The effect of PRP on microenvironment in deep partial-thickness burn wounds.The expression of growth factors EGF,bFGF and VEGF in the tissues was quantified by ELISA.3.Clinical Study of PRP in Repairing the Wounds of Split-Thickness Skin Graft Donor Sites(1)Patients grouping.Patients receiving split-thickness skin graft were selected from the author's hospital between June 2013 to December 2015.Autologous PRP was prepared by two-step centrifugation.Part 1:using self-control,patients were divided into PRP group and control group according to a random principle.Part 2:Patients treated with antiplatelet drug(aspirin,100 mg/day,lasting for more than 6 months)were selected as aspirin group.The blank group was used as a control PRP group.Both of group were treated with PRP.(2)The effect of PRP on the proliferation of ESCs in the donor sites.Acupuncture biopsy of the wound was performed for CK19 staining on the 10th day.(3)The effect of PRP on wound healing of donor sites.The healing time of the wound were recorded.The visual analogue score(VAS)was used to evaluate the pain index of the patients on the 7th,10th,and 14th days.The vancouver scar scale(VSS)was used to evaluate the scar of the wounds 1 and 3 months later.(4)The effect of aspirin on the role of PRP during wound healing process.The healing time,VSS scores and bleeding complication of the wound in two groups were recorded.4.Statistical analysis was performed using statistical software(SPSS 17.0,IBM,Chicago,IL).The data was expressed as mean ± standard deviation.The t test was used for comparison between the two groups,and the one-way analysis of variance(ANOVA)was used for the comparison between multiple groups.P<0.05 was judged to be statistically significant.Results1.Experimental Study of PRP in Regulating the Biological Behavior of Heat-Injured Epidermal Stem Cells(1)Cultivation and identification of ESCs.Observed through a microscope,the ESCs were seen to be paving stone-like on the 5th day.A large number of cells could be seen to fuse into pieces on the 14th day.The surface markers CK19 and ?1 integrin of ESCs were counted by flow cytometry.The positive rates of CK19 and ?1 integrin were 90.72%and 92.83%,respectively.(2)The effect of PRP concentration on the proliferation of ESCs.?PRP preparation:The normal whole blood platelet count was(280.67±54.56)×109/L,and the PRP platelet count was(1195.00±262.50)× 109/L.The platelet count in PRP was 4 times greater than that in normal whole blood.? CCK-8 test showed that with the increase of the concentration,PRP promoted the proliferation of ESCs,but after increasing to a certain concentration(10%-20%)(Vol/Vol),there was no significant statistical difference between the two volume fractions of PRP in promoting the proliferation of ESCs.(3)The effect of PRP on the biological behavior of heat-injured ESCs.?Flow cytometry showed that the apoptosis rate of the PRP group was significantly lower than that of the control group(9.77±0.47%,31.07±0.72%,P<0.05),PRP inhibited the apoptosis of heat-injured ESCs.?CCK-8 test showed that the cell OD value of the PRP group was significantly higher than that of the control group(0.49±0.01,0.23±0.01,P<0.05),PRP promoted the proliferation of heat-injured ESCs.?The cell scratch experiment showed that the migration area of the PRP group was better than that of the control group(86.19±1.90,71.49±5.31,P<0.05).?The western blot showed that compared with the control group,the expression of pAKT in the PRP group increased.After the addition of PI3K inhibitor LY294002,the expression of pAKT decreased,indicating that the PI3K/AKT signaling pathway might be involved in the process of PRP affecting the proliferation and apoptosis of ESCs.?Flow cytometry showed that the apoptosis rate of ESCs after the application of PI3K inhibitor LY294002 was higher than that of the non-added group(9.77±0.47%,30.90±1.31%,P<0.05).?CCK-8 test showed that the OD value of ESCs after the application of PI3K inhibitor LY294002 was lower than that of the non-added group(0.49±0.01,0.25±0.02,P<0.05).2.Experimental Study of PRP in Regulating Epidermal Stem Cells to Repair Deep Partial-Thickness Burns in Pigs(1)Establishment of deep partial-thickness burn model.?A deep partial-thickness burn model was established.H&E staining and HMGB1 staining results showed that the deep dermis was damaged after heat-injury.? PRP preparation.The normal whole blood platelet count was(356.50±26.63)× 109/L,and the PRP platelet count was(1123.83±94.65)×109/L.The platelet count in PRP was 3 times greater than that in normal whole blood.(2)The effect of PRP on deep partial-thickness burn wound healing in pigs.?The average wound healing time in the PRP group was shorter than that in the control group(16.67±1.75,19.33±1.63 days),(P<0.05).?At the 4th week after PRP treatment,the thickness of the epidermal layer in the PRP group was(100.47±19.03)?m,which was higher than the control group(82.10±16.09)?m(P<0.05).The thickness of the dermis in the PRP group was(2060.00± 163.16)?m and the control group was(1996.67±244.22)?m.There was no significant statistical difference between the two groups(P=0.24).(3)The effect of PRP on cell proliferation and apoptosis in deep partial-thickness burn wounds.?Ki67 staining results showed that the average scores of Ki67(+)cells in the PRP group and the control group were(2.50±0.51,1.90±0.66,P<0.05),(3.30±0.47,2.83±0.75,P<0.05)at the first and second weeks after treatment,respectively.?CK19 staining showed that the average scores of CK19(+)cells in the PRP group and the control group were(0.73±0.69,0.23±0.43,P<0.01),(2.10±0.66,1.37±0.89,P<0.01)at the first and second weeks after treatment,respectively.The results showed that the application of PRP promoted the proliferation of ESCs in vivo.?TUNEL staining showed that the average scores of apoptotic cells in the PRP group and the control group were(1.03±0.77,2.40±0.62,P<0.01)at the first week after treatment.Cell apoptosis in wound tissues in the PRP group was significantly reduced.?Western blot showed that in the first week after treatment,compared with the control group,the expression of pAKT in the PRP group increased under in vivo conditions.(4)The effect of PRP on blood vessel formation in deep partial-thickness burn wounds.The ?-SMA staining results showed the average scores of ?-SMA(+)vessels in the PRP group and the control group in the first and second weeks after treatment were(1.60±0.68,0.57±0.63,P<0.05),(2.53±0.73,1.83±0.65,P<0.05),respectively.The blood vessel score of the PRP group was higher than that of the control group.(5)The effect of PRP on microenvironment in deep partial-thickness burn wounds.The ELISA results showed the expressions of EGF in the PRP group and the control group in the first and second weeks after treatment were(643.14±61.81,331.56±63.70,P<0.01)(pg/ml),(801.13±88.08,622.59±19.59,P<0.01)(pg/ml);bFGF expression was(510.56±56.77,276.13±22.90,P<0.01)(pg/ml),(757.07±68.23,558.65±70.85,P<0.01)(pg/ml);VEGF expression was(808.45±49.51,314.16±93.23,P<0.01)(pg/ml),(1104.32±120.80,802.73±37.45,P<0.01)(pg/ml).The expression levels of growth factors in the PRP group were higher than those in the control group.3.Clinical Study of PRP in Repairing the Wounds of Split-Thickness Skin Graft Donor Sites(1)Patients grouping.? There were 15cases in each group.There were no significant differences in age,gender and wound area of donor site.?PRP preparation.In the control PRP group,the normal whole blood platelet count was(231.80±67.98)×109/L and the PRP platelet count was(954.53±329.93)×109/L.The platelet count in PRP was 4 times greater than that in normal whole blood.In the aspirin group,the normal whole blood platelet count was(244.27±52.73)×109/L and the PRP platelet count was(984.33±329.93)×109/L.The platelet count in PRP was 4 times greater than that in normal whole blood.(2)The effect of PRP on the proliferation of ESCs in the donor sites.CK19 staining results showed that the average scores of CK19(+)cells in the PRP group and the control group were(1.03±0.67,0.67±0.55,P<0.05).PRP promoted the proliferation of ESCs in the donor sites.(3)The effect of PRP on wound healing of donor sites.?The healing time of the PRP group were(17.47±2.17)days while the healing time of the control group were(20.60±3.40)days.The healing time of the PRP group was significantly shorter than that in the control group(P<0.05).?On the 7th,10th and 14th days after the operation,the VAS scores of PRP group(5.67±0.98,3.20±0.86,1.00±0.54,respectively)were statistically significantly lower than the control group(7.33±0.90,4.33±0.72,1.67±0.72,respectively)(P<0.01).?In the 1st and 3rd months after the treatment,the total VSS scores of PRP group(5.60±1.45,3.93± 1.94,respectively)were statistically significantly lower than those of the control group(7.87±2.01,5.87±2.42,respectively)(P<0.05).(4)The effect of aspirin on the role of PRP during wound healing process.?The healing time of aspirin group were(18.00±1.77)days while the healing time of the PRP group were(17.47±2.17)days.There were no significant differences between the two groups(P=0.47).?In the 1st and 3rd months after the treatment,there were no significant differences in the total VSS scores(P=0.10 and P=0.15)between the PRP group and control group.?The use of aspirin did not increase the risk of bleeding in donor site treated with PRP.Conclusions1.In vitro conditions,PRP promoted the proliferation of ESCs,and 10%volume concentration was the optimal concentration.2.In vitro conditions,PRP promoted the proliferation and migration of heat-injured ESCs and inhibited their apoptosis.3.PRP regulated the apoptosis and proliferation of heat-injured ESCs by up-regulating the expression of pAKT through PI3K/AKT signal pathway.4.In vivo conditions,PRP promoted the proliferation of ESCs in deep partial-thickness burn wounds,inhibited cell apoptosis,up-regulated the expression of pAKT,and changed the expression of growth factors(EGF,bFGF,VEGF)in the wound microenvironment.5.PRP promoted blood vessel formation in deep partial-thickness burn wounds of Bama pigs.6.Autologous PRP promoted deep partial-thickness burn wound healing of Bama pigs and improved the quality of healing.7.Autologous PRP promoted the proliferation of ESCs,shorten the healing time,alleviate the pain during the dressing process,and reduce scar development in split-thickness skin graft donor sites.8.Continuous aspirin use would not increase bleeding risk or postpone the healing process of the donor sites treated by PRP. |
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