| Background:Tissue engineering technology-based keratinocytes sheet transplantation provides a new method for repairing wounds.Conventional autologous keratinocytes sheet transplantation has high preparation cost,relatively long preparation cycle(3 to 4 weeks),unstable transplant take rate,and risk of virus transmission.This study was based on the traditional in vitro construction theory of keratinocytes sheet.It attempted to use low-temperature oxygen plasma-modified polyurethane membranes as carriers,and used allogeneic fibroblasts derived from young children's foreskin tissue as the trophoblast without growth arrest.The bovine serum-free culture system efficiently expanded human allogeneic keratinocytes,so as to rapidly constructed cell sheets containing allogeneic keratinocytes and fibroblasts,and further observed their treatment effect in partial-thickness burn wounds through a single-center,prospective randomized controlled trial.Part I:Construction of cell sheet containing human keratinocytes and fibroblasts based on low temperature oxygen plasma modified polyurethane membraneObjective:To construct a cell sheet containing human-derived keratinocytes and fibroblasts using a polyurethane membrane modified by low-temperature oxygen plasma as a carrier.Method:In order to realize the in vitro construction of human keratinocytes and fibroblasts sheet with high biological activity,the research team extracted primary keratin formation from the foreskin tissue obtained from the foreskin of neonates to 7-year-old children.Cells and fibroblasts were further subcultured to establish a human-derived keratinocyte and fibroblast bank.To ensure the safety of tissue sources,the donor must undergo medical examinations,surgical examinations,foreskin fungal smears,hematuria,liver and kidney function,HIV1/HIV2,hepatitis B two and a half,HCV,cytomegalovirus,EB virus,syphilis,herpes simplex virus(HSV)and other pathogenic tests.At the same time,the requirements for the preparation of carrier materials with strong drainage and high biocompatibility were put forward,and the equipment company was subjected to laser drilling and low-temperature oxygen plasma surface modification treatment of the polyurethane membrane.After completing the above steps,human-derived fibroblasts were seeded on the plasma surface-modified polyurethane membrane at 2×10~5/cm~2.After the cells were adhered,we continued to seed keratinocytes in a ratio of 8:1?10:1 to fibroblasts,and observed the growth and adhesion of cells on various surfaces.After the cells are completely confluent,the preparation of the cell sheet is completed,and the cell sheet is subjected to histological sectioning and HE staining.Results:The research team designed and completed the preparation of a cell sheet containing human-derived keratinocytes and fibroblasts using a low-temperature oxygen plasma-modified polyurethane membrane as a carrier.The results show that the polyurethane membrane is biocompatible after being modified by low-temperature oxygen plasma.When human-derived fibroblasts are seeded at a density of 2×10~5/cm~2,the ratio of the two cells is 1:8?1:10,the keratinocyte colony formation area can reach more than80%after the cells are completely confluent The whole process of preparing the cell sheet is about 2?3 days.That is,a cell sheet containing human-derived keratinocytes and fibroblasts has been efficiently prepared in vitro.Histological examination of the cell sheet longitudinal section The cell sheet has 1 to 3 layers of cells,with clear boundaries between keratinocytes and fibroblasts.Fibroblasts secrete a large amount of extracellular matrix to encapsulate keratinocyte colonies.Conclusion:The research team developed a cell sheet using low temperature oxygen plasma surface modified polyurethane membrane as a carrier,and containing human keratinocytes and fibroblasts.During the preparation of the membrane under the condition of a specific seeding density ratio,no excessive growth of fibroblasts was found,and the cell sheet can be quickly prepared under the condition that the cell seeding density is appropriately increased.Part II:Safety of cell sheet containing human-derived keratinocytes and fibroblasts,detection of infectious disease risk,and ethical review of clinical trials.Objective:To detect the biosafety of human-derived keratinocytes and fibroblast-containing cell sheet and the risk of infectious disease transmission,and submit relevant materials to the ethics review department of this unit for ethical review of clinical trials.Method:According to the National Standard for Biological Evaluation of Medical Devices of the People's Republic of China(GB/T16886.5,GB/T16886.10),a cell sheet containing human-derived keratinocytes and fibroblasts was selected as the test object.A delayed hypersensitivity test,a skin irritation test,and a cytotoxicity test were performed.After the primary keratinocytes and fibroblasts of each skin donor were successfully cultured,the 0th generation cell culture fluid was taken to the hospital laboratory for infectious disease risk examination(Hepatitis B,Hepatitis C,HIV,Syphilis).Then submit the clinical research plan and safety evaluation materials to the unit's ethics committee for approval.Results:The results of biosafety testing and infectious disease risk testing show that the cell sheet products are non-cytotoxic,have no skin irritation to the test animals,and have no delayed hypersensitivity reactions.After reviewing the cell sheet clinical trial protocol and related safety test data,the ethics committee of the First Affiliated Hospital of Naval Medical University agreed to adopt a clinical trial protocol of cell sheet containing human keratinocytes and fibroblasts.Part III:Clinical study of cell sheets containing allogeneic keratinocytes and fibroblasts for the treatment of partial-thickness burn woundsObjective:To evaluate the efficacy and safety of cell sheets containing allogeneic keratinocytes and fibroblasts in the treatment of partial-thickness degree burn wounds.Methods:Cell sheets containing allogeneic keratinocytes and fibroblasts were constructed using polyurethane biofilm as a carrier.From April 2016 to December 2017,the Department of Burn Surgery of the First Affiliated Hospital of Naval Military Medical University recruited patients with acute partial-thickness degree burn wounds meeting the inclusion criteria for this prospective positive self-control clinical trial.A group of 40 acute partial-thickness degree burn wounds were planned.The selected single wounds were not less than 10 cm×10 cm and not more than 5%total body surface group(TBSA).The selected wound was divided equally into two areas,and randomly recruited into cell sheet group and conventional treatment group by computer random method.The wounds in cell sheet group were covered by inner cell sheet and outer sterile gauze.Depending on the wound healing and exudation,the outer sterile gauze was replaced every 1 to 3 days after the treatment was started,and the cell sheet was replaced every 7 days.The wounds in conventional treatment group were covered by inner sulfadiazine silver cream gauze and outer sterile gauze,and the dressing was changed every 2 to 3 days depending on wound exudation.On treatment day 5,7,10,and 14,the healing rates of wounds in the two groups were calculated.Complete wound healing time,the total number of dressing changes,and the status of wound infection during treatment were recorded.Visual analogue scale was performed at the first dressing change.Six to 12 months after injury,scar formation of patients was followed up.Safety indicators included vital signs and laboratory tests and adverse reactions were observed.Measurement data are expressed by median and interquartile range(M(P25%,P75%)).Data were processed with Wilcoxon rank sum test,and Bonferroni correction.Results:(1)The prepared cell sheet had a diameter of about 8 cm and size of about49 cm2 per piece,containing 2 or 3 layers of cells.(2)A total of 43 patients were enrolled,of whom 3 patients fell off.Among the patients who completed the treatment,there were22 males and 18 females,aged 1?57 years old,with total burn area of 2%to 26%TBSA.(3)treatment day 5,7,10,and 14,the healing rates of wounds in cell sheet group were significantly higher than those in conventional treatment group(P<0.05).The complete wound healing time in cell sheet group was 7(5.75,8.5)d,which was significantly shorter than 11(7,14)d in conventional treatment group(Z=4.219,P<0.05).The total number of dressing changes of wounds in cell sheet group was 1(1,2)times,which was significantly less than 6(4,7)times in conventional treatment group(Z=5.464,P<0.05).(4)The wounds in cell sheet group in 31 patients had healed before the first replacement of cell sheet,and the pain visual analogue scores of wounds in the first dressing change in cell sheet group of9 patients were 1(0,1)points,and the pain visual analogue scores of wounds in the first dressing change in conventional treatment group of 40 patients were 2(1,3)points.There was no obvious infection in the wounds of 40 patients in the 2 groups before the wound healing.After the trial,9 patients were followed up.In 6 patients,no scar formation was found in the wounds of cell sheet group and conventional treatment group.The color of wounds in cell sheet group was consistent with normal skin and there was only a small amount of pigment deposition in the wounds of the conventional treatment group.In 3patients,the wounds in cell sheet group only had pigment deposition,while the scars of the wound in conventional treatment group were obvious.(5)The abnormal fluctuations of vital signs such as body temperature,blood pressure,heart rate,respiratory rate and laboratory examination indexes of all patients during treatment were alleviated by the rehabilitation of burns and wound healing.No obvious adverse reactions or abnormalities related to the cell sheet treatment were observed.Conclusions:The cell sheets containing allogeneic keratinocytes and fibroblasts can reduce the number of dressing changes,accelerate wound epithelialization,shorten wound healing time,reduce dressing pain in the treatment of partial-thickness burn wounds and may reduce scar hyperplasia because of rapidly wound healing.Its clinical application is simple,safe,and effective. |
PDF Full Text Request