Nghiên cứu tác dụng điều trị tại chỗ của gel ceri nitrat 2,2% trên vết thương bỏng do nhiệt

MINISTRY OF EDUCATION MINISTRY OF NATIONAL AND TRAINING DEFENCE VIETNAM MILITARY MEDICAL UNIVERSITY NGUYEN THANH CHUNG STUDY ON TOPICAL TREATMENT EFFECT OF CERIUM NITRATE 2.2% GEL ON THERMAL BURN WOUNDS PhD MEDICAL DISSERTATION HA NOI - 2021 MINISTRY OF EDUCATION MINISTRY OF NATIONAL AND TRAINING DEFENCE VIETNAM MILITARY MEDICAL UNIVERSITY NGUYEN THANH CHUNG STUDY ON TOPICAL TREATMENT EFFECT OF CERIUM NITRATE 2.2% GEL ON THERMAL BURN WOUNDS Specialty: Surgery Code: 9720104 PhD MEDICAL DISSERTATION SCIENTIFIC INSTRUCTORS: Assoc Prof NGUYEN NGOC TUAN, MD PhD DO LUONG TUAN, MD PhD HA NOI - 2021 DECLARATION I declare that this is my own work, and the research results presented in the dissertation are truthful, objective, and have never been used in any other studier I hereby declare that all help in conducting the research has been acknowledged, and the information in this dissertation has been cited Hanoi, 2021 Author NGUYEN THANH CHUNG ACKNOWLEDGMENTS During the process of studying and completing this dissertation, I have received valuable guidance and help from teachers, scientists, departments, faculties, agencies, and units I would like to thank the Party Committee and Board of Directors of Le Huu Trac National Burn Hospital, relevant organization, Post Graduate Department - Vietnam Military Medical University for creating faverable conditions and allowing me to study, research and complete the dissertation I would like to express my deep gratitude to my two supervisors: Senior Colonel Assoc.Prof.Dr Nguyen Ngoc Tuan and Senior Colonel Dr Do Luong Tuan who wholeheartedly helped me throughout the process of studying, implementing and completing the dissertation I would like to thank my family and friends who encouraged and supported me doing the study and completion of my dissertation Author Nguyen Thanh Chung TABLE OF CONTENTS DECLARATION ACKNOWLEDGMENTS TABLE OF CONTENTS APPENDICES LIST OF ABBREVIATIONS LIST OF TABLES LIST OF DIAGRAMS LIST OF FIGURES INTRODUCTION CHAPTER 1: OVERVIEW 1.1 CLASSIFICATIONS OF BURN DEPTH 1.1.1 Superficial burns 1.1.2 Deep burns 1.2 PROCESS OF BURN WOUND HEALING 1.2.1 The hemostasis 1.2.2 Acute phase 1.2.3 The proliferative phase 1.2.4 Maturation phase, scar formation 1.3 THE BURN WOUND INFECTION AND TOPICAL DRUGS FOR TREATMENT OF THE BURN WOUND INFECTION 11 1.3.1 Burn wound infection 11 1.3.2 Drugs to treat burn wound infection 16 1.4 CERIUM NITRATE AND APPLICATIONS IN BURN TREATMENT 20 1.4.1 Cerium and Cerium nitrate overview 20 1.4.2 Some biological effects of Cerium 21 1.4.3 The application of cerium nitrate in burn treatment 22 1.4.4 Toxicity and adverse effects of Cerium nitrate 34 1.4.5 Cerium nitrate gel 35 CHAPTER 2: SUBJECTS AND RESEARCH METHODS 37 2.1 SUBJECTS 37 2.1.1 Subjects of drug toxicity study 37 2.1.2 Research subjects of antibacterial effect 37 2.1.3 Research subjects of experimental burn treatment 38 2.1.4 Research subjects of clinical treatment of burns 38 2.1.5 Research materials 39 2.2 RESEARCH METHODS 41 2.2.1 Study methods for acute toxicity of Cerium nitrate 41 2.2.2 Study method semi-chronic toxicity of Cerium nitrate 42 2.2.3 Research methods for skin irritation of Cerium nitrate 44 2.2.4 Evaluation of the antibacterial ability of Cerium nitrate 47 2.2.5 Method to evaluate the therapeutic effect of Cerium nitrate gel on experimental burns 50 2.2.6 Methods of assessing the clinical effectiveness of Cerium nitrate gel 57 2.2.7 Data processing methods 61 2.2.8 Research ethics 61 2.2.9 Limitations and difficulties of the research 62 CHAPTER 3: RESULTS 64 3.1 TOXICITY AND ANTIBACTERIAL EFFECTIVENESS OF CERIUM NITRATE 64 3.1.1 Acute toxicity of Cerium nitrate gel on experimental animals 64 3.1.2 Semi-chronic toxicity of Cerium nitrate gel on white rats 67 3.1.3 Study result of skin irritation of Cerium nitrate gel 70 3.1.4 The in vitro antibacterial ability on some bacterial species 72 3.2 TOPICAL EFFECTS OF CERIUM NITRATE GEL 75 3.2.1 Effect of burn wound treatment on experimental mice 75 3.2.2 Treatment effect on burn wounds in clinical 87 CHAPTER 4: DISCUSSION 105 4.1 ACUTE TOXICITY, SUBACUTE TOXICITY, AND SKIN IRRITATION OF CERIUM NITRATE 105 4.1.1 Acute toxicity of cerium nitrate gel 105 4.1.2 Semi-permanent toxicity of the cerium nitrate gel 106 4.1.3 Skin irritation of cerium nitrate gel 107 4.2 ASSESSMENT OF THE ANTIMICROBIAL EFFECTS OF CERIUM NITRATE GEL 107 4.2.1 Causes of burn wound infection 107 4.2.2 In vitro antibacterial effect of cerium nitrate gel 111 4.2.3 The minimal inhibitory and complete bactericidal concentration of cerium nitrate gel 113 4.2.4 Antibacterial effect of cerium nitrate gel on experimental and clinical burn wounds 115 4.3 TREATMENT EFFECTS OF CERIUM NITRATE GEL ON EXPERIMENTAL AND CLINICAL BURN WOUNDS 118 4.3.1 Anti-inflammatory and anti-edema effects of cerium nitrate 118 4.3.2 The drying effect for necrosis of cerium nitrate 122 4.3.3 Immunological effects of cerium nitrate gel 125 4.3.4 Safety of cerium nitrate gel 127 4.3.5 Initial evaluation of the clinical effect of cerium nitrate gel 130 CONCLUSION 137 RECOMMENDATIONS 140 LIST OF PUBLISHED ARTICLES RELATED TO THE DISSERTATION REFERENCES APPENDICES LIST OF ABBREVIATIONS No Abbreviations Full text Aci baumannii Acinetobacter baumannii BSA Body surface are ECM Extracellular Matrix E coli Escherichia coli FGF Fibroblast growth factor EGF Epidermal growth factor ICU Intensive Care Unit K pneumoniae Klebsiella pneumoniae LPC Lipid protein complex 10 MBC Minimum Bactericidal Concentration 11 MIC Minimum Inhibitor Concentration 12 MMP Matrix Metalloproteinase 13 MPS Mucopolysaccharide 14 OECD Organization for Economic Co-operation and Development 15 P aeruginosa Pseudomonas aeruginosa 16 PDGF Platelet derived growth factor 17 S aureus Staphylococcus aureus 18 S epidermidis Staphylococcus epidermidis 19 SSD Silver Sulfadiazine 20 SB Standard Basis 21 TGF β Transforming growth foctor-β 22 VEGF Vascular endothelial growth factor LIST OF TABLES Tables Name of Tables Page 2.1 Evaluation and classification of skin irritation in rabbits 46 3.1 Death rate of mice after 14 days using orrally Cerium nitrate gel 64 3.2 Some manifestations of rats after drinking cerium nitrate gel 65 3.3 The changes in mice weight when drinking Cerium nitrate gel 67 3.4 Changes of hematological and blood biochemical indicators of mice 68 3.5 The irritation of Cerium nitrate gel on healthy rabbit skin 70 3.6 Effect of Cerium nitrate gel on negative gram bacteria 72 3.7 Effect of Cerium nitrate gel on positive gram bacteria 73 3.8 The relationship between the counted bacteria and drug concentrations along contact time 74 3.9 Weight changes (g) of the mouse during research 75 3.10 Local manifestation of experimental burn wound 76 3.11 Changes in burn size through treatment time 80 3.12 Experimental burn wound healing time 81 3.13 Percentage of positive bacteria isolated in burn wounds 82 3.14 The percentage of bacterial strains on burn wounds 82 3.15 Density of bacteria on the wound surface with positive cultures 83 3.16 Changes in some hematological indexes of experimental rats 84 3.17 Changes in some biochemical indicators of mice 85 3.18 Some characteristics of the study group 87 3.19 Progression of deep dermal burn necrosis 89 3.20 Necrosis progression of deep dermal burn wounds 90 3.21 Percentage of positive cultures on deep dermal burn wounds 93 3.22 The percentage of bacterial species in the deep dermal burn wound 93 3.23 The density of bacteria/cm2 of deep dermal burn wounds 94 3.24 Changes in hematological indices in patients with deep dermal burns 95 3.25 Changes in blood biochemistry index in patients with deep dermal burns 95 3.26 Cytological changes on the surface of deep dermal burns 96 3.27 Local progression of deep burn wounds 97 3.28 Progression of local necrosis of the deep burn wound 98 3.29 Percentage of deep burn wounds with positive cultures 101 3.30 The proportion of bacterial species in deep burns 101 3.31 Number of bacteria per cm2 of deep burn wound 102 3.32 Changes in hematological parameters in patients with deep burns treated with Cerium nitrate 102 3.33 Blood biochemistry indices in patients with deep burns treated with Cerium nitrate 103 3.34 Histopathological changes of deep burn wounds 104 LIST OF DIAGRAMS Diagram 4.1 Name of Diagram Page Effect of cerium nitrate 127 spectrum of action of several antimicrobial creams against isolated microorganisms in a burn centre Burns, 18(1): 39-44 79 Fraser J F., Bodman J., Sturgess R., et al (2004) An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream, 1% silver sulphadiazine cream and a silver coated dressing Burns, 30(1): 35-41 80 Helvig E I., Munster A M., Su C T., et al (1979) Cerium nitratesilver sulfadiazine cream in the treatment of burns: a prospective, randomized study Am Surg, 45(4): 270-2 81 Monafo W W., Tandon S N., Ayvazian V H., et al (1976) Cerium nitrate: a new topical antiseptic for extensive burns Surgery, 80(4): 465-73 82 Fox C L Jr., Monafo W W Jr., Ayvazian V H., et al (1977) Topical chemotherapy for burns using cerium salts and silver sulfadiazine Surg Gynecol Obstet, 144(5): 668-72 83 Ross D A., Phipps A J., Clarke J A (1993) The use of cerium nitrate-silver sulphadiazine as a topical burns dressing Br J Plast Surg, 46(7): 582-4 84 Bowser B H., Caldwell F T., Cone J B., et al (1981) A prospective analysis of silver sulfadiazine with and without cerium nitrate as a topical agent in the treatment of severely burned children J Trauma, 21(7): 558-63 85 W Boeckx, M Focquet, M Cornelissen, et al (1985) Bacteriological effect of cerium-flamazine cream in major burns Burns Incl Therm Inj, 11(5): 337-42 86 Marone P., Monzillo V., Perversi L., et al (1998) Comparative in vitro activity of silver sulfadiazine, alone and in combination with cerium nitrate, against staphylococci and gram-negative bacteria J Chemother, 10(1): 17-21 87 M Vehmeyer-Heeman, T Tondu, E Van den Kerckhove, et al (2006) Application of cerium nitrate–silver sulphadiazine allows for postponement of excision and grafting Burns, 32: 60–63 88 Nguyễn Ngọc Tuấn, Phan Thị Thục Trang (2019) Đánh giá tác dụng điều trị dung dịch Ceri Nitrat 0,04M vết thương khâu kín vùng cho da khâu kín sau cắt sẹo Tạp chí Y học Thảm họa Bỏng, 4: 59-67 89 Kremer T., Hernekamp F., Riedel K., et al (2009) Topical application of cerium nitrate prevents burn edema after burn plasma transfer Microvasc Res, 78(3): 425-31 90 Hernekamp J F., Harenberg P S., Lehnhardt M et al (2012) Microvascular effects of burn plasma transfer and therapeutic options in a rat model Handchir Mikrochir Plast Chir, 44(4): 209-19 91 Peterson V M., Hansbrough J F., Wang X W., et al (1985) Topical cerium nitrate prevents postburn immunosuppression J Trauma, 25(11): 1039-44 92 Sparkes B G (1993) Mechanisms of immune failure in burn injury Vaccine, 11(5): 504-10 93 Fang Y., Chen Y., Wei D (1996) Effects of wound wet dressing with cerium nitrate on cell-mediated immunity after severe burn Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 12(4): 265-7 94 Qian L W., Evani S J., Chen P., et al (2020) Cerium nitrate treatment provides eschar stabilization through reduction in bioburden, DAMPs, and inflammatory cytokins in a rat scald burn model J Burn Care Res, 41(3): 576-584 95 Sengezer M., Deved M., Eski M (1998) Cerium nitrate bathing prevents TNF-α elevation following burn injury (experimental study) Ann Medit Burns Club, 11(4): 227-231 96 Deveci M., Eski M., Sengezer M., et al (2000) Effects of cerium nitrate bathing and prompt burn wound excision on IL-6 and TNFalpha levels in burned rats Burns, 26(1): 41-5 97 Nguyễn Như Lâm, Trần Đình Hùng (2017) Biến đổi nồng độ số cytokin huyết bệnh nhân bỏng có biến chứng ARDS Tạp chí Y học Thảm họa Bỏng, 2: 12-16 98 Boeckx W., Blondeel P N., Vandersteen K., et al (1992) Effect of cerium nitrate-silver sulphadiazine on deep dermal burns: a histological hypothesis Burns, 18(6): 456-62 99 Eski M., Deveci M., Celiköz B., et al (2001) Treatment with cerium nitrate bathing modulate systemic leukocyte activation following burn injury: an experimental study in rat cremaster muscle flap Burns, 27(7): 739-46 100 Hadjiiski O G., Lesseva M I (1999) Comparison of four drugs for local treatment of burn wounds Eur J Emerg Med, 6(1): 41-7 101 Lansdown A B., Myers S R., Clarke J A., et al (2003) A reappraisal of the role of cerium in burn wound management J Wound Care, 12(3): 113-8 102 Scholten-Jaegers S M., Nieuwenhuis M K., Baar M E V., et al (2017) Epidemiology and outcome of patients with burns treated with cerium nitrate-silversulfadiazine J Burn Care Res, 38(1): e432-e442 103 De Gracia C G (2001) An open study comparing topical silver sulfadiazine and topical silver sulfadiazine-cerium nitrate in the treatment of moderate and severe burns Burns, 27(1): 67-74 104 Cobrado L., Azevedo M M., Silva-Dias A., et al (2012) Cerium, chitosan and hamamelitannin as novel biofilm inhibitors J Antimicrob Chemother, 67(5): 1159-62 105 Teixeira S R Z., dos Reis E., Apati G P., Meier M M., et al (2019) Biosynthesis and functionalization of bacterial cellulose membranes with cerium nitrate and silver nanoparticles Materials Research, 22(1): e20190054 106 Ramenzoni L L., Weber F E., Attin T., et al (2017) Cerium chloride application promotes wound healing and cell proliferation in human foreskin fibroblasts Materials, 10(573): 1-9 107 Nur-Ahmad L., Mohammad-Javad F D C., Ali-Asghar S K., et al (2019) Comparing the effects of silver sulfadiazine and cerium nitrate silver sulfadiazine on burn-wound healing and survival rate of rat animal model J Surg Med, 3(6): 433-436 108 Vitse J., Tchero H., Meaume S., et al (2018) Silver sulfadiazine and cerium nitrate in ischemic skin necrosis of the leg and foot: Results of a prospective randomized controlled study Int J Low Extrem Wounds, 17(3): 151-160 109 Darres A., Delaval R., Fournier A., et al (2019) The effectiveness of topical cerium nitrate-silver sulfadiazine application on overall outcome in patients with calciphylaxis Dermatology, 235(2): 120-129 110 Wassermann D., Schlotterer M., Lebreton F., et al (1989) Use of topically applied silver sulphadiazine plus cerium nitrate in major burns Burns, 15(4): 257-60 111 Kistler D., Hafemann B., Schoenenberger G A., et al (1990) Increased survival rates by topical treatment of burns with cerium nitrate Eur Surg Res, 22(5): 283-90 112 Scheidegger D., Sparkes B G., Luscher N., et al (1992) Survival in major burn injuries treated by one bathing in cerium nitrate Burns, 18(4): 296-300 113 Vehmeyer-Heeman M., Van Holder C., Nieman F., et al (2007) Predictors of mortality: a comparison between two burn wound treatment policies Burns, 33(2): 167-72 114 Kath M A., Shupp J W., Matt S E., et al (2011) Incidence of methemoglobinemia in patients receiving cerium nitrate and silver sulfadiazine for the treatment of burn wounds: a burn center's experience Wound Repair Regen, 19(2): 201-4 115 Ha L Y., Woollard G A., Chiu W W (2015) Falsely raised whole blood chloride caused by systemic absorption of cerium nitrate cream for burns Ann Clin Biochem, 25(2): 288-92 116 Ramesh A., Ratla N N., Indukuri R., et al (2014) Acute and sub-acute oral toxicity assessment of the cerium oxide nanoparticles in wistar rats International Journal of Phytopharmacology, 5(1): 46-50 117 Wu Y., Tang X., Yang W., et al (2019) Subchronic toxicity of cerium nitrate by 90-day oral exposure in wistar rats Regul Toxicol Pharmacol, 108: 104474 118 Boye T., Terrier J P., Coillot C., et al (2006) Cerium-induced granulomatous dermatitis Ann Dermatol Venereol, 133(1): 50-2 119 Nguyễn Ngọc Tuấn cs (2014) Nghiên cứu bào chế dung dịch Ceri nitrat gel Ceri nitrat, bước đầu đánh giá tác dụng chế phẩm vết thương bỏng Báo cáo tổng kết đề tài cấp thành phố Hà Nội 120 OECD guideline for testing chemicals (2001) OECD/OCDE 423; Acute oral toxicity - Acute toxic class method 121 Reagan-Shaw S., Nihal M., Ahmad N (2007) Dose translation from animal to human studies revisited The FASEB Journal 22: 659–661 122 Nguyễn Thượng Dong cs (2006), Phương pháp nghiên cứu tác dụng dược lý thuốc từ dược thảo, Nhà xuất Khoa học Kỹ thuật 123 OECD guideline for testing chemicals 407 (2008) Repeated dose 28day oral toxicity study in rodents: 1-13 124 OECD guideline for testing chemicals (2015) OECD/OCDE 404 Acute dermal irritation/Corrosion 125 Nguyễn Thị Tỵ (1989), Tác dụng điều trị chỗ vết thương bỏng thực nghiệm tinh dầu tràm bước đầu ứng dụng lâm sàng, Luận án phó tiến sĩ khoa học Y Dược, Học viện Quân y, Hà Nội 126 Nguyễn Ngọc Tuấn (1998), Nghiên cứu đặc điểm lâm sàng tác dụng điều trị chỗ vết bỏng vơi tơi nóng thuốc mỡ Maduxin, Luận án Tiến sĩ Y học, Học viện Quân y 127 Bộ môn Vi sinh - Trường Đại học Y Hà Nội (2007), Vi sinh Y học, Nhà xuất Y học, Hà Nội 128 Rosenkranz H S (1979) A synergistic effect between cerium nitrate and silver sulphadiazine Burns, 5: 278-81 129 Schuenck R P., Dadalti P., Silva M G., et al (2004) Oxacillin- and mupirocin-resistant Staphylococcus aureus: in vitro activity of silver sulphadiazine and cerium nitrate in hospital strains J Chemother, 16(5): 453-8 130 Silva-Dias A., Miranda I M., Branco J., et al (2015) In vitro antifungal activity and in vivo antibiofilm activity of cerium nitrate against Candida species J Antimicrob Chemother, 70(4): 1083-93 131 Hirakawa K (1983) Determination of silver and cerium in the liver and the kidney from a severely burned infant treated with silver sulfadiazine and cerium nitrate Radioisotopes, 32: 59-65 132 Oen I M., Baar M E V, Middelkoop E., et al (2012) Effectiveness of cerium nitrate-silver sulfadiazine in the treatment of facial burns: a multicenter, randomized, controlled trial Plast Reconstr Surg, 130(2): 274e-283e 133 de Graaf E., Baar M E V., Baartmans M G A., et al (2017) Partialthickness scalds in children: A comparison of different treatment strategies Burns, 43(4): 733-740 APPENDIX ILLUSTRATING IMAGES Bacterial culture images determine the minimum bactericidal concentration E coli (raw) E.coli after hours E.coli after hours E.coli after 24 hours S aureus (raw) S aureus after hours S aureus after hours S aureus after 24 hours P aeruginosa (raw) P aeruginosa after hours P aeruginosa after hours P aeruginosa after 24 hours PL1 Colonies of E coli, S aureus and P aeruginosa at the time of exposure to cerium nitrate gel Anatomical image of deep burn wound PL2 Morphology of tissue structure of deep burns in area A before drug application Thrombolytic necrosis of the skin surface, in which there are many N cells (blue arrows) Coagulation necrosis reaches the dermis including the hair follicles (red arrows) and sweat glands The fibrous connective tissue is hyalinized, the blood vessels are hemorrhagic, necrotic, and the interstitial tissue has little edematous fluid HE 10X and 40X; (Template number: D1532A - T0) PL3 Morphology of tissue structure of deep burns in area A on the rd day of application The skin is completely necrotic to the dermis layer (black arrow), the hair follicles are also necrotic, some blood vessels are distorted, the erythrocytes contain red blood cells (blue arrows), the fibers are tightly adherent The necrotic surface shows no inflammatory cell infiltration HE 20X and 40X; (Template number: D1532A – T3) PL4 Morphology of tissue structure of deep burns in area A on the 7th day of application The wound is still abundant (black arrow), the necrotic surface has fibrin fibers (green arrow), the sub-necrotic tissue is infiltrated with many inflammatory cells such as N leukocytes, leukocytes (yellow arrow ), interstitial tissue rich with edematous fluid, and fibrin fibers HE 10X and 40X; (Template number: D1532A – T7) PL5 Morphology of tissue structure of deep burns in area B before drug application Superficial fibrin necrosis with few N white blood cells (black arrows), dermis with sweat glands with visible sweat platelets (blue arrows), and infiltrating lymphocytes around the gland The connective tissue in the dermis is hyalinized HE 20X and 40X; (Template number: D1532B - T0) PL6 Morphology of tissue structure of deep burns in area B on the rd day of application Necrosis of the entire epidermis to the dermis (black arrows), the surface is infiltrated with inflammatory cells such as N white blood cells (blue arrows), the connective tissue in the dermis is mildly edematous, fibroblasts are hyaline chemistry (yellow arrow), blood vessels are less likely to become congested HE 20X and 40X; (Template number: D1532B – T3) PL7 Structural morphology of deep burns in area B on the th day of application Total necrosis from the epidermis to the subcutaneous fat layer of the dermis (black arrow), necrotic subcutaneous tissue with many inflammatory cells, which are infected N-leukocytes, white blood cells (red arrows), blood vessels necrosis (green arrow), interstitial fluid edema, fibrin fibers HE 20X and 40X; (Template number: D1532B – T7) APPENDIX RESEARCH PROFILE APPENDIX LIST OF RESEARCH PATIENTS APPENDIX MINUTES OF THE ETHICS COUNCIL IN BIOLOGY RESEARCH APPENDIX STANDARDS OF US PRODUCTS FACILITIES ... IRRITATION OF CERIUM NITRATE 105 4.1.1 Acute toxicity of cerium nitrate gel 105 4.1.2 Semi-permanent toxicity of the cerium nitrate gel 106 4.1.3 Skin irritation of cerium nitrate gel ... effects of cerium nitrate 118 4.3.2 The drying effect for necrosis of cerium nitrate 122 4.3.3 Immunological effects of cerium nitrate gel 125 4.3.4 Safety of cerium nitrate gel ... concentration of cerium nitrate gel 113 4.2.4 Antibacterial effect of cerium nitrate gel on experimental and clinical burn wounds 115 4.3 TREATMENT EFFECTS OF CERIUM NITRATE GEL ON