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

VIETNAM MILITARY MEDICAL UNIVERSITY NGUYEN THANH CHUNG STUDY ON TOPICAL TREATMENT EFFECT OF CERIUM NITRATE 2.2% GEL ON THERMAL BURN WOUNDS PhD... VIETNAM MILITARY MEDICAL UNIVERSITY

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

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

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

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 1

CHAPTER 1: OVERVIEW 3

1.1 CLASSIFICATIONS OF BURN DEPTH 3

1.1.1 Superficial burns 3

1.1.2 Deep burns 4

1.2 PROCESS OF BURN WOUND HEALING 5

1.2.1 The hemostasis 6

1.2.2 Acute phase 6

1.2.3 The proliferative phase 7

1.2.4 Maturation phase, scar formation 9

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

3 ECM Extracellular Matrix

5 FGF Fibroblast growth factor

6 EGF Epidermal growth factor

9 LPC Lipid protein complex

10 MBC Minimum Bactericidal Concentration

11 MIC Minimum Inhibitor Concentration

12 MMP Matrix Metalloproteinase

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

21 TGF β Transforming growth foctor-β

22 VEGF Vascular endothelial growth factor

LIST OF TABLES

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 1 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 Name of Diagram Page 4.1 Effect of cerium nitrate 127

LIST OF FIGURES

1.1 Pathophysiology of wound healing 10

1.2 The wound covered by membrance created by Flammacerium containing calcium 30

1.3 Two ways causing dermal Calcification in wounds treated with Cerium nitrate 31

2.1 Trial drug and placebo drug 39

2.2 Automated hematology testing machine 40

2.3 Semi-automatic biochemistry analyzer 40

2.4 Creating experimental burn instrument 52

2.5 Biopsy device (Biopsy Punch) used in research 55

2.6 Olympus CX41 optical microscope with photo shooting system 56

2.7 Research designed diagram 63

3.1 Morphology of liver structure, kidney and spleen of rat after 14 days drinking Cerium nitrate gel HE; 100X 66

3.2 Rat liver, kidney and spleen structure after 4 weeks of drinking Cerium nitrate gel He, (400x) 69

3.3 Rabbit skin at the times of exposure to Cerium nitrate gel 71

3.4 The chat of sterile ring diameter of drugs with gram negative bacteria 72

3.5 Chart of sterile ring diameter of Cerium nitrate gel with bacteria 73

3.6 Image of injury after post burns 76

3.7 Image of injury after 7 days of treatment 78

3.8 Image of injury after 14 days of treatment 78

3.9 Image of injury after 21 days of treatment 79

3.10 Image of injury after the treatment was cured 79

3.11 Fire deep dermal burn injury on the 3rd day after burn 91

3.12 Deep dermal burn injury in the first week of study 91

3.13 Deep dermal burn injury in zone A, 7th day of study 92

3.14 Deep dermal burn injury on 16th day 92

3.15 Deep dermal burn injury on 18th day 92

3.16 Applying the drug to treat deep burns on the right leg on the first day of the study 99

3.17 Characteristics of deep burn injury on the 6th day of study 99

3.18 Necrotomy and skin grafting for deep burn lesions 100

3.19 Results of skin grafting for deep burns 100 4.1 Nuclear medicine images of mice on the 3rd and 7th day after the burn 124

PL1 Colonies of E coli, S aureus and P aeruginosa at the time of exposure

to cerium nitrate gel

PL2 Morphology of tissue structure of deep burns in area A before drug

application

PL3 Morphology of tissue structure of deep burns in area A on the 3rd day

of application

PL4 Morphology of tissue structure of deep burns in area A on the 7th day

of application

PL5 Morphology of tissue structure of deep burns in area B before drug

application

PL6 Morphology of tissue structure of deep burns in area B on the 3rd day of

application

PL7 Structural morphology of deep burns in area B on the 7th day of application

INTRODUCTION

Burn is an acute injury to the body caused by heat, chemicals, electricity, and radiation Burn injuries disrupt body functions and systemic responses to protect and heal The process from the time of burn, may arise systemic dysfunction and changes at the burn site manifested by pathological syndromes appearing with regularity called “burn disease” [1] Burns are the leading cause of injury, with more than 300,000 deaths globally each year The rate of burns in developing countries is significantly higher than in developed countries The average number of victims per year in Russia is 170.000, and in the UK it is 140,000 [2], [3]

Burn infection is a common complication, adversely affecting the healing process, prolonging the treatment time, if the infection is severe, it also affects the whole body Wet necrotic burns are severe due to the necrosis and absorption into the body, causing a state of toxicity, wet necrosis is also a nutritional environment for bacteria to grow, causing local and systemic infections, and slowing down the healing process, and are prone to secondary necrosis [4], [5]

Despite advances in resuscitation, nutrition, and surgery, mortality in severe burns remains high, especially in developing countries The main cause

is an infection, and multi-organ failure, which are mainly causes from burn injuries The basic principle of treatment for extensive burns is surgical, intervention and to cover with autologous skin However, due to the lack of autologous skin and temporary skin substitutes as well as difficulties in resuscitation during and after surgery in patients with extensive burns, inhalation injury, the treatment of extensive burns is still difficult The reasonable solution is partial surgery, each stage along with minimizing the bad progress in the remaining deep burns [3]

Cerium nitrate is an effective antibacterial product for local treatment

of burn wounds, be cause it neutralizes the effect of burn toxins, dries up wet necrosis, and contributing to reduce burn mortality [6], [7] Experimental research by Eski M., et al (2012) showed that cerium nitrate was effective in preventing the progression to necrosis in the stasis area of burn injury [6] According to Jakupec M A., et al (2005), cerium nitrate in combination with silver sulfadiazine has been used for local treatment of extensive burns that cannot be removed early Beside direct antiseptic effect, cerium also helped to prevent post-burn sepsis and systemic inflammatory response by immobilizing burn toxins [7] Cerium nitrate also had other advantages of high safety and poor absorption into the body

Currently, in developed countries, the use of cerium nitrate preparations

is increasingly widespread, thus increasing the possibility of saving the lives

of patients with extensive burns However, the cost of importing cerium nitrate products is quite high compared to the income of Vietnamese people

In order to meet the urgent need in treating burn wounds, Le Huu Trac National Burn Hospital has researched and prepared a 2.2% cerium nitrate gel product that met basic standards To be able to apply cerium nitrate gel in the treatment of burn wounds in clinical practice, it is necessary to study and evaluate the toxicity and effect of the gel on burn wounds Therefore, we conducted a study

on the topic: "Study on the local therapeutic effects of cerium nitrate 2.2% gel

on thermal burn wounds" with the following two objectives:

1 To evaluate the toxicity in experimental animals and antibacterial activity of cerium nitrate gel.

2 To evaluate the local therapeutic effects of cerium nitrate gel on experimental burns and initially clinical evaluation.

CHAPTER 1 OVERVIEW

1.1 CLASSIFICATIONS OF BURN DEPTH

There are many ways to classify the degree of burn injury based on clinical symptoms, anatomical damage, and reconstruction process Basically, burn injuries can be divided into two main groups: superficial burns and deep burns [1], [3]

1.1.1 Superficial burns

Superficial burns (Partial thickness burn) include acute post-burn dermatitis, epidermal burns, and dermal burns Symptoms on the skin are blisters Burns can heal spontaneously by epithelialization from germ layer epithelial cells, epithelial cells of sebaceous glands, hair follicles, and sweat glands

- First-degree burns (acute post-burn dermatitis): Injury to the superficial layer of the epidermis (the stratum corneum) Clinical manifestations are erythema, swelling, burning pain After 2 - 3 days, the lesion peels off the superficial layer of the epidermis, leaving no skin pigmentation disorder

- Second-degree burns (Epidermal burns): Injury to the epidermis, but the germ cell layer and basement membrane are mostly intact Clinically, they are thin dome blisters, containing clear or pale yellow fluid After 3 - 4 days, the burning fluid is partially absorbed, and the volatile part forms albumin, which solidifies in the burn node It heals in 1 - 2 weeks, leaving no scars

- Third degree burns (dermal burn): Burn injury to the entire epidermis

to a part of the dermis, but the skin appendages deep in the dermis are mostly intact Burns of the dermis are divided into two groups:

Superficial dermal burn: Necrosis of the entire epidermis, damage to the dermis papilla, but the skin appendages (hair roots, sweat glands .) remain intact

Deep dermal burns: Damage to the deep dermis, leaving only the posterior part of the sweat glands

Clinical: Third-degree burns manifest mainly as blisters or necrotic skin patches The dermis burn is a thick dome, and the fluid is cloudy, red, and blood red The pain is still there but reduced Burns of the dermis can heal after 15-30- 45 days, leaving soft scars, pale in color compared to neighboring healthy skin, with small holes and holes in close inspection [3], [8]

The course of dermal burns is often complicated The ability to heal burns

is lost if the epithelial islands of the residual skin appendages become necrotic secondary to purulent inflammation, due to circulatory disturbances (pressure)

is not seperated, so infection is easy to develop and spread Wet necrosis is a favorable environment for bacteria to grow, especially when purulent inflammation occurs (corresponding to the systemic response is often severe) The process of dry necrosis: does not disintegrate, but dries out and then falls off the whole mass, forming granulation tissue Dry necrosis can turn into wet necrosis and vice versa Deciduous necrosis will form granulomatous tissue When necrosis falls off, fever can be reduced by 1 - 30 [3], [8]

Burn necrosis is a favorable environment for the growth of bacteria, and is also a source of toxicity for the body Tissue damage also causes a systemic inflammatory response syndrome with excessive releasing inflammatory mediators, cytokines, enzymes, and metabolic products such as serotonin, free oxygen radicals, and metabolic products of arachidonic acid (prostaglandin, leukotriene), kinins (Prekallikrein, bradykinin, neurokinin A, B), tumor necrosis factor α (TNF α) [8], [9].

The lipid-protein complex (LPC) is formed from burned skin, with a high molecular weight (3,000,000 Da), which is generated by thermal polymerization of 6 polypeptides of the skin (each of these polypeptides is not toxic poison) is also known as burn toxin LPC is 1,000 times more potent than endotoxin LPC inhibits cell growth and division, increases cell membrane permeability, causes cell damage, causes hemolysis LPC causes systemic inflammatory response leading to multi-organ dysfunction, multiple failure organs, and immunodeficiency LPC is absorbed into the blood in the first hours after the burn, increasing gradually in the following days LPC is primarily responsible for the death of patients with severe burns Therefore, when there is burn necrosis, one of the active treatment measures is to actively remove the necrotic tissue as soon as possible, taking advantage of covering the burn wound This is a proactive measure to eliminate the cause

of local and systemic disorders

1.2 PROCESS OF BURN WOUND HEALING

Depending on the area, the depth of the burn, the resistance of the body, burns basically evolve in 4 stages: hemostasis, acute, reconstruction and scar formation These four phases are intertwined and influence each other [8], [9]

1.2.1 The hemostasis

The hemostatic phase, which occurs immediately after burn, involves vasoconstriction, platelet activation and aggregation, and the release of coagulation and growth factors (such as Platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor β (TGFβ)) by platelets, keratinocytes, and macrophages cells and fibroblasts, leading to fibrin clot deposition at the site of injury, which serves as a temporary substrate for the subsequent phases of wound healing [8]

1.2.2 Acute phase

Acute phase has manifestations of acute inflammation, exudation, purulent inflammation, necrotic shedding, and burn wound cleaning This phase begins with a circulatory response, expressed in microvascular reactions: congestion, vasodilation, increased permeability leading to drainage

of inflammatory exudates and edema In the burn area, there is an inflammatory cell response: neutrophils, followed by macrophages, and later, lymphocytes Inflammatory cells are responsible for removing necrosis, killing bacteria, initiating and regulating wound healing This stage, depending on the area and depth of the burn injury, can last 3 - 7 days or overlap with stage 3 The inflammatory response occurs within the first 72 hours after the injury, in which the vascular response occurs within the first minutes of the inflammatory phase This is followed by the migration of inflammatory cells (after 2 - 4 hours) and fibroblasts (after 2 hours) [8], [9]

The inflammatory response is the coordinating activation of signaling pathways that regulate levels of inflammatory mediators in local tissue cells and blood mobilized inflammatory cells The inflammatory response process can be summarized as follows: 1) modeled cell surface receptors that recognize harmful stimuli; 2) inflammatory pathways are activated; 3)

inflammatory markers are released, and 4), inflammatory cells are mobilized [8], [9]

1.2.3 The proliferative phase

The proliferative phase includes the proliferation of cells, including epithelialization, connective tissue formation, and wound contraction

- Epidermal burns heal themselves by the process of epidermal regeneration, which originates from germ cells The regeneration of dermis burns originates from the epithelial cells remaining in the skin appendages, which combine with epithelialization from the margin to cover the burn

- With deep burns of the entire skin, regeneration after necrosis falls, the primary processes are granulation tissue formation followed by epithelialization from the wound margin or covering the granulation tissue with skin grafts Granulation usually begins on days 3 - 4, and is completed

by day 21 post-burn Granulomatous tissue is composed of neovascularization, new cells, and matrix During wound healing, fibroblasts are activated, proliferate and resynthesize fibronectins, followed by extracellular proteins including collagen, elastin, and glycosaminoglycans Granulomatous tissue is a neoplastic connective tissue, which is the basis for epithelialization When epithelialization covers the granulation tissue, the regeneration process is terminated

The proliferative phase is the stage of the formation of new tissues Besides initiating the inflammatory response through interaction with leukocytes, microvascular endothelial cells play a key role in the proliferative repair phase The formation of new capillaries from pre-existing ones (angiogenesis) is necessary to restore oxygen and provide necessary nutrients

to the newly formed granulation tissue in the wound Vascular stimulants including growth factors, most notably Vascular endothelial growth factor (VEGF), Fibroblast growth factor - 2 (FGF-2), PDGF factor and components

of factor TGFβ, chemokines and angiogenic enzymes (notably serine proteinase thrombin), endothelial-specific receptors and adhesion molecules, such as integrins, many of which are released during the inflammatory phase

of repair [10], [11]

The construction of a vascular network requires sequential steps that include increased microvascular permeability, the release of proteinases from activated endothelial cells with local degradation of the basal membrane surrounding existing blood vessels, migration and budding of endothelial cells into the interstitium, endothelial cell proliferation, and differentiation into mature blood vessels, followed by degeneration and shrinkage of new vessels upon tissue regeneration [ 10], [11], [12]

The formation of a lumen may include fusion of the plasma membrane

of individual cells and/or neighboring cells, as well as the formation of large intracellular vacuoles followed by the fusion of airspace cells to form ring cells, which eventually fuse to form seamless capillaries The capillaries then become stabilized as endothelial cells interact with the new basement membrane within 24 hours of new angiogenesis and through the mobilization

of extravascular and smooth muscle cells During wound healing, this angiogenic response results in a vascular density that exceeds that of the capillaries in normal, undamaged tissue and gives the granulation tissue a red granular appearance Angiogenesis stimuli are downregulated and/or local concentrations of anti-angiogenic factors (thrombospondin, INF-α, protein 10/CXC motif chemokine 10, and Sprouty 2) are increased and most vascular networks are increased Recently formed cells rapidly shrink through matrix-degrading metalloproteinase (MMP) activity, specifically MMP-1 and MMP, and selective cell death of endothelial cells The color of the wound fades as the rich capillary layer disappears from the granulation tissue [12], [13], [14]

Shortly after penetrating the wound, fibroblasts synthesize and secrete collagen and mucopolysaccharide (MPS) into the matrix, which plays an important role in wound healing To close the defect in the epidermis, the keratinocytes at the wound edge must first loosen their adhesions to each other and to the basement membrane, as well as develop the flexibility needed

to move across the matrix Many regulatory organs play important roles in the regulation of keratinocyte proliferation and migration during epithelialization; they include chemokines, cytokines, integrins, keratin, ECM (Extracellular Matrix), and MMP molecules [15], [16], [17], [18]

1.2.4 Maturation phase, scar formation

The maturation phase is the longest phase of the burn wound healing process, lasting 12 - 24 months or more During the scar reconstruction phase, there is a reduction of collagen, gradual transformation of fibrous tissue into a fatty layer, and fibrous tissues are rearranged and oriented Collagen remodeling includes collagen accumulation by fibroblasts and collagen breakdown by collagen enzymes Collagenase plays a decisive role in maintaining the normal physiological ratio between collagen types in scar tissue [17], [18], [19]

In the scar formation phase, at first the scar volume is large, slightly firm, thick, and the scar surface is higher than the skin surface and adheres to the adjacent tissue, and is less mobile In the 2nd month, the scar surface gradually returns to the surrounding skin surface, by the 3rd - 4th month, the scar is mobile on the subcutaneous layer, if it goes well, the scar color is close

to the normal skin color, with smooth and soft scar surface In the callus, the interstitial fluid and MPS decrease, and the collagen fibers regenerate and rebuild stably (Figure 1.1) [8], [16], [17], [18]

Figure 1.1 Pathophysiology of wound healing

* Source: Jeschke M.G., et al (2020) [8]

The process of burn injury involves a series of physiological and biochemical processes occurring in the body for the purpose of repairing and regenerating new tissues to heal the wound For superficial burns, second-degree burns are self-healing by the process of regenerating the epidermis, which originates from the division of keratinocytes in the basal cell layer and differentiation into the spinous, granular, and transparent layers., stratum corneum For third-degree burns with complete damage to the epidermis and part of the dermis, burn regeneration depends on epithelialization from epithelial islets of skin appendages (hair ducts, sebaceous glands, glands, etc.) perspiration) and from the epidermal portion of the margin of healthy skin surrounding the burn For deep burns (grade IV, degree V), the burn wound healing process is much more complicated After the burn necrosis has fallen off or has been excised, granulation tissue is formed and epithelialization

from the epidermis of the healthy skin margins spreads to cover the granulation tissue (if the burn diameter is less than 5 cm) or close the granulation tissue with skin grafts The biology of deep burn wound healing includes many overlapping phenomena, with the latter following the former, most notably inflammation, proliferation, and scar remodeling

Thus, the biological process of burn wound healing has coordination between factors, and only one stitch in this process malfunctions or works poorly, which will slow down the burn wound healing process or form an overgrown scar Many authors have noted that because burn damage causes very strong destruction of the dermal matrix, and the arrangement of collagen fibers is not orderly, and proteases have little effect in the area of coagulation necrosis due to burns Therefore, burn scars are usually hypertrophic scars or keloid scars [17], [18], [19]

1.3 THE BURN WOUND INFECTION AND TOPICAL DRUGS FOR TREATMENT OF THE BURN WOUND INFECTION

1.3.1 Burn wound infection

1.3.1.1 Pathophysiology of burn wound infection

Skin is a tough, flexible tissue membrane that covers the entire human body The skin has the function of protecting the body against harmful influences of the external environment and against the invasion of bacteria thanks to the stratum corneum layer forming a biological barrier, insulating and retaining water for the body When a burn injury occurs, the skin layer is damaged, and the protective barrier of the body is not intact, making wound conditions for the bacteria resident on the patient's skin, and the environment, especially the hospital environment, easily penetrates into subcontinuous organs and systemic organs through the burns wound [3], [20], [21], [22]

- Burn necrosis is a favorable environment for the growth of bacteria

- The burn injury area is poorly nourished because the microvessels are blocked by thrombosis, and the edematous fluid is stagnant, obstructing circulation Inflammatory mediators have a detrimental effect on the adjacent healthy tissue, facilitating the penetration of bacteria from the surface of the burn through the hair follicles, and the sweat glands into the healthy tissue surrounding the burn [3], [20]

- In the burned area, the bactericidal process of leukocytes is affected due to low oxygen saturation, decreased local circulation, and cellular immune function (macrophages, IL-1, IL-6 ) is also hindered [21], [22]

- Systemic disorders (especially deep and extensive burns) cause plasma loss leading to a decrease in the body's resistance

Infection is a major complication and is responsible for 50% - 60% of deaths in burn patients [3] Therefore, the local burn wound and the systemic status are closely related to the bacterial infection at the burn injury site

Survived Gram-positive bacteria after thermal injury, is deeply located

in sweat glands and hair follicles and strongly invades the wound surface in the first 48 hours unless topical antibacterial agents are used After 5 - 7 days, these wounds are then infected with other bacteria, including gram-positive bacteria, gram-negative bacteria, and yeasts derived from the animal's gastrointestinal and upper respiratory tract microbiota of the host and/or from the hospital environment or from the hands of healthcare workers Over the past several decades, gram-negative bacteria have emerged as the most common etiology of invasive infections owing to their virulence factors and antibiotic resistance characteristics If the host's defenses and therapeutic measures (removal of necrotic tissue and wound closure) are inadequate or delayed, the bacteria will invade the subcontinuous tissue [23], [24], [25], [26], [27], [28]

The formation of biofilm from the bacteria in the wound environment reduces the effectiveness of systemic or local antibiotics against the bacteria [29], [30], [31], [32]

In burn wounds, biofilm can grow within 48 - 72 hours Factors that delay biofilm formation may be related to the microorganism's need for additional nutrients, consist of the immune system and wound cleaning The bacteria in biofilms often undergo a phenotypic change whereby virulence factor production by the organism is altered and metabolic and motility rates are reduced [33], [34], [35] ], [36]

When the biofilm is formed, it prevents the antibiotic from coming into contact with bacteria, especially in the center of the wound substrate Biofilm has the ability to protect bacteria against antibodies, antibiotics, disinfectants, and inflammatory macrophages Wounds with biofilm can be effectively treated with a combination of debridement, wound washing, and dressing change to remove biofilm from the wound, prevent new bacteria from entering, and destroy remaining bacteria remains at the wound bed [27]

1.3.1.2 The causes

The leading cause of infection in burn wounds is Staphylococcus

aureus (S aureus), some recent studies show that the leading causes of death

from bacterial infections today are drug-resistant bacteria, including

Pseudomonas and Acinetobacter [37], [38], [39], [40], [41], [42]

* Bacteria:

- S aureus is still the main cause of burn wound infection Staphylococcus

produces toxic products such as proteinase, collagenase and hyaluronidase that invade tissues and spread hematogenously, causing local and systemic

infections Methicillin-resistant S aureus is currently one of the leading

organisms causing invasive infections in burns with infection rates greater than 50% [38], [39], [40], [41], [42]

- Enterococcus: The importance of Enterococci has been brought to the

fore with the emergence of vancomycin-resistant Enterococcus A

comparison of the causes of death from sepsis between the decades

(1989-1999) and (1999-2009) shows that the rate of Enterococci infection had

decreased sharply (25% to 2%), but the mortality rate due to

vancomycin-resistant Enterococcus was higher than that of methicillin-vancomycin-resistant S aureus (58% vs 33%) [37], [40], [43], [44]

Shokoohizadeh L., et al (2018) analyzed 340 bacterial samples isolated

from burn wounds and found that the rate of Enterococci was 16.4% (n=56)

In which, Enterococcus faecalis was in 35/56 samples (62.5%) and

Enterococcus faecium was in 21/56 samples (37.5%) More than 20% (n=5)

of E faecium samples were resistant to vancomycin The authors suggested that the emergence of strains of E faecium resistant to the study vancomycin

was a risk factor for burn centers [44]

- Pseudomonas is the most common pathogen in burn wounds Wound infections caused by P aeruginosa often have characteristics of yellow/green

color and a rotten fruit odor [45], [46], [47], [48], [49], [50]

- Acinetobacter: the infection rate of burn wounds is second only to P

aeruginosa, this bacterium is capable of infecting patients because of its

ability to survive in both dry and wet conditions as well as on both animate and inanimate objects, whether metal or plastic [47], [50], [51], [52], [53] Bayram Y., et al (2013) isolated 250 strains of bacteria from the burn wound

of 179 patients and found that the highest proportion was Acinetobacter

baumannii (Aci baumannii) (23.6%), followed by P aeruginosa (12%), S aureus (11.2%), E coli (10%) [45]

- Enterobacteriaceae (E coli, Klebsiella, Enterobacter, Serratia, Proteus): associated with bacterial infections in burn patients, often causing

pneumonia and urinary tract infections

- Anaerobes: Anaerobic bacteria are now rarely a cause of invasive

infection in burns The most common isolated anaerobic organisms were

Bacteroides and Fusobacterium spp [22], [43]

Branski L.K., et al (2009) studied 398 patients with severe burns (>40% of body surface area) and found that Gram-negative bacteria were the most common cause of infection in burn patients Colistin was a highly

effective antibiotic against multi-resistant Pseudomonas and Acinetobacter The main fungal pathogens in burn patients were Candida spp., Aspergillus

spp., and Fusarium spp [23]

Wang L F., et al (2014) studied on 1.914 bacteria samples isolated from burn patients and found 1.355 Gram-negative and 559 Gram-positive

species The top eight bacteria strains were Aci baumannii, P aeruginosa, S

aureus, E coli, S epidermidis, K pneumoniae, Enterobacter cloacae and Enterococcus The prevalence of drug resistance in Aci baumannii, P aeruginosa, S epidermidis and S aureus was 52.2%; 21.7%; 27.8%, and

33.3% respectively of all patient samples [52]

Forson O A., et al (2017) cultured of 50 samples from 50 burn patients and found 43 positive samples (86%) and 7 negative samples (42%) The

authors isolated 9 different strains of bacteria, mainly Pseudomonas sp (30.2%), followed by Acinetobacter (20.9%), Proteus mirabilis (16.3%),

Enterobacter sp (11.6%), Klebsiella sp (7.0%), Citrobacter sp (4.7%), Klebsiella oxytoca (4.7%), Proteus vulgaris (2.3%), and S aureus (2.3%)

Most of the bacteria were antibiotic resistant [5]

* Fungi / yeast:

Fungal colonization has become an increasing problem because of the advent of topical antimicrobials and the uncontrolled use of broad-spectrum antibiotics This resulted in an increased fungal infections, which are

associated with higher mortality regardless of the degree of burn, inhalation injury or patient age [48], [54], [55], [56], [57]

Ballard J., et al (2008) studied on 6.918 patients in 15 burn treatment facilities and found that 435 patients (6.3%) had positive fungal cultures

Fungi included Candida (85%), non-Candida yeasts (21%), Aspergillus

(14%), other molds (9.0%) and other fungi (1.4%) Patients with advanced age, large burn area, and respiratory burns had a higher risk of fungal infections, higher rates of positive fungal cultures, and significantly increased mortality (21.2%) Positive cultures obtained from wounds were most common, followed by respiratory, urine and blood samples [55]

Pham Phuoc Tien et al (2015) studied on 858 patients with severe burns for 3 years (2012 - 2014) and found that 18/166 blood cultures were positive

(10.8%) In 18 cases, Candida Albicans species still predominated (55.6%), but have a change in epidemiology, Non-Candida albicans species was increasing

in prevalence (44.4%) and was gradually decreasing to become the main cause

of blood fungal infections, the mortality rate is 50% [56]

Nguyen Thai Ngoc Minh et al (2019) studied on 30 patients with severe burns diagnosed with invasive fungal infections and found that

Candida still had a high rate of 90%, and the rest was Aspergillus (10%) The

C albicans accounted for only 30%, and the rest was C non albicans

Patients with invasive fungal infections were cultured at different sites The first week was the highest positive rate in waste: urine 75% and feces 82% Especially there was a 33% positive rate in biopsied tissue samples and 20%

in blood samples At the second and third-week, positive cultures reached a higher rate in patients diagnosed with invasive fungal infections [57]

1.3.2 Drugs to treat burn wound infection

The ideal antibacterial drugs should meet the following criteria: No or little pain for the patient when used; Effective with bacteria that cause burn

wound infection; The rate of drug resistance is low; No local and systemic allergies, few side effects, no harm to healthy tissue; Capable of penetrating deep into necrosis; Easy to use, easy to store, a rich source of medicinal herbs, low cost Some drugs to treat local infections of burn wounds today are [1], [3], [58], [59], [60], [61], [62]:

* Silver sulfadiazine 1% (Silvaden, silvirin, silver sulfadiazine):

Silver sulfadiazine (SSD) is a silver salt formed by reacting silver nitrate with sodium sulfadiazine dissolved in a water-soluble cream base, having a concentration of 1% The drug has a molecular weight of 375,14 g/mol; and water solubility of 1/105 After applying SSD to the burn wound, the drug acts as a storehouse of released silver ions attached to tissue proteins, and free silver ions are maintained at concentrations toxic to bacteria The silver concentration is equivalent to the amount of silver present in a 0.5% silver nitrate solution (30 mFq/l) SSD is a competitive inhibitor with folic acid, causing damage to bacteria cell membranes The anti- bacteria effect of the drug is due to many mechanisms: SSD acts on the cell membrane to damage the membrane, and silver accumulates in the DNA molecule, and weakens the replication of bacteria [1], [59], [61]

SSD is a topical antibacterial agent that has been widely used for the prophylaxis and treatment of bacterial burn infections for more than 50 years The drug has a broad antibacterial spectrum, strong effect on many gram-positive bacteria, prevents and slows the infection of burns by gram-negative

bacteria, works against the Candida albicans, penetrates into the burn necrotic

tissues does not cause pain, but reduces white blood cells in the blood [58], [60], [62]

However, the drug still has some disadvantages: The drug softens the necrosis, and reduces the destruction and shedding of necrosis The ability of

the drug to penetrate into the necrosis is poor, so the drug has poor control of infection in deep burns, with bacteria developing in the necrotic sublayer The drug is less effective in burns with more than 60% of the body surface area It should not be used in pregnant and lactating women (causes neonatal jaundice), hepato-nephrotoxicity (breastfeeding infants) [1], [58], [59]

SSDs can cause side effects such as reducing the epithelialization of the burn, prolonging the wound healing process, and even causing a change in burn depth in the diffuser If applied on a large scale, silver penetrates into the blood, toxic to the liver, kidneys and can cause allergies The use of SSDs in combination with systemic antibiotics (aminoglycosides) can lead to the

emergence of resistant strains of bacteria such as Enterobacter, Klebsiella, and Pseudomonas, which grow rapidly in burns [1], [58], [62]

* Silver nitrate 0.25-0.5%:

The drug has a broad antibacterial spectrum, especially with P

aeruginosa, it is less allergic, but can cause low Na+, Cl- level in the blood It

is used by gauze soaked on the burn wound, then every 2 hours watering on the gauze, and changing the bandage daily The drug does not cause discomfort and skin sensitization [3], [58]

* Boric acid 2-4% solution, 5-10% ointment or crystal form: has a good

effect on P aeruginosa [3]

* Sulfamilon (mafenid):

The cream drug has a good anti-infective effect (on the metabolism of

bacteria), against P aeruginosa, gram-positive bacteria and Clostridia, but has a limited effect on S aureus and Fungi The drug quickly penetrates into

the burn necrosis, and has little effect on burns with significant pus or serum The drug causes local pain lasting 1-2 hours, and allergies, as well as inhibits carbonic anhydrase leading to metabolic acidosis [1], [3], [58]

* Nitrofurazone:

The drug inhibits the enzymes of bacteria related to metabolism with a

broad spectrum in both gram-positive and negative bacteria, little effect on P

aeruginosa, ineffective on fungi or viruses, and appears resistant to S aureus

and E coli The drug has the ability to penetrate deep into the necrosis, so it is

used to treat deep burn infections caused by bacteria Undesirable effects: Atopic dermatitis, kidney failure [3]

* Chlorhexidine:

The drug acts on bacteria by the mechanism of destroying the structure

of bacteria The drug has a broad antibacterial spectrum, and resistance has

appeared in some strains of Pseudomonas and Proteus The drug is used to

treat moderate and mild burns, combined with SSD to increase the effect of the drug The drug can cause local pain, and acute nephrotoxicity [1], [58], [59], [60]

* Povidone - iodine:

The drug is used as an iodine storehouse, when used, it will continue to release iodine, which has effective antiseptic properties, quickly killing bacteria, viruses, fungi, and some protozoa Two mechanisms of action include free iodine to kill bacteria and iodines bound in the polymer as a reserve source When in contact with skin and mucous membranes, iodine separates polymers from time to time, and free iodine combines with OH-groups that can oxidize amino acids in enzymes and microbial protein structures to inactivate and destroy those enzymes and proteins, consequent destruction of bacterial proteins and DNA Most of the bacteria in the vegetative process are destroyed in less than 1 minute The drug is effective against gram-negative bacteria and some fungi, but the drug dries up the burn necrosis and causes stinging when applied [1], [58], [59], [60]

1.4 CERIUM NITRATE AND APPLICATIONS IN BURN TREATMENT 1.4.1 Cerium and Cerium nitrate overview

Cerium is a lanthanum metal, which has an atomic number of 58, and

an atomic mass of 140,116, is a solid, silver-white color Cerium has properties similar to alkaline earth metals, and often coexist with earth alkaline metals in nature

In the past, cerium was known as a rare earth element The term "rare" derives from the ancient practice that they could only be extracted from rare minerals in the soil But so far, studies of rare earth elements have determined that they are relatively common in the earth's crust Cerium is the most abundant of the rare earth elements, making up about 0.0046% of the earth's crust by weight Rare earth elements are widely distributed in the world, abundant in China, the USA, Russia, Australia, South Africa, Thailand, India

In Vietnam, according to the assessment of geological scientists, the amount of rare earth is about 10 million tons, scattered in ore mines in the Northwest and

in the form of black sand distributed along the coasts of the central provinces Rare earth is a precious resource of our country and its estimated reserve ranks third in the world The biggest difficulty in rare earth mining is that it requires a high-tech process, especially on a large scale [63], [64]

Rare earth elements and their compounds are increasingly widely used

in many fields of science and technology Among them, cerium is the only metal with trivalent and is abundant in nature Cerium comes in many forms,

of which the nitrate salt form is used medicinally with many effects on burn patients in several burn centers in Europe and North America

Cerium nitrate is a nitrate salt of the rare earth element cerium The chemical formula of cerium nitrate is Ce(NO3)3.6H2O Scientific name: Cerium(III) nitrate hexahydrate Molecular weight: 434,23 grams Cerium nitrate is a fine, colorless, odorless powder Cerium nitrate as well as rare

earth nitrate salts are very soluble in water and have moderate solubility in solvents such as ethanol, ketone, ester but insoluble in alkali [65], [66]

A solution of cerium nitrate at a concentration of 100g/L with an aqueous solution at 25°C has a pH of 3.7 Cerium nitrate is soluble in water,

at 25°C, the solubility is 1754 grams/L Cerium nitrate interacts with strong reducing agents, strong acids, and cyanides Cerium nitrate has very low toxicity, and LD50 in rats orally is 4200mg/kg body weight When cerium nitrate was applied to rabbit skin, no significant irritation was observed [67]

The initial application of cerium nitrate in medicine was in the form of hydrophilic cream cerium nitrate The results show that the drug strongly inhibits Gram-negative bacteria, in contrast to SSD being dominant on Gram-positive bacteria [66], [68], [69], [70] Currently, cerium nitrate is widely used

as a combination of cerium nitrate (0.05M) with SSD (0.03M) (brand name as Flammacerium) Cerium nitrate can be used as a solution (0.04 M) to bathe the patient and wash the burn wound daily [71], [72], [73]

1.4.2 Some biological effects of Cerium

As with the lanthanides, the biological role of cerium remains to be studied Ceri is not capable of penetrating the intact cell membrane of mammals but can penetrate into the cytoplasm of injured cells [74]

* Interactions with calcium-dependent systems:

The interaction of the lanthanide group (including cerium) on the calcium system depends on the concentration that stimulates or inhibits

Calcium activates trypsinogen to trypsin, which stabilizes the substrate

In contrast, lanthanide inhibits other calcium-dependent processes, including activation of factor X, phospholipase A2 and the cellular Ca2+-ATPase channel Calcium plays an important role in keratinocyte maturation and wound healing Cerium has the ability to modulate calmodulin/Calcium effects during wound regeneration [75]

* Cell interaction:

The lanthanides are not directly permeable to mammalian cell membranes but interact with calcium-dependent transmembrane transporters, thereby possibly interacting with intracellular factors Lanthanides inhibit cell-coupled secretion as inhibit angiotensin secretion, including aldosterone secretion from the adrenal cortex and trypsin secretion from the pancreas (Evan C H 1983) Cerium (and lanthanum) inhibit the degrading effect of mast cells, inhibit the secretion of histamine from mast cells and basophils, and inhibit the membrane ATPase pump This effect is also seen in cutaneous Langerhans cells (Gruner S 1991, 1992) [76], [77] This is the basis for the use of cerium as a topical treatment for atopic eczema (inhibiting histamine secretion), a disease with dysfunction in the antigen presentation of Langerhans cells and the secretion of histamine from mast cells Other studies show that collagen and G-actin polymerization was influenced by lanthanides

1.4.3 The application of cerium nitrate in burn treatment

1.4.3.1 Antibacterial effect of cerium nitrate

* In vitro studies:

In vitro studies had shown that cerium nitrate had an inhibitory effect

on bacteria It should be noted that because of the effect of cerium on protein coagulation, it affects the results of agar sterility formation when studying antibacterial effects in vitro

According to Burkes S., et al (1947), cerium nitrate had a broad inhibitory effect on bacteria but depended on pH, and had a strong effect in

acidic environments Cerium nitrate inhibited Pseudomonas growth at concentrations of 0.001 and 0.004 M, deactivated Escherichia and Salmonella

at concentrations ~ 0.005 M with S aureus concentrations almost 2 times Antifungal effect on 35 common fungi of cerium nitrate with concentration as

compared to that of bacteria [72]

Sobek J M (1968) found that Cerium entered the cytoplasm of E coli

(in stark contrast to mammalian cells), inhibited cellular respiration, and inhibited glucose and oxygen metabolism Morphologically, the cell wall was intact but was bulging when viewed under the electron microscope The author also noted similar effects on fungi, in which the fungal membrane was modified and broken [73]

Herruzo C R., et al (1992), Fraser J F., et al (2004) both recognized

cerium nitrate as one of the best topical antibacterial agents against S aureus and P aeruginosa [78], [79]

* Synergistic effect with Silver sulfadiazine:

SSD cream which has been used since 1968, and is effective against

many types of bacteria, including Staphylococci, Pseudomonas and fungi

Currently, SSD is still the mainstay of treatment for burn infections However, due to widespread use and the disappearance of drug assistance after a long time, SSD has little or no effect in treating infections in patients with burns over 60% of the BSA To overcome this situation, researchers have combined SSD with cerium nitrate (for example, a preparation called Flammacerium) [80]

Many studies have demonstrated that cerium nitrate lead to increased antibacterial activity of SSD drugs

Monafo W W., et al (1976) used cream and cerium nitrate solution for topical treatment of burn wounds for 60 patients found that cerium nitrate had

a strong disinfecting effect on burn wounds, especially gram-negative bacteria and fungi [81]

Fox C L Jr., et al (1977) and Ross D A., et al (1993) reported that SSD-cerium nitrate reduced mortality by more than 50% in patients with severe burns due to a significant reduction in infection [82], [83]

Bowser B H., et al (1981) evaluated the effect of SSD when combined with cerium nitrate on 31 pediatric patients with severe burns and recorded positive results [84]

Boeckx W (1985) noted that treatment with cerium flammazine (specifically containing Cerium nitrate + SSD) in patients with severe burns contributed to a reduction in mortality, a reduction in the risk of infection,

especially a reduction in the incidence of P aeruginosa and S aureus infections [85]

Marone P., et al (1998) noted the synergistic effect of cerium nitrate with SSD with bacteria in vitro and clinical It should be noted that because of the effect of ceri on protein binding, it may affect the results of agar ring formation when studying antibacterial effects in vitro [86]

Vehmeyer - Heeman M (2006) noted that the synergistic effect of cerium nitrate with SSD increased the success rate of necrotectomy and taken grafted skin, reduced the risk of infection [87]

1.4.3.2 Anti-inflammatory effects of cerium nitrate

The anti-inflammatory effects of cerium nitrate were recognized by many authors: it inhibits the degrading effect of mast cells, inhibits the secretion of histamine from mast cells and basophils, and inhibits the ATPase pump of the cell membrane In addition, cerium nitrate also has anti-inflammatory effects, reducing albumin leakage from the vascular and anti-edema [76], [77], [88], [89], [90]

1.4.3.3 Immunological effects of Cerium nitrate

Burns reduce and suppress the immune system, thus increasing the risk

of infection; they also affect the immune system of the skin, causing a variety

of pathophysiological disorders after injury Immune dysfunction in burn patients is closely associated with mortality, increasing the risk of death from multiple organ failure An experimental study on injecting allogeneic skin

into the abdomen of healthy mice caused 80% mortality after 10 days, while injection of healthy skin did not cause this phenomenon Clinical studies have shown that early necrolysis reduced mortality Thus, burn necrosis is mainly responsible for immunodeficiency [65]

* Cell-mediated changes in immunity:

LPC plays an important role in causing immunodeficiency in burn patients, and disrupting T-lymphocyte types Post-burn cell-mediated immunity has been implicated in an increase in the rate of infection [91], [92], [93]

Cerium nitrate significantly increased the T-cell helper/suppressor ratio, which contributed to a reduction in mortality Peterson V M., et al (1985) found that after 14 days, the greatest burn immunodeficiency, cerium nitrate treatment restored 90% of CMI, to 99% in the Cerium nitrate-SSD group (CMI: sensitive marker of cell-mediated immunity, reflecting the integrity of the T-cell mechanism) This effect was observed when cerium nitrate was administered early after the burn (preferably within the first 2 hours) [91]

Sparkes B G (1993) evaluated the response of lymphocytes taken from burn patients within a few weeks after the burn (burn area >30%) and found that cerium nitrate had the effect of immobilizing LPC in the burn wound and preventing the infiltration of it enters the circulatory system A study on 10 burn patients who were bathed in cerium nitrate found that T-lymphocyte activity was normal but not inhibited [92]

Research by Qian L W., et al (2020) in experimental burn animals treated with cerium nitrate found that at day 1 and 7 post-burn, concentrations

of substances associated with skin damage formed from burn injury (DAMPs: damage associated molecular) patterns) in the blood were significantly reduced (p<0.05) Burn injury also increased levels of hyaluronan, an extracellular matrix component at day 7 post-burn Treatment with cerium nitrate also significantly reduced hyaluronan levels (p<0.05) [94]

* Effects on cytokine:

Physiological changes in burn wounds are not only characterized by heat but are also closely related to the acute inflammatory process After the thermal burn, a rapid cytokine response occurs (cytokine cascade) Excess of certain cytokines and activation of leukocytes and endothelial cells cause overproduction of inflammatory activators A variety of events can lead to systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (Acute Respiratory Distress Syndrome - ARDS), multiple organ dysfunction syndromes (MODS), multiple organ failure (MOF), even leading

to death Burn necrosis under the influence of cytokines interacts with the body to affect a variety of circulatory and metabolic changes [95], [96], [97] Cytokines are intercellular signaling proteins or peptides that regulate inflammatory responses after injury and are important regulators of infection resistance in heat-burn patients The most important cytokines are IL-2, IL-6, Granulocyte Colony Stimulating Factor (G-CSF), IL-8, and TNF-α in burn patients TNF-α is secreted by activated macrophages, and TNF-α also regulates the production of several other cytokines It also enhances endothelial adhesion to leukocytes, stimulates neutrophils and monocytes to adhere to phagocytosis, oxidation, and degradation TNF-α may be considered the most important cytokine involved in the systemic inflammatory response and multi-organ failure following severe trauma Both TNF-α and IL-6 are thought to be important prognostic indicators after thermal burn IL-2 as a growth factor for all T lymphocyte populations, activates natural killer cells, produces B-Cell antibodies, and increases cytokines such as IL-1, TNF-α, TNF-β, and Interferon-γ It plays a central role in the regulation of the immune response and acts as a marker of T-cell-mediated Immunity significantly increased after thermal burns [95], [96], [97]

Bathing patients after burns with cerium nitrate has been used clinically Bathing with cerium nitrate solution has the effect of activating

Lymphocyte function, and maintaining a higher level of IL-2 Receptor activity than traditional treatment It also maintains the in vitro cellular IL-2 inducing effect, which is similar in the modulation of proinflammatory cytokine levels such as IL-6 and TNF-α in mice [95], [96]

Sengezer M., et al (1998) investigated the effect of cerium nitrate bath

on serum TNF-α levels in burned rats and found that at day 3 post-burn, the control group's TNF-α levels were significantly different compared with the experimental group The concentration of TNF-α in the control group was 24.6, and in the experimental group was 19.3 (p<0,001) On day 7 after the burn, the TNF-α concentration in the control group was 25.4 and in the cerium nitrate group was 14.6 (p<0,001) The authors suggested that treatment with cerium nitrate prevented the increase in TNF-α levels in the early stages of burns This

is done by preventing toxic substances derived from the burn wound from entering the bloodstream and thereby improving survival [95]

The experimental studies of Eski M., et al (2012) showed that using cerium nitrate solution to wash the wound after burns can prevents the development of necrosis of the zone of stasis, contributes to injury recovery, shortens the time of epithelial regeneration The mechanism of cerium nitrate is to reduce leukocyte activation, reduce pro-inflammatory cytokines, and reduce macromolecular escape, thereby reducing edema Cerium nitrate binds to LPC, reduced phagocytic activity, and reduced cytokine secretion, thereby reducing the systemic inflammatory response The time of epithelialization of the study area using cerium nitrate compared with the control area was also faster [6]

Qian L W., et al (2020) found that cerium nitrate treatment significantly reduced the levels of cytokines (p<0.0001 for IL-1β and MIP-1α; p<0.01 for GRO-KC and IL-10) in the burn skin region The authors suggested

that cerium nitrate was effective in reducing the increased proinflammatory cytokines in burned tissues [94]

* Enhance immune effects in patients with burns:

The immune-enhancing effects of cerium nitrate in burn patients are mainly related to burn toxin inactivation and bacterial inhibition [94], [96], [97]

Deveci M., et al (2000) noted the experimental prevention of post-burn immunodeficiency of cerium nitrate Burning animals given cerium nitrate baths reduced TNF and IL-6 concentrations comparable to the removal of necrotic tissue [96]

Qian L W., et al (2020) studied the experimental animal and found that burn injury increased myeloperoxidase activity in burned tissue, reflecting tissue neutrophil infiltration Cerium nitrate treatment reduced myeloperoxidase activity from 93.84 ± 31.35 to 51.3 ± 5.6 (47 % reduction) Cerium nitrate also reduced the concentration of Xanthine oxidase, the enzyme that catalyzes the formation of free oxygen radicals that cause post-burn edema (p<0.001) [94]

1.4.3.4 Effects on necrosis of Cerium nitrate

* The effect on drying necrosis:

Burn necrosis is the source of local and systemic disorders With wet burn necrosis lesions, local and systemic processes are often more severe than dry necrosis The current trend is to use methods to convert wet necrosis to dry necrosis Some measures to dry burn necrosis [98], [99], [100] as followed:

- The simple measure is to completely open the burn, which can be combined with physical measures such as drying the wound, and applying betadine solution many times This measure requires environmental factors to ensure sterility and can only be done in large centers