Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Abbasidezfouli, Samin
Master of Science - MSc
University of British Columbia
Background: Based on the WHO report in 2018, 11 million burn injuries occur worldwide annually, 180,000 of which are lethal. Many studies over the past few years have demonstrated that infection and delayed wound healing are responsible for 42%–65% mortality in burn victims. Hence, the development and application of an antibacterial nutritional liquid scaffold in large burn injuries could reduce the infection rate, accelerate wound healing, and ultimately, save lives. MeshFill is a nutritional liquid scaffold found to improve wound healing and esthetic outcomes upon application in a split-thickness meshed skin graft (STMSG) in a porcine model. To expand the efficacy of MeshFill and address the prevention of infections due to wound contamination, our group added antimicrobial properties by replacing polyvinyl alcohol (PVA), a component in the MeshFill complex, with Povidone-iodine (PVP-I). Unlike silver and antibiotics, low concentrations of iodine are not toxic to human cells, and there is no potential for bacterial resistance. Experimental approach: We assessed the antibacterial effect of various ratios of PVP-I/PVP scaffoderm on both gram-positive and gram-negative bacteria during the 4- and 24-hour time points. The cytotoxicity of the three best ratios of PVP-I/PVP scaffoderm was assessed in vitro, while the wound healing effects in terms of antibacterial activity were then evaluated in vivo using relevant rat models. Result: In terms of antibacterial activity, 150% and 200% PVP-I/PVP scaffoderm was shown to kill gram-positive and gram-negative species most effectively in comparison with other groups. Moreover, 100% and 150% PVP-I/PVP scaffoderm showed the best healing results in vivo and did not delay the wound healing process. Conclusion: Scaffoderm containing 150% PVP-I/PVP is most efficacious in terms of antibacterial activity in full-thickness wounds and the promotion of wound healing. The incorporation of PVP-I into MeshFill may be an effective method to prevent impaired wound healing caused by bacterial infection.
Evaluating the anti-bacterial and wound healing promoting effects of PVP/PVP-I containing liquid scaffold in vitro and in vivo. by Samin Abbasidezfouli A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2023 © Samin Abbasidezfouli, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Evaluating the anti-bacterial and wound healing promoting effects of PVP/PVP-I containing liquid scaffold in vitro and in vivo. submitted by Samin Abbasidezfouli in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Dirk Lange, Associate Professor, Department of Urologic Sciences, UBC Supervisor David Granville, Professor, Department of Pathology and Laboratory Medicine, UBC Supervisory Committee Member Erin Brown, Clinical Professor, Department of Surgery, UBC Additional Examiner iii Abstract Background: Based on the WHO report in 2018, 11 million burn injuries occur worldwide annually, 180,000 of which are lethal. Many studies over the past few years have demonstrated that infection and delayed wound healing are responsible for 42%–65% mortality in burn victims. Hence, the development and application of an antibacterial nutritional liquid scaffold in large burn injuries could reduce the infection rate, accelerate wound healing, and ultimately, save lives. MeshFill is a nutritional liquid scaffold found to improve wound healing and esthetic outcomes upon application in a split-thickness meshed skin graft (STMSG) in a porcine model. To expand the efficacy of MeshFill and address the prevention of infections due to wound contamination, our group added antimicrobial properties by replacing polyvinyl alcohol (PVA), a component in the MeshFill complex, with Povidone-iodine (PVP-I). Unlike silver and antibiotics, low concentrations of iodine are not toxic to human cells, and there is no potential for bacterial resistance. Experimental approach: We assessed the antibacterial effect of various ratios of PVP-I/PVP scaffoderm on both gram-positive and gram-negative bacteria during the 4- and 24-hour time points. The cytotoxicity of the three best ratios of PVP-I/PVP scaffoderm was assessed in vitro, while the wound healing effects in terms of antibacterial activity were then evaluated in vivo using relevant rat models. Result: In terms of antibacterial activity, 150% and 200% PVP-I/PVP scaffoderm was shown to kill gram-positive and gram-negative species most effectively in comparison with other groups. Moreover, 100% and 150% PVP-I/PVP scaffoderm showed the best healing results in vivo and did not delay the wound healing process. iv Conclusion: Scaffoderm containing 150% PVP-I/PVP is most efficacious in terms of antibacterial activity in full-thickness wounds and the promotion of wound healing. The incorporation of PVP-I into MeshFill may be an effective method to prevent impaired wound healing caused by bacterial infection. v Lay Summary Large burn injuries and chronic wounds are frequently colonized or infected. Bacterial infections can be life-threatening in larger wounds. Moreover, these types of wounds require more time to heal completely. A delay in the healing process further increases the risk of infection. The goal of this study is to provide a liquid scaffold possessing antibacterial properties in order to accelerate the healing process and prevent bacterial infections in wound sites. vi Preface This thesis is original and unpublished. Samin Abbasidezfouli was involved in designing, conducting, and analyzing the research experiments under the direct guidance of Dr. Dirk Lange and Dr. Aziz Ghahary with the help of Dr. Lange's and Dr. Ghahary’s Lab members at the University of British Columbia. In terms of in vivo experiments, Dr. Ida Hassanpour, a veterinarian, and Dr. Nafise Amiri conducted the surgeries on rats. The protocol for the in vivo experiments was conducted in agreement with protocols approved by the University of British Columbia Animal Care Committee in accordance with the Canadian Guidelines on Animal Care. The protocol number is A17-0292. Dr. Sahand Ghaffari also helped me with performing the antibacterial experiments. All PVP-I/PVP scaffoderm samples were prepared by Dr. Ruhangiz Kilani. vii Table of Contents Abstract……………..……………..…………….… .………………………………………..…iii Lay Summary………………….....………………...……….………...………...……………….v Preface………………………….……..….........…...…………………………………………....vi Table of Contents.……………………………….............…………………………......…….....vii List of Figures…………………...………..…………………..……………..…………..……....xi Acknowledgements………….……….…………..……....……………………………...……..xiii Dedication…………....……………...…….…..…....…………………………………………..xiv Chapter 1: Background..................……………………..………….……………………............1 1.1) Burn injuries…………………………………………………………………………..…2 1.1.1) Burn injury classifications……………………………………………………………2 1.1.2) Existing treatment for full-thickness burn injuries…………………………..……..3 1.2) Physiology of normal skin…………………….……………………………………….....4 1.3) Bacterial infection in burn patients………………………………………………….…...6 1.4) MRSA (Methicillin resistant Staphylococcus aureus)……………………………….….8 1.5) Pseudomonas aeruginosa (P.A) ………………………………………………………….8 viii 1.6) Antimicrobial agents……………………………………………………………………...8 1.6.1) Antibiotics………………………………………………………………………………9 1.6.2) Silver nanoparticles (AgNPs)…………………………………………………………..9 1.6.3) Povidone-iodine (PVP-I)……………………………………………………………..10 1.6.4) Hydrogels embedded with antibacterial agents …………………………………….11 1.7) Wound healing phases…………………………………………………………………..12 1.8) What is a nutritional liquid scaffold (MeshFill)?...........................................................13 1.9) Hypothesis……………………………………………………………………………….15 1.10) Objectives………………………………………………………………………………..15 Chapter 2: Materials and Methods…………………...……………………………………….16 2.1) Cell culture medium preparation…………………………………………………………16 2.2) Evaluation of cell viability in PVP-I/PVP Scaffoderm………………………………….16 2.2.1) Cell harvesting………………………………………………………………………..16 2.2.2) Gel preparation……………………………………………………………………….16 2.2.3) Live and dead assay…………………………………………………………………..17 2.3) Fibroblasts migration assessment in PVP-I/PVP scaffoderm…………………………..17 2.4) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm...18 2.4.1) Sample preparation…………………………………………………………………..18 2.4.2) Bacterial part………………………….……………………………………………...19 2.5) Evaluation of the antimicrobial activity of 150% PVP-I/PVP scaffoderm on various doses of MRSA and P.A………………………………………………………………………..19 2.6) Scaffoderm preparation…………………………………………………………………...20 2.7) Release study……………………………………………………………………………….20 ix 2.7.1) Standard solution…………………………………………………………………...…21 2.8) In vivo experiments……………………………………………………………………..21 2.8.1) Animal model wound generation and treatment…………………………………...21 2.8.2) Postsurgical Evaluation………………………………………………………………23 2.8.3) Histology………………………………………………………………………………23 2.8.4) H&E staining for paraffin-embedded sections……………………………………..24 2.8.5) H&E staining process………………………………………………………………...24 2.8.6) Immunofluorescence staining………………………………………………………..24 2.8.7) Masson’s Trichrome staining ……………………………………………………….25 2.9) Statistical analysis………………………………………………………………………….26 Chapter 3: Results………………………………………………………………………………27 3.1) Evaluation of cell viability in PVP-I/PVP scaffoderm…………………………………..27 3.2) Fibroblasts migration assessment in PVP-I/PVP scaffoderm…………………………..29 3.3) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm on MRSA……………………………………………………………………………………………30 3.4) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm on P.A……………………………………………………………………………………………….33 3.5) The antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of MRSA and P.A…………………………………………………………………………………………..36 3.6) Release study………………………………………………………………………….……39 3.7) Wound healing study……………………………………………………………………...40 3.7.1) H&E staining ……………………………………………………………………….42 3.7.2) Masson’s trichrome staining…………………………………………………….….44 x 3.7.3) Immunofluorescent staining………………………………………………………...48 Chapter 4: Discussion…………………………………………………………………………..51 Chapter 5: Conclusion……………………………………………………………………….…55 Bibliography………………………………………………………………………………….…57 xi List of Figures Figure 1. Normal skin layers……………………………………………………………………5 Figure 2. Antimicrobial mechanism of action of PVP-I………………………………….…..11 Figure 3. Blinded assessment of wounds to evaluate the aesthetic outcomes of the healing process using MSTSG+ MF/ MSTSG in a pig model………………………………….……..14 Figure 4. Fibroblasts viability in various ratios of PVP-I/PVP scaffoderm………………...28 Figure 5. Fibroblasts viability in various ratios of PVP-I/PVP scaffoderm after 4 days which was assessed using the live and dead assay. ………………………………….………..29 Figure 6. Migration of fibroblasts in different ratios of PVP-I/PVP scaffoderm. 0% is the control group which is just PVP. ……………………………………………………….……..30 Figure 7. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on MRSA. ………………………………………………………………………………………………..….32 Figure 8. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on MRSA. ……………………………………………………………………………………….……….…..33 Figure 9. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on Pseudomonas aeruginosa (P.A). ……………………………………………………………….34 Figure 10. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on Pseudomonas aeruginosa (P.A)…..……………………………………………….……………35 Figure 11. Antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of P.A. ……………………………………………………………………………………………………37 xii Figure 12. Antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of MRSA (USA300). ………………………………………………………………………………….……38 Figure 13. Release study on 150% PVP-I/PVP scaffoderm at different time points.............39 Figure 14. Representative digital images of top view of wounds at different time points….40 Figure 15. Changes in percentage of wound area with different treatments at various time points. …………………………………………………………………………………………...41 Figure 16. H&E staining of thin sections of the skin from experimental groups of rats; 14 days’ time point. ………………………………………………………………………………..43 Figure 17. H&E staining of thin sections of the skin from experimental groups of rats; 17 days’ time point..………………………………………………………………………………..44 Figure 18. Masson’s trichrome staining of thin sections of the skin from experimental groups of rats; 14 days’ time point.………………………………………………………..…..46 Figure 19. Masson’s trichrome staining of thin sections of the skin from experimental groups of rats; 17 days’ time point.……………………………………………………………47 Figure 20. K14 staining on skin samples collected from rats with full thickness wounds treated with PVP, 100%, 150%, and 200% PVP-I/PVP scaffoderm ……………………….49 Figure 21. K14 staining on skin samples collected from rats with full thickness wounds treated with PVP, 150% PVP-I/PVP scaffoderm ……………………………………………50 xiii Acknowledgments I would like to take this opportunity to express my deepest gratitude to all those who have contributed to the completion of this thesis. First and foremost, I would like to thank my thesis supervisor, Dr. Dirk Lange, for their guidance, support, and patience throughout the entire research process. Their invaluable insights and feedback have been instrumental in shaping the direction of this project. I am also grateful to my previous supervisor Dr. Aziz Ghahary for providing me with the opportunity to conduct this research in the first place and helping me with the overall design of this project. My sincere gratitude also goes to Dr. David Granville for reviewing my thesis and providing me with valuable advice toward the improvement of my thesis. I would also like to express my appreciation to my Lab colleagues, Nafise, Sahand, Ruhangiz, Ida, Demian, Roman, Sarah, Dalia and Khaled, for their stimulating discussions, encouragement, and assistance during the course of my studies. Their unwavering support has been an immense source of motivation. I would also like to thank my husband, Armin, who always believed in me and helped me to be a better version of myself. Also, I am grateful to my parents, Naser and Sepideh, as well as my siblings, Saba and Sobhan, who have supported me throughout my academic journey. Their encouragement, understanding, and patience have been invaluable during the ups and downs of the research process. Lastly, I am grateful to everyone who has contributed to this thesis, directly or indirectly. Without their support, this project would not have been possible. Thank you all. xiv Dedication Dedicated to all firefighters and people suffering from full-thickness burn injuries and chronic wounds. 1 Chapter 1: Background Given the significant fluid loss associated with injury and the likelihood of systemic immune response leading to sepsis and organ failure, burn injuries are considered one of the most physiologically challenging injuries an individual can sustain 1,2,3,4. Although burn injuries are decreasing in frequency in high-income countries, the number of burn victims remains high in low- and middle-income countries 5. Based on a WHO report, in 2018, 11 million burn injuries occurred worldwide annually, 180,000 of which are lethal 1. The number of children dying from burn injuries is 11 times higher in low-income countries than in high-income ones 6. In 2016, in the United States, approximately 486000 individuals sought medical care for burn injuries, roughly 40000 of which required hospitalization 7. Hospitalized patients with large burn injuries are at high risk of getting infected by hospital-associated infections, especially those caused by highly resistant bacterial species. Many recent studies have demonstrated that infection is responsible for 42%–65% of the mortality observed in burn victims 7. Moreover, the mortality rate of burn patients with microbial infections is three times higher than that of uninfected patients 8. In addition, based on an autopsy study, multiple organ failure accounted for almost 70% of deaths in burn victims, of which 51% was due to sepsis 9. The rate of wound healing is another vital factor in the survival of burn victims 10,11. For instance, a healing rate of 2%/day on day 20 post-burn resulted in 100% survival. In contrast, the mortality of patients with a slower healing rate (i.e., -2%/day) was 100% 11. Given that healing rate is a key determinant of survival, accelerated wound healing of burn wounds is crucial. The ability to achieve a healed burn wound injury and prevent secondary infection are two essential aspects of reducing mortality in burn patients. Hence, antibacterial nutritional liquid 2 scaffolds in severe burn injuries may reduce infection rate, accelerate wound healing, and ultimately save lives. 1.1) Burn injuries Various factors need to be determined to classify severe burn injuries. For example, patient age, the percentage of total body surface area burned (%TBSA), depth and type of burn, and whether some particular body parts get involved, like genitalia, face, eyes, and inhalation parts 12. According to the Provincial Clinical Practice Guidelines for the Management of Major Burn Trauma in British Columbia, major burns are defined as: • Partial and/or full thickness burns ≥ 20% of the total body surface area (TBSA) at any age. • Partial and/or full thickness burns ≥10% TBSA in children (≤10 years old) or elderly (≥50 years old) • Full-thickness burns ≥ 5% TBSA at any age • Burns involving the face, hands, feet, genitalia, joints • Electrical burns • Chemical burns • Inhalation injury associated with burn injury • Burns associated with major trauma 13. 1.1.1) Burn injury classifications Based on the depth of burn injuries, they can be classified as: 1. Superficial or first degree in which only the epidermis is damaged. 2. Partial thickness or second-degree in which the injury extends to dermoepidermal junction into the dermis (it can involve a small or large amount of dermis). 3. Full-thickness or third/fourth-degree in which the injury extends through the entire dermis, and it can even engage the lower layers (subcutaneous tissue) as well 14. 3 1.1.2) Existing treatment for full-thickness burn injuries Five phases should occur in severe burn care and treatment. The first step is initial assessment and prioritization, in which the cause of the burn gets removed; airway, breathing, circulation, and other injuries get evaluated. The second phase is to focus on fluid resuscitation to manage hypovolemia. The wound should get debrided, grafted, and covered to accelerate healing and prevent infections in the third step. The fourth phase is to support and provide critical care like nutritional support and treating hospital complications. If the patient survives, the final step is to rehabilitate and enable the patient to return to regular life mentally and physically 1. Topical antimicrobial agents and prophylactic systemic antibiotics can be utilized as a nonsurgical burn treatment to prevent sepsis in acute burn injuries. Topical agents are accessible in different forms, such as creams, ointments, liquids, and impregnated dressings 1. Autologous split-thickness skin graft (STSG) is the gold-standard burn wound reconstruction, in which the uninjured skin (partial thickness) is harvested using a dermatome and transferred to the wound site. If the burn area is large, and there is insufficient intact skin, the harvested skin is meshed to be expanded and to cover larger areas. Although the meshed skin can cover a larger region, it requires secondary healing in the interstices, which slows healing and results in poorer aesthetic outcomes 1. A number of artificial skin substitutes have been utilized over the years that provide either temporary or permanent coverage for open wounds in cases where autologous skin is insufficient 1. Some of the commercially available skin substitutes are 1. CEA (Cultured Epithelial Autograft) which consists of human keratinocytes embedded in a fibrin mesh 1. 2. Integra is a Dermal regenerative matrix combined of two layers, dermal and epidermal-like components 15. 3. MySkin is suspended CEA, but in comparison to CEA, it is faster to produce and easier to use 4 in wound site 16. 4. SkinGun is epidermal cells and stem cells 1. 5. Epifix is a compound of dehydrated amniotic and chorionic membranes comprising collagen, connective tissue, cytokines, and growth factors 17. Apart from the advantages of using these substances, the main disadvantage of using them is being costly. Another disadvantage is that there is no Randomized Controlled Trial for these products 1. 1.2) Physiology of normal skin The skin is the largest organ of the human body and consists of three layers. The outer layer is called the epidermis, which is continually renewing. There are two main types of cells in the epidermis called keratinocytes and dendritic cells, and the majority of the cells in this layer are comprised of keratinocytes. The epidermis consists of four layers named 1. the basal cell layer, 2. the squamous cell layer, 3. the granular cell layer, and 4. the cornified or horny cell layer, which is comprised of dead epithelial skin cells 18. Beneath the epidermis, there is a thicker layer called the dermis. Blood vessels, nerve endings, oil and sweat glands, and hair follicles are located in this layer. The two main components of this layer are collagen and elastic tissue, which provides much of the observed mechanical and structural aspects of the skin. Collagen is a major structural protein for the entire body and accounts for 70% of the skin’s dry weight. There are thicker clusters of collagens in the adult dermis compared to infants. In fact, the infant dermis consists of more fibroblasts which integrate the procollagen molecules and make collagen fibrils 18. The last layer, which is located beneath the dermis, is called the subcutaneous layer. This layer consists of fat tissue and functions as a store of energy and a foundation for the dermis 18. 5 The skin functions mainly as an initial barrier and protects the body from pathogens, UV light, chemicals, and physical injury 19. It is the first organ to detect pain, temperature, touch, and deep pressure. In addition, it lets the body move freely. Water, urea, and ammonia are released during exocrine activity. Skin secretes sebum, sweat, and pheromones, as well as performs important immunologic functions by secreting bioactive substances such as cytokines and therefore develops immunity against pathogens 20. It also maintains the homeostasis of the body by regulating temperature and protecting from dehydration by controlling the amount of water released into the environment 19. Skin also serves as a large storage facility for the body, with the deepest layer of skin capable of storing water, fat, and metabolic products. It also produces hormones and hormone-like substances, such as sex steroids, melatonin, and vitamin D, that are essential for the entire body 21,22. Created with BioRender.com Figure 1. Normal skin layers 6 1.3) Bacterial infection in burn patients Injuries that damage the skin, such as third-degree burn injuries and other full-thickness wounds, result in the loss of the normal protective functions of the skin 23. The loss of these protective functions increases the risk of secondary infections, as well as the loss of fluid and temperature homeostasis 24. Both gram-positive and negative bacteria can cause infections 25. Among these bacteria, Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens causing such infections 26. Burn wound infections remain a risk for these individuals until the damaged skin is replaced with skin grafts or heals secondarily. The natural history of bacterial colonization and infection follows a typical pattern of early colonization with gram-positive organisms, followed by the subsequent replacement by gram-negative organisms 7,27,28,29,30. Gram-positive bacteria that survive the thermal damage, such as staphylococci situated within sweat glands and hair follicles in deeper parts of the skin, heavily proliferate within the first 48 hours unless topical antimicrobial agents are applied 24. Also, during the first few days after burn injury, endogenous gram-negative bacteria derived from the patient's gastrointestinal flora translocate and can become resident in extraintestinal sites, such as the burn wound 24,31. Yeast and fungal infections normally occur later as a result of broad-spectrum antibiotic therapy 24,32. The surface of deep partial-thickness and all full-thickness burn wounds is a desirable niche for the colonization of microbes because it is a protein-rich area containing avascular necrotic tissue (eschar) 24,33. Microorganisms typically colonize the surface of a burn wound prior to the invasion of viable subcutaneous tissue 34. Lack of blood vessels in the wound site results in the shortage of immune cells in that area due to the impairment of host immune cell migration and release of toxic substances by eschar tissue which interferes with the local host immune responses 24. 7 The classification of burn wound infections is based on the appearance of the burn wound and/or eschar, time of occurrence, and mortality rate 25. With early excision and skin grafting, new definitions for burn wound infections have been introduced by a subcommittee of the American Burn Association's Committee on the Organization and Delivery of Burn Care 35. Different types of burn wound infections, including impetigo, cellulitis, invasive infection, and Burn-related surgical wound infection, are explained below 25. Burn wound impetigo: Impetigo is defined as the loss of epithelium from an earlier re-epithelialized surface like grafted burns, partial-thickness burns or a donor site after getting healed 25,36. This type of infection may or may not be associated with systemic signs of infection, such as hyperthermia or leukocytosis 35. Burn-related surgical wound infection: Surgical wound infections occur in non-healed areas that have not been epithelialized yet. These types of infections show a lack of wound coverage, changes in wound appearance, and erythema in the intact surrounding skin 25. Burn wound cellulitis: Burn wound cellulitis is caused when the infection expands into the healthy skin and soft tissues surrounding the burn wound or donor site 25. This type of wound infection involves the dermis and subcutaneous tissues 37. Cellulitis is thought to be caused primarily by Gram-positive cocci such as Streptococcus spp. and Staphylococcus aureus 37. 8 Invasive infection in unexcised burn wounds: Unexcised deep partial-thickness or full-thickness burn wounds are more prone to develop invasive infections 24,38. Gram-negative bacteria or filamentous fungi are the most common causes of this type of infection 39. These infections may demonstrate a rapid change in appearance or character of the burn wound, like separation of the eschar, changes in the colour (dark brown, black, or violaceous discoloration) of it or increased erythema in the surrounding area 24,40, 41. 1.4) MRSA (Methicillin resistant Staphylococcus aureus) Staphylococcus aureus is a gram-positive coccus 42. Methicillin-resistant S. aureus (MRSA), particularly, is a dangerous organism because of its resistance to antibiotics, typically effective against staphylococcal species and the ability to form biofilm 42. MRSA infection can be challenging to treat, ultimately resulting in hematogenous spread and potential sepsis 42,43. 1.5) Pseudomonas aeruginosa (P.A) Pseudomonas aeruginosa is a Gram-negative rod-shaped opportunistic pathogen. The term “opportunistic” is used because Pseudomonas aeruginosa rarely infects healthy people, but it can be serious pathogen for individuals with compromised immune systems such as individuals with untreated HIV, cystic fibrosis and other individuals experiencing an immunocompromised state, including major burn wound injuries. These bacteria are often resistant to various types of antibiotics and therapeutic agents, which is why they are difficult to treat 44. 1.6) Antimicrobial agents 9 Many different antimicrobial agents can be utilized in the management of burn wound colonization and infection. Systemic antibiotics, as well as topical agents, such as AgNPs (silver nanoparticles), and Povidone-iodine (PVP-I) may be used in the treatment of burns. 1.6.1) Antibiotics Although antibiotics have had strong antibacterial effects and have been used worldwide to treat bacterial infections, they will not be effective on some particular bacterial infections anymore because of their antibiotic resistance. From an evolutionary point of view, bacteria use two main genetic mechanisms to adapt to the antibiotic “attack”, 1. gene mutations which are often associated with the mechanism of action of the compound, and 2. gaining of external DNA coding for resistance determinants through horizontal gene transfer (HGT). Apart from antibiotic resistance issues, when it comes to viral infections, antibiotics are completely inefficient 45. In addition, systemic antibiotics are not routinely used unless specifically required because they lead to the transition from gram-positive colonization to gram-negative colonization, which makes treatment of severe infections more challenging, and in some types of wounds, such as burn injuries, the antibiotics cannot reach to the necrotic burn eschars as there is no circulation to this tissue. Moreover, systemic antibiotics are associated with many side effects as well. In terms of topical antibiotics, apart from the fact that they induce bacterial resistance, the number of exciting topical antibiotics is much smaller, including bacitracin, neomycin, polymyxin, mupirocin, and fusidic agents, in comparison with the broad number of antibiotics that are used systematically 46. Because of these reasons, antibiotics cannot be considered a proper choice of antimicrobial agent. 1.6.2) Silver nanoparticles (AgNPs) 10 AgNPs exhibit antibacterial activity on both gram-positive and gram-negative bacteria, and because of that, they have been used in different therapeutic fields like drug delivery systems, dental applications, and wound healing 47,48. However, apart from AgNPs benefits, they have some disadvantages too. They can contaminate the aquatic and terrestrial environments as well as enter the food chain 49. Moreover, it has been proven that AgNPs (20,50,80 nm) have skin sensitization 50. All in all, it is better to look for another antibacterial agent with less or no toxicity effects. 1.6.3) Povidone-iodine (PVP-I) Povidone-iodine (PVP-I) is a broad-spectrum antimicrobial agent that has been safely used in clinical settings to control and prevent infections for many years 51,52. PVP-I is effective on both gram-positive and gram-negative bacteria, fungi, and viruses 53. PVP-I kills pathogens via various modes of antibacterial mechanisms. Overall, iodine is released from the PVP-I complex, and it can penetrate microorganisms rapidly and then, oxidizes fatty acids, proteins, and nucleotides which can eventually lead to the death of cells. Given the broad range of antibacterial mechanisms, no resistance to PVP-I has been reported in bacteria to date 54. 11 Created with BioRender.com Figure 2. Antimicrobial mechanism of action of PVP-I Although high concentrations of PVP-I can be toxic for human fibroblasts, it has been proven that a low concentration of PVP-I (<1%) has no adverse effects on host cells while retaining its antibacterial properties 55. In addition, PVP-I increases the healing quality in wound sites 54. 1.6.4) Hydrogels embedded with antibacterial agents Hydrogels are utilized during the first hours after a small superficial burn injury to cool the burn wound and reduce the pain. However, as they do not have antimicrobial characteristics, they would not be anticipated to reduce the risk of wound infection. Hydrogels may incorporate antibacterial agents or active substances, including ammonium salts, nanocrystal silver, zinc, growth factors, 12 cytokines, or cells, as well as natural agents, such as honey or herbs. Incorporated hydrogels may have antimicrobial properties or stimulate wound healing 56. Khan et al. (2023) studied the antibacterial activity and wound-healing properties of Chitosan hy-drogels embedded with Aloe vera and Ofloxacin (OX). Based on their studies, while the addition of 0.5 mg of OX prevented bacterial colonization and infections, it had additional benefits, in-cluding good angiogenic activity as well as better fibroblast proliferation among other concentra-tions, including 2.5 mg and 5 mg OX hydrogels. Therefore, based on this study, OX-incorporated chitosan/Aloe vera-based hydrogels can be promising wound dressings for further clinical use 57. Wang et al. (2021) also studied the antimicrobial activity of polysaccharide-based hydrogel dressing containing plant essential oil, including eucalyptus, ginger, and cumin. Based on this study, hydrogel containing essential oils had antimicrobial activity against Staphylococcus aureus and Escherichia Coli 58. 1.7) Wound healing phases Wound healing is a complicated process that naturally happens in response to tissue injuries in human bodies. The wound-healing process is an interaction between various cell types, cytokines, mediators, and the vascular system. This process has three main phases, which happen sequentially and overlap each other 59: 1. Hemostasis/ Inflammation phase: Following a skin injury, the exposed sub-endothelium, collagen, and tissue factor will activate platelet aggregation, resulting in degranulation and the release of chemotactic factors (chemokines) and growth factors (GFs) to form the clot, and all of the preceding procedures will achieve successful hemostasis. Neutrophils, the first cells to appear at the site of an injury, clean debris and pathogens to create a favourable condition for wound 13 healing. Following that, macrophages accumulate, aiding the phagocytosis of bacteria and causing tissue damage. The hemostasis/ inflammatory phase frequently takes a few days to complete 59,60. 2. Proliferative phase: This phase is distinguished by an accumulation of numerous cells and abundant connective tissue. Fibroblasts, keratinocytes, and endothelial cells are all present in the wound. Extracellular matrix (ECM) components such as proteoglycans, hyaluronic acid, collagen, and elastin form granulation tissue to replace the original clot formation. Many cytokines and GFs are involved in this phase. This stage lasts for several days or weeks 59. 3. Maturation and remodeling phase: The final step requires a precise balance of apoptosis of existing cells and cell production. Gradual degradation of abundant ECM and immature type III collagen, as well as the formation of mature type I collagen, are crucial in this phase, which can last months or years. Any deviation during this phase could result in excessive wound healing or a chronic wound 59. Fibroblasts play an important role in tissue repair, secreting growth factors, cytokines, collagens, and other extracellular matrix (ECM) components from the late inflammatory phase until the ultimate reepithelization of the wound. Simultaneously, fibroblast migration and proliferation play critical roles in the healing process by initiating the proliferative phase of repair 61. 1.8) What is a nutritional liquid scaffold (MeshFill)? MeshFill (MF) is a collagen-glycosaminoglycan- based scaffold cross-linked with polyvinyl alcohol (PVA)- borate, which is a reconstitutable liquid scaffold. To produce MeshFill (MF), Type I bovine collagen is mixed with chondroitin-6-sulphate and then neutralized with 1 N NaOH. Collagen is cross-linked utilizing 0.02% v/v glutaraldehyde. Next, a combination of PVA, sodium borate decahydrate and ascorbate is added to generate the scaffold. Lastly, to get a powdered form 14 of MF, this mixture is lyophilized and minced. MeshFill (MF) is liquid in low temperatures (4-8oC), and it is jellified within the wound site. Previous studies have documented that MeshFill improves the rapidity of wound healing and the ultimate aesthetic outcomes upon application to a meshed split-thickness skin graft (STMSG) in a large animal pig model 62. To assess the aesthetic outcomes, two factors have been measured: 1. appearance and 2. Contracture 62. Figure 3. Blinded assessment of wounds to evaluate the aesthetic outcomes of the healing process using MSTSG+ MF/ MSTSG in a pig model. Blinded assessment of wounds showed significantly higher scores on the clinical appearance of the MSTSG+MF group in comparison with the MSTSG photos (74.00 ± 4.51 vs. 63.33 ± 4.60%, p < 0.05). In addition, contractures in wound sites were significantly decreased in the MSTSG+MF group compared to that of the MSTSG images (28.50 ± 3.50 vs. 40.67 ± 4.64%, p < 0.01) 62. MeshFill does not possess intrinsic antibacterial properties. One of the potential risks during skin graft healing or the healing of other wounds is the disruption of the process by bacterial 15 colonization and infection. To mitigate this risk, we developed a hydrogel with the added antimicrobial agent povidone-iodine. 1.9) Hypothesis We hypothesize that the addition of PVP-I into MeshFill would prevent bacterial growth in vitro, and the application of that on full-thickness wounds would improve the healing outcome in vivo in rat models. 1.10) Objectives 1. Evaluating the efficacy of various ratios of PVP / PVP-I liquid scaffold on cell viability in a 3D cell culture. 2. Testing the antibacterial activity of various ratios of PVP / PVP-I liquid scaffold in vitro. 3. Investigating the efficacy of various ratios of PVP / PVP-I liquid scaffold on fibroblast proliferation in a 3D cell culture. 4. Evaluating wound epithelialization by using H&E staining of tissue sections. 5. Evaluating the application of various ratios of PVP / PVP-I liquid scaffold on healing outcome in delayed wound healing in rats. 16 Chapter 2: Materials and Methods 2.1) Cell culture medium preparation 55 mL of Fetal Bovine Serum (FBS) [Gibco™ 25200056] and 5.5 mL of Antibiotic-antimycotic (100X) [Gibco™ 15240062] were added to DMEM (Dulbecco's Modified Eagle Medium) [Gibco™ 15240062], and they were mixed completely to make 10% FBS-DMEM. 2.2) Evaluation of cell viability in PVP-I/PVP Scaffoderm 2.2.1) Cell harvesting To harvest cells, the medium was discarded from a flask containing primary Human Dermal Fibroblasts (HDF) cell lines (derived from foreskin during the circumcision process, obtained from Vancouver General Hospital (VGH)), and then, it was washed with PBS two times. A proper amount of Trypsin-EDTA (0.25%), phenol red, [Gibco™ 25200056] (5 mL for big flasks and 1.5-2 mL for smaller ones) was next added to the flask in order to detach cells from the surface area. When all cells were detached, 10 mL of 10% FBS-DMEM was added to deactivate trypsin. All cells plus medium were then transferred to a falcon tube and centrifuged at 1500 rpm, 4 ℃ for 5 minutes. After centrifugation, the supernatant was discarded, 8 mL medium was added to the pellet, and the cells were kept on ice. 2.2.2) Gel preparation A specific amount of cold ddH2O was added to the tubes containing PVP-I/PVP scaffoderm powder; then, they were vortexed until they were dissolved completely (gels should be kept cold during the process). Next, they were centrifuged at 1000 rpm, 4 ℃ for 1 minute to get rid of bubbles. 150,000 cells were then added to each Eppendorf tube (we prepared 6 of them) and 17 centrifuged at 1500 rpm, 4 ℃ for 5 minutes. Next, the supernatant was discarded, and 600 μL of gel was added to the pellet. They were mixed by pipetting and then, it was centrifuged at 1000 rpm, 4 ℃ for 1 minute. 300 μL of the solution was then added to each well (use a 48-well plate), and the plate was put in an incubator at 37 ℃ until the wells were cast. When they were cast, 1 mL of 10% FBS-DMEM was added to each well, and the plate was put back into the incubator at 37 ℃ for 4 days. After 4 days, Live and dead assay was performed. 2.2.3) Live and dead assay [As live and dead assay is sensitive to light; all steps should be performed in darkness.] Firstly, calcein AM and ethidium homodimer-1 [Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells, 30002] should be thawed. Then, Ethidium homodimer-1 (EthD-1), Calcein AM, and PBS (1X) should be mixed together with the ratio of 2 μL:1 μL:1000 μL respectively in a tube which is wrapped in foil to make the cell staining solution. Next, the culture medium was removed from the cells, and they were washed with PBS (1X) twice. Afterward, 500 μL of staining solution was added to each well containing cells. Then, the plate was wrapped in foil and put in an incubator (37 ℃) for 50 minutes. After the incubation period, alive cells were seen in green and dead ones in red under a fluorescent microscope. The number of both alive and dead cells was counted, and the percentage of alive cells was calculated. 9 experimental groups were used in this evaluation, including 0% (PVP), 10%, 20%, 30%, 40%, 50%, 75%, 100%, and 150% PVP-I/PVP scaffoderm. PVP was considered the control group. 2.3) Fibroblasts migration assessment in PVP-I/PVP scaffoderm PVP-I and PVP gels were prepared as described earlier, and they were kept on ice. Next, different ratios of PVP-I/PVP scaffoderm (100%, 80%, 40%, 20%, and 0% PVP-I/PVP 18 scaffoderm) were made by mixing different amounts of PVP-I and PVP-40 gels. After making experimental samples, all tubes containing the sample were centrifuged to get rid of bubbles and kept on ice. 1.5 mL of each sample group was added to a 12-well plate, after which the plate was put in an incubator (37 ℃) until they were cast. Meanwhile, after harvesting cells, the specific number of fibroblasts corresponding to 150,000 cells were added to 6 different Eppendorf tubes followed by centrifugation at 1500 rpm, 4 ℃ for 5 minutes. After centrifugation, the supernatant was discarded, and 300 μL of each experimental sample was added to the pellet, and they were kept on ice. After all gels existing in each well were cast, an 8mm hole was created in the middle of each well using an 8mm punch biopsy, and the gel plus all liquid soluble existing inside the hole were discarded. Next, 100 μL of the prepared cell solution was added into the hole in each well. The plate was then put in the incubator (37 ℃) until all gels were cast, after which 2mL of cell culture medium was added to each well, and the plate was kept in the incubator for a week. Following the incubation period, the Live and dead assay was performed on all wells, and they were evaluated under the fluorescent microscope to assess the migration of fibroblasts from the center of the well (hole) to the sides. 2.4) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm 2.4.1) Sample preparation The specific amounts of PVP-I scaffoderm and PVP-40 scaffoderm powder were measured, and they were added to Eppendorf tubes [each sample had 80 mg powder in total, but with different ratios.]. 950 μL of cold LB broth was added to each Eppendorf tube and vortexed [samples should be kept cold during the process]. The samples were centrifuged at 1000 rpm,4 ℃ for 1 minute to get rid of bubbles. Samples were kept on ice. 19 2.4.2) Bacterial part Bacterial strains were cultured and sub-cultured at least 24 hours prior to the experiments. Optical density (OD595) measurement and CFU counting methods were used to determine the starting CFU/mL concentrations of the bacteria. The corresponding volume was determined for the specific starting CFU/200 μL (105 CFU/200 μL) based on the OD and CFU counts. 50 μL of MRSA (MRSA USA 300 GFP) stock solution with the specific starting CFU/200 μL was added to each sample, and they were vortexed. 200 μL of each sample was added to wells [ 48-well plate was used]. The plate was put in a shaker incubator for 4 and 24 hours at 120 rpm. After 4 and 24 hours of incubation, the following steps proceeded: 200 μL of cold PBS was added to each well; the gels were disrupted, and 20 μL of each well was taken out; then, serial dilution was made by adding 180 μL of PBS. Next, 10 μL of various dilutions of each group was dropped on an LB agar plate, and CFU counting was done after 14-18 hours. Eight experimental groups were used for the test, with a 4-hour time point, including PVP, 25%, 50%, 75%, 100%, 150%, 200%, and a control group which was just LB broth plus bacteria. Three experimental groups were also used for the test, with a 24-hour time point, including control, PVP, and 150% PVP-I/PVP scaffoderm. The same experiments were done by using Pseudomonas aeruginosa. 2.5) Evaluation of the antimicrobial activity of 150% PVP-I/PVP scaffoderm on various doses of MRSA and P.A The same procedure of gel preparation and bacterial experiment were performed for this experiment, but this time, the antibacterial activity of 150% PVP-I/PVP scaffoderm was evaluated on different doses of bacteria. As the process explained earlier, the corresponding 20 volume was determined for the specific starting CFU/200 μL (105 CFU/200 μL) based on the OD and CFU counts, after which various doses of MRSA and P.A were made by making specific dilutions (105, 104, 103, 102, and 101). 2.6) Scaffoderm preparation Type I bovine collagen (5005; Advanced Biomatrix, San Diego, CA) was mixed with chondroitin-6-sulphate (C4384; Sigma Aldrich, St. Louis, MO) to a final concentration of 3 mg/mL followed by neutralizing with 1 N NaOH. Collagen was cross-linked with the use of 0.02% v/v glutaraldehyde (G5882; Sigma Aldrich). A combination of PVP-I (PVPI-100G; Sigma Aldrich), sodium borate decahydrate (31457; Sigma Aldrich), and ascorbate (PHR1008; Sigma Aldrich) was added to make the scaffold and to make a powdered form of PVP-I scaffoderm, the mixture was lyophilized and minced. For the PVP-40 group, instead of PVP-I, PVP-40 (PVP40, Sigma Aldrich) was added to the combination in the last step. Powdered scaffoderm was reconstituted in distilled water; it was kept on ice to hinder gelation, and it was warmed up by placing it in a 37°C incubator for 15 minutes to help with the polymerization of collagen. Each millilitre of scaffoderm contains 80 mg of powder, and PVP-I contains 10% iodine. Having said that, 100% PVP-I/PVP scaffoderm samples contain 0.06 mg/mL iodine, 150% PVP-I/PVP scaffoderm samples contain 0.09 mg/mL iodine, and 200% PVP-I/PVP scaffoderm contains 0.12 mg/mL iodine. 2.7) Release study 1 mL of cold ddH2O was added to 80 mg of PVP-40 and 150% PVP-I/PVP scaffoderm powder, and they were centrifuged at 1000 rpm, 4 ℃ for 1 minute to get rid of bubbles. All gels were 21 transferred to a falcon tube, and they were put into an incubator (37 ℃) for 20 minutes to get solidified. Next, 12 mL of PBS was added to each falcon tube, and they were placed in a shaker incubator (37 ℃). After each time point, 10 mL of PBS was transferred to an empty falcon tube, and it was replaced with fresh PBS; then, the falcon tubes were put back in the shaker incubator, and the collected samples were kept in a refrigerator (4 ℃). After all samples were collected (Time points in which samples were collected were: 2, 4, 8, 12, and 24 hours), they were sent to be analyzed by ICP-OES [10 mL of pure PBS was used as a blank.]. 2.7.1) Standard solution 20 mg of Potassium iodide (KI) was added to 20 mL of ddH2O (1mg/mL of iodine concentration), and it was diluted (1:2) to make standard samples with a concentration of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, and 0.03125 mg/mL. 2.8) In vivo experiments 2.8.1) Animal model wound generation and treatment The protocol for the experiments below was conducted in agreement with protocols approved by the University of British Columbia Animal Care Committee (protocol number: A17-0292) in accordance with the Canadian Guidelines on Animal Care. The aseptic surgical method was followed for all processes, and all animals received humane care during the experiments. Isoflurane and CO2 asphyxia were used to anesthetize and euthanize all rats, respectively 63. 20 Sprague Dawley (SD) female rats weighing 298-388 g were ordered from Charles River Laboratories (Wilmington, MA). In each cage, two rats were housed prior to wounding, and they were fed an unlimited amount of rodent chow and water. Standard conditions were applied to all cages which were kept at an institutional animal facility with a 12-hour light-dark cycle 64. The 22 rats were accommodated in these conditions during the 1-week acclimation period 64. However, they were housed individually during the 2-week experimental period due to observed disruption of their cage mates’ dressings after experimental wounding. All wounding and post-operative care was performed by a veterinary surgeon. Rats were weighed before starting the surgical operation. Next, they were anesthetized using 2% isoflurane in 100% oxygen at a flow rate of 1.0 L/minute during the entire surgical process. After anesthesia induction, 1 mg/kg Meloxicam (Metcam) and 10 mL/kg LRS (Lactated Ringer’s Solution) were injected subcutaneously to control pain and maintain the water and electrolyte balance, respectively. Upon injection of Meloxicam and LRS, the dorsal surface of each rat was shaved and disinfected using three sequential scrubs of 4% chlorhexidine gluconate and 70% ethanol. Two sterilized silicone splints (Grace Bio-Labs, Bend, OR) with an 8 mm and 12 mm inner and outer diameter, respectively, were located around the pre-defined parathoracic wound area and secured utilizing six outer sutures (4-0 Prolene Nylon Suture, Ethicon LLC, Cornelia, GA) in order to overcome the problem of wound contracture in rodent wounds 65. Two full-thickness, 8-mm wounds, one on each side of the midline, were created on the shaved dorsal hemithorax of each rat utilizing an 8-mm biopsy punch (Acuderm, Fr. Lauderdale, FL). The scaffoderm samples were kept in liquid form on ice prior to wounding, then removed and kept at 25ºC for five minutes prior to applying to the wound site. Then, a specific ratio of PVP-I/PVP scaffoderm was applied to the wound area, and after 5-10 minutes, it polymerized, after which a semi-occlusive dressing (Tegaderm, 3M, St. Paul, MN) was applied to the wound. A subsequent layer of sterile gauze was placed over the wound, and a silicone ring to provide additional protection of the wound site. This was further reinforced with a secondary layer of dressing (3M Coban) over the Tegaderm one in order to hinder splint removal by rats and wound 23 healing process delay, and finally, a layer of leucoplast tape kept all layers in place. Subsequently, one rat was housed per cage, located on a warm water circulation veterinary blanket, and monitored carefully until full recovery from the anesthesia. The no-treatment (NT) group received dressing only. Other experimental groups received PVP, 100%,150%, or 200% PVP-I/PVP scaffoderm. 2.8.2) Postsurgical Evaluation All groups of rats were monitored every day in terms of general health condition and weight pursuant to protocols. In addition, all of them were anesthetized to evaluate the wound areas on days 0, 3, 7, 10, and 14 during bandage substitution in terms of splint stability, inflammation, and infection symptoms. During these time points, the wound sites were imaged using a digital camera. All rats were euthanized on day 14, and a 12-mm circular biopsy punch was used to harvest all wound sites for tissue analysis. The same experimental procedure was performed using PVP and 150% PVP-I/PVP scaffoderm plus no treatment group over a 17-day period. We decided to perform this experiment on 150% PVP-I/PVP scaffoderm, and not 100% because it had the best antibacterial effect as well as acceptable wound healing outcomes. 2.8.3) Histology Tissues were fixed in 10% formalin overnight, and paraffin blocks were sectioned with 5 μm thick by using a microtome (TBS, SHUR/Cut TM 2500) for histological assessment. 24 2.8.4) H&E staining for paraffin-embedded sections The sectioned slides were heated at 60 ℃ for 1-2 hours. Slides were washed in xylene I, II, and III for 10 minutes each. Then, they were washed in ethanol (100, 95, 95, 80, and 70%) for 5 minutes each. Next, they were washed in tap water for 5 minutes. 2.8.5) H&E staining process Slides were immersed into hematoxylin solution (Harris, modified, Sigma HHS16) for 5 minutes, and next, they were washed under running tap water for 10 minutes. Next, they were dipped into 0.1% (W/V) HCl (Sigma-Aldrich, 258148) for 1 minute. After that, they were dipped into tap water 3-4 times. They were then immersed in 0.1% NaHCO3 (Sodium bicarbonate (ThermoFisher Scientific, S233-500) 1g/1L dH2O) for 1 minute, followed by immerging into tap water 3-4 times. They were next dipped into Eosin Y aqueous solution (Sigma HT110216) for 1-3 minutes. Then, they were washed in ethanol 95% and 100% for 3 and 4 minutes, respectively. Afterward, they were washed in Xylene I and II for 5 minutes each. Lastly, a drop of Xylene-based mountain solution was put on samples, after which were covered with a coverslip and they were left overnight to dry. The next day, they were assessed under the light microscope (Nikon, Eclipse 80i). 2.8.6) Immunofluorescence staining Samples were heated in an oven at 60 ℃ for 2 hours or overnight. Then, they were de-paraffinized and rehydrated like the first steps in H&E staining process. Next, they were steamed in antigen retrieval solution (1.47 gr Sodium Citrate (Fisher Chemicals, S279-3) in 500 mL dH2O, pH 6.0) for 20 minutes, followed by passive cooling for 30 minutes. Next, they were washed under running tap water for 5 minutes. After they dried, the sections were labeled with water blocking pen. 25 Processing for the intracellular antigen K14 Samples were washed with 0.5% Saponin (Sigma Aldrich, 47036) solution (in water) for 5 minutes to penetrate the cell membrane. The next step is washing samples with PBS-T 3 times in a shaker for 5 minutes each (1mL of tween-100 was added to 1L of PBS to make a PBS-T solution.). Samples were then washed with 5% BSA-PBS-T block for 1 hour. Anti-Cytokeratin 14 antibody [abcam, LL002] and 2% BSA-PBS-T buffer (1 μL: 500 μL) solution was then added to samples (covering them completely); they were kept at 4 ℃ overnight. The next day, samples were washed with PBS-T 3 times in a shaker for 5 minutes each. Alexa Fluor 488 goat anti-mouse antibody (ThermoFisher, Alexa Fluor 488) conjugated with fluorescent dye in 2% BSA was then added to samples, and they were incubated for 1 hour in the dark. After incubation, they were washed with PBS-T 3 times in the dark for 5 minutes each. Finally, a small drop of DAPI solution was added to each section and covered with a long coverslip in the dark; they were stored at 4 ℃ for 2-3 hours or overnight (light sensitive), and they were then evaluated under a fluorescent microscope (ZEISS, Observer. D1). 2.8.7) Masson’s Trichrome Staining Samples were heated in an oven at 60 ℃ for 2 hours or overnight. Then, they were de-paraffinized and rehydrated like the first steps in the H&E staining process. Next, the slides were put in Bouin’s solution (Sigma HT101128) at 56 ℃ for 1 hour, followed by washing the slides under running tap water for 10 minutes. Then, the slides were dipped into dH2O for 1 minute; after they dried, the sections were labelled with water blocking pen. Weigert’s Iron Hematoxylin solution (Sigma Aldrich, HT1079) [1:1 ratio of part A and part B] was then added to the tissue 26 samples covering them completely for 15 minutes. The solution was then discarded, and samples were washed under running tap water for 10 minutes. After that, they were dipped into distilled water for 1 minute, followed by adding Biebrich scarlet-acid fuchsin solution (Sigma Aldrich, HT151) to the slides. After 1 minute, the slides were dipped into distilled water 3 times, followed by adding Phosphomolybdic-phosphotungstic acid solution (1:1:2 dH2O) to them on a shaker; this step was done 3 times, each for 10 minutes. Then, samples were covered with Aniline blue solution for 15 minutes. They were next dipped into distilled water (one quick dip only), after which 1% acetic acid solution was added to them, and they were kept in the solution for 4 minutes. Next, they were immersed in (95% and 100%) Ethanol, respectively, each for 1 minute, followed by immerging into Xylene I, II, and III for 3 minutes each. Finally, one drop of Permanent Mounting Medium (VectaMount®, H-5000-60) was added to the tissue samples and covered with a long coverslip; they were kept under a fume hood to dry overnight and were evaluated under a light microscope (Nikon, Eclipse 80i) a day after. 2.9) Statistical analysis Statistical analysis was conducted using IBM SPSS Statistics software version 27. One-way ANOVA with multiple comparisons was performed on all data. Post hoc analysis was performed using Tukey’s method. Statistical significance was set at p < 0.05. Standard deviations were calculated using Excel 2016. Image J software was used to measure the wound area resulting from wound healing experiments. 27 Chapter 3: Results 3.1) Evaluation of cell viability in PVP-I/PVP scaffoderm The Live and dead assay results demonstrate that there is no toxicity associated with 75% PVP-I/PVP scaffoderm and lower ratios in comparison with the control group, which is just PVP (0%) because the cell viability is more than 80%. However, 100% and 150% PVP-I/PVP scaffoderm demonstrated a reduction in the cell viability (57% and 71%, respectively). Statistical analysis showed no significant reduction in the cell viability in 10%, 20%, 30%, 40%, 50%, and 75% ratios of PVP-I/PVP scaffoderm experimental groups compared to 0% (p > 0.05). However, there is a significant decrease in the percentage of viable cells in 100% and 150% ratios of PVP-I/PVP scaffoderm compared to 0% (p < 0.05). Based on the outcomes, it can be concluded that all ratios of PVP-I/PVP scaffoderm lower than and equal to 75% appear safe to use and do not significantly interfere with fibroblast proliferation. In addition, images from live and dead assays illustrate that the morphology of fibroblasts in lower concentrations < 50% ratio of PVP-I/PVP scaffoderm is different from higher concentrations like 75% and 100%. Although the morphology of cells in higher concentrations is circular, it doesn’t mean that they are dead. Interestingly, the morphology of cells in 150% PVP-I/PVP scaffoderm looks more filamentous than 75% and 100%. 28 97.13 96.77 96.03 93.8 89.4881.25 84.2556.8771.750204060801000% 10% 20% 30% 40% 50% 75% 100% 150%Cell viability (%)Various ratios of PVP-I/PVP scaffodermFigure 4. Fibroblasts viability in various ratios of PVP-I/PVP scaffoderm Different ratios of PVP-I/PVP scaffoderm were made by adding the specific amount of cold ddH2O to particular amount of PVP-I/PVP scaffoderm powder which has been kept on ice, then 600 μL of it was added to 150000 fibroblasts and mixed. Next, 300 μL of each ratio was added to a well in a 48-well plate. After that, the plate was put in an incubator in 37 ℃ until the gel casts. 1 mL 10% FBS-DMEM was then added to each well. Afterwards, the plate was kept in the incubator in 37 ℃ for 4 days. Finally, the fibroblasts viability was assessed by using live and dead assay after 4 days. Data are shown as mean ± SE of samples and are representative of ten repetitions (n=10) of experiment. The experiment was performed on different Fibroblasts derived from different people (F33, F103 and F3). ***p < 0.001. *** *** 29 0% 10% 20% 30% 40% 50% 75% 100% 150% Figure 5. Fibroblasts viability in various ratios of PVP-I/PVP scaffoderm after 4 days which was assessed using the live and dead assay. Images were taken with a fluorescent microscope. 3.2) Fibroblasts migration assessment in PVP-I/PVP scaffoderm The outcomes show that fibroblasts were able to migrate in various ratios of PVP-I/PVP scaffoderm. As is apparent in Figure 6, the morphology of migrated fibroblasts in the greater ratios changed to circular from the fibre form found for lower ratios and the control group (PVP). 30 0% 20% 40% 80% 100% Figure 6. Migration of fibroblasts in different ratios of PVP-I/PVP scaffoderm. 0% is the control group which is just PVP. 3.3) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm on MRSA Results from the antibacterial experiments on MRSA demonstrate that increasing the ratio of PVP-I/PVP scaffoderm, results in increased antibacterial activity. Based on Figure 7, 150% and 31 200% PVP-I/PVP Scaffoderm had the most antibacterial effect on MRSA by leading to around a 2-log reduction in the number of bacteria compared to the control group after 4 hours of incubation. Statistical analysis demonstrates that all groups were able to significantly reduce the number of bacteria in comparison with the control group (p < 0.001). However, within different ratios of PVP-I/PVP scaffoderm, 150% and 200% are the two groups that reduce the number of MRSA colonies most significantly compared to the PVP group (p < 0.001). However, the results related to the experiment with the same procedure on MRSA, but with 24 hours of incubation showed that 150% PVP-I/PVP scaffoderm led to nearly a 1-log reduction in the number of bacteria in comparison with the control group. Statistical analysis revealed that both PVP and 150% PVP-I/PVP reduced the number of bacteria significantly compared to the control group (p < 0.05 and 0.001, respectively). (Figure 8) 32 7.426.42 6.39 6.17 5.996.055.27 5.3102468Control PVP 25% 50% 75% 100% 150% 200%log cfu/mlVarious ratio of pvp-i/pvp scaffodermAfter 4 hours incubation*** Figure 7. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on MRSA. MRSA (USA300) was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 4 hours. CFU counting was performed after 14-18 hours, and data are shown as mean ± SD of samples, representative of at least 4 repetitions (n=4) of experiment. The result shows that 150% and 200% PVP-I/PVP MF have almost the same antibacterial effect on MRSA by almost 2 log reduction of bacteria compared to the control group. ***p < 0.001. *** *** *** *** *** *** 33 3.4) Evaluation of the antimicrobial activity of various ratios of PVP-I/PVP scaffoderm on P.A The results show that as the ratio of PVP-I/PVP scaffoderm increases, its antibacterial activity increases as well. 150% and 200% PVP-I/PVP scaffoderm had the most antibacterial effect on P.A with an almost 2-log reduction in the number of bacteria within a 4-hour period. Statistical 10.36 109.37024681012Control PVP 150%log cfu/mlvarious ratios of pvp-i/pvp scaffodermAfter 24 hours incubation***Figure 8. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on MRSA. MRSA (USA300) was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 24 hours. CFU counting was performed after 14-18 hours, and data are shown as mean ± SD of samples, representative of at least 2 repetitions (n=2) of experiment. The result shows that 150% PVP-I/PVP scaffoderm has almost 1 log reduction of MRSA compared to control group. * p < 0.05, ***p <0.001. * 34 analysis demonstrated that all groups were able to reduce the number of bacteria significantly in comparison with the control group (p < 0.001). (Figure 9) However, the outcomes resulted from the same experimental procedure on P.A, but within 24 hours of incubation, demonstrated that 150% PVP-I/PVP scaffoderm resulted in around 1 log reduction in the number of bacteria compared to the control group. Statistical analysis showed that this reduction in the number of bacteria is significant (p < 0.01). (Figure 10) 7.376.696.21 6.05 5.815.55 5.37 5.1102468Control PVP 25% 50% 75% 100% 150% 200%LOG CFU/mlvarious ratios of PVP-I/PVP scaffodermAfter 4 hours incubationFigure 9. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on Pseudomonas aeruginosa (P.A). P.A LUX was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 4 hours. CFU counting was performed after 14-18 hours, and data is shown as mean ± SD of samples, representative of at least 4 repetitions (n=4) of experiment. The result shows that 150% and 200% PVP-I/PVP scaffoderm have almost the same antibacterial effect on P.A by almost 2 log reduction of bacteria compared to control group. ***p < 0.001. *** *** *** *** *** *** *** 35 Our antibacterial studies demonstrate that 150% and 200% PVP-I/PVP scaffoderm have acceptable antibacterial effects on both gram-positive (MRSA) and gram-negative (P.A) bacteria after 4 hours of incubation. 10.7 10.219.750246810Control PVP 150%log cfu/mlvarious ratios of pvp-i/pvp scaffodermAfter 24 hours incubationFigure 10. Antibacterial studies with various ratios of PVP-I/PVP scaffoderm on Pseudomonas aeruginosa (P.A). P.A LUX was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 24 hours. CFU counting was performed after 14-18 hours, and data are shown as mean ± SD of samples, representative of at least 2 repetitions (n=2) of experiment. The result shows that 150% PVP-I/PVP scaffoderm has almost 1 log reduction of P.A compared to control group. ** p < 0.01. ** 36 3.5) The antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of MRSA and P.A As is apparent in Figure 11, the number of P.A colonies decreased in the PVP and 150% PVP-I/PVP scaffoderm groups compared to the control group. Since the purpose of adding iodine to MeshFill was to reduce bacterial contamination, which generally involves lower bacterial numbers, we chose to assess the efficacy of PVP/PVP-I to reduce a broad range of bacterial numbers. When a dose of 101 P.A was used as the initial inoculum, an almost 4-log reduction was observed by either PVP or 150% PVP-I/PVP. When a higher bacterial inoculation was used (102-104) only a 1-log reduction was observed for PVP alone, while an almost 2-log reduction was observed in the 150% PVP-I/PVP group. In addition, the reduction in both PVP and 150% PVP-I/PVP was around 1.5-log within 105 doses. Based on the statistical analysis, the logs of reduction were significant in both PVP and 150% PVP-I/PVP compared to the control group (p < 0.01) within various doses of P.A. 37 Figure 11. Antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of P.A. P.A LUX was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 4 hours. CFU counting was performed after 14-18 hours, and data are shown as mean ± SD of samples, representative of at least 3 repetitions (n=3) of the experiment. The result demonstrates that 150% PVP-I/PVP scaffoderm can lead to nearly 2 log reduction in most of the various doses of P.A. ** p < 0.01, *** p < 0.001. As is apparent in Figure 12, the number of MRSA colonies decreased in the PVP and 150% PVP-I/PVP scaffoderm groups compared to the control group. When a 101 dose of MRSA was used, an almost 4-log reduction was observed by utilizing either PVP or 150% PVP-I/PVP groups. However, this reduction reached nearly 1-log by using the PVP within 103 to 105 doses of MRSA, and almost 2-log in the 150% PVP-I/PVP group within the same doses of bacteria. The reduction in 150% PVP-I/PVP was around 3-log within the 102 doses. Based on the statistical analysis, the 3.935.236.057.18.204.084.756.116.8303.364.255.196.77012345678910^1 10^2 10^3 10^4 10^5LOG CFU/mlVarious doses of P.AcontrolPVP150%*** *** *** *** *** *** *** *** ** 38 reduction was significant in 150% PVP-I/PVP compared to the control group (p < 0.001) within various doses of MRSA. Based on the results shown in Figures 11 and 12, 150% PVP-I/PVP scaffoderm is able to significantly reduce the number of both gram-positive and gram-negative bacteria compared to the control group, and it demonstrated greater antibacterial effects than PVP. 3.994.626.156.628.0303.734.785.626.9101.444.154.896.28012345678910^1 10^2 10^3 10^4 10^5LOG CFU/mlVarious doses of MRSAControlPVP150% Figure 12. Antibacterial effect of 150% PVP-I/PVP scaffoderm on various doses of MRSA (USA300). MRSA was cultured and sub-cultured prior to experiments, and starting CFUs were determined. Experimental groups were added to 48-well plates and incubated for 4 hours. CFU counting was performed after 14-18 hours, and data are shown as mean ± SD of samples, representative of at least 4 repetitions (n=4) of experiment. The results demonstrate that 150% PVP-I/PVP scaffoderm can lead to a near 2 log reduction in most of the various doses of MRSA. ** p < 0.01, *** p < 0.001. *** *** *** *** ** 39 3.6) Release study The release study result reveals that 4 hours post-casting of the gel, nearly 70% of iodine is released from the 150% PVP-I/PVP complex, while after 8-10 hours, most of the existing iodine in the gel was released. It means that most of the iodine is released over the first 4 hours, leaving little iodine to be released after 8-10 hours. Figure 13. Release study on 150% PVP-I/PVP scaffoderm at different time points. The solidified 150% PVP-I/PVP scaffoderm was immersed in PBS and placed in a shaker incubator at 37 ℃. At different time points (2, 4, 8, 12, and 24 hours), samples were collected, and ICP-OES analysis was performed. The Y axis shows the percentage of iodine that has been released from 150% PVP-I/PVP scaffoderm and accumulated over a 24-hour period. The outcome reveals that after 8-10 hours, most of the existing iodine in the gel was released. 033.3369.9991.1 93.3294.4301020304050607080901000 5 10 15 20 25 30% of occumulation of Iodinetime points (hours)40 3.7) Wound healing study Day 3 Day 7 Day 14 Day 17 Figure 14. Representative digital images of top view of wounds at different time points. Full-thickness 7mm excisional wounds were created in the back of rats (two wounds /rat). To overcome the problem of wound contracture in rodent wounds, we used a silicone-splinted wound model. Five experimental groups (3 rats /group) remained untreated (control) or tested with either PVP alone or PVP-I with different concentrations:100%, 150%, and 200% that are related to scaffoderm containing 0.06, 0.09, and 0.12 mg of Iodine in 1000 cc of final scaffoderm respectively. The wound healing process was monitored every 3 days for up to 2 weeks. NT, PVP and 150% were monitored for up to 17 days. Wounds were photographed digitally to document changes in clinical appearance (redness, swelling, inflammation) and the rate of wound closure. The fastest wound closure was observed in the 100% group, while 200% showed slower healing compared to the other treatment groups during 2 weeks. No treatment PVP 100% 150% 200% 41 As is apparent in Figure 14, wounds treated with 100% PVP-I/PVP scaffoderm showed faster wound closure and had the lowest area of the wound among other experimental groups during 14 days. During this time, wounds treated with 150% PVP-I/PVP scaffoderm showed the second-greatest wound closure. Based on the outcomes shown in Figure 15, it is apparent that 200% PVP-I/PVP scaffoderm didn’t accelerate the wound healing closure after 2 weeks in comparison with the No treatment group. Although 100% PVP-I/PVP scaffoderm had the best wound healing outcomes, we decided to continue the experiment with 150% PVP-I/PVP scaffoderm for up to 17 020406080100Day 0 Day 3 Day 7 Day 10 Day 14 Day 17Wound area (%)Time after wounding (day)NTPVP100%150%200%Figure 15. Changes in percentage of wound area with different treatments at various time points. Changes in the percentage of wound area in the no treatment group (NT) and wounds treated with either PVP alone or PVP-I with different concentrations:100, 150, and 200 percent are shown on days 0, 3, 7, 14, and 17. 42 days because this ratio had the best antibacterial effects. The results reveal that this ratio of scaffoderm was able to reduce the wound size after 17 days compared to the control group. Based on the statistical analysis performed on the 100% and 150% groups, in 14 and 17 days after wounding, none of the experimental groups reduced the wound size significantly compared to the control group and did not have any deleterious effect on the wound healing process either. 3.7.1) H&E staining Histological outcomes demonstrate that re-epithelialization was achieved after 14 days in wound sites treated with 100% and 150% PVP-I/PVP scaffoderm. Re-epithelialization and wound closure were not observed in other groups. (Figure 16) Figure 17 shows the wound healing outcomes in wound sites treated with the PVP and 150% PVP-I/PVP scaffoderm after 17 days. Aside from re-epithelialization in the 150% PVP-I/PVP experimental group, hair follicles and sweat glands had re-formed within the dermal layer. In addition, the wound size was reduced in 150% compared to the control group after the same period. 43 No treatment PVP-40 100% 150% 200% Figure 16. H&E staining of thin sections of the skin from experimental groups of rats; 14 days’ time point. Arrows indicate a re-epithelialized wound area (C and D). A B C D E 44 No treatment PVP-40 150% Figure 17. H&E staining of thin sections of the skin from experimental groups of rats; 17 days’ time point. In image C, the arrow indicates a re-epithelialized wound area. 3.7.2) Masson’s trichrome staining Masson’s trichrome staining validated the results obtained from H&E staining. Re-epithelialization occurred in both wound sites treated with 100% and 150% PVP-I/PVP scaffoderm over a 2-week period in comparison with the no-treatment group. A B C 45 In Figure 19, the re-epithelialized wound and regenerated sweat glands were apparent in H&E staining, while the blue staining represents collagen formation in the wound area which represents the mechanical strength of the wound site. Collagen is also an important regulator of several processes in wound healing, such as inflammation and angiogenesis 66. No treatment PVP-40 A B 46 100% 150% 200% Figure 18. Masson’s trichrome staining of thin sections of the skin from experimental groups of rats; 14 days’ time point. Arrows indicate a re-epithelialized wound area (C and D). C D E 47 No Treatment PVP-40 150% Figure 19. Masson’s trichrome staining of thin sections of the skin from experimental groups of rats; 17 days’ time point. Arrows indicate a re-epithelialized wound area (B and C). C A B 48 3.7.3) Immunofluorescent staining K14 is an indicator of re-epithelialization, which shows the migration of keratinocytes from the intact skin near the wound site to the center of the wound area. As is apparent in Figure 20, keratinocytes migrated toward the wound area, and re-epithelialization occurred in both wound sites treated with 100% and 150% PVP-I/PVP scaffoderm. Comparing both Figures 20 and 21, it is apparent that wound sites treated with PVP re-epithelialized after 17 days, while they were not re-epithelialized at the 14-day time point. Also, the thickness of the epithelial layer in wound sites treated with 150% PVP-I/PVP scaffoderm reduced from 14 days to 17 days time point which means that the re-epithelialized layer is getting mature. 49 Figure 20. K14 staining on skin samples collected from rats with full-thickness wounds treated with PVP, 100%, 150%, and 200% PVP-I/PVP scaffoderm (B, C, D, and E). A is a control group in which wound areas weren’t treated with anything. Keratinocytes are shown in green. Arrows indicate re-epithelialized wound areas (C and D). Figure 21 shows that after 17 days, wounds in the no-treatment groups were re-epithelialized. As is apparent, keratinocytes migrated to the wound area, and re-epithelialization has taken place. A B C D E 50 However, comparing the wound area with the wound edge, it can be observed that keratinocytes are not differentiated completely, and re-epithelialization is not mature. Figure 21. K14 staining on skin samples collected from rats with full-thickness wounds treated with PVP, 150% PVP-I/PVP scaffoderm (B and C). A is a control group in which wound areas weren’t treated with anything. Keratinocytes are shown in green. Arrows indicate re-epithelialized wound areas (A, B, and C). B C A 51 Chapter 4: Discussion The purpose of this study was to find the optimum ratio of PVP-I/PVP scaffoderm, as a liquid scaffold loaded with an antibacterial agent (Iodine), to accelerate the healing process and prevent bacterial infections in wound sites. This study revealed that the optimum ratio of PVP-I/PVP scaffoderm which is 150%, can prevent infections originating from both gram-positive and gram-negative bacteria for a period of 4 hours and can also improve and accelerate the wound healing process in full-thickness wounds. Although 100% PVP-I/PVP scaffoderm didn’t delay the wound healing process in full-thickness wounds, since it was less effective than 150% PVP-I/PVP scaffoderm in terms of antibacterial effects, we chose 150% as the optimum ratio that can prevent bacterial infection and accelerate wound healing. Balin and Pratt (2002) have shown that using a high concentration of 10% PVP-I on wound sites can inhibit fibroblast growth and delay wound healing 67. However, Kaspar, Schwarz, Claes, and Ignatius (2006) confirmed that using lower concentrations of PVP-I (<1%) is not associated with toxic effects on chondrocytes 68. Our studies demonstrated that using 150% PVP-I/PVP scaffoderm which contains a very small amount of Iodine (0.09 mg/mL), does not interfere with the migration of fibroblasts, but based on the in vitro toxicity test, it seemed that 150% PVP-I/PVP scaffoderm shows slight toxicity. However, based on the in vivo wound healing studies, it seems that 150% PVP-I/PVP scaffoderm does not interfere with the growth of fibroblasts and the wound healing process. It is not surprising to see slight differences between the in vivo and in vitro results because in vitro models provide researchers with a controlled and isolated environment, while running experiments on in vivo models are associated with more factors and biological variables that may 52 affect the outcomes. Although some of these variables are controlled by researchers, some others cannot be controlled by scientists. Koerner, George, Kissam, and Rosco (2018) have confirmed that povidone-iodine with a concentration of 0.25% was able to kill bacteria, including Moraxella nonliquefaciens, Propionibacterium acnes, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella oxytoca, and Pseudomonas aeruginosa over 30 seconds of exposure 69. Our studies demonstrated a 2-log reduction in the number of MRSA and P.A over a 4-hour period using the PVP-I/PVP scaffoderm. However, no antibacterial activity was observed after 24 hours. To better understand this, a release study was conducted to see the rate of released iodine. Based on this study, it has been observed that over the first 8 hours, 91% of iodine existing in the scaffoderm has been released into the environment. Hence, this is the reason why we see a loss in antibacterial activity beyond 4 hours. Gilmore, Reid, and Strokon (1977) assessed the effect of dry powder povidone-iodine (Disadine D.P.) on wound healing experimentally and clinically 70. They created a 4 cm vertical mid-line incision through all layers of the anterior abdominal wall on 72 female Wistar rats and evaluated the wound healing process after 7 and 28 days. Based on their studies, dry powder povidone-iodine didn’t interfere with wound healing mechanically, microscopically, or histologically. In addition, Goldenheim (1993) evaluated the effect of four forms of PVP-I, including PVP-I solution, PVP-I skin cleanser/surgical scrub, PVP-I ointment, and PVP-I cream, on wound healing. In this study, 10% PVP-I solution was applied to rat, guinea pig, rabbit, or pig wounds, and the outcomes demonstrated that this solution didn’t reduce the tensile strength of the wound and didn’t delay re-epithelialization 71. Our studies also revealed that full-thickness wounds treated with 100% or 150% PVP-I/PVP scaffoderm, both re-epithelialized after 2 weeks. 53 However, the wound area in the other three groups, including no treatment, PVP, and 200% PVP-I, didn’t re-epithelialize during the same period. Furthermore, wounds treated with 150% PVP-I/PVP scaffoderm not only accelerated the wound healing process and re-epithelialization but also, after 17 days, hair follicles formed in the wound sites compared to the control group, which shows that the maturation process has started. Also, more collagen was observed in the wound site, which can be one of the indicators of mechanical strength in the wound area. Although neither of the 100% and 150% PVP-I/PVP scaffoderm reduced the wound size significantly, both helped to accelerate wound healing. Based on a review article, povidone-iodine accelerates wound healing due to the following reasons: 1. Wide spectrum of antimicrobial activity, 2. Preventing biofilm formation, 3. Lack of resistance, 4. Favourable tolerability, and 5. Preventing the effect of excessive inflammation caused by both microbes and the host response 53. Based on bacterial tests, both PVP and 150% PVP-I/PVP scaffoderm were able to reduce the number of bacteria significantly. Although the reduction is not significant biologically, it is significant statistically. Despite the fact that 1- or 2-log reduction is not significant biologically, it is still enough to prevent the colonization of bacteria in the wound site. As far as we know, PVP itself doesn’t have any antibacterial effect, and iodine is the antibacterial agent. However, since PVP led to a reduction in bacteria, it may be due to the change in the form of the scaffoderm from liquid to gel. One of the limitations in performing this study was that we couldn’t assess the true efficacy of PVP-I/PVP scaffoderm to inhibit colonization since we have no in vivo model to accurately mimic this because bacterial colonization occurs with a low amount of bacteria, and since in in 54 vivo studies immune system is able to clear the low amount of bacteria, so the outcomes would not represent the true efficacy of this scaffoderm. In addition, although rat models are used as a proper animal model for wound healing studies, wound healing in rats is not the same as in humans, so more experiments on bigger animal models like pigs and then clinical trials on humans, need to be performed. 55 Chapter 5: Conclusion Based on in vitro studies, 150% PVP-I/PVP scaffoderm is capable of preventing bacterial growth and infection in the first 4 hours after applying the scaffoderm, but it cannot be considered a treatment after the infection happened. Based on the results, both 100% and 150% PVP-I/PVP scaffoderm had positive effects on the wound healing process, but neither of them accelerated the wound healing process significantly. On the other hand, both ratios reduced the number of bacteria significantly after 4 hours incubation period, but 150% showed better antibacterial effects than 100%. Therefore, we chose 150% PVP-I/PVP scaffoderm over 100% ratio one as it showed better antibacterial effects and it had acceptable wound healing outcomes. Due to having a limited number of PVP-I/PVP scaffoderm samples, we couldn’t run more in vivo wound healing-related experiments on 100% and 200% PVP-I/PVP scaffoderm at 17 days time point, and we just decided to choose the best ratio of PVP-I/PVP scaffoderm that has the best antibacterial activity and acceptable wound healing effects to carry out more experiments on the wound healing aspect of that. This is why in figure 14. the images for 100% and 200% are not applicable. Furthermore, 150% PVP-I/PVP scaffoderm could accelerate the wound healing process in full-thickness wounds and help wound closure, and re-epithelialization happens faster compared to the No-treatment group. Based on the results derived from different staining on experimental groups of skin tissues, it is apparent that the maturation phase started earlier in the wound areas treated with 150% PVP-I/PVP scaffoderm, and the re-epithelialized area of this group is stronger than the control group due to the emigration of collagen to this site. 56 Overall, after the 150% PVP-I/PVP scaffoderm was applied on the full-thickness wound site, iodine starts to release from the povidone-iodine reservoir immediately. Released iodine kills the microbes, including gram-positive and gram-negative bacteria, promptly. In addition, iodine and metabolite iodide have anti-inflammatory effects through radical scavenging activity and destruction of the pathogenic factors, cytokines, and tissue-destructive enzymes. It was also shown that PVP-I has anti-inflammatory effects by impacting the human inflammatory effector cells and inflammation mediators 72. Therefore, this scaffoderm creates an ideal setting for cell growth by inhibiting microbial infection and excessive inflammation, which eventually leads to wound healing. In conclusion, 150% PVP-I/PVP scaffoderm is able to prevent infection and accelerate the wound healing process in full-thickness wounds. 57 Bibliography 1. Jeschke, M. G., van Baar, M. E., Choudhry, M. A., Chung, K. K., Gibran, N. S., & Logsetty, S. (2020). Burn injury. Nature Reviews Disease Primers, 6(1), 1-25. 2. Smolle, C., Cambiaso-Daniel, J., Forbes, A. A., Wurzer, P., Hundeshagen, G., Branski, L. K., Huss, F., & Kamolz, L. P. (2017). 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Lange, Dirk (Microbiologist)
Evaluating the anti-bacterial and wound healing promoting effects of PVP/PVP-I containing liquid scaffold in vitro and in vivo
Text