The Effects Of Lysozyme-balsa Dressing On Wound Healing And The Underlying Mechanism

Backgroud:Patients with large skin defects urgently require effective coverage of the wound surface to prevent bacterial invasion and loss of mucus,water electrolytes,and energy.A wound dressing can reconstruct the skin barrier and accelerate wound healing,thus creating conditions appropriate for subsequent surgery.An ideal wound dressing should have excellent mechanical properties,proper water and air permeability,and outstanding biological compatibility.Infections and imbalance in inflammatory responses retard or prevent wound healing.An ideal wound dressing should also provide an aseptic microenvironment for the wound.Natural materials,including chitosan,chitin,glucans,celluloses,alginate,and fibroin,are thought to be ideal materials for preparing wound dressings,because of their high bioavailability and good biological compatibility.As such,promoting healing of wound infections by a combination of the natural materials and various antibacterial agents,growth factors,or other chemical substances has become a focus of research in the field of wound repair.Wood is a renewable resource characterized by low density,high modulus,strength,and toughness,and low heat-conductivity coefficients.Balsa is the lightest commercial timber in the world.It is used to make various structures,because of its low density,uniform texture,low heat-conductivity coefficient,ease of processing,and good volume stability.Studies have shown that balsa can be modified to form novel transparent lightweight structures,maintaining its cellulose structure while permitting light transmittance up to 90%.It is relatively inexpensive,with high air permeability and transparency.It also has functions relevant to wound-surface dressings.Lysozyme is a small monomer protein,containing 129 amino acid residues,with anti-inflammatory,antiviral,antibacterial and antihistamine activities.Currently,it has been widely used in antibacterial practices.The bactericidal mechanism is mainly manifested as biochemical function,that is,to catalyze and hydrolyze the peptidoglycan layer which is the basic component of cytoderm,and to destroy the cytoderm of the microorganism,thus achieving bacteriolysis.Lysozyme is an ideal medical enzyme,which can deactivate virus by interacting with virus protein with negative charge.When used as an enzymatic antibiotic,it can participate in the metabolism of mucopolysaccharide and have an anti-inflammatory effect.It can also make various acidic substances inactivate the inflammatory reaction,enhance the curative effect of antibiotics and other drugs,improve the tissue metabolism of mucopolysaccharide,thus reducing inflammation and achieving tissue repair.Therefore,lysozyme molecules,as non-specific immune factors in normal body fluids and tissues,have a variety of pharmacological effects.They participate in the immune response of the body,and play an important role in maintaining the physiological balance of the body.In the second chapter of this paper,the balsa structure was successfully optimized by using lignin removal techniques and dopamine glue bonding method was adopted to adhere different concentrations of lysozyme to it.Meanwhile,scanning electron microscopy was used to observe its micro-structure,Fourier transform infrared spectroscopy and contact angle to detect its chemical and physical properties,drug-loading rate and entrapment efficiency to detect the loading condition of lysozyme,and drug release test to detect the sustained release effect of lysozyme.Staphylococcus aureus and escherichia coli were cultured together to detect its antibacterial activity in vitro,and CCK-8 method was used to detect its inhibition on cell proliferation in vitro.Finally,mice infected wound models were established to observe the general healing conditions of mice’s wounds.The length of newborn epithelium was detected by haematoxylin-eosin(HE)staining.In the third chapter of this paper,with the successful preparation of new type of lysozyme-balsa dressing,translucent technique was adopted to optimize balsa structure without lignin and cinnamic acid,caffeic acid and cumaric acid were used as modifiers,which were compared and screened out for the best one.In the fourth chapter of this paper,we will make clear the mechanism of new type of dressing promoting wound healing.On the basis of adopting the same biological evaluation method with raw materials,RT-PCR,WB,scratch assay,cytoskeleton staining were used for further research.It was concluded that modified lysozyme-translucent balsa can effectively promote healing of infected wound and influence relevant molecule mechanism.ObjectiveIn this study,It is to discuss the preparation of lysozyme-balsa dressing and its influence on healing of infected wound.And to optimize preparation process of relevant materials,prepare modified lysozyme-translucent balsa dressing and study its molecular mechanisms,thus providing a new measure for the clinical treatment of infected wound.Methods1.Preparation of lysozyme-balsa dressingThe original balsa structure was optimized by using lignin removal techniques and dopamine glue bonding method was adopted to adhere different concentrations of lysozyme.2.Characterization of lysozyme-balsa dressingScanning electron microscopy was used to observe its micro-structure,Fourier transform infrared spectroscopy to observe whether related substances were adhered and contact angle to detect changes in hydrophilicity and hydrophobicity of dressings.Drug-loading rate and entrapment efficiency were used to detect the loading condition of lysozyme,and drug release test to detect the sustained release effect of lysozyme.3.The effect of different concentrations of lysozyme-balsa dressing on wound healingStaphylococcus aureus and escherichia coli were co-cultured together to detect the antibacterial activity of dressings,and then CCK-8 method was used to detect its biotoxicity by co-culture of dressings and fibroblasts.Finally,full-thickness skin defect infection mice models were constructed according to in vitro experiment results to detect the influence of dressings on wound healing rate,complete healing period.HE staining was carried out to explore changes in length of newborn epithelium.4.Preparation of modified lysozyme-translucent balsa dressingTranslucent-balsa was made by MMA and cinnamic acid,caffeic acid and cumaric acid were used as modifiers.Bacteria circle,minimal inhibitory concentration,bacterial co-culture,proper pH+temperature+time,determination ofhydrophobicity and secondary structure were used to screen out the best modifier.5.Characterization of modified lysozyme-translucent balsa dressing and its biological functionLysozyme standard curve was made,dressing loading rate and entrapment efficiency were dtermined and their release curve in vitro was determined.Staphylococcus aureus and escherichia coli were co-cultured together to detect the antibacterial activity of dressings,and then CCK-8 method was used to detect its biotoxicity by co-culture of dressings and fibroblasts.Finally,full-thickness skin defect infection mice models were constructed according to in vitro experiment results to detect the influence of dressings on wound healing rate,complete healing period.HE staining was carried out to explore changes in length of newborn epithelium.6.Mechanism of modified lysozyme-translucent balsa dressing promoting wound healingRT-PCR was used to detect the influence of dressings on Wnt3 a,β-catenin,and PCNA mRNA,the influence of dressings on cell migration in 24 h was observed by using MTT assay.WB was adopted to detect changes of signal pathway related proteins including Wnt/β-catenin and migration proteins including F-actin,vinculin.Results1.Characterization of lysozyme-balsa dressing1.1 lysozyme-balsa dressing was successfully prepared and they were divided into Control group(balsa),group A(dopamine glue bonding),group B(5 mg/ml lysozyme),group C(10 mg/ml lysozyme),group D(15 mg/ml lysozyme)and group E(20 mg/ml lysozyme).1.2 Under high power scanning electron microscopy,conglobate lysozyme spherical particles and the bedding of dopamine colloid layer.Fourier transform infrared spectroscopy showed that lysozyme had been adhered to and relevant absorption peaks were detected.Contact angle test indicated that with the gradual increase of lysozyme concentration,the hydrophilia of materials also increased gradually.2.The effect of different concentrations of lysozyme-balsa dressing on wound healing2.1 In vitro experiment showed that entrapment efficiency of lysozyme-balsa dressing could reach 60%,48 h release curve of dressings in group E could reach 92.3%.The bacteriostasis test showed that lysozyme-balsa dressing could effectively inhibit the growth of staphylococcus aureus and escherichia coli(p<0.05)and only group E had slight inhibitory effect on the growth of fibroblast at 7 d(p<0.05).2.2 In vivo experiment indicated that the 7-day wound healing rates of mice in group A-E were 30.7%,38.3%,50.7%,61.2%,61.9% and 62.4%(p<0.05)and the complete healing period of group D and group E were the shortest(p<0.05).At 7 d,the length of newborn epithelium in group A-E were 631.7 μm,702.5 μm,759.4 μm,825.3 μm,831.7 μm and 836.6 μm(p<0.05).3.Preparation of modified lysozyme-translucent balsa dressing3.1 Research on enzyme activity indicated that p-Coumaric acid were the best in antibacterial circle diameter,minimal inhibitory concentration,bacterial co-culture and other indexes,better than caffeic acid and cinnamic acid(p<0.05)and p-Coumaric acid was also the best one in surface hydrophobic index and secondary structural stability.Besides,p-Coumaric acid-lysozyme had the best effect at the temperature of 45 ℃,pH=6 and reaction time of 30 min.3.2 The research was re-designed according to modified dressings and there were four groups: group A(balsa),group B(translucent balsa),group C(translucent lysozyme-balsa)and group D(translucent balsa-p-Coumaric acid-lysozyme).4.The characterization and biological function evaluation of translucent balsa-modified lysozyme dressing.4.1 Material characterization showed that the loading rate and entrapment efficiency of group D were all better than that of group C(p<0.05)and the 72 h release rate of group C and group D were 85.2% and 93.8%.Group A was lower than group D in tensile strength(p<0.05)and D≈C≈B>A in water vapor permeability(p<0.05).4.2 Biological assessment indicated that D > C > B ≈ A(p<0.05)in the anti-bacterial activity to escherichia coli and staphylococcus aureus after 24 h while the toxicity test with fibroblast showed that all groups had no evident cytotoxicity(p<0.05).After 7 d,the wound healing rates of mice were 30.6%,48.3%,56.7%,70.9% and 79.2%(Control group,group A-D)and D > C > B > A > Control in the length of newborn epithelium(p<0.05).5.Mechanism of modified lysozyme-translucent balsa dressing promoting wound healing5.1 Cell proliferation mechanism: RT-PCR showed that D > C > B≈ A > control(p < 0.05)in proliferation of Wnt3 a,β-catenin and PCNA mRNA and detection of Wnt/β-catenin signal pathway by WB found that D > C > B ≈ A > Control(p< 0.05)in expression of proliferin PCNA and D < C < B ≈ A < Control(p< 0.05)in Axin expression,but there was no difference in expression of GSK-3β between the different groups(p>0.05).When Wnt/β-catenin signal pathway inhibitor XAV939 was added,the expression of Wnt3 a,β-catenin and PCNA proteins decreased while Axin2 expression increased(p<0.05).5.2 Cell migration mechanism: The 24 h migration test showed that D > C > B ≈ A > Control(p<0.05)in migration rate.The influence of WB on migration related protein F-actin and vinculin showed that D > C > B ≈ A > Control(p<0.05),with a consistent trend.ConclusionFrom the research results of this subject,the following conclusions can be obtained:1.lysozyme-balsa antibacterial dressing was successfully prepared by using lignin removal techniques and dopamine glue bonding method and related material characterization confirmed that lysozyme of proper concentration was successfully adhered.2.lysozyme-balsa can effectively inhibit the activity of staphylococcus aureus and escherichia coli,with low biological toxicity,and can effectively promote the healing of infected wound and the growth of newborn epithelium.3.Enzyme activity experiment showed that p-Coumaric acid was better in all indexes,so MMA and p-Coumaric acid were used to modify and prepare modified lysozyme-translucent balsa.4.Translucent lysozyme-balsa can effectively inhibit the activity of staphylococcus aureus and escherichia coli in vitro,with good appearance,and has stronger and more stable effect on gram-negative bacteria.Besides,it can also effectively promote the healing of infected wound and the growth of newborn epithelium.5.Related mechanism research indicated that modified lysozyme-translucent balsa dressing can promote cell proliferation and migration,thus promoting wound healing.