In Situ Synthesis Of Nano Zinc Oxide/Bacterial Cellulose Membrane And Exploration In Vitro And In Vivo For Wound Dressing

Background and ObjectivesThe wound healing process is a very complex and dynamic process.It is affected by various local and systemic factors that can hamper the wound healing,such as hypothermia,pain,infection,radiation,tissue oxygen tension,the overall health or disease state of the individual,age and poor nutrition.Among these,wound infection is the major concern in the wound care management,especially in certain wounds,such as diabetic foot ulceration,chronic wounds and burn,which are associated with high volume of exudates and susceptible to microbial infection.Therefore,it is important to treat the wound immediately with appropriate care,and the proper dressing has to be selected.Bacterial cellulose(BC)is a natural polymer material which possesses many excellent physical and chemical properties such as good mechanical properties,high crystallization,gas permeability,translucency,plasticity,biodegradability and histocompatibility.Thus BC is an ideal material for wound dressing.Although BC is an ideal material for wound dressing,BC exhibits no antibacterial activity.Zinc oxide nanoparticles(nZn O)have been documented to possess antibacterial activity by the mechanism of ROS,the free zinc ions and destroy of bacterial cell wall because of electrostatic interaction and mechanical damage.However,in vitro genotoxic effect of n ZnO on human keratinocytes,neural cells,and epidermal cells has been reported.Till date,several methods,such as co-precipitation method,chemisorbed method,and in situ synthesis,have been reported for synthesis of nZn O/BC nanocomposites.The common concern about these n Zn O/BC nanocomposites is the poor stability of combination of Zn O and BC,which would influence the cytotoxicity of nZn O/BC nanocomposites.In the present work,we develop an advantageous method to in situ synthesize n Zn O/BC nanocomposite membrane(n Zn O/BCM),namely in situ synthesis of n ZnO using the chemically modified BCM as template.Then,we are also focusing on the stability,and designing the nZn O/BCM with appropriate concentration of nZnO,which possesses both antibacterial activity and favorable biocompatibility,and evaluating the role of the n Zn O/BCM for both antibacterial activity and thereby its role in infected wound healing.Method:1.Preparation of nZn O/BCM by in situ synthesis and characterization of physical properties of nZn O/BCM1.1.n ZnO/BCM was prepared via the method of in situ synthesis,taking the chemically modified BCM as template.The preparation process was conducted in two steps:chemical modification and in situ synthesis of n ZnO.Chemical modification:Firstly,BCM was swelled in 5.0(wt/v)%Li Cl in DMAc,which was stopped by pure DMAc.Then,the MA was added into the system and then maintained the reaction at 60°C for 3 h under mild magnetic stirring.In situ synthesis of n ZnO:nZn O were in situ synthesized by hydration method.n ZnO were synthesized by the reaction of zinc acetate and sodium hydroxide using chemical modified BCM as template.The binding stability of n ZnO of the n ZnO/BCM was tested.1.2.The structure of n ZnO/BCM were observed by scanning electron microscope(SEM).1.3.Measurement of the mechanical properties and water vapor transmission rates(WVTR)of the membranes.2.Screening of Zn O/BCM with appropriate nZn O content and evaluation of biosafety of n ZnO/BCM2.1 Evaluation of antibacterial activity of the membranesThe antibacterial activity of 35 wt.%,25 wt.%,15 wt.%and 5 wt.%n Zn O/BCM was tested by shaking flask method.The bacteria were co-cultured with the membranes for24h at 37°C in a shaking incubator.2.2 Cytotoxicity test of the membranesThe cytotoxicity of aforementioned samples was detected by MTT assay according to protocol of ISO 10993-5:2009 Biological Evaluation of Medical Devices_Part 5-In Vitro Cytotoxicity Test.L929 cells were cultured with the extracts of the membranes.The n Zn O/BCM,which had both antibacterial activity and no cytotoxicity,was selected.2.3 Skin irritation testSkin irritation of the selected membranes was tested according to the protocol of ISO10993-10:2009 Biological Evaluation of Medical Devices_Part 10:Stimulation and Delayed Type Hypersensitivity Test.BCM was taken as negative control.20%SLS was taken as positive control,and albino rabbits were used as animal model.The samples were attached to the skin of rabbits.Erythema,edema and other reactions of skin were observed after 1,24,48 and 72 h,respectively.2.4 Histocompatibility evaluationThe local reaction experiment after implantation was carried out to evaluate the histocompatibility of the selected membranes.3.The effects of the selected nZnO/BCM on the infected wound healingA mouse full thickness skin defect infected wound model,infected with 5×10~7cfu Staphylococcus aureus and 5×10~7cfu Escherichia coli,was used to study the effects of n Zn O/BCM on the wound healing and wound contraction;the number of the bacteria of wound tissue homogenate was quantified by the standard plate count methods;the number of inflammatory cell infiltration and the length of the newly formed epithelium was determined based on the hematoxylin and eosin(HE)section.Results:1.Preparation of n ZnO/BCM by in situ synthesis and characterization of physical properties of nZn O/BCM1.1 The carboxyl group-containing side chain groups were successfully introduced into BCM by chemical modification.1.2 The chemically modified BCM enhanced binding stability of nZn O.After autoclave sterilization and extraction,only 8.98%of n ZnO was released.1.3 The average tensile strength,Young's modulus,the elongations at break and the WVTR of nZn O/BCM were 29.68MPa,2387.41MPa,1.30%and 2856.60 g/m2·day.2.Screening of Zn O/BCM with appropriate nZn O content and evaluation of biosafety of n ZnO/BCM2.1 The antibacterial rates of 35wt.%,25wt.%,15wt.%and 5wt.%nZn O/BCM against Staphylococcus aureus were 100.00%,99.98%,99.48%,and 96.73%,respectively.The antibacterial rates of 35wt.%,25wt.%,15wt.%and 5wt.%n ZnO/BCM against Escherichia coli were 99.50%,97.33%,93.10%,and 60.33%,respectively.2.2 The relative growth rate of L929 cells cultured in the extraction of 35wt.%,25wt.%,15wt.%and 5wt.%n ZnO/BCM was 4.43%,3.60%,0.90%and 109.08%,respectively.The selected 5wt.%nZn O/BCM had antibacterial activity and no cytotoxicity.2.3 The 5wt.%n ZnO/BCM was non-irritant to skin of New Zealand white rabbit,and favorable tissue compatibility was demonstrated for 5wt.%nZn O/BCM in a rat subcutaneous embedding model.3.The effects of the 5wt.%nZn O/BCM on the infected wound healing3.1 The analysis of the bacteria number in the wound showed that 5wt.%n Zn O/BCM could restrain bacteria growth.3.2 The quantitative analysis of the number of inflammatory cell infiltration demonstrated that 5wt.%nZnO/BCM could alleviate the inflammation reaction.3.3 The wound healing experiment results demonstrated that the wound healing was accelerated and the wound closure time was shortened when 5wt.%n Zn O/BCM was applied on the wound.3.4 The wound re-epithelialization was also demonstrated enhanced when 5wt.%n Zn O/BCM was applied on the wound.Conclusion:1.A novel n Zn O/BCM composite membrane prepared by innovative techniques possesses good nZnO bonding stability.2.The selected 5wt.%nZn O/BCM,which exhibits favorable physical properties,biosafety and antibacterial activity,is in line with the basic requirements of wound dressings;3.It is confirmed that 5wt.%n ZnO/BCM could inhibit bacterial growth in vivo,alleviate the inflammation reaction,and promote the infectious wound healing.And the5wt.%nZnO/BCM may serve as a kind of promising wound dressing.4.The present research lays a good theoretical,technological and practical basis for the further development of nZnO/BCM as medical dressings for the prevention and control of wound infections.