Biomimicry Of Oil Infused Layer On 3D Printed Poly(Dimethylsiloxane): Non-fouling, Antibacterial And Promoting Infected Wound Healing

Background:Of the numerous threatens to skins,burn injury is a most dreadful healthy problem due to bacterial infection,which can damage both epidermis and dermis layers of human's skin,resulting in deeper wounds and even death.Therefore,preventing burn wound from invasive infection is of great significance to accelerate wound healing.Meanwhile,non-fouling is very important for wound dressing,because changing dressing may result in painful and harmful to neo-generated epidermis due to the adhesion of dressing to wound.Wound dressing in burn wound is to keep wound clean and protect the wound from the external contamination.The traditional wound dressings such as cotton gauze and bandages can fulfill surfacing barrier to external agitation,but in fact,the efficiency of reducing bacterial infection is of below expectation.Plus,they are prone to adhere due to wound exudate soaking into dressing fibers,which leads to non-tolerable pain in dressing change.The natural and synthetic alternatives using chitosan,alginate,silicone,polyacrylamide and polylactic acid are soft,biocompatible and able to absorb wound exudate thus maintaining desirable humidity for wound healing.Wound dressing to efficiently battle bacterial infection,adhesion and clearing microorganisms remains a challenge.Usually,incorporated antimicrobial agents include such as Iodine,triclosan,quaternary ammonium?QA?salts,silver,and N-chloramines.Polydimethylsiloxane?PDMS?,a hydrophobic,non-toxic,biocompatible polymer with rubber-like elasticity,holds a competitive place in the fabrication of medical devices.As biomimicry of insect shell in-out of liquid surface,PDMS was rendered a slippery surface by perfusing with a myriad quantity of a lubricating liquid.An anti-bacterial dressing cannot neglect bacterial attachment and biofilm formation.We thus employ non-fouling,lubricant-infused slippery polymers as proof-of-concept medical materials that are based on oil-infused polydimethylsiloxane?iPDMS?for this purpose.3D printing,an additive manufacturing technology,“draws”patterns on a substrate from bottom to up to patterned manipulated 3D shapes.Bioink and high-resolution mechanics render 3D printer to have more potential in bioengineering such as biosensors and tissue-engineering scaffolds and customized wound dressing.Objective:This study verified firstly whether iPDMS/AgNPs possessed non-fouling and antibacterial capacity from in vitro and in vivo.Staphylococcus aureus and Escherichia coli were chosen as the model bacteria for the antibacterial and non-fouling properties investigation.We detected the biocompatibility of iPDMS/AgNPs to mammalian cells and organs.Finally,iPDMS/AgNPs was applied to the murine full-thickness skin defect infected wound model to estimate the antibacterial and biocompatible characteristics in vivo.Materials and methods:MaterialsPolydimethylsiloxane?PDMS?and curing agent were purchased from Tianying Technology Industry Co.,Ltd,China.5 Cst silicone oil was bought from Beijing HagebesTechnology Co.,Ltd.Silver nanoparticles are bought from Dongge Nano Technology Co.,Ltd.3D Bioprinter was from HKable Biotechnology Co.Ink formulationThe printable ink was prepared by mixing the prepolymer agent with the curing agent in a mass ratio of 1:1,after thorough stirring,0.5%,2.5%?silver/total weight?of silver nanoparticles was added and then the mixture was sonicated in an ice bath for 1 hour.The mixture was kept in a hot-water bath at 50°C for 20 minutes for pre-curing to obtain the ink.3D printingAll wound dressing were architectured using a 3D bioprinter?HKable 3D?.The ink was carefully loaded into a 5 ml of extrusion syringe,which was fixed on the conveyor for extrusion through a 20G flat tip needle.During printing,different desired 3D shapes were encased in a glass slide according to gcode generated on Computer Aid Design?CAD?.After printing,the samples were kept on a hotbed?80??for 2 hours for a complete curing.Preparation of iPDMSAfter curing,the 3D printed PDMS membranes were washed double distilled water for3 times and rinsed with ethanol and dried.They were then transferred to a glass container full of 5 Cst silicon oil for 18 hours.Rheology CharacterizationTo study the printable ability of the ink,the dynamic oscillatory frequency sweep of pre-curing PDMS was performed for a constant frequency of 10 rad/s to study the storage moduli?G'?and loss moduli?G''?using a TA Discovery Hybrid Rheometer.Two different temperature were 50oC and 80oC;the dynamic viscosity was also observed in this experiment.Scanning electron microscopy observation?SEM?and Energy Dispersive Spectrometer observation?EDS?The morphological characteristics of the iPDMS/AgNPs samples mesh were observed by Scanning electron microscopy observation?SEM?and Energy Dispersive Spectrometer observation?EDS??Hitachi,S-3400N,Japan?.Briefly,the specimens were firstly washed carefully with deionized water,dried and sputter-coated with Au for 3 minutes,then observed under vacuum condition by SEM and EDS.Scanning transmission electron microscopy?STEM?observationA scanning transmission electron microscope?Zeiss LIBRA 200 FEG,200 kV?was used to detect the detailed morphology and size distribution of the AgNPs,and The compositional analysis was characterized by Energy Dispersive Spectrometer observation.Briefly,after the PDMS/AgNPs films were ultrasonicated in DDH₂O,a drop of the prepared solution was deposited on a carbon-coated copper grid and dried at room temperature prior to the STEM analysis.Ag+release testTo investigate the release of silver ions from the PDMS/AgNPs films,specimens?n=3?were each cut into?10×10mm?pieces and immersed in 6mL of phosphate-buffered solution?PBS?at 37°C in darkness.The total supernatant of each group was harvested at each determined points,i.e.day 1,3,5 and 7 after PDMS/AgNPs membrane immersed in PBS,then analyzed by inductively coupled plasma and atomic emission spectrometry?ICP-AES,Leeman,USA?.Contact Angle CharacterizationAn optical contact angle meter?JY-PHA,Shengding Co,.LTD.?was used to measure water static contact angles on flat substrates.The excess lubricant was removed from all iPDMS samples before testing.For every sample,15?l deionized water droplet was added on the substrate surfaces and the contact angle was obtained using JY-PHA software.AnimalsThe BALB/c mice?male,18-25 g?and transgenic neonatal mice of green fluorescent protein?GFP?were obtained from the Experimental Animal Department?EAD?of the Third Military Medical University?TMMU?.All the animals were raised in plastic cages individually for 7 days before the experiments.All the animals were fed with free access to both autoclaved standard rodent chow and water.Living conditions of all the animals were under standardized conditions?relative humidity:50%;circadian rhythm:12 h;and room temperature:25°C?.All of the animal experiments were performed according to the guidelines of TMMU.Cytotoxicity testCell cultureFibroblasts were extracted from green fluorescent protein?GFP?transgenic neonatal mice as described by Cheng et al.Briefly,skin tissue was obtained from transgenic neonatal mice,then washed three times with PBS,afterwards adding dispase II 0.5 mg/mL?Sigma,USA?and keeping at 4°C overnight to separate the dermis and epidermis.After removing the epidermis,the dermis samples were minced and digested in 1 mL of trypsin 0.25 mg/mL?Boster,China?for 10 minutes.Then,3 mL Dulbecco's Modified Eagle's Medium?DMEM,Gibco,USA?was added to interdict the digestion.Then the mixture solution was centrifuged at a speed of 1000 rpm for 6 minutes.Finally,the cells were incubated and collected in DMEM,streptomycin?100mg/mL?and penicillin?100 U/mL?at 37°C in a 5%CO₂ incubator.CCK-8 assayThe third-generation fibroblasts were seeded on 96-well plates in 3×10³/100?l/well.Afterward,the culture medium was replaced by the leach liquor extracting from CTPM and HTPM.The untreated medium was used as the blank control group.The biocompatibility of the materials was estimated by CCK-8?Dojindo,Kyushu,Japan?proliferation assays respectively on day 1,3,5 and 7 after seeded.Briefly,at each time point,replaced the medium with Dulbecco's modified Eagle medium,and CCK-8 solution10?l was added in each well,then incubated for 2h at 37°C.The mean value of optical density?OD?was at 450 nm reading by the assay reader of enzyme-linked immunosorbent?Thermo Varioskan Flash,USA?.Non-fouling assayThe normal Staphylococcus Aureus and Escherichia Coli were used for the non-fouling assay.To start with,the bacterial concentration was amplified?shake overnight?to 1×10⁹ CFU/ml,then adjusted to 1×10⁴ CFU/ml.The mesh were punched to 6mm diameter discs.After sterilized by 75%alcohol at room temperature for 20 min,the mesh was washed with PBS for three times.For bacterial adhesion and developing,200 ul dilution bacteria liquid was joined into the hole of containing the material and was incubated at 37°C for 1.5 h.Then the membrane was washed with PBS for one time and sticked to the bottom of the dish flatly.The preheated cooling to 45°C of LB AGAR was poured into the hole of containing the material.After solidification,the dish was developed for the night at 37°C.Last,the dishes'images were taken photos and counted.The test of antibacterial activity in vitroThe normal Staphylococcus Aureus and Escherichia Coli were used for the test of antibacterial activity in vitro respectively.First,bacterial stock 200mL was mixed with Luria-Bertani?LB?medium five mL,then shaked at 37°C at 200 rpm overnight.LB bacterial suspension was diluted to the predetermined initial concentration?OD=0.07;optical density at 600 nm?,then 500mL bacterial solution was added to each well of 24-well plate.Six groups membranes were punched into 10×10 mm in size discs,then sterilized by 75%alcohol for 30 minutes,followed by PBS washing for three times.Finally,the six groups membranes were immersed into the bacterial solution,incubated in the shaker incubator at 50 rpm at 37°C for 24 h.100mL of bacterial solution was transferred into the96-well plate,and the OD₆₀₀00 value were measured.Wound infection modelsTo test the potential roles in wound healing,wound bacterial counting,epithelialisation and granulation tissue were measured in a defect wound model of murine full-thickness skin.First,the BALB/c mice were anesthetized with 1%pentobarbital by intraperitoneal injection,then the dorsal surface of the mice was shaved by a razor and cleaned with 75%alcohol.Full-thickness wound at a size of 0.6cm in diameter at both sides of the mice back were created by a punch.We then prepared the microbial concentration of 10⁸CFU/mL of a mixture of standard Staphylococcus aureus and Escherichia coli.15 to 20 ul bacterial suspension was added to the wound hole.Finally,a standard 0.6 cm diameter meshwas placed close to the hole to represent the day 0 wound area.A digital camera was used to monitor the wound contour by taking pictures.Eight groups were prepared:negative control?not bacteria covered by the negative pressure membrane??A?,positive Control?adding bacteria covered by the negative pressure membrane??B?,PDMS?C?,PDMS+oil?D?,PDMS+0.5%AgNPs?E?,PDMS+2.5%AgNPs?F?,PDMS+0.5%AgNPs+oil?G?,and PDMS+2.5%AgNPs+oil?H?,respectively.Then we glued the adhesiv e biological membrane?Negative Pressure Wound Therapy Kit?NPWT-1?,China?on the surface of each wound as fixed membrane.The wound healing evaluationThe wounds were taken pictures on day 1,3,5 and 7 post-wounding,and measured by IPP 6.0 software.The wound healing rate was calculated by the formula:The wound healing rate=?O-R?/O×100%.O represents original wound area,and R represents residual wound area on the determined point.The bacterial wound countThe exudate?1 ul?of wound infection models was taken at day 3 and 7 post-wounding.After diluted 10⁴ times,the exudate was cultivated overnight.Then the wound bacterial colony was taked photos and counted.Hematoxylin-eosin?H&E?stainingAfter 3 and 7 post-wounding,the wound tissues were harvested and fixed in paraformaldehyde 4%,embedded in paraffin,sliced,and stained by Hematoxylin-eosin?H&E?.The new epithelial length and the granulation tissue thickness were measured by Image J.As most of the infected wounds had little granulation tissue on day 3post-wounding,thickness had histologically measured just on day 7 post-wounding.Statistical analysisWe expressed all data as the mean±SD?n=5?and analyzed using one-way ANOVA.Results:1.To investigate the printable ability of the ink,the dynamic oscillatory frequency sweep of pre-curing PDMS was performed for a constant frequency of 10 rad/s to study the storage moduli?G'?and loss moduli?G''?.it is advised that G''curve was higher than G'curve at the beginning but G'went up rapidly and exceeded G'',which indicates the transformation from liquid to solid occurring very fast.Also,in the temperature of 80°C,viscosity surged since the very beginning and reached its plateau in 500s.Therefore,This is a good indication for ink deposition onto substrate and printing can be heated to 80°C to facilitate the ink cured and to maintain a stable shape.2.Meanwhile,for the convenience of surface infusion,we considered using 5 Cst silicone oil for their similarity to PDMS in structure and polarity but better fluidity than PDMS.The 3D architecture of iPDMS/AgNPs wound dressing was obtained by using a versatile 3D bioprinter?3D HKable??.iPDMS/AgNP could be folded and stretched easily.It suggests that iPDMS/AgNP membrane have good flexibility.PDMS membranes decorated with silicon oil showed a smooth surface without bumps.AgNPs dotted the surface of oil-infused PDMS membranes with the average diameter 14.8 nm of the bump of the surface in keeping with the size of AgNPs?less than 100 nm?of the report.The dispersive energy analysis of iPDMS/AgNPs shows that 1.1%Ag was found using EDS.The morphology and distribution of AgNPs were further investigated by scanning transmission electronic microscopy?STEM?.AgNPs were typically spherical and uniformly distributed on the PDMS surface,and the average diameter of AgNPs was 14.8nm,as determined by an Image J analysis.Together with STEM,the energy-dispersive X-ray spectroscopy?EDS?analysis of the PDMS/AgNPs nanocomposite demonstrated the presence of Ag.3.The release profile of silver ions from PDMS/AgNPs was examined at days 1,3,5and 7 in PBS using ICP-AES.Silver ions were rapidly released from PDMS/AgNPs at day1,which was subsequently followed by a relatively slow and sustained release.Briefly,the concentration of released silver ions was approximately 0.42mg/mL at day 1.Furthermore,constant silver ion release could still be observed at day 7,suggesting that PDMS/AgNPs had a prolonged and steady antibacterial activity.4.iPDMS/AgNPs with different AgNPs concentrations shows little inhibitory effect on cell proliferation from day 1 to day 7?p>0.05?.We found that iPDMS/AgNPs had no obvious cytotoxic effect on fibroblasts by the CCK-8 assay.The results are a coincidence with the previous study that cytotoxicity of AgNPs was only found at high concentration.5.An important indicator in anti-bacterial is the bacterial attachment and subsequent biofilm formation on medical devices.Two standard drug-resistant bacteria?Staphylococcus aureus and Escherichia coli?are common used in the clinical wound for in non-fouling assay.Obviously,the groups decorated with silicon oil had low bacteria colonies than the other groups.Notably,the groups infused with silicon oil had a better ability against adherence against S.aureus and E.coli than the corresponding group,which may efficiently improve wound healing microenvironment.6.The groups with 2.5%AgNPs had lower OD value than other groups,indicating a stronger capacity against Staphylococcus Aureus or Escherichia Coli respectively.Our research results showed that iPDMS/AgNPs in vitro could protect effectively infected wounds,which may efficiently promote wound healing as previous studies that the better antibacterial activity against S.aureus than E.Coli of mesh may attribute to the different cellular wall structures between S.aureus and E.Coli.7.To further test iPDMS/AgNPs'effect on the wound healing,the number of the bacterial colony,wound infection,the new epithelial length and the granulation tissue thickness were investigated in a murine full-thickness skin defect wound model.On day 3and seven post-wounding,the wound healing rate of E,F,G and H groups were higher than that of group B?p<0.05?.On day 7 post-wounding,the wound healing rate of group H is higher than that of group A?p<0.05?,which revealed iPDMS/AgNPs could significantly promote wound healing compared with other groups,consistent with previous study.At day 3 and 7 post-wounding,the number of the bacterial colony of group B was higher than that of A,E,F,G,and H group?p<0.05?,which shows iPDMS/AgNPs have effective antibacterial activity in vivo.The wound healing was decided by the re-epithelialization for the compact-skin species.The histological analysis shows that the neo-epithelial length and the granulation tissue thickness were obviously increased in the presence of for iPDMS/AgNPs.In short,iPDMS/AgNPs can promote neo-epithelial and granulation tissue formation to promote accelerated wound healing,which due to excellent biocompatibility,a better ability against adherence,and effective antibacterial activity of the iPDMS/AgNPs,with the same with previous studies.Conclusion:We developed a novel wound dressing iPDMS/AgNPs using 3D printing method and biometics of the oil-infused layer to cater for different requirements from patients in burn injuries.The eco-friendly iPDMS/AgNPs novel wound dressing not only exhibited excellent biocompatibility,longer service longevity,a better ability against adherence,and effective antibacterial activity but also could effectively promote the wound healing.In a nutshell,iPDMS/AgNPs can potentially be served as an ideal wound dressing in the near future.