Preparation And Healing-promoting Mechanism Of Chitosan/silk Proteins Composite Enhanced Adhesive Gel

The skin is the body’s organ in direct contact with the outside,and its thermoregulatory and sensory functions to external stimuli are essential to protect the body from external injuries.Damage to the skin is inevitable in daily life,and millions of people suffer from skin breakdown defects every year due to car accidents,surgical trauma,and chronic diseases.As a result,wound management has also become a major challenge worldwide.Conventional wound dressings cannot achieve tight adhesion to wounds in a dynamic environment and perform poorly in promoting wound healing.Various synthetic dressings with high adhesion have poor biocompatibility and tend to cause wound tissue rejection.New highly adhesive wound dressings with good biocompatibility can provide a better healing-promoting microenvironment for various types of damages,which is of great importance for better and faster healing of wounds.In this thesis,a chitosan/silk protein composite reinforced viscous gel was prepared using a formic acid/calcium chloride solubilization system.This silk protein-based strong viscous gel was composed of an orderly assembly of the functional matrix element of silk protein and two composite reinforcers（chitosan non-fabric reinforcer and inorganic nano calcium carbonate reinforcer）.The following results were obtained:（1）Exploration of optimal preparation conditions for chitosan/silk proteins composite enhanced adhesive gelThe dissolution and regeneration of the silk protein is the key to applying silk protein-based materials,and its dissolution method will directly affect the properties of silk protein-based viscous gels.Based on this,this study was conducted to dissolve the silk protein by formic acid/calcium chloride dissolution system.There are many factors affecting the dissolution process,and it is the key of this study to evaluate the degree of influence of each factor and to obtain the best preparation conditions for highly viscous silk protein-based gels.To this end,the optimal preparation conditions of chitosan/silk proteins composite enhanced adhesive gels were investigated in this paper,and the following conclusions were obtained:In this study,silk protein was dissolved by a certain formic acid/calcium chloride solution concentration.Then a specific area of chitosan nonwoven fabric（CS,50 g/m²）was added to the dissolved silk protein solution as reinforcement and placed in a fume hood until the formic acid evaporated to obtain silk protein composite reinforced gel（MSC）.Coated-Ca CO₃ was obtained by forming an enteric polymer coating layer outside the calcium carbonate nanoparticles through electrostatic adsorption.The chitosan/silk protein composite enhanced viscous gel MSC@Ca CO₃was obtained.Preparation was obtained through single-factor experiments and response surface analysis MSC@Ca CO₃.The single-factor experiments and response surface analysis showed that（1）in the system of dissolving silk protein in formic acid/calcium chloride solution,the optimal conditions were 5.5 wt%of calcium chloride,8.3 wt%of silk protein and 86.1 wt%of formic acid;（2）in the process of spraying Coated-Ca CO₃,the mass fraction ratio between formic acid/calcium chloride was 7.82:2.18.The concentration of Coated-Ca CO₃ dispersion was 1.3 mg/m L,1 m L was taken and sprayed on the gel surface.Chitosan/silk fibroin composite enhanced viscous gel obtained under the optimal preparation conditions MSC@Ca CO₃ The adhesion performance is215.49±4.87 k Pa,the optimal viscosity when the data regression model reaches an ideal state.By comparing the predicted value with the actual experimental value,the error does not exceed 10%,indicating that the model data is reasonable and reliable.（2）Physicochemical properties of chitosan/silk proteins composite enhanced adhesive gelTo further investigate the physicochemical properties of chitosan/silk proteins composite enhanced mucoadhesive gels,their properties such as apparent morphology,compositional structure,adhesion properties,mechanical properties,self-healing properties,and in vitro model construction were characterized in this section,and the following results were obtained:Chitosan/silk fibroin composite enhanced viscous gel（MSC@Ca CO₃）due to the incorporation of chitosan non-woven fabric,it contains a large number of fiber structures inside.The average particle size of nano calcium carbonate particles sprayed on the surface of the gel is96.40±4.23 nm.Coated Ca CO₃ is obtained by coating nano porous Ca CO₃ with enteric polymer Eudragit L100-55.The average thickness of the enteric polymer coating layer is 15.00±3.46 nm.Scanning electron microscope display MSC@Ca CO₃.A large amount of Coated Ca CO₃ can be seen on the surface,indicating the successful loading of Coated Ca CO₃.At the same time,the viscosity test results show that MSC@Ca CO₃’s viscosity reaches 192.74±6.84 k Pa,and tensile tests show that its breaking strength reaches 100.33±7.35 k Pa.In addition to skin tissue,MSC@Ca CO₃ also exhibits adhesion to various substrates,including metal,rubber,plastic,glass,and other biological tissues.The results of the self-healing test showed that two gels of different colors were cut and placed together.After 30 min,the gels started to bond with each other,and after60 min,the boundary between the two different colored gels became blurred,and the different colors eroded each other.This indicates that MSC@Ca CO₃ can heal without external intervention or cracks,even under tension perpendicular to the cutting surface.Subsequently,the ability of MSC@Ca CO₃ to seal the broken tissue was confirmed by a series of in vitro models.（3）Healing-promoting properties of chitosan/silk proteins composite enhanced adhesive gelTaking advantage of the large number of pores on the surface of Nano-Ca CO₃,we encapsulated exogenous growth factor（a FGF）into Nano-Ca CO₃ to obtain a nano-calcium carbonate particle a FGF-Ca CO₃ containing acidic fibroblast growth factor（a FGF）,which was then coated with a FGF-Ca CO₃ by a p H-responsive enterolysis polymer Eudragit L100-55（which produces dissolution at p H>5.5）to coat a FGF-Ca CO₃ to obtain Coated-Ca CO₃.Coated-Ca CO₃was dispersed into FA-Ca solution sprayed on the surface of MSC@Ca CO₃ to obtain a highly viscous filamentous protein gel loaded with growth factor a FGF-MSC@Ca CO₃.The biocompatibility of a FGF-MSC@Ca CO₃ as a wound dressing was evaluated by hemolysis rate test,cellular assay and in vitro degradability.Experimental results show different concentrations of a FGF-MSC@Ca CO₃.The hemolysis rate did not exceed 4%,and the cell survival rate was greater than 90%.In vivo muscle implantation experiments,a FGF-MSC@Ca CO₃ had completely degraded by day 77.In addition,in vitro degradation experiments showed that a FGF-MSC@Ca CO₃’s degradation rate on the 21st day was 60.61±2.24%,indicating good stability.The above experiments show that a FGF-MSC@Ca CO₃ has excellent biocompatibility and can be applied to human skin tissue.To verify a FGF-MSC@Ca CO₃ To explore the healing-promoting effect of wounds,a model of animal back skin trauma was constructed.In the skin damage model,on day 3,there were inflammatory reactions such as redness and swelling on the surface of the commercial control group a FGF-MSC@Ca CO₃.Although there was no significant wound contraction in group A,there was no residual blood clot on the surface and no inflammatory reaction,indicating that growth factors had a certain promotion on the subsequent wound recovery rate.On day 13,a FGF-MSC@Ca CO₃’s healing efficiency reached 99.41±2.35%,superior to the commercial control Tegaderm^TM（94.14±2.68%）.In the wound closure model,a FGF-MSC@Ca CO₃,Within 11days,the wound was closed entirely without significant redness,swelling,or inflammation.There were no scars at 18 days,while in the commercial control group(ergo^?),there were still substantial scars at 18 days.Histopathological results also showed that a FGF-MSC@Ca CO₃ was effective in reducing abscess reaction at the wound site.The results from animal models indicate that a FGF-MSC@Ca CO₃ has excellent healing-promoting effects on skin breakdown and wound closure.In summary,this thesis presents a systematic study on the physicochemical properties and healing-promoting properties of chitosan/serine protein composite enhanced adhesive gels loaded with growth factors and pioneers a serine protein-based adhesive gel synergistically enhanced by chitosan fabric and calcium carbonate nanoparticles,and further promotes the healing-promoting effect of the adhesive gel by loading exogenous growth factors.This dressing provides a new idea for developing novel dressings that combine strong adhesion and mechanical properties and promote rapid skin wound healing and offers a unique reference for pioneering the transformation of promoting wound dressings from experimental research to clinical application.