Application Of Multifunctional Biomass Microgel Particles In Wound Healing

Part ?.Preparation and characteristic of multifunctional biomass particlesObjective: To prepare the multifunctional biomass hydrogel microspheres,screening out suitable biomass raw materials and characterizing the preparation process of microspheres by scanning electron microscope and ordinary optical microscope.Methods: Firstly,silica microsphere templates were prepared by microfluidic device using silica colloidal crystal particles(SCCBs).Then chitosan(CS)inverse opal microcarriers were prepared by the Yin-Yang template method.In this process,the viscometer was used to screen the appropriate chitosan solution concentration.Next,the CS scaffold was immersed in a temperature-responsive N-isopropylacrylamide(NIPAM)gel.After a while,the NIPAM hydrogel was polymerized by ultraviolet light irradiation.Finally,the CS scaffold was taken out of the NIPAM hydrogel,peeled off cleanly,and the ideal multifunctional biomass hydrogel microcarrier was obtained.Results: Through the viscometer screening,chitosan at 4% concentration was employed to prepare multifunctional biomass hydrogel microcarrier.From the results of scanning electron microscopy,it was observed that the SCCBs self-assembled into intact hexagonal closest packed structure on the surface and inside of microcarriers,indicating its space utilization rate reached the maximum.It was also observed that the self-assembled SCCBs showed a three-dimensional spherical shape from the optical microscope.In addition,after infiltration chitosan gel,a layer of gel-like substance was observed on the surface of SCCBs,indicating that the chitosan gel had fully pouring into the pores of the microsphere template.On the other hand,we also observed that the microspheres showed the highly ordered,three-dimensional porous microstructure,confirming the preparation of chitosan inverse opal scaffold.Scanning electron microscopy showed that the pores of the chitosan inverse opal scaffold were sufficiently filled with gel-like substances,which confirmed the infusion of temperature-sensitive hydrogels and the acquisition of multifunctional biomass hydrogel microspheres.After removing the SCCBs template,we observed changes in the color of the microspheres at this time.During the generating process,the reflection peak and color of particles in different stages showed the corresponding changes.Conclusions: The selected chitosan solution has suitable permeability and mechanical properties,which can ensure the stability of the chitosan inverse opal scaffold structure and the infusion of the temperature-sensitive NIPAM gel.From the macroscopic results and reflection peak data,the real development of chitosan inverse opal scaffolds and multifunctional biomass microparticles was obtained.Part ?.Drug encapsulation and controlled release experimentsObjective: In order to confirm the temperature-responsive property of overall scaffold caused by NIPAM hydrogel components,the effects of drug loading and temperature-responsive controlled release of multifunctional biomass hydrogel microspheres were explored.Methods: Different NIPAM pre-gel solution were previously prepared and mixed with FITC-BSA solution that was selected as model drug.After the preparation of drug loaded particles,for the temperature depending release experiment,the samples were suffered with different stimulates of temperature.In addition,fluorescent pictures of the particles were detected.Results: Confocal layer-by-layer scanning pictures showed that microcarriers could achieve full loading of FITC-BSA model drug.As a result of natural drug release,the microcarriers can continue to act until 30 th days.The particles could response to the surrounding temperature,then release the inner activities by shrinkage which was caused by the NIPAM hydrogel.This shrinkage was related to the concentration of NIPAM hydrogel.Additionally,under the drug releasing,the particles exhibited different colors and possessed relevant reflection peak.Conclusions: Microcarriers can sufficiently load drugs,and release encapsulated drugs in response to the stimuli of temperature.Under temperature stimulation,the drug release of the microcarriers was related to the concentration of the NIPAM gel.The development of NIPAM with higher concentration,the less drug releasing under the action of temperature stimulus.Instead,the lower NIPAM concentration meted to more drug release.In addition,it should be noted that the shrinking transformation of the hydrogel would result in changes in the distance between adjacent nanopores and the average reflective index of the whole particles.These results indicated that the biomass particle-based drug delivery system is advanced and intelligent and can monitor the drug release in real time.Part ?.In vitro antibacterial test of multifunctional biomass particlesObjective: In order to confirm the antibacterial properties of the overall scaffold endowed by the chitosan hydrogel component,in vitro antibacterial effect of the multifunctional biomass hydrogel microspheres was explored.Methods: Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were chosen as the bacteria in the antibacterial examinations.Subsequently,the isolates of bacteria were incubated with different CS inverse opal scaffolds fabricated by different chitosan solution.After 24 hours,the results of bacterial survival were recorded by confocal microscopy.Results: Compared with the control group,it was clearly observed that the number of dead bacteria on the CS scaffolds with the three different concentrations was increased both for E.coli and S.aureus.In addition,with improvement of CS concentration,the corresponding bacteria reduction rate also increased.Particularly,when the concentration of CIOS reached 4%,there were almost no living bacteria present on the scaffold,which displayed the best antibacterial ability.Conclusions: The chitosan hydrogel microcarrier scaffold had excellent antibacterial effects.And its action was related to the original chitosan concentration,showing a concentration-dependent role.In a word,with the chitosan higher concentration,the antibacterial effect increases.The reason for this phenomenon could be attributed to the greater number of inherent positive charges resulting from the higher CS concentration,so that 4% CIOS displayed best antibacterial activity.Part ?.Preparation of acute wound infection model and effectiveness of multifunctional biomass particles for wound healingObjective: In order to evaluate the effective of multifunctional biomass hydrogel microcarriers in wound repair and explore its potential mechanism of action,the animal experiments were approved.Methods: All the rats suffering from an infectious wound were randomly divided into four groups.These groups were treated with the different interventions.Among these groups,the PBS group was defined as the control group,and the other treatments were considered the experimental groups.Images of the infectious wounds in each group were photographed on days 0,3,5,and 7.Results: Clearly,the degree of tissue regeneration in the experimental groups was better than the control group.The minimum thickness of granulation tissue was observed in the PBS group.The thicknesses of granulation tissue in the CS inverse opal film and CIOS+hydrogel groups were better than the PBS group.The maximum thickness of granulation tissue was observed in the CIOS+hydrogel+FGF group.Compared with the PBS group,TNF-? and IL-6 were down-regulated in the CS inverse opal film,CIOS+hydrogel,and CIOS+hydrogel+FGF groups,because the CS played a role in the protection against bacterial infection.Moreover,it was also observed that the CIOS+hydrogel+FGF group had the greatest collagen deposition,followed by the CIOS+hydrogel group,then the CS inverse opal film group,and the lowest was the PBS group,which was clearly consistent with the findings of the histological analysis.It was found that these structures were densely distributed in the CIOS+hydrogel+FGF group,whereas little positive staining of CD31 and ?-SMA was detected in the PBS group.In the CIOS+hydrogel and CS inverse opal film groups,the vascular structures were present at a moderate level.Conclusions: Chitosan inverse opal scaffold had obvious effect in alleviating inflammation.Its slow-releasing of growth factor can promote the formation of new blood vessel tissue and granulation tissue in wounds.Among them,the multifunctional biomass hydrogel microcarrier group was capable of slow-release growth factor and antibacterial properties originated from chitosan.The advantages of chitosan inverse opal scaffold and temperature-responsive drug releasing,could promote the best effect of wound tissue repair.