Preparation Of Porous Microspheres Based On Collagen/Bacterial Cellulose And Its Drug Adsorption/Release Behavior

As a component of animal tissue,Collagen?COL?demonstrates many excellent features,such as good degradability,biocompatibility and low activity of antigen.Bacterial cellulose?BC?,with its high crystalinity,three-dimensional mesh structure,good biocompatibility and degradability,has attracted extensive attention in the fields of biological medicine.Based on the good application prospect of tissue engineering technology in the field of medicine and the advantages of porous microspheres as tissue engineering scaffolds,the COL/BC functional porous microspheres with multi-structure and multi-component were prepared using COL and BC as raw material.At the same time,the adsorption/release behaviors of the porous microspheres were studied to elucidate their mechanisms.The primary application of the porous microspheres in bone tissue engineering was also discussed.The main contents include:?1?The type?COL was extracted from pig skin using acid expansion-pepsin hydrolysis method and analyzed by modern analytical testing technology such as SDS-PAGE,FT-IR,XRD,TGA,SEM and CP/MSA ¹³C NMR.The extraction process was optimized as follows:the degreasing agent was isopropanol,w_{leather piece}:V_degreasant=1:10,w_pepsin=5.0 wt%,the hydrolysis time was 60h,and the concentration of NaCl was 1.1 mol/L.Under this condition,the extraction rate of COL was 86.63%.The extracted COL had a certain fiber structure.The chemical groups were confirmed to the characteristic functional groups of COL,which was composed of 2?1 chains and 1?₂ chain as[??₁?₂??₂?₁],and was consistent with the typical peptide chains structure characteristics of type I COL.?2?A strain G.xylinus ZHCJ618?GenBank no.MG451840?with the BC yield of 3.56 g/L was isolated from kombucha,and it was identified as Gluconacetobacter xylinus by morphology of colony and cell,physiological and biochemical properties and 16S rRNA sequence comparative analysis.The analysis results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that,the product BC had a typical three-dimensional multi-layered three-dimensional network structure,and showed good heat resistance and high crystallinity.The BC fermentation medium was optimized by the response surface experiments as:sucrose 39.0 g/L,peptone 20.0 g/L,magnesium sulfate 1.0 g/L,sodium citrate3.5 g/L,xanthine 1.0 g/L,ethanol 1.0%?v/v?mL,pH 6.0.Under this conditions,the BC yield reached 6.15 g/L,which was 72.8%higher than that before optimization.?3?BC was oxidized to prepare 2,3-dialdehyde bacterial cellulose?DABC?by NaIO₄ using malaprade reaction.The preparation process of DABC was optimized by orthogonal experiment as:reaction temperature 40?,reaction time 14 h,w_NaIO4:w_BC=5:10,w_BC=0.4 wt%,pH 6.0,under this conditions,the molar concentration of the aldehyde group was 2.10 mmol/100 g,the recovery rate of DABC was 75.2%.Analyzing results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that-OH in BC molecules was successfully oxidized into-CHO,and the degree of oxidation of BC increased with the extension of oxidation time.The crystal characteristics of BC was not influenced by oxidation process,however partial cellulose chain of BC was fractured,the fibers began to curl,and molecules of cellulose became disordered,which caused crystallinity decreased,and the reduction degree of crystallinity was associated with the degree of oxidation.Also the thermal stability of BC was reduced after oxidation,but not by much.And it still had a three-dimensional network structure.?4?COL/DABC composite material?CDABC?was prepared by schiff-base reaction.The preparation process was optimized by orthogonal experiment as:reaction time 5 h,reaction temperature 30?,w_DABC:w_COL=1:1,under this conditions,the consumption rate of aldehyde group was 86.9%.Analyzing results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that schiff-base reaction was conducted successfully between DABC and COL.The crystallization characteristics of CDABC remained same as DABC,but the crystallinity was reduced.The number of holes in CDABC was reduced,however it still presented a three-dimensional network structure.And the thermal stability of CDABC was was not significantly reduced.?5?COL/BC porous microspheres were prepared using CDABC as raw material by template method combined with reverse phase suspension regeneration method and analyzed by SEM,particle size analysis and N₂adsorption–desorption experiments.The preparation process was optimized as:w_CDABC=4.0 wt%,w_{polystyrene template}=2.5 wt%,w _tween-80:w_span-80=1:3,w_surfactant=100wt%,w_IL:w_HD=1:11,emulsifying time 2 h,the surface of COL/BC porous microspheres prepared under this condition was rough and full of pores,and there were a large number of pore structures in the interior,the particle sizes of COL/BC porous microspheres were ranging from 8 to 12 m,S_BET=123.4 m²/g,V_Pore=0.59 cm³/g,D_BJH=198.5 nm.The adsorption isotherm to N₂ was belonged to type?,which conformed to the adsorption characteristics of mesoporous materials.And BSA molecules could be spread into the internal and attach to the inner wall of their pores through the process of drug loading with a drug-loading rate of 309.8 mg/g.?6?The porous microspheres were loaded with BSA as a model drug.The adsorption/release behavior of COL/BC microspheres was studied with drug-BSA as a model protein.The fitting results showed that,within the temperature range of 303.15³13.15 K,the linear Langmuir equation could better describe the adsorption equilibrium relationship between COL/BC porous microspheres and BSA molecules.The pseudo-second model can explain and predict the membrane diffusion kinetic process of microsphere adsorption on BSA better.Fitting curve of Kannan-Sundaram diffusion model showed that during the adsorption process,the adsorption rate was affected by both membrane and intra-particle diffusion.The results of adsorption thermodynamics indicated that,during the adsorption process,?H⁰=0.10 J/mol>0,which showed that the adsorption process is endothermic reaction.Within the adsorption temperature range of 283.15³13.15 K,?G⁰<0,and|?G⁰|showed a trend of increase with the increase of temperature,showed that the process is spontaneous,and the spontaneous tendency became greater with the increase of temperature.?H⁰=0.10 J/mol<84 KJ/mol,?G⁰ was between 18.47²0.43 KJ/mol,which showed that the adsorption was mainly a physical adsorption process.The results of release behavior of COL/BC porous microspheres showed that most of BSA loading into COL/BC porous microspheres gave priority to adsorbing on the inner wall of the pores,and the other was wrapped inside the mesh of polymer material,which was similar to framework drug loading system.The release of BSA in COL/BC porous microspheres would be affected along with the increase of ion strength in the damaged tissue changes of ionic strength,while the small change of temperature and pH of damaged tissue fluid had little influence on the drug release behavior of porous microspheres,and their drug release kinetic processes could be well described with the first-order release model.?7?The primary application of the COL/BC porous microspheres in bone tissue engineering was discussed using mouse MC3T3-E1 cells.The results showed that the COL/BC porous microspheres had a good biocompatibility.When the MC3T3-E1 cells were grown on COL/BC porous microspheres and COL/BC/BMP-2 porous microspheres,more calcium nodules were generated,the protein expression level of active factors such as Runx2,ALP,OPN,OCN and?COL was higher.Which showed that the COL/BC/BMP-2 porous microspheres with a multistage structure and component could effectively promote the adhesion,proliferation and bone differentiation of MC3T3-E1 cells,the structure design of the porous microspheres confirmed to desired effect well.Through this study,the COL/BC/BMP-2 porous microspheres with a multistage structure and component was designed and prepared;The mechanism of drug adsorption/release of COL/BC porous microspheres was clarified,and the equilibrium relationship between drug adsorption and release was established;The primary application of the COL/BC porous microspheres in bone tissue engineering was discussed.The study provides a theoretical basis for the application of porous microspheres in the slow/controlled drug release technology and chemical adsorption technology.A new type of porous microsphere material was provided for tissue engineering,drug sustained release,chemical adsorption and other fields,and a theoretical and technical basis were also provided for high value transformation of COL and BC.