Preparation Of Biochemical Eggshell Membrane And Its Preliminary Study As GTR Membrane

In the clinical practice on periodontal tissue repair and regeneration, the lackof periodontal support tissue is one of the main causes of failure. How to preparethe guided tissue regeneration membrane with excellent performance is still achallenge we are facing. Natural egg shell membrane has good microbial barrierproperties, biocompatibility and the porous network structure with mutualpermeability. In addition, one side of the membrane is dense and the other side isosteoporosis, so it is similar to the guided tissue regeneration (GTR) membrane.But because of poor mechanical strength, natural eggshell membrane cannot bedirectly used for GRT film. Synthetic polymers, such as poly (lactic-co-glycolicacid (PLGA), polycaprolactone (PCL), etc., have high mechanical strength andgood spinnability, but their biocompatibilities are worse than natural polymers.Compared with other film-forming technology, electrospinning technology canprepare a membrane with good morphology, which was similar to intercellular substance. So, the electrospun membrane was more conducive to cell adhesion,growth and differentiation. Based on the GTR membrane performancerequirements, this study combines hydrolysis of acid solution, electrospinning,and porogen leaching technology together, to develop a new GRT membrane withboth excellent biocompatibility and good mechanical strength.This study contains four parts:1. Preparation and physical and chemical properties of SEP/PLGA fibermembraneSoluble eggshell membrane protein (SEP) was extracted from naturaleggshell membrane using hydrolysis of acid solution. Then different ratios ofelectrospun SEP/PLGA nanofiber membrane were prepared, The blendmembrane was observed and characterized with scanning electron microscope(SEM), contact angle, tensile mechanical tests and Fourier transform infraredspectroscopy measurement.The results indicated that interconnected porous network structures wereshowed in nanofiber membranes of different ratios. Moreover, infraredspectroscopy proved that both polymers were blended successfully. The tensilestrength and elongation at break increased were increased along with PLGAadded, and there was a significant difference between different ratios (P <0.05).With the increase of the SEP content, the contact angle became small whilehydrophilicity was improved, but there was no significant difference betweendifferent ratios (P>0.05). These meant that SEP/PLGA fiber membrane had acertain mechanical strength and hydrophilic.2. Degradation in vitro of SEP/PLGA fiber membraneThe SEP/PLGA fiber membrane was immersed in simulated body fluidsolution (SBF) for degradation, and after degradation for2,4,6weeks, its structure, surface morphology and fracture strength was detected.In conclusion, the quality and fracture strength of the SEP/PLGA membranedecreased with the extension of degradation time. In the first two weeks ofdegradation, the speed decreased faster, while the speed slower from2w to6w. Inthe degradation of6weeks, SEP/PLGA nanofibers swell and became adhesive,but there was still whole structure and a certain mechanical strength.3. Cytocompatibility of SEP/PLGA fiber membranePeriodontal ligament cells (PDLC) were cultured on SEP/PLGA membrane.Then the cytocompatibility of membrane was evaluated by3-(4,5-dimethylthiazol-2-yl)-2,5-dimphenyltetrazolium bromide (MTT) test.As a result, PDLC cells extended fully on the surface of SEP/PLGAmembrane, and the cells connected each other via pseudopodia. MTT test showthat PDLC cells had strong proliferative capacity in the fibrous membrane.4. Preparation and cytocompatibility of bilayer asymmetric SEP/PLGA-PCLMembraneBilayer asymmetry fiber membrane that consisted of a porous layer and adense layer was prepared by continuous electro-spinning combined porogenleaching method. The porous layer was SEP/PLGA fiber membrane with largepore size while the dense layer was PCL fiber membrane with small pore size.The pore size of bilayer fiber membrane was measured and calculated usingImageJ software. PDLC were cultured on the bilayer membrane and their growthon the porous layer and the dense layer could be observed. Then thecytocompatibility of the bilayer fiber membrane was evaluated by MTT testThe result showed that the pore sizes of SEP/PLGA layer (porous layer) andthe PCL layer (dense layer) were respectively21±1.3μm and3.3±0.4μm. PDLCcells cultivated in the bilayer fiber membrane could only grow along the surface of the dense layer, whereas can grow into the pores of the porous layer with alarge number.The research explored the feasibility of preparing SEP/PLGA-PCL bilayerasymmetry fiber membrane, and studied its physicochemical properties,degradation in vitro and biocompatibility properties. The prepared bilayerSEP/PLGA-PCL membrane could be used as a potential guided tissueregeneration membrane.