The Fundamental Researches About DOX-loaded Nanofibrous Membrane Fabricated By Sequential Electrospinning For GTR/GBR Application

Guided tissue regeneration(GTR) and Guided bone regeneration(GBR), one of the effective methods for the treatment of periodontal disease caused by periodontal tissue damage, can effectively promote the repair and regeneration of the periodontal Tissue.The oral environment is filled with microorganisms, and the bacterial plaque could continuously form on the teeth, which may have a negative effect on the surgical treatment outcome. Traditional GTR/GBR only have the effect of mechanical barrier and physical isolation, and can't effectively inhibit the growth of oral bacteria and it's colonization on the root and periodontal tissue, which is a not ignorable defect for the repair and regeneration of periodontal tissue. An antibiotic-loaded GTR/GBR membrane, the combination of traditional GTR/GBR technical and local antibiotic therapy, could place a barrier over the denuded root surface and the debrided periodontal defect, on the other hand consistently release antibiotic to eliminate microorganism infection and inhibit the bacterial colonization, for the aim of providing a good environment for the repair of periodontal tissue repair and regeneration.Compared with self-organization, phase separation etc., electrospinning has some advantages, such as convenient, economical, easy to operate, for preparing drug-loaded GTR/GBR membrane. Furthermore, electrospinning nanofibers have the remarkable characteristics of good permeability, high surface area-to-volume, adjustable surface morphology and the high biocompatibility which can possibly enhance the adhesion, proliferation and growth of cells. However, there is an obvious initial burst phenomenon for the drug release for drug-loaded nanofibers fabricated by blending electrospinning, which is not conducive to cell attachment, growth and proliferation. After years of technological development, the sequential electrospinning has been widely used to the study and application of drug delivery, which could forming a multilayer nanofibrous membrane by a variety of electrospinning solution in a certain order. For multilayer nanofibrous membrane, the nanofibers on surface directly contact with tissue and could protective drug loaded in the intermediate, to improve biocompatibility of the drug-loaded membrane and reduce the initial burst release of the drug.In this study, we used the biodegradable polymer materials Poly ?-caprolactone(PCL) as the carrier to fabricate a multilayer drug-loaded nanofibrous membrane by the sequential electrospinning, in which the PCL-gelatin(PCL-GE) was as the surface nanofibers and the PCL-doxycycline hydrochloride(PCL-DOX) was as the intermediate layer. And evaluate its application to antibiotic-loaded GTR/GBR membrane according to some fundamental researches. Purposes:Used the biodegradable polymer materials PCL as the carrier to fabricate a multilayer drug-loaded nanofibrous membrane by the sequential electrospinning, which has both anti-bacterial and guided tissue regeneration function. In this multilayer membrane, the antibacterial drug-doxycycline hydrochloride(DOX) was loaded in intermediate layer, and the natural polymer materials-gelatin(GE) was added to the surface nanofibers to benefit the cell attachment and growth. Further, the morphology, structure, physical and chemical properties, drug release kinetics, cell compatibility and antimicrobial activity against oral bacteria were evaluated to explore the feasible and effective for it as a new antibacterial GTR/GBR membrane.Methods: 1. Prepared the PCL-GE/PCL-DOX/PCL-GE multilayered nanofiber membrane, in which the PCL combined with GE(PCL-GE) as the surface layer and the DOX-loaded PCL(PCL-DOX) nanofiber as the intermediate layer. Investigating the surface morphology of each layer and the multilayered fibrous structure by Scanning electron microscope(SEM). The surface hydrophilic of PCL-GE, PCL-DOX, and PCL-GE/PCL-DOX/PCL-GE was analyzed by contact angle measurements;The existence form of DOX in the fibers after electrospinning was detected by differential scanning calorimetry(DSC). 2. Determine the detecting wavelength for DOX and draw the standard curve between DOX concentration and optical absorption value. Further, analysis the drug release kinetics of PCL-GE/PCL-DOX/PCL-GE nanofiber membrane during 28 day experiment. 3. Seeded the 4th generation human periodontal ligament cells(h PDLCs) on PCL-GE/PCL-DOX/PCL-GE nanofibrous membrane to culture, and analysis the cell viability by Alarma Blue test. Further observed the cell attachment and morphology by laser scanning confocal microscope(LSCM) and SEM. 4. Determine the minimum inhibitory concentration(MIC) of DOX against Actinobacillus actinomycetemcomitans(Aa) and Porphyromonas gingivalis(Pg) by semi-dilution assay. And analysis the antibacterial effect of DOX-loaded nanofibrous membrane by agar diffusion test. Results: 1. PCL-GE/PCL-DOX/PCL-GE nanofibrous membrane showed significant Tri-layers structure, in which the DOX-loaded PCL fibers was entrapped in the intermediate layer. Each layer of fibers was intertwined without obvious stratification, and all of the fibers was smooth without significant beaded and other defects. The DSC analysis showed that the semi-crystalline DOX was distributed in nanofibers as an amorphous molecules. The contact angle measurement results show that the natural polymer materials-GE and hydrophilia DOX can significantly improve the hydrophilicity of PCL fibers. 2. In the range of 0.5ug/ml-0.2mg/ml, there was a linear correlation(y=9.9492x+0.0006, R2=1) between the concentration of DOX and its optical absorption value at 350 nm wavelength. Compared with single layer PCL-DOX nanofibers, the drug in tri-layers was released more slowly and stable with a decreased initial burst effect, and could sustained release up to 28 day.The minimum DOX concentration of eluent throughout the experimental is 0.9923?g/ml for tri-layer membrane and 3.1388?g/ml for PCL-DOX membrane. 3. Alarma Blue showed the h PDLCs viability and growth on tri-layers membrane was better than PCL, PCL-DOX membrane, and had no difference with PCL-GE group. LSCM and SEM showed that there was obvious pseudopod closely attached to the fiber for seeded on PC-GE and tri-layers membrane. While h PDLCs on PCL-DOX showed synapse-like stretch on the surface of nanofibers. 4. The MIC of DOX against Aa and Pg was 0.125?g/ml and 0.0625?g/ml respectively, which is lower than the DOX concentration of elution in drug release test before. And the DOX-loaded nanofibers show an antibacterial activity against Aa, Pg. Conclusion: In this test, the PCL-GE/PCL-DOX/PCL-GE nanofiber membrane we fabricated by sequential electrospinning was conducive to cell attachment, growth and proliferation; DOX in electrospinning nanofibrous membrane could be sustained release with a relative stable concentration to inhibit the colonization of periodontal pathogen above 28 day. As the combination of GTR/GBR technology and drug therapy, PCL-GE/PCL-DOX/PCL-GE nanofiber membrane could do some efforts to promote the repair and regeneration of periodontal tissue.