Preparation And Bioactivities Of Electrospun PBC Nanofiber Membrane And Hybrid PBC Nanofiber Membrane Of Sustained Release Growth Factors In Platelet

In the clinical work of stomatology,alveolar bone defect is often faced,such as from chronic apical periodontitis, periodontitis or the extracted teeth.The alveolar bone defect not only seriously affects the function of teeth, butalso does harm to the dentures and implants.Many ways have been adopted to promote bone formation. Scholarsimplant the biological materials with a function of bone guiding and releasingcomplex growth factors. This method has become common due to its stableeffect and being atraumatic.Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) the secondgeneration of platelet concentrates, contain high concentration of cytokines,which are involved in the process of wound healing. In particular, they play animportant role in bone regeneration. They constitute a system of regulation oftissue repair function not only by their respective role but also by promotion,cooperation, regulation of each other. They have a good effect on tissue healing,regulating inflammation and anti-infection.However, the clinical effects of PRP still have controversy. A lot ofopposite results have been reported. Many studies show that PRP and PRFresults are unsatisfactory. The study found that the growth factor, with a veryshort half-life, can not meet the requirements of the osteogenesis. Therefore, tomaintain a sustained release of growth factors, we need to find suitable scaffoldmaterials.In the study of osteoblasts, organic polymer is one of the hot topics ofresearch. The ideal scaffold material should be easy to load growth factors and have a sustained release effect. It should also protect biological activities ofgrowth factors during the process. Thus it can constantly promote cell growthand tissue regeneration. Poly butylenes succinate(PBS) is degradable aliphaticpolymer, which has good cell and tissue compatibility and good biodegradation,and can be decomposed into carbon dioxide and water by a variety ofmicrobiological, animal and plant enzymes. PBS is a typical completebiodegradable polymer material. The study of PBS application has attractedwide attention as a potential biomedical polymer material.Nanofibers are widely applied in a lot of medical domains, such as in thedrug release, artificial skin, wound healing, teeth enhancement and so on,dueto their high surface area and high microporosity. Electrospinning technologyis one of the most simple and effective methods to manufacture nanofibers. Atpresent the preparation of nanometer-sized polymer fibers using on theelectrostatic spinning is the most simple and effective method. In recent years,nanofiber polymers by electrospinning method has been applied to the bonetissue engineering field.PBS nanofibers electrospinning method has been reported, but the studieswere focused on the conditions of spinning, spinning parameters, mechanicalproperties, thermal resistance and modification. Its basic research as bonesubstitute materials are rarely reported. Nanofibers prepared by electrospinningwith platelet plasma and its effect on osteoblasts have not been reported.This study explored the manufacture of PBS nanofiber membrane andhybrid PBS nanofiber membrane with platelet-rich plasma (PBS/Platelet) byelectrospinning. It evaluated surface properties, hydrophilicity, tensile strength,the bioactivities of PBS and PBS/Platelet nanofiber membrane on theproliferation, differentiation of human osteosarcoma cell lines with MG63. Itdetected osteoblast-related genes by real-time PCR and protein expressionrelated to the osteogenesis by Western Blot. 1Preparation and characterization of PBS and PBS/Platelet nanofibermembraneObjective: To investigate the electrospun PBS optimal concentration, andthe process parameters; the feasibility of electrospinning hybird nanofibermembrane with platelet-rich plasma and PBS. To explore the characterization,hydrophilicity and tensile strength of the two scaffolds.Methods: Electrospun nanofiber membrane was made with PBS dissolvedby chloroform. PRP was added to the PBS solution which was then vibratedthrough ultrasonic to become emulsion.The emulsion was Electrospun. Thematerial surface properties was observed by scanning electron microscopy.Hydrophilicity and tensile strength were tested.And then the degradation of thetwo materials was observed in vitro.Results: The fiber membrane has the characteristics of uniform textureand good elasticity through the observation of naked eyes. Under a scanningelectron microscope, the diameter of PBS nanofiber was about200nm~900nm,full of pores connected with each other. The effects were similar betweenPBS/Platelet nanofiber membrane and PBS nanofiber membrane. The diameterof fiber was about100nm~1000nm. PBS hydrophilic angle was68.81±0.3,which demonstrated better hydrophilicity. The tensile strength of the fibermembrane was2.04±0.65~2.25±0.72(MPa). Elongation at Break was284.5±17.41~297.53±25.13(%). By adding the platelet rich plasma, the elasticmodulus of the nanofibers decreased, with a slight increase in elongation atbreak.2In vitro degradation of two nanofiber membrane and in vitro release ofthe PBS/Platelet nanofiber membraneObjective: To understand in vitro degradation capacity of the material andthe release characteristics of growth factors in platelet in the material.Methods: In vitro, the material degradation in the buffer PBS has been evaluated during12weeks.Platelets contained large quantities of growth factors,among which PDGF was one of the most important. In vitro release andduration of PDGF were tested by ELISA, reflecting release of the material.Results: The PBS/Platelet nanofiber membrane metabolism was slightlyfaster during the first three weeks, growing into a linear metabolism model laterthen, and finally achieving23.80%at the end of the12thweek. The pure PBSnanofiber degradation was slow, weight loss rate being5.20%at the end of the12thweek.In the experiment, PDGF release was most obvious on the first day, therelease concentration being58.8%, with a significant burst release phenomenon.The second week witnessed PDGF release up to66.1%, the seventh week72.4%. This showed that the PBS scaffold material loading platelet was able tomaintain the local cytokines concentration after the initial burst release, with asustained release effect. However, further studies are required on how toachieve the desired controlled release.3The effect of PBS nanofiber membrane and hybrid nanofiber mem-brane on the proliferation and differentiation of MG63Objective: In a model with the human osteoblast-like cells MG63cells-asthe bone cells, the effect of osteoblast cells of scaffold material has beenevaluated on its biological characteristics, so as to understand the role ofmaterial in osteogenesis.Methods: MG63, cultured routinely, was inoculated in cell culture plate.The hemolytic properties and biological activity of the scaffolds have beenevaluated synthetically by means of hemolytic behavior of the material, surfacecells fluorscent staining of scaffold material, MTT, and ALP activity detection.Results: The hemolysis rate of scaffold material was0.1475%, far lessthan5%of the national standards, demonstrating that the scaffold material wassafe and without acute hemolytic activity. The osteoblast proliferation of scaffold material has been promoted to some extent.4The effect of PBS and PBS/Platelet nanofiber membrane on the osteo-blast-related gene and protein of MG63cellObjective: In a model with the human osteoblast-like cells MG63cells-asthe bone cells, the effects of scaffold material were explored onosteogenesis-related gene and protein expression.Method: MG63was cultured routinely and inoculated on the materialsurface. The termination of culture occurred on the first, third and fifth day,respectively, followed by digestion of cells, extraction of RNA and protein.Real-time PCR method was used for detection of gene expression of Runx2,COL1α1,OPN and protein expression levels of OPN, SP7were detected byWestern Blot method.Results: The Real-time PCR results showed the changes of Runx2geneexpression in MG63cells: Runx2gene expression of the blank group wassignificantly higher than that of PBS scaffold material and PBS/Plateletscaffold group on the first day; low expression could be observed on the fifthday in the blank group, while significantly higher gene expression occurred inPBS group and PBS/Platelet group. Gene expression in the blank group haskept reducing from the first to fifth day. The changes of COL1α1geneexpression in MG63cells were as follows: gene expression was not high on thefirst and third days, with no significant differences among groups; geneexpression of the blank group on the fifth day was significantly higher than thaton the first and third days; the expression of the PBS group and PBS/Plateletgroups were significantly higher than the blank group. OPN gene expression inMG63cells has experienced the following changes: gene expression of theblank group was significantly higher than that of the PBS group andPBS/Platelet group on the first day, while on the fifth day gene expression ofthe PBS and PBS/Platelet group was significantly higher than that of the blank group;The Western Blot test results showed that: protein expression of SP7hadno significant differences among groups on the first day; gene expression of thePBS group and PBS/Platelet group was significantly higher than that of thecontrol group on the third and fifth days; that of PBS/Platelet group wassignificantly higher than that of PBS group on the fifth day; the expression ofthe control group declined on the first, third and fifth days.The protein expression of OPN has undergone the following changes: theexpression of the blank group decreased gradually, while the expression ofPBS/Platelet group showed a trend of gradually increasing on the first, thirdand fifth days; PBS group had the lowest protein expression on the first dayand was significantly different from that of the PBS/Platelet group; theexpression of PBS group and PBS/Platelet Group was significantly differentfrom that of the control group on the third day; the expression of PBS andPBS/Platelet group was significantly different from that of the control group onthe fifth day, and there was also a significant difference between PBS/Plateletgroup and PBS group.In summary, PBS can be fabricated into nanofibers at the appropriateconcentration under the proper conditions, and be successfully loaded with PRP.PBS nanofibers and PBS/Platelet nanofiber plays a role in promoting theproliferation of osteoblasts, increasing osteogenesis-related gene and proteinexpression. In this study, PBS as bone scaffold material was discussed andmore effective methods were explored for the clinical application of PRP andPBF.