Enhanced Osteogenesis Of Polymeric Electrospun Nanofibers Loaded With RhPTH (1-34) In Vitro

Background and Objective:As oral implantology is increasingly sophisticated,dental implants have become the important choice for the patients with dentition defect or edentulous. The success ratio of dental implant is one of the most important concerns focused by clinical workers. One of the key factors of implant failure is bone defects. Hence, improving the bone quality and mass around the implant are the critical keys to maintain the success of oral implant. A variety of measures will be taken to increase the bone mass around the implants clinically, but they have their own drawbacks:the shortcomings of autogenous bone graft are the limited of resources,the rapid absorption of autogenous bone,the poor compatibility of allograft bone with human body, unfavourable postoperative reaction and more healing time need for allograft bone,etc.Parathyroid hormone(PTH) is a single polypeptide containing 84 amino acids, the N-terminal 1-34 amino acids have biological activities,PTH acts as a promoting bone formation drug to increase bone formation and density by increasing the number of osteoblasts, prolonging life span of osteoblasts, promoting the bone formation, reducing the apoptosis of osteoblasts[1-3]. rh PTH(1-34) is the only drug in the treatment ofosteoporosis which was approved by FDA in the United States in 2002.As be a kind of polypeptide drug, it is back of targeting because of general administration, and it requires repeated injections [4-5].These shortages limit further clinical applications of rh PTH(1-34).Electrospinning as a new drug carrier, it can encapsulate and release drugs slowly, so as to improve the bioavailability of drugs. Therefore, we consider to use electrospinning to load rh PTH(1-34) in the oral implant osteogenesis. Hence, we maked PLLA(poly L-lactic acid) as a drug delayed-released carrier to load rh PTH(1-34). Regulating the release rate to achieve the purpose of sustained releasing rh PTH(1-34). Then observed the effects of the polymeric electrospun nanofibers loaded with rh PTH(1-34) on mouse osteoblasts(MC3T3-E1)’ proliferation and differentiation in Vitro, and discussed the osteogenesis mechanism of polymeric electrospun nanofibers loaded with rh PTH(1-34).Methods:W/o emulsion was applied in this experiment, trace syringe was used to form PLLA/rh PTH(1-34) fiber membrane under 20 k V high voltage static electricity, used scanning electron microscopy(SEM) to observe the morphology of the fiber, purple light spectrophotometry was applied to mensurate the release curve at 280 nm. The experiments were divided into 7 groups: control group(a), intermittent dosing group(dosing at the first 6 hours per day)(b), one-time dosing group(c), the pure fiber materials group(d), and the three experimental groups with10-8, 10-9, 10-10 mol/L rh PTH(1-34)(e,f,g). MTT and ALP were individually measured to assess the proliferation and differentiation effects of the PLLA/rh PTH(1-34) on mouse osteoblasts(MC3T3-E1).Results:1. PLLA fibers presented criss-crossing, overlapping and even thickness under SEM, with the diameters in the range of 400-600 nm.The morphology of PLLA/rh PTH(1-34) was consistent with PLLA. The addition of rh PTH(1-34) didn’t affect the PLLA slow-release uniform fiber morphology. Rh PTH(1-34) drug particles were not emerged at the spinning fiber surfaces.2. The release of the model drug(containing drug BSA) of electricity spinning fiber in vitro was relatively much in 1d,at 30% of the total releasing amount. From 2d to 7d it reached a steady releasing stage,at 55% of the total releasing amount. From 7d to 21 d, the releasing amout Tended to be stable, at 14% of the total releasing amount.The duration time of drug releasing reached up to 21 days.3. The proliferation of MC3T3-E1 under PLLA/rh PTH(1-34) fiber environment: at 1d、2d、3d, the OD value of e、f、g was respectively statistically higher than the OD value of group a、c、d(P<0.05);at 1d、2d,the OD value of e、f、g was respectively lower than the OD value of group b(P<0.05); at 3d, the OD value of group f and b were not significantly different, whereas, the OD value of group e and g were significantly lower than group b; at 1d、2d、3d, the OD value of f wasstatistically higher than the OD value of group e、g(P<0.05);there was no significantly difference in other groups(P>0.05).4. The cell differentiation detected by ALP: the OD value of group b was significantly higher than group a、c 、d、e and g(P<0.05);there were not significantly different in group b and f(P>0.05);the OD value of e、f、g was respectively statistically higher than the OD value of group a、c、d(P<0.05); the OD value of group f was higher than group e and g(P<0.05); there was no significantly difference in other groups(P >0.05).Conclusion:1. PLLA fibers presented criss-crossing, overlapping and even thickness with the diameters in the range of 400-600 nm. The morphology of PLLA/rh PTH(1-34) was consistent with PLLA.2. The duration time of drug releasing loaded by PLLA/rh PTH(1-34) fiber reached up to 21 days.3. PLLA/rh PTH(1-34) fiber could significantly enhance the proliferation and differentiation of MC3T3-E1, showing the same effects as the method of intermittent dosing of drug.It theoretically proved the feasibility of sustained releasing rh PTH(1-34) loaded by PLLA.