| Bone defects arising from damage and disease are common phenomenon in clinicalpractice. The smaller defects will heal spontaneously, while the larger bone defectsmeaning 'critical sized defects' requied the artificial implants to repair. Althoughautologous bone graft is considered the gold standard in clinical practice, limitedavailability, additional surgery, and potential donor site morbidity limited its widelyapplication. To solve the problem, xenogenic grafts are used to repair the bone defectsclinically due to their ready availability and good osteoconductivity as a result of theirorigin from natural bone tissues of animals, i.e., Bio-Oss derived from bovine femur,and OsteoBiol derived from porcine bone. Nevertheless, the disadvantage of theseproducts are their time-consuming manufacturing process and high price. Therefore,extensive research effort is focused on synthetic materials. Especially, the norganicand organic composite materials in composition and structure similar natural boneextracellular matrix have been widely applied in the bone repair and regenerativemedicine, and have achieved lots of good results. Numerous researches have shownthat collagen and apatite composite materials have good biological effects both invitro and in vivo. In this study, the application of composite nanofibers includinggelatin and thicalcium phosphate (β-TCP) in bone repair and their osteoinductionmechanisms were investigated.In the present study, gelatin/β-TCP composite nanofibrous membranes werefabricated via electrospinning with β-TCP nanoparticles content of0,5,10, and20wt%respectively. Scanning electron microscope (SEM) observation showed thatuncrosslinked nanofibers present a homogeneous fiber morphology with the range of200–400nm in diameter for all kinds of nanofibers. This indicated that theincorporation of β-TCP could not affects the fiber diameter. The random arrangedand interlaced fibers formed many pore. Pure gelatin nanofibers presented a smoothsurface morphology, while composite nanofibers incorporated β-TCP showedgranule-like surface appearance. Meanwhile, the apparent granule appearance on thesurface of composite nanofibers increased with the increasing amount of β-TCPnano-particles. Transmission electron microscope (TEM) image showed that islets ofβ-TCP nano-particles were enveloped into gelatin matrices. The diameter of fiber increased clearly distribute to fiber swelling in the procedure of crosslinking treatment,while the pore size decreased significantly in comparison with the as-electrospun one.A phenomenon was observed that thfebers were curled and conglutinat ed with eachother throughout the membrane after being crosslinked. This indicated that all thenanofibrous memberanes were crosslinked absolutely. The apatite particles wereremained on the surface of composite nanofibers. It suggested that gelatin/β-TCPcomposite nanofibrous membranes have some surface roughness, which will benefitcell adhesion and proliferation. The structure integrity and functional groups ofgelatin and β-TCP were retained during the procedure of electrospinning. The resultsof protein adsorption analysis showed that the amount of serum protein adsorptiononto the composite nanofibrous membranes increased with the amount ofincorporated β-TCP, and presented a manner of time dependence within24h.The results of calcium ion release test showed that calcium ions released obviouslyafter incubation for one day when the culture medium was not changed. At the7thday, the concentration ofcalcium ions release in20wt%β-TCP containing compositenanofibrous membranes reached the saturation state, while the other two groupsreached the saturation concentration after14days. Moreover, numerous apatiteparticles deposited on the surface of20wt%β-TCP membrane materials from SEMimages. When the medium was changed regularly, calcium ions continue to releaseafter7days for20wt%β-TCP membrane materials. From this phenomenon, it couldbe inferred that a balance between dissolution of β-TCP and formation of mineralitedepositions was detected.When MG-63osteoblast cell line and primary cultured rats bone marrowmesenchymal stem cells (rBMSCs) were seeded on the composite nanofibrousmembranes respectively for1and7days, SEM images and cell attachment efficiencyanalysis showed that four kinds of nanofibrous membranes are beneficial to celladhesion and proliferation. For the composite nanofibers incorporated β-TCP groups,plenty of extracellular matrix secreted on the surface of cells and formed apparentprotruding cellular processes. Laser confocal microscope observation showed thatcytoskeletal organization and cell spreading area on the composite nanofibrousmembranes increased with the amount of incorporated β-TCP. ELISA tests showedthat cells in20wt%β-TCP group exhibited highest ALP activity, indicating higheramount of incorporated β-TCP enhanced higher ability of osteogenic differentiation of MG-63cells. These results showed that composite nanofiber membranes with20wt%β-TCP could promote effectively the adhesion and proliferation of osteoblasts.Furthermore, the third generation of rBMSCs were cultured on the pure gelatin and20wt%β-TCP membrane materials respectively after7,14and21days. Real-timequantitative PCR detection results show that the composite nanofibrous membranescontaining20wt%β-TCP not only can promote the upregulation expression ofosteoblast differentiation key transcription factors RUNX-2, type I collagen, BMP-2and bone gamma-carboxyglutamate protein (BGLAP), but also activate the calciumsensitive receptor (CaSR) during the experiment period, suggesting the osteoinductionmechanisms of gelatin/β-TCP composite nanofibrous membrane were related to theactivity of CaSR and BMP-2signaling pathway.On the basis of in vitro studies, pure gelatin nanofibrous membranes and compositenanofibrous membranes with20wt%β-TCP were further implanted into rabbitmandibular critical size defect models (8mm in diameter) respectively. Thecommercial collagen membranes were used as control. At4and12weekspost-operation, anatomical observation, Micro-CT detection, H&E staining andimmunohistochemical analysis showed that the bone repair efficiency of pure gelatinnanofibrous membranes was close to that of commercial collagen membranesregarding new bone morphology, callus formation, bone mineral density, bone volumeratio, and bone remodeling degree. By comparison, gelatin/β-TCP compositenanofibrous membranes had better bone repair quality, and formed good boneintergration with host bone at12weeks post-operation. Bone mineral density ofgelatin/β-TCP composite nanofibrous membranes group was close to that of normalbone tissue, sugessting the gelatin/β-TCPcomposite nanofibrous membranes had wellguid bone regeneration effects. These results demonstrated that electrospungelatin/β-TCP composite nanofibrous membranes could be used to bone repairtreatment, and the results would provide the feasible and theoretical guidance forfurther bone regeneration research.
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