| Background and Objectives:In recent years,bone tissue engineering has become a research hotspot in the treatment of bone defects because of its unique advantages such as abundant tissue sources and no secondary infection.The scaffold is one of the important factors in bone tissue engineering,which should have three-dimensional porous interconnected structure,good biocompatibility and biodegradability,effective loading and sustained release of growth factors,good bone conductivity and bone induction for bone regeneration.Therefore,how to construct scaffolds conforming to the above principles is a hot research topic in bone tissue engineering research,and endowing scaffolds with good osteogenic activity(osteoinductivity and osteoconductivity)is the difficulty and emphasis in the design of bone tissue engineering scaffolds.Polycaprolactone(PCL)is a kind of organic macromolecule material.The PCL electrospun nanofiber scaffolds have good biocompatibility,mechanical strength,porous interconnected structure,high specific surface area and can load many kinds of growth factors.It can simulate the natural extracellular matrix structure of bone tissue.However,its weak hydrophilicity and the acidity of degradation products limit their use.Nano-hydroxyapatite(nHA)is the main inorganic component of bone matrix,which has good affinity with cell adhesion and plays an important role in the differentiation and mineralization of osteoblasts.At the same time,its alkaline properties can effectively neutralize the acid production during the degradation of organic polymers and make it act as ideal conductive material for bone,however,its strength is low and fragile,which limits its application.Recent studies have shown that nano-hydroxyapatite can be combined with organic polymer materials by phase separation and electrospinning technology to form polymer nanocomposites containing nano-scale bioceramics,which can can better exert the advantages of the material of ceramic materials and polymer materials.Bone regeneration is a process of new bone formation,which requires the participation of multiple growth factors.Nel-like molecule 1(NELL-1)is a newly discovered growth factor that promotes bone formation and participates in the recruitment,proliferation,differentiation,mineralization of osteoblasts and the interaction between cells and intercellular matrix.NELL-1 is located in the downstream of Cbfal/Runx2.Compared with BMP-2,NELL-1 does not have ectopic osteogenesis and induces denser bone.The goal of bone tissue engineering is to reconstruct and regenerate extracellular matrix microenvironment system and bone biochemical signal molecule system,so as to achieve bone tissue regeneration quickly.Based on the above understanding,this study is divided into three parts to construct bioactive scaffold materials.In the first part,the NELL-1-loaded aggregates were prepared by desolvent method,and then chitosan was coated and stabilized by electrostatic adsorption to form chitosan nanospheres,forming the first barrier for growth factor release.In the second part,nano-hydroxyapatite and PCL with different weight were prepared into polymer nano-composites containing nano-bioceramics by electrospinning technology,in order to provide new scaffolds with bone conductivity for bone defect repair.In the third part,the chitosan microspheres and nHA were embedded into the polycaprolactone polymer fiber scaffold by electrospinning technology to form a double-layer protective barrier released by growth factors,and the nanofiber scaffolds were given osteoconductive and osteoinductive properties so as to construct the biomimetic microenvironment required for bone tissue regeneration,which can provide new scaffold materials and growth factor drug loading and release methods for bone defect repair.The research objectives of this study are as follows:(1)To construct nanoparticles loaded with NELL-1 protein to form the first barrier of NELL-1 growth factor released,and to detect the nanoparticles characterization,drug release and bioactivity in vitro.(2)To fabricated PCL composite fiber scaffold material rich in nano-hydroxyapatite in order to impart bone conductivity to the material,to detect and evaluate the physical-chemical characterization and biocompatibility of materials with different nHA content,and investigate the effects of materials on adhesion,proliferation,differentiation and expression of osteogenic related genes(RUNX2,OPN,COL1)of MC3T3-E1 precursor osteoblasts.(3)To construct composite fiber scaffolds rich in nano-hydroxyapatite and sustained-release NELL-1 protein thus to form a double-layer protective barrier for growth factor released,and to endow nanofiber scaffolds with bone conductivity and osteoinductivity.Then to evaluate the physical-chemical characterization and biocompatibility of the scaffolds,and to detect the amount of NELL-1 protein sustained-release from the material.To investigate the effects of composite scaffolds on adhesion,proliferation,differentiation and mRNA expression of osteoblast-related genes(RUNX2,OPN,COL1)in MC3T3-E1 precursor osteoblasts.Materials and Methods:(1)Firstly,NELL-1 loaded chitosan nanospheres(Chi/NNPs)were prepared by desolvation method and electrostatic self-assembly technique.The materials were characterized by transmission electron microscopy,nano-particle size analyzer,ART-FTIR and CLSM.The encapsulation efficiency and in vitro protein release of the microspheres were detected by NELL-1 Elisa Kit,and the biological activity of NELL-1 was released by detecting the expression of alkaline phosphatase of mouse osteoblasts.(2)Composite fibrous scaffold materials were prepared from nano-hydroxyapatite with different content and polycaprolactone by electrospinning technology.The results were characterized by SEM,TEM,tensile strength test,ART-FTIR,contact angle meter and hydrophilicity test.The adhesion of MC3T3-E1 on the scaffolds was evaluated by CCK-8 kit and CLSM.Alizarin red staining,ALP kit and RT-PCR were used to evaluate the effects of different scaffolds on osteogenic differentiation and mineralization of MC3T3-E1 cells.(3)Chi/NNPs and nHA were embedded into polycaprolactone fiber scaffolds by electrospinning.The scaffolds were characterized by SEM,TEM,tensile strength test,contact angle tester and hydrophilicity test.NELL-1 Elisa Kit was used to detect the sustained release of NELL-1 from the scaffolds in vitro.The CCK-8 kit,CLSM and SEM were used to evaluate the adhesion,growth,extension of MC3T3-E1 cells and material toxicity of the scaffolds.The effects of different scaffolds on the osteogenic differentiation and mineralization of MC3T3-E1 cells were evaluated by ALP kit and RT-PCR.Results:The first part of the experiment:TEM results show that Chi/NNPs and Chi/BNPs can form nano-microsphere structure,the surface is obviously coated by chitosan and the shape of microsphere is stable,spherical,smooth and non-agglomerated.ART-FTIR shows that the characteristic peaks of chitosan and BSA proteins can be observed in the spectrum of chitosan microsphere.Under the CLSM,the chitosan-coated nanoparticles were uniformly dispersed on the glass piece without obvious agglomeration and the green fluorescent chitosan was evenly distributed on the BSA particles and formed a polymer.The particle size results showed that the Chi/BNPs particle size was 378.726±13.140 nm,and the Chi/NNPs(0.075%,0.15%,0.3%)particle sizes were 368.663±15.470 nm,382.881 ± 18.767 nm,and 390.480±11.465 nm,respectively.The particle size did not differ but was larger than the diameter of the BSA aggregates(P<0.05).The PDI values in the Chi/BNPs group were 0.378±0.029,and the Chi/NNPs(0.075%,0.15%,0.3%)groups were 0.379±0.017,0.388±0.039,and 0.462±0.030,respectively,both less than 0.5,the PDI value of BNPs was 0.669±0.072.The potentials of Chi/NNPs(0.075%,0.15%,0.3%)are +25.03±1.42mV,+30.27±1.80mV,+31.03±2.05 mV,respectively.Chi/BNPs and BNPs+29.03.03±2.15mV,+23.067±2.54mV,respectively.Chi/NNPs(0.075%,0.15%,0.3%)were 88.500±3.629%,89.302±3.816%and 87.833±3.500%,respectively.Statistical analysis showed no difference between the groups(P>0.05).The cumulative release curve in vitro showed that NELL-1 could be sustained for more than 192h,and the initial release was not obvious.There was a similar release curve between 0.15%chitosan group and 0.3%chitosan group.There was no statistical difference between these two groups.Compared with the fresh NELL-1 solution,the ALP activity of NELL-1 released from Chi/NNPs(0.15%)0～2d was 93.18±8.99%,3～5d was 85.35±5.84%,and 6～8d was 82.67±.8.74%.Statistical analysis showed that the biological activity of NELL-1 released from Chi/NdxNPs was higher than that of the negative control group(Chi/BNPs)during the entire release period(P<0.05).The biological activity of fresh NELL-1 solution was higher than the released NELL-1 solution,however there was no statistical difference(P>0.05).The second part of the experiment:SEM showed that the fabricated fiber scaffolds were disordered three-dimensional staggered structure,fiber and fiber interlaced into a network.The diameters of electrospun fibers in PCL group,PCL+10%nHA group,PCL+20%nHA group,PCL+30%nHA group and PCL+40%nHA group were 550.90 ±37.43 nm,572.44±35.27 nm,581.38±40.58 nm,617,18±30.17 nm,630.02±60.53 nm,respectively.TEM showed that the surface of pure PCL was smooth,while that of nHA-rich PCL was slightly rough,and especially in PCL+30%nHA group and PCL+40%nHA group there were more nHA aggregation in the fibers,and the roughness of the surface was more obvious.The results of ART-FTIR showed that the representative functional groups of nHA were OH-1 and PO43-,and the main functional groups of pure PCL were C=O,C-H and C-O.The main functional groups of nHA and PCL can be found in PCL with different contents of nHA.It can be seen that nHA can be embedded in PCL during electrospinning,and the structure and composition of PCL and nHA have not changed significantly.The results showed that the tensile strength of PCL group was the highest,reaching 14.98±1.13 MPa.With the increase of nHA addition,the tensile strength of nHA rich PCL gradually decreased.The tensile strength of the PCL+40%nHA group was the lowest,which was as low as 6.02 ±0.98 MPa.Contact angle test found that the contact angle of PCL was large,with the increase of nHA content,the contact angle of composite scaffolds decreased significantly,and the hydrophilicity of PCL+30n%nHA group and PCL+40n%nHA group increased significantly.The results of further water absorption test showed that with the increase of nHA content,the water absorption rate of the material increased significantly.Compared with PCL group,the water absorption rate of PCL+30n%nHA group and PCL+40%nHA group increased significantly,and there was statistical significance.The results of CLSM showed that with the different content nHA added to PCL,the number of cell adhesion increased significantly especially in PCL+30%nHA group and PCL+40%nHA group was the largest.In other words,the introduction of hydroxyapatite into PCL can significantly improve cell adhesion and extension.The results of CCK-8 indicate that the addition of nHA to PCL can significantly improve the growth and proliferation of cells on the scaffold,or that the composite scaffold material with nHA added to PCL is non-toxic to MC3T3-El cell.Alizarin red staining results show that mineralization is more obvious with the increase of nHA content than pure PCL fiber scaffolds,which shows that there are more calcium deposits on the fiber scaffolds.The number and size of calcium nodules on PCL+30%nHA and PCL+40%nHA scaffolds were significantly higher than those in pure PCL scaffolds.The addition of nHA could promote the expression of ALP in MC3T3-E1 cells,especially in PCL+30%nHA group and PCL+40%nHA group.The results of RT-PCR indicated that the expression of RUNX2 mRNA in the groups of PCL composite fiber scaffold containing nHA was significantly increased at 4 days,and the higher the nHA content is,the higher the expression of RUNX2 mRNA is.At 21 days,the expression of RUNX2 mRNA in the groups of PCL composite fiber scaffold containing nHA decreased significantly,and the higher the nHA content,the more obvious the low expression of RUNX2 mRNA.The expression of COL1 in the PCL composite fiber scaffold containing nHA increased gradually over time(7d,14d,21d),and its expression increased significantly especially in PCL+30%nHA group and PCL+40%nHA group.The expression of OPN mRNA in PCL+30%nHA group and PCL+40%nHA group was increased at 14 days after inoculation.On the 21st day,the expression of OPN was significantly increased in each group containing nHA(except 10%nHA group),and the expression of OPN mRNA in PCL + 30%nHA group and PCL + 40%nHA group with higher nHA content increased more significantly.The third part of the experiment:As shown in the SEM,the electrospinning scaffolds of each group presented a disorder structure,and the fibers interlaced with fibers.The diameters of electrospun PCL fibers in pure PCL group,PCL/nHA group,PCL/BNPs group,PCL/NNPs group and PCL/nHA/NNPs group were 550.90±37.43 nm,617.18±30.17nm,680.23±29.36 nm,689.36±37.04 nm,702.72±34.24 nm,respectively.The embedding of the microspheres significantly increases the diameter of the PCL fiber.TEM results indicate that the pure PCL fiber scaffold material has a relatively uniform thickness and a smooth fiber surface.The nHA-rich PCL fiber scaffold has a certain agglomeration.The surface of the electrospun fiber in which the nanospheres(BNPs or NNPs)are embedded has a slight protrusion and a thickened diameter.The tensile strength results show that the addition of nHA or the addition of microspheres reduces the tensile strength of the fiber.The results of contact angle and water absorption showed that the contact angle of all the groups except PCL group decreased significantly(less than 90°),and the hydrophilicity increased significantly after 3 minutes of ddH2O added.The water absorption of each group of electrospun fibers of PCL containing nHA or nanospheres was significantly increased.The results of in vitro drug release showed that the PCL/nHA/NNPs group could release the drug at a low concentration for 30 days and the total release amount was over 90%.The release profile of the PCL/NNPs group was similar to that of the PCL/nHA/NNPs group,which significantly reduced the burst release of the drug.The CLSM microscopic observation shows that the MC3T3-E1 cells were adhered to the scaffold,and the number of cells in the groups containing nHA or microspheres was bigger than the other group.Scanning electron microscopy showed that MC3T3-E1 cells had extended on the scaffolds with pseudopodia,good morphology and large spreading area.The results of CCK-8 showed that nHA and nanospheres embedded in PCL did not reduce the biocompatibility of the scaffold material,and significantly promoted cell adhesion and proliferation.ALP detection showed that each group containing nHA or NNPs microspheres significantly promoted ALP expression in MC3T3-E1 cells.The results of RT-PCR showed that the expression of COL1 mRNA in PCL/nHA group,PCL/NNPs group and PCL/nHA/NNPs group was significantly higher than that in PCL group and PCL/BNPs group at 7 days after inoculation(P<0.05).The expression of COL1 mRNA in PCL/nHA group,PCL/NNPs group,PCL/nHA/NNPs group was higher than that in pure PCL group and PCL/BNPs group at 14 and 21 days after inoculation.The expression of COL1 mRNA in PCL/nHA/NNPs group was also higher than that in PCL/nHA group(P<0.05).At 14 days after inoculation,the expression of OPN mRNA in PCL/nHA group,PCL/NNPs group,PCL/nHA/NNPs group was higher than that in pure PCL group and PCL/BNPs group(P<0.05),but there was no significant difference between pure PCL group and PCL/BNPs group(P>0.05).At 21 days after inoculation,the expression of OPN mRNA in PCL/nHA group,PCL/NNPs group,PCL/nHA/NNPs group was higher than that in pure PCL group and PCL/BNPs group.The expression of OPN mRNA in PCL/nHA/NNPs group was also higher than that in PCL/nHA group.The expression level of RUNX2 mRNA in PCL/nHA group and PCL/nHA/NNPs group was significantly higher than that in the other three groups when MC3T3-E1 cells were inoculated for 7 and 14 days(P<0.05).There was no significant difference among the other three groups(P>0.05).The expression of RUNX2 mRNA in PCL/nHA group and PCL/nHA/NNPs group was significantly lower than that in the other three groups at 21 days after inoculation(P<0.05).There was no significant difference among the other three groups(P>0.05).Conclusion:(1)Chitosan was successfully coated on the surface of BSA aggregates to form nanospheres,which were stable in shape,spherical,smooth,non-agglomerated,moderate in particle size,high in encapsulation efficiency.It could form a protective barrier for growth factor release,so the release of the NELL-1 protein is preferably sustained and the released NELL-1 showed high biological activity.(2)The PCL composite scaffolds embedded with nHA have three-dimensional cross-linking structure,uniform fiber diameter distribution,uniform morphology,nHA wrapped in PCL fiber,good hydrophilicity,good tensile strength(except PCL +40nHA group)and good biocompatibility.The high content of nHA composite fiber scaffolds(PCL + 30%nHA group and PCL + 40%nHA group)is more conducive to the adhesion and proliferation of MC3T3-E1 cells,and significantly promotes the expression of ALP and osteogenic related genes(RUNX2,OPN,COL1)mRNA in MC3T3-E1 cells,which indicated that these scaffolds could promote MC3T3-E1 cells osteogenic differentiation,maturation and mineralization.(3)The PCL fiber scaffolds embedded with nHA and NELL-1 microspheres were successfully encapsulated in PCL fibers with three-dimensional interlacing network,which had good wettability,tensile strength and biocompatibility,and could significantly promote the adhesion and growth of MC3T3-E1 cells on the scaffolds.Chitosan microspheres and PCL fiber scaffolds can form a double-layer protective barrier for the release of NELL-1,which can effectively prolong the release time of NELL-1 growth factor.The released NELL-1 protein can increase the expression of ALP,indicating that the released growth factors maintain good biological activity.PCL fiber scaffolds embedded with nHA and NELL-1 microspheres can promote the expression of osteogenic related genes(RUNX2,OPN,COL1)in MC3T3-E1 cells and promote the osteogenic differentiation and maturation of MC3T3-E1 cells.The composite fiber scaffolds provide ideal scaffolds for the repair of bone defects. |
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