The Effect Of Nano-ZnO Surface Modification On Osteoinductivity Of Hydroxyapatite Whisker

Objective:As a kind of bioactive ceramic,hydroxyapatite（HA）has been widely used in bone defect repair in clinic.Hydroxyapatite whisker（HAw）is a kind of HA with high aspect ratio and is similar to the morphology of mineral composition in natural bone.Due to the special morphology and characteristics,HAw used as strengthening and toughening phase can effectively absorb the crack propagation energy through whisker bridging and whisker drawing,leading to an improvement of the mechanical properties of matrix materials.The elastic modulus,compressive strength and fracture toughness of bioactive glass,dental repair materials or calcium and phosphorus（Ca-P）scaffolds could be significantly enhanced by incorporating HAw into the matrix materials or by generating HAw in situ on the surface of Ca-P scaffolds.The osteoinductivity of HAw remains controversial.Studies reported that HAw inhibited the alkaline phosphatase（ALP）activity,calcium deposition,osteogenic marker genes expression of osteoblasts or bone marrow mesenchymal stem cells（BMSCs）.The presence of HAw generated in situ on the surface of Ca-P scaffolds significantly reduced new bone formation inside the scaffolds after canine intramuscular implantation.How to increase the osteoinductivity of HAw to improve the applicability of HAw in bone defect repair is the focus of this study.Surface modification is an effective way to improve the biological performance of HAw.Polymer materials modified HAw or porous scaffolds fabricated by metallic elements magnesium or strontium doped HAw showed superior osteoinductivity.Zinc（Zn）is an essential factor for bone development.Zn or Zinc oxide（ZnO）doped biomaterials can promote the differentiation of mesenchymal stem cells or osteoblasts,increase chondrogenesis and endochondral osteogenesis,and induce the formation of new bone in bone defect through the precipitation of zinc ions(Zn²⁺).At the same time,Zn is crucial to the antioxidant function of human body,which can counteract cellular oxidative stress induced by exogenous materials or endogenous diseases through scavenging intracellular reactive oxygen species（ROS）or enhancing cellular antioxidant defenses,thus increasing the osteogenic activity of osteoblasts or BMSCs.In addition,compared with polymer materials or metallic elements magnesium and strontium,ZnO has good antibacterial ability,the applicability of biomaterials in bacteriological environment can be improved by the incorporation of ZnO.HAw modified with nano-zinc oxide（nano-ZnO）particles on the surface was fabricated in this study.The influence of hydroxyapatite sphere（HAs）and HAw on osteoblasts membrane integrity was compared to explore the reason of HAw’s low biocompatibility.The effects of nano-ZnO particles modified hydroxyapatite whisker（nano-ZnO/HAw）on Porphyromonas gingivalis（P.g）viability and biofilm formation,compressive strength of calcium sulfate,osteoblasts proliferation and differentiation,and new bone formation in bone defects were verified.In order to find a kind of bone defect repair material with good antibacterial property and osteoinductivity,which is similar to the morphology of mineral composition in natural bone and possesses well ability to improve the mechanical property of matrix material for clinic.Methods:1.HAw was used as substrate material,HAw modified with 1%,5%and 10%nano-ZnO particles were fabricated by sol-gel method（mass ratio）.The microstructure,elemental compositions,and phase compositions of nano-ZnO/HAw was identified and analyzed by scanning electron microscopy,energy dispersive spectroscopy and X-ray diffraction analysis.2.Calcium sulfate was used as matrix material,10%volume ratio of HAs,HAw and10%nano-ZnO/HAw were incorporated into calcium sulfate respectively,pure calcium sulfate was served as control.The maximum compressive strength of calcium sulfate in each group was detected by mechanics experimental machine.3.The suspension of HAs,HAw,1%nano-ZnO/HAw,5%nano-ZnO/HAw and10%nano-ZnO/HAw were co-cultured with P.g suspension in EP tubes or on the surface of stainless-steel sheet respectively,P.g suspension cultured without material was used as control.After 24 h,P.g suspension in EP tubes of each group was diluted 10,100,1000 times and inoculated on brain heart infusion broth-ager plate,the colony growth of P.g was observed after 10 d.The bacteria adhering to the surface of stainless-steel sheet were stained by crystal violet,and the dyes attached to the biofilm were eluted by anhydrous ethanol,the amount of bacterial biofilm formation on the surface of stainless-steel sheet was evaluated according to the color depth of the eluent.4.Osteoblasts were co-cultured with the suspension of HAs and HAw,or with the suspension of HAw,1%nano-ZnO/HAw,5%nano-ZnO/HAw and 10%nano-ZnO/HAw respectively,osteoblasts cultured without material was used as control.After the corresponding time point,cell viability was detected by CCK8,alkaline phosphatase activity of osteoblasts was detected by ALP staining,osteogenic marker genes expression of osteoblasts,including ALP,Runt-related transcription factor 2（RUNX2）,Collagen I（COL I）,Osteocalcin（OCN）and Osterix（OSX）were detected by Real-time PCR.5.Osteoblasts were co-cultured with the extract of HAs and HAw,or with the extract of HAw,1%nano-ZnO/HAw,5%nano-ZnO/HAw and 10%nano-ZnO/HAw respectively,cells cultured alone was used as control.After the corresponding time point,cell viability was detected by CCK8,alkaline phosphatase activity of osteoblasts was detected by ALP staining.6.A cylindrical non-penetrating bone defect with a diameter of 3 mm and a depth of 3mm was prepared on lateral condyle of rat femur.HAs,HAw,5%nano-ZnO/HAw and10%nano-ZnO/HAw were grafted into the bone defect respectively,the bone defect grafted without material was used as blank control.After 4 w,the rat femur specimens were scanned by Micro-CT.Three-dimensional reconstruction was performed and three-dimensional tomography was extracted of each specimen.The components in cylindrical bone defect consistent with the density of bone tissue was reconstructed by image analysis software.The percent bone volumes（BV/TV）,trabecular number（Tb.N）,trabecular thickness（Tb.Th）and trabecular separation（Tb.Sp）of the components in cylindrical bone defect consistent with the density of bone tissue were quantitatively calculated to analyze the bone regeneration within the bone defect.The femur specimens were decalcified,embedded and sliced,Hematoxylin-Eosin（H&E）staining was performed to observe the new bone tissue in bone defect.7.Osteoblasts were co-cultured with the suspension of HAs,HAw and 10%nano-ZnO/HAw（with or without 1%nano-ZnO/HAw and 5%nano-ZnO/HAw）respectively,cells cultured without material was used as control.After the corresponding time point,scanning electron microscopy and energy dispersion spectroscopy were used to observe and analyze the effect of the physical interaction between HAw and osteoblasts on cell morphology.Lactate dehydrogenase（LDH）release assay was performed to compare the LDH content in cell supernatant among each group.Cell apoptosis rate was detected by flow cytometry.8.The concentration of Zn²⁺in the suspension of 10%nano-ZnO/HAw was measured,and same concentration of Zn²⁺solution was prepared and added to the suspension of HAw.The suspension of HAs,HAw,HAw+Zn²⁺and 10%nano-ZnO/HAw were co-cultured with osteoblasts respectively.The intracellular ROS level was detected by flow cytometry,the expression of nuclear factor-erythroid 2-related factor 2（Nrf2）in nucleus was detected by western bolt,the differentiation level of osteoblasts was detected by ALP staining and Real-time PCR.9.The experimental data was analyzed statistically.One-way analysis of variance（ANOVA）was used for the comparison among multiple groups,and t-test was used for the comparison between two groups.P<0.05 was considered statistically significant.Results:1.HAw modified with 1%,5%and 10%nano-ZnO particles were successfully fabricated by sol-gel method,nano-ZnO particles were uniformly distributed on the surface of HAw.The element compositions of HAw were Calcium（Ca）,Phosphorus（P）and Oxygen（O）,the phase composition of HAw was HA.The elemental compositions of nano-ZnO/HAw included Ca,P,O and Zn,and the phase compositions of nano-ZnO/HAw were HA and ZnO.2.Compared with pure calcium sulfate,the compressive strength of calcium sulfate incorporated HAs had no obvious improvement,but the compressive strength of calcium sulfate incorporated with HAw was significantly improved.Calcium sulfate incorporated with 10%nano-ZnO/HAw had the highest compressive strength among all the groups.3.The P.g viability and biofilm formation had no significant difference among the groups of control,HAs and HAw.Compared with HAw,1%nano-ZnO/HAw did not reduce the P.g viability and biofilm formation,while 5%nano-ZnO/HAw and 10%nano-ZnO/HAw significantly inhibited P.g viability and biofilm formation.4.Compared with control or HAs group,the suspension of HAw significantly inhibited the proliferation,ALP activity and osteogenic marker genes expression of osteoblasts.Compared with pure HAw,the suspension of nano-ZnO particles modified HAw significantly improved the proliferation,ALP activity and osteogenic marker genes expression of osteoblasts.5.Compared with control or HAs group,the extract of HAw had no significant effect on osteoblasts proliferation and ALP activity.Compared with HAw,the extract of5%nano-ZnO/HAw and 10%nano-ZnO/HAw significantly improved the proliferation and ALP activity of osteoblasts.6.Micro-CT results showed that,compared with blank group,the bone defect grafted with HAs exhibited high-density images.Compared with HAw,the imaging density of the bone defect grafted with HAs,5%nano-ZnO/HAw and 10%nano-ZnO/HAw increased,the total volume and BV/TV of the components in bone defect consistent with the density of bone tissue increased significantly,while the Tb.N and Tb.Th showed an increasing trend,the Tb.Sp showed a decreasing trend.H&E staining showed that,new bone formation of bone defect grafted with HAs was higher than that of blank group.Compared with HAs,HAw decreased the new bone formation.Whereas,the new bone formation of 5%nano-ZnO/HAw and 10%nano-ZnO/HAw group was higher than HAw group,which was close to that of HAs group.7.Osteoblasts punctured by HAw and 10%nano-ZnO/HAw could be observed under scanning electron microscopy.LDH content in the supernatant of osteoblasts co-cultured with HAs had no difference compared with control group.Compared with control or HAs group,the released LDH and cell apoptosis rate increased when osteoblasts co-cultured with HAw,while the surface modification of 5%and 10%nano-ZnO particles significantly reduced the LDH leakage and cell apoptosis induced by HAw.8.Compared with HAs,HAw significantly improved the intracellular ROS level and reduced the endonuclear Nrf2 expression of osteoblasts.The surface modification by10%nano-ZnO particles reduced the intracellular ROS and the inhibitory effect on endonuclear Nrf2 expression of osteoblasts caused by HAw.Compared with HAw,the intracellular ROS level showed a decreased trend,the endonuclear Nrf2 expression increased,and the differentiation of osteoblasts improved when cells co-cultured with HAw+Zn²⁺.Conclusion:1.Nano-ZnO/HAw served as strengthening phase,the enhancement effect of HAw on compressive strength of calcium sulfate was further improved due to the presence of nano-ZnO particles on the surface of HAw.HAw modified with nano-ZnO particles possessed favorable antibacterial ability which could inhibit the viability and bacterial biofilm formation of P.g.2.HAw has poor osteoinductivity,which inhibited osteoblasts differentiation and new bone formation in bone defect.The surface modification of nano-ZnO particles improved the inducibility of HAw on osteoblasts differentiation and new bone formation in bone defect,5%nano-ZnO/HAw and 10%nano-ZnO/HAw had good osteoinductivity.3.HAw punctured osteoblasts membrane and caused mechanical damage to cells,resulting in the reduction of osteoblasts proliferation and differentiation.The"nano-ZnO particles layer"on the surface of HAw effectively prevented the integrity of osteoblasts membrane from being destroyed by HAw,and reduced the mechanical damage caused by HAw to cells.At the same time,the released Zn²⁺from nano-ZnO particles increased the endonuclear Nrf2 expression,reduced the intracellular ROS level and promoted the differentiation of osteoblasts.The inhibitory effects of HAw on osteoblasts proliferation and differentiation were rescued through the both aspects.