A Method To Visually Observe The Degradation-diffusion-Reconstruction Behavior Of Hydroxyapatite In The Bone Repair Process

Background and Objective:In recent years,nanostructured hydroxyapatite(HAp)has been widely recognized as a scaffold material for bone tissue engineering because of its good osteoinduction and biodegradation.However,in the bone repair process the degradation process and distribution of HAp in the cavity of the bone defect are still unclear.Terbium(Tb),as one of the lanthanide elements,has good biocompatibility and fluorescence stability.It has been widely used in bioluminescence labeling.To study the behavior of HAp in the process of bone repair intuitively,HAp/Tb-HAp nanowires(NWs)membrane with concentric circle structure(CCS)was designed,and the inner circle and outer ring were constructed with Tb-HAp NWs and HAp NWs,respectively.HAp/Tb-HAp CCS membrane had both good osteogenic ability and high fluorescence visibility.HAp/Tb-HAp CCS membrane was implanted into the rat calvarial defect model to study the degradation-diffusion-reconstruction behavior of HAp in the process of bone defect repair in vivo by tracking the fluorescence distribution.Therefore,this study provided an intuitive method to observe the degradation-diffusion-reconstruction behavior of HAp nanomaterials in the bone repair process.Materials and Method:1.Synthesis and characterization of HAp NWs and Tb-HAp NWs membranes.HAp NWs and Tb-HAp NWs membranes were synthesized by the hydrothermal method.Phase analysis of HAp NWs and Tb-HAp NWs were conducted by X-ray diffraction(XRD).Qualitative analysis of HAp NWs and Tb-HAp NWs was carried out by Fourier transform infrared spectroscopy(FTIR).The morphology and lattice characteristics of HAp NWs and Tb-HAp NWs were observed under the scanning electron microscope(SEM)and transmission electron microscope(TEM).The excitation and emission spectra were measured by a fluorescence spectrophotometer(FLS).The doping concentration of Tb was analyzed by an electron probe microanalyzer(EPMA).2.In vitro detection of biocompatibility,osteogenic differentiation,and biodegradation of HAp NWs and Tb-HAp NWs membranes.2.1.Rat bone mesenchymal stem cells(BMSCs)were implanted on HAp NWs and Tb-HAp NWs membranes,and implanting BMSCs on the blank tissue culture plate(TCP)was used as the negative control to observed the cell morphology under SEM,performed live-dead cell staining,and cell activity was measured by cell counting kit 8(CCK8).2.2.BMSCs were inoculated onto HAp NWs and Tb-HAp NWs membranes,and osteogenic induction culture was carried out with implanting BMSCs on TCP as the negative control.The osteogenic potential in vitro was determined by alkaline phosphatase(ALP)activity and quantitative real-time polymerase chain reaction(qRT-PCR)gene expression.2.3.Choose the same quality HAp NWs and Tb-HAp NWs membranes compared with sintered HAp.Three samples from each group of HAp NWs,Tb-HAp NWs and sintered HAp were immersed in'sterile phosphate buffered solution(PBS)of different pH values(5.6 and 7.4).At different time points,equal volume samples were collected and replaced with fresh PBS,and then methylene blue method calcium detection kit was used to determine the content of Ca2+ ion released by each group of materials.The concentrations of Tb3+and Ca2+ ions in Tb-HAp NWs were determined by inductively coupled plasma optical emission spectroscopy(ICP-OES).3.The HAp/Tb-HAp CCS membrane was designed to facilitate visual observation of the bone repair process in vivo.3.1.The HAp/Tb-HAp CCS membrane was synthesized by cutting and inlaying method,inserting a Tb-HAp inner circle with a diameter of 3 mm into a HAp outer ring with an outer diameter of 5 mm and an inner diameter of 3 mm to prepare a HAp/Tb-HAp CCS membrane.3.2.Thirty-six 8-week-old male Wistar rats were selected to establish a 5-mm-diameter bilateral rat calvarial defects model.HAp NWs membrane or HAp/Tb-HAp CCS membrane was filled in the right side while the left side was empty as NC.After the operation,the samples of the HAp/Tb-HAp group were placed.The fluorescence distribution of the samples was observed at an excitation wavelength of 470-490 nm under a fluorescence microscope,and the relative fluorescence intensity was measured by ImageJ.3.3.Micro-computed tomography(micro-CT)was used to scan the calvarial bone in three dimensions,and avatar software was used to reconstruct the calvarial bone in three dimensions,and ImageJ was used to analyze the quality and quantity of newly formed bone tissue in the defect area.3.4.The samples were made into paraffin sections and stained with hematoxylin and eosin(H&E)and Masson's trichrome.The new bone in the defect area was observed under the microscope and measured with Image-Pro-Plus 6.0 software.Results:1.HAp NWs and Tb-HAp NWs films were successfully prepared.XRD patterns confirmed the crystal phases of HAp and Tb-HAp nanocrystals.Both HAp NWs and Tb-HAp NWs had typical diffraction peaks.FTIR showed that HAp NWs and Tb-HAp NWs had the intrinsic peak of HAp at 3752 cm-1(-OH)and 1029 cm-1(PO43-).SEM results showed that the length of HAp NWs was about 200 ?M.After Tb doping,the length of Tb-HAp NWs was about 100 ?M.Therefore,Tb doping could reduce the length of HAp NWs.The width of HAp NWs and Tb-HAp NWs were about 10 nm,which showed that Tb doping had little effect on the width of NWs.Under 488 nm excitation,Tb-HAp NWs could emit 543 nm light.The molar ratio of Tb/Ca obtained by EPMA quantitative analysis was?3%.2.HAp/Tb-HAp CCS membrane had good biocompatibility,could significantly promote the osteogenic differentiation of BMSCs and could be biodegraded.2.1.The adhesion between BMSCs and HAp NWs or Tb-HAp NWs was observed by SEM.The cells grew well and had a lot of pseudopods.The results of live/dead cell staining and CCK8 showed that BMSCs showed good activity on HAp NWs and Tb-HAp NWs membranes compared with the TCP.2.2.After 7 days of osteogenesis induction,the ALP activity of BMSCs cultured on HAp NWs and Tb-HAp NWs membranes was significantly higher than that of BMSCs cultured on TCP(P<0.0001).The expression of osteocalcin(OCN),bone sialoprotein(BSP)and ALP on HAp NWs and Tb-HAp NWs membranes was significantly up-regulated(P<0.0001)compared with TCP after 7 and 14 days of osteogenesis.2.3.The results of calcium ion detection kit showed that in the PBS at pH 5.6 and 7.4,Ca2+ ions were continuously released from HAp NWs membranes,Tb-HAp NWs membranes,and sintered HAp.Compared with pH 7.4,the degradation rate of materials in the PBS at pH 5.6 was faster.At the same time,the degradation rate of HAp NWs and Tb-HAp NWs was significantly faster than that of sintered HAp.The results of ICP-OES analysis of Tb-HAp NWs showed that with the increase of Ca2+ ion concentration,the concentration of Tb3+ ion increased simultaneously at pH 5.6 and pH7.4,indicating that the simultaneous release of Tb3+ ion showed that the change of fluorescence distribution could be used to evaluate the degradation behavior of HAp in the process of bone repair.3.HAp/Tb-HAp CCS membrane was successfully synthesized and used to observe the degradation-diffusion-reconstruction behavior of HAp in the process of repairing bone defects in vivo.3.1.The boundary between the inner circle of fluorescent Tb-HAp and the outer ring of dark HAp can be observed obviously in the initial state when the samples of the HAp/Tb-HAp CCS membrane were placed under the fluorescence microscope with the excitation wavelength of 470-490 um.Four weeks later,the green fluorescence region began to invade the outer ring of HAp.The fluorescence intensity of the inner circle of Tb-HAp was the strongest,followed by that of the outer ring of HAp,while the surrounding bone tissue had no fluorescence.After 8 w,the fluorescence intensity of the HAp outer ring was significantly higher than that of 4 w,and the boundary between the HAp outer ring and origin bone was clear.After 12 w,there was no significant difference of the fluorescence intensity between the outer ring of HAp and the inner circle of Tb-HAp.The results showed that Tb3+ions diffused uniformly to the whole defect area and as a fluorescent indicator visualized the degradation-diffusion-reconstruction behavior of HAp in the process of bone repair.3.2.Micro-CT results showed that the bone healing of the experimental group with HAp NWs membrane and HAp/Tb-HAp CCS membrane was significantly better than that of NC group at 4,8 and 12 w,and there was no significant difference between the two experimental groups.3.3.H&E and Masson trichrome staining showed that the amount and maturity of bone repair in the HAp membrane and HAp/Tb-HAp CCS membrane group were significantly higher than that in the NC group at 4,8 and 12 weeks.Conclusion:HAp/Tb-HAp CCS membrane composed of the inner circle of Tb-HAp NWs and the outer circle of HAp NWs was successfully prepared.The degradation-diffusion-reconstruction behavior of HAp in the process of the bone repair was observed.Both Tb-HAp NWs and HAp NWs had high biocompatibility,could induce osteogenic differentiation of BMSCs and could be biodegraded in vitro.In vivo study of the rat calvarial defect repair model showed that the central fluorescence gradually diffused outwards until it was evenly distributed in the bone defect area,indicating that HAp first degradation,then the degraded products diffused,and finally formed new HAp to repair the bone defect.Besides,micro-CT and histological analysis showed that the doping of Tb did not affect the process of bone repair.Therefore,this study provides a new research method for visualizing the degradation-diffusion-reconstruction behavior of HAp in the process of bone repair and evaluating the osteogenic ability of HAp-based materials.