Fabrication And Surface Modification Of Porous Polyetheretherketone Microspheres For Bone Repair

Purpose of the study: Fabrication and surface modification of porous polyetheretherketone(PEEK)microspheres for bone repair.Materials and Methods: 1.In our previous work,smooth PEEK microspheres(S-MS)have been prepared by liquid-liquid phase separation method.In this study,porous PEEK microspheres(PMS)were fabricated via hydroxylation of S-MS.Scanning electron microscope(SEM)observation,Fourier transform infrared spectrometer(FTIR)analysis,mercury intrusion porosimetry,water contact angle,protein adsorption capability and cytotoxicity tests by extracts were then performed.Mineralized extracellular matrix(m ECM)could be formed on the surface of P-MS by mineralizing culture and repeated decellularization technique.The m ECM was then observed by SEM,and it was qualitatively and quantitatively analyzed by energy dispersive X-ray spectrometer(EDX)and thermal gravimetric analyzer(TGA)respectively.2.The in vitro and in vivo bone regeneration capability of P-MS alone and with m ECM coating were then evaluated.Calcium-AM staining and SEM observation were used to assess the adhesion and ECM secretion of MC3T3-E1 cells on different microsphere surfaces.DAPI staining and CCK-8 assay were used to evaluate cell proliferation.In vitro bone regeneration capability of different samples was assessed by alkaline phosphatase staining(ALP),ALP activity,alizarin red s(ARS)staining,calcium deposition assay and gene expression level of Runx2 and Col-1.The calvarial defects of rats were created to further evaluate the bone repair of different samples through micro-CT reconstructive analysis,hematoxylin and eosin(H&E)and Masson staining.3.Insulin-like growth factor 1(IGF-1)and bone morphology protein 2(BMP-2) were immobilized on P-MS through the adhesion characteristics of dopamine(DA)coating.The change of color and surface morphology of P-MS with DA coating were observed by stereomicroscope and SEM,the composition and proportion of surface elements by X-ray photoelectron spectroscopy(XPS),and hydrophilicity by contact angle test.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the immobilized amount and 14 days released behavior of growth factors on the microspheres.In vitro bone regeneration capability of different samples was assessed by ALP staining,ALP activity,ARS staining,calcium deposition assay and gene expression level of Runx2,OPN and OCN.The calvarial defects of rats were created to further evaluate the bone repair of different samples through micro-CT reconstructive analysis,H&E and Masson staining.Results: 1.SEM observation shows that P-MS maintains spherical shape after hydroxylation and the surface pore structure is uniform.Compared with the S-MS,FTIR shows that the carbonyl group was significantly reduced in the P-MS and the alcoholic hydroxyl group was newly formed.As measured by mercury porosimeter,PMS had a porous rate greater than 90% and an average diameter of 569.72 nm.Compared with S-MS,the water contact angle of P-MS is significantly reduced,and protein adsorption capacity is improved.After mineralizing culture and repeated decellularization treatment,nitrogen,calcium and phosphorus were found on the surface of microspheres by EDX.Through SEM observation,it is found that with the increase of decellularization cycles,the m ECM on the surface of P-MS was denser and more uniformly distributed.The results of TGA show that the mass loss of microspheres at 400 degrees can increase from 6.41% to 12.38% with the increase of decellularization times.The cell viability of 100% and 50% extracts of P-MS was greater than 85% and 95%,respectively.2.When microspheres were co-cultured with MC3T3-E1 cells,at each prescribed time point,Calcein-AM staining,DAPI staining,CCK-8 assay and SEM observation were carried out.Compared with S-MS,the results show that more cells could be found on the surface of P-MS,cells were typically flat,highly elongated with more protruding filopodia.The cell adhesion status is better and the secretion of ECM more vigorous.With the increase of decellularization times,the number of cells adhered to the surface of P-MS surface and the absorbance value by CCK-8 method to detect cell proliferation increased continuously.After ALP and ARS staining,the P-MS presented deeper color than S-MS,the activity of ALP,calcium deposition analysis,Runx2 and Col-1 expression levels have also been significantly improved.When the surface of P-MS was coated with m ECM,the above-mentioned in vitro bone regeneration capability was further improved.After 4 weeks of implantation,micro-CT reconstruction shows that in the m ECM coated microspheres group,the newly formed bone almost completely covered the defected area.After 8 weeks of implantation,it exhibited superior bone thickness and density than other groups.The fraction of bone volume was 42.19 ± 2.80% and 65.19 ± 1.63%,respectively,which was also significantly higher than other groups.Histochemical staining found that in the S-MS group,the microsphere was mainly wrapped with fiber tissue and could not form a strong bond between the newly formed tissue and the material itself,resulting in microspheres missing in sections.However,P-MS was mainly wrapped with new bone tissue which is closely bonded with materials and there is no microsphere missing.Masson staining indicated that the tissue around m ECM coated microsphere was mainly dense mature bone and more blood vessels could be found around it.3.When DA coating was fabricated on P-MS,the color turned from white to uniform dark gray and SEM observation shows no significant change of surficial morphology.Elemental analysis shows that nitrogen appeared on the surface of the microsphere after DA coating,and quantitative analysis showed that the proportion of nitrogen increased from 1.58% to 1.71% after IGF-1 immobilization.The DA coating reduces the water contact angle of P-MS from 65.88 ± 4.55 degrees to 51.08 ± 3.7 degrees.ELISA assay has confirmed that DA coatings could successfully immobilize growth factors on P-MS and present a relatively slower release behavior within 14 days.After the microspheres were co-cultured with MC3T3-E1 cells,at each prescribed time point,Calcein-AM,DAPI staining and CCK-8 assay indicated that the microsphere immobilized with IGF-1 showed better cell adhesion status and larger cell numbers.CCK-8 assay shows that the cell proliferation rate of the microsphere immobilized with dual growth factors was slightly lower than that of the microsphere immobilized with IGF-1 alone,and the difference was not significant,but significantly higher than that of the other groups.After ALP and ARS staining,microsphere immobilized with BMP-2 presented deeper color,but the difference between BMP-2 alone and dual group was not obvious.ALP,calcium quantitative analysis,Runx2,OPN and OCN expression levels were significantly higher at 14 days in dual growth factors group.Micro-CT reconstruction shows that the dual growth factors group not only completely covered the defect area at 8 weeks,but also had a high bone density,and the center of the bone defect area was almost as high as the defect edge.Bone connections can be observed on coronary CT images above the X-Ray permeable PEEK microsphere,forming a "bone bridge" structure that connects the edge of the bone defects to the newly formed bone in the central area.The fraction of bone volume of the dual growth factors group at 8 weeks was 85.79 ± 6.20%,significantly higher than that of the other groups.Histochemical staining observation shows that in the dual growth factors group,the newly formed structure is denser and mainly made up of mature bone tissue,blood vessels are also densely distributed.Conclusions: 1.P-MS was successfully fabricated from S-MS by hydroxylation treatment.2.The size of the micropores on the surface of P-MS can be regulated by adjusting exchange solution composition and temperature.3.Through mineralizing culture and repeated decellularization technique,m ECM could be deposited on P-MS.With the increase of decellularization times,m ECM is denser and more uniformly distributed.4.Compared with S-MS,P-MS is more conducive to cell adhesion,proliferation and ECM secretion.5.P-MS coated with m ECM further promotes cell adhesion,proliferation,ALP activity,calcium deposition and bone-related gene expression level.The P-MS group shows better bone repair outcomes in rat calvarial defects than S-MS.6.IGF-1 and BMP-2 could be immobilized on P-MS by DA coating and presents sustained release behavior within 14 days.7.Compared with the application of single growth factor,the microsphere immobilized with dual growth factors improved its in vitro osteogenic capability and calvarial defects repair in rats.Innovations in this study: 1.The surface topology and chemical composition of microsphere could be modified simultaneously in one step through hydroxylation.2.Mineralized ECM could be successfully coated on the surface of P-MS through mineralizing culture and repeated decellularization technique.3.IGF-1 and BMP-2 are jointly used to promote bone repair and can reduce the dose of single growth factor.