Synthesis And Biological Study Of Graphene-incorporated Polyamide 66 Functional Nanocomposites And Nano-hydroxyapatite Composites

1 Background Ideal orthopedic implant materials should have excellent mechanical properties,biocompatibility and osteoinductivity.At present,titanium alloy,stainless steel and other metal is the most common clinical material of internal fixation implants.As a result of "stress shelter" effect of high rigid metal materials leading to implant loosening as well as complications such as metal allergy and peri-implant inflammation,many patients need a second surgery to take out the internal fixation implants,increasing patients’ risks and consuming valuable medical resources.Degradation products of poly(lactic acid)(PLA)and other biodegradable materials such as magnesium alloy lead to local inflammatory response and even subcutaneous emphysema,which limit the application of such materials.Nano materials and nano technology is widely used in many fields,which is also the research focuses in biomedical field.Main work of our research team is focused on the synthesis of nanomaterials and its application in bone tissue engineering for basic and clinical research.Graphene is one of the most popular nanomaterials in the past decade.Due to its striking characteristics and excellent performance,graphene has been widely concerned in physics,material science,chemistry,energy,biomedical and other fields.Graphene/polymer nanocomposites is one of the most promising applications of graphene.Even a small amount of added graphene can significantly enhance the mechanical,electrical,thermal and other aspects of properties of polymer materials.Polyamide 66(PA66),which is also known as nylon 66,is widely used in a variety of applications because of its abrasion resistance,chemical resistance as well as excellent mechanical properties.PA66 also exhibits similarity to collagen protein in chemical structure and possess good biocompatibility without foreign body reaction according to our previous studies.In this paper,in combination with the research background of our team,we cooperate PA66 with graphene to prepare graphene-incorporated polyamide 66 nanocomposite(G/PA66),and evaluate its bioactivity and biocompatibility as an application for orthopedic implant materials in vitro and in vivo.2 Objectives(1)Preparation of graphene-incorporated polyamide 66(G/PA66).(2)Characterization of G/PA66.(3)In vitro study of G/PA66 composites for bone marrow mesenchymal stem cell adhesion,proliferation,differentiation.(4)In vivo evaluation of bioactivities and biocompatibility of G/PA66 bone screws.3 Method(1)The G/PA66 composite was prepared by solvent blending.G/PA66 screws and specimens for mechanical property testing were prepared by an injection-molding machine and hot pressed into specimens with a controlled shape.G/PA66 substrata was prepared by a spin-coater.G/PA66 was characterized by atomic force microscopy(AFM),raman spectroscopy,field emission scanning electron microscopy(FESEM),X-ray diffraction(XRD)observation,and mechanical properties evaluation.The loading capacities of the composites for dexamethasone and β-glycerolphosphate were determined by spectrophotometry.(2)C3H10T1/2 cells were cultured on G/PA66 substrata,and the adhesion as well as proliferation of the cells were evaluated by SEM,immunofluorescence microscopy,and quantitative real-time polymerase chain reaction(q RT-PCR)analysis.The cell cycle of C3H10T1/2 cells was determined by flow cytometry.Cytotoxicity studies of the G/PA66 composites was performed by indirect CCK-8 assay for periods of 1,2 and 3 days.(3)To further evaluate the effect of G/PA66 for the osteogenesis differentiation of C3H10T1/2 cells,calcium deposits and adipogenesis was analyzed by alizarin red staining and oil red staining.The gene expression of osteoblast markers was evaluated by q RT-PCR.(4)G/PA66,PA66 and stainless steel bone screws were implanted into dogs for in vivo study.The peri-implant bone formation was evaluated by micro computed tomography(micro-CT)and histomorphometry.The maximum removal torque value of the breakage of the bone-implant interface was measured.Serum was collected for analyzed of alanine aminotransferase,a marker for liver function;creatinine,a marker for kidney function;and C-reactive protein(CRP),a marker of systemic inflammation.Sections of organs(liver,spleen,kidney and brain)were stained with hematoxylin and eosin.5)The G/n HA/PA66 composite was prepared by solvent blending and injection molding and characterized by SEM,XRD.Mechanical properties such as the tensile strength and elastic modulus were tested by a mechanical testing instrument with a testing speed of 5 mm min-1.(6)The in vitro biocompatibility of G/n HA/PA66 was evaluated by CCK-8 analysis and fluorescence staining.The osteogenesis promoted by G/n HA/PA66 was determined by quantitative evaluation of Alizarin red staining and q RT-PCR test.G/n HA/PA66 screws were implanted into femoral condyle of dogs to evaluate the in vivo biocompatibility.The same surgical procedure were done in the sham operation control group,without implantation of screws.The animals were sacrificed 24 weeks after operation and were collected as specimens.4 Results(1)Unique characteristics of the nano scale topography was observed by AFM on surface of G/PA66,with a significantly increased roughness.The Raman spectrum of G/PA66 and graphene had two peaks which was known as the G and D bands(~1600cm-1 and ~1350cm-1 respectively),and no peaks was observed on Raman spectrum of PA66.The diffraction peak in graphene is too weak to be visible when drawn together with XRD pattern of graphite.The XRD pattern of G/PA66 is almost the same with that of PA66,without a graphite layer structure peak at 26° or diffraction peaks derived from graphene,indicating the exfoliated feature of graphene nanosheets in the polymer matrix.Graphene dispersed well in PA66 matrix,which significantly increased the tensile strength,yield strength and elasticity modulus by 53.41%,48.31% and 81.13% respectively compared with PA66.(2)C3H10T1/2 cells on G/PA66 exhibited improved spread and a rough surface with extracellular matrices containing globules of mineral deposits after an initial culture time of 4 h,in contrast to PA66,on which small elliptical-shaped and relatively smooth cells were observed.After 24 h of culture time,the elliptical morphology still dominated the appearance of C3H10T1/2 cells on the PA66 substratum,whereas the surface of the G/PA66 substratum was covered with a sheet-like and flattened confluent layer,implying enhanced attachment to and interaction with the composite surface.Greater cytoskeletal organization(f-actin)and more focal adhesions(vinculin)were observed in C3H10T1/2 cells cultured on G/PA66 substratum than on PA66 substratum at both 4 h and 24 h of culture.The m RNA expression levels of α1 integrin and cx43 were significantly upregulated in C3H10T1/2 cells on the G/PA66 substratum(P<0.05).(3)The flow cytometry results demonstrated that cells were promoted to S and G2/M phase by the G/PA66 nanocomposite substratum;81.07%,7.16% and 11.77% of cells in the PA66 group and 62.49%,11.98% and 25.52% of cells in the G/PA66 group were in G0/G1,S and G2/M phases,respectively.The m RNA levels of cyclins A2,B1,D1 and E1 of cells on G/PA66 were significantly increased compared to the control(P<0.05).(4)Cytotoxicity studies of the G/PA66 composites was performed by indirect CCK-8 assay for periods of 1,2 and 3 days.For all culture periods,cell viability was reduced in the positive control group.No cytotoxicity was observed in the G/PA66 group or the control group because no reduction in cell viability was observed.(5)After 14 days of culture on PA66 or G/PA66,in the presence of G/PA66 and osteogenic medium,calcium deposits were significantly increased compared with the other groups.Quantification analysis confirmed these observations,revealing significantly increased absorbance at 450 nm for cells cultured with G/PA66 compared to PA66(P<0.05).The m RNA levels of ALP,OC and COL-I of cells on G/PA66 were significantly increased,and PPARγ was notably decreased in the osteogenic medium environment after incubation for 7 or 14 days(P<0.05).The loading capacities of the G/PA66 composites for several osteogenic chemical inducers were determined.The adsorption isotherms indicated a significantly improved loading capacity of G/PA66 for the chemical agents.(6)A decreased propensity of adipogenic differentiation was observed among cells differentiated on G/PA66,with a significantly lower amount of lipid deposits than cells differentiated on PA66.Quantification analysis indicated that G/PA66 suppressed adipogenic differentiation,with 25.48±13.99 μm2/cell,whereas PA66 enhanced adipogenesis,with 142.0±50.73μm2/cell.(7)The bone-implant interface was observed 4 weeks after surgery.A higher degree of bone-implant contact was observed on G/PA66(76.95±4.67%),and the surrounding trabeculae grew toward and finally enclosed the implant surface.The new bone contact with the stainless steel surface was indirect with a BIC of 52.14±9.43%(P<0.05).The bone volume fraction(BV/TV)was significantly increased in the G/PA66 group compared to the control(PA66 group).This increase in BV/TV comprised a 33.3% increase in trabecular number(Tb.N),a 44.1% increase in trabecular thickness(Tb.Th)and a 32.2% decline in trabecular separation(Tb.Sp)(P<0.05).The removal torque was tested 1 week and 8 weeks after operation.At the earlier time point,the G/PA66 screws exhibited a slightly but not significantly higher removal torque.At the later time point,8 weeks after operation,the removal torque was significantly increased for both groups(P<0.05).(8)No significant changes in either marker of organ toxicity was found((P>0.05))and no gross abnormalities were observed in any analyzed tissue.Inflammatory granulocytes occupied small focal areas within the peri-implant tissue of stainless steel screws.(9)Well dispersion of graphene and n HA in PA66 matrix was confirmed by SEM and XRD.The tensile strength and elastic modulus of the composites were significantly improved by 49.14% and 21.2% respectively compared with n HA/PA66(P<0.05).(10)CCK-8 assays were performed after the cells were co-cultured with the G/n HA/PA66 composite for 1,3 and 7 days.The results demenstrated that the OD value of each group increased over time,and the group with graphene additives in n HA/PA66 exhibited higher metabolic activity than that of n HA/PA66(P<0.05).Cell morphology was observed by immunofluorescence staining after co-culturing with G/n HA/PA66 for 4,24 and 72 hours.Cells co-cultured with G/n HA/PA66 exhibited a higher density and a significantly clearer cytoskeleton.Calcium salt deposition of C3H10T1/2 cells during osteogenic differentiation was detected by an alizarin red staining assay.After 7 and 14 days,a large area of orange staining was observed in the surrounding material,indicating precipitations of calcium salt.The quantitative detection results showed that graphene-reinforced n HA/PA66 co-cultured cells produced more calcium salt precipitation(P<0.05)than the control group(n HA/PA66).At the molecular level,after the cells were co-cultured with the composite materials for 7 or 14 days,the ALP and OC expression was significantly up-regulated(P<0.05)in G/n HA/PA66 co-cultured cells compared with that of the control group(n HA/PA).16 weeks after G/n HA/PA66 was implanted in vivo,histological observation was performed for the liver,spleen,kidney,brain and tissues around implants using HE staining demonstrated no significant abnormalities.5 Conclusions In this study,G/PA66 was prepared by solvent blending with significantly improved mechanical properties and surface roughness of the material.Adhesion,proliferation as well as osteogenic differentiation was promoted by G/PA66 in vivo.Good biocompatibility and enhanced bone formation around the G/PA66 were observed in vivo.All acquired data support the potential use of G/PA66 composites in bone tissue engineering and regenerative medicine applications.