Apatite/laminin Coating To Regulate Behaviors Of Neural Cells

In the process of repair and regeneration after nerve injury,the proliferation of neural cells and neural stem cells(NSCs)is very important.In recent years,it has been proposed that the microenvironment of damaged nerves had great influence on neural cells and NSCs.With the rapid development of tissue engineering technology,biomaterials with good biocompatibility and biodegradability immobilized a series of bioactive molecules on the surface are used to create biological environments that can regulate behaviors of neural cells and NSCs,which has been an effective strategie to promote nerve repair.In the present study,diverse methods,such as physical adsorption,covalent chemical bonding and some suitable loading carrier have been applied to immobilized biological molecules on the surfaces of substrates.However,immobilizing biological molecules through physical adsorption is unsatisfied,like low surface adsorption efficiency and initial rapid diffusion of bioactive molecules from the surface of the material.Typical chemical approach requires tedious crosslinking procedures with organic chemical solution,which may destruct surface feature of substrates and denature immobilized molecules.Therefore,it is crucial to develop a simple and effective immobilization strategy.Apatite,a naturally occurring inorganic material,is currently widely used for biomedical applications due to its nontoxicity,excellent biocompatibility,biodegradability,and favorable affinity to plenty molecules.In this study,apatite coating was prepared on medical titanium by alkali treatment and biomimetic precipitation.Laminin(LN),one of the ECM components which is continuously synthesized after nerve injury,was immobilized on apatite coating by coprecipititation to obtain a functional coating(apatite/LN).The microcosmic morphology,chemical composition and physicochemical properties of the coating were measured and the amount and retention of immobilized LN were investigated.Further,we cultured PC12 and NSCs on apatite and apatite/LN to determine their effects on cell adhesion,proliferation,differentiation and also investigated the cytotoxicity of apatite and apatite/LN in vitro.The details and results of the study are as follows:1)A porous and unifor apatite coating can be prepared on titanium by biomimetic mineralization.The surface morphologies of apatite and apatite/LN were revealed by field emission scanning electron microscopy(FE-SEM).During the biomimetic precipitation,flake-like structure was observed on the surface,and the size of the flake has increased gradually.Both biomimetic apatite and apatite/LN displayed homogeneous porous structure composed of straight and sharp units.With further coprecipitating LN with apatite,the flake size of apatite/LN(9.79 ± 0.69 ?m)were slightly larger than that of biomimetic apatite(8.16 ± 0.87 ?m).2)Detection of nitrogen by energy dispersive spectrometer(EDS)and X-ray photoelectron spectroscopy(XPS)and detection of amide groups by attenuated total reflection-fourier transform infrared spectroscopy(ATR-FTIR)showed the chemical composition of apatite and apatite/LN.The amount of the immobilized LN within the apatite/LN coating was quantified using the Micro-BCA protein assay quantification kit,which increased from approximately 1.43 ?g to nearly 4.55 ?g with increasing initial concentration of LN from 5 ?g/mL to 40 ?g/m L.The results suggested that LN can be immobilized on the coating successfully.3)According to the investigation of physicochemical properties of the coating,Apatite/LN coating(9.57 ?m)was slightly thicker than apatite coating(8.79 ?m).The bonding strength of apatite/LN and apatite coating were comparable,which were 11.06 MPa and 10.11 MPa,respectively.In addition,the water contact angle of the surface of apatite/LN and apatite coating were tested,which suggested that the hydrophilic property was effectively improved with LN immobilization.4)Degradation in vitro of apatite/LN coating in phosphate buffered saline(PBS)was studied.The flake of apatite/LN was destroyed slightly after degradation for 28 days.After 90 days,the flake-like structure almost degradated.The release in vitro of LN within the apatite/LN coating was at a slow rate,and the released amount was about 20.72%.5)Cell experiments of PC12 and NSCs showed that the apatite/LN coating,with excellent biocompatibility,can effectively promote adhesion,proliferation and synapse formation of neural cells and NSCs,Moreover,NSCs were perefered to differentiate into neurons on the apatite/LN coating.