Nano Functional Surface Modification For Bone Repair Materials

The poor osteoinductivity of bone repair materials and the postoperative infection are the main challenges in clinics. It is important to prepare the bone repair materials with good biocompatibility through surface modifications. Most previous studies on surface modifications focused on changing the micro/nano structures or physical-chemical properties of bone repair materials, so as to endow materials with osteoinductivity. The objectives of this study is to modify the surfaces of bone repair materials with multi-functions, including osteoinductivity, antibacterial and stimuli-responsive properties. The main contents include the following aspects:(1) Bone morphology protein-2 (BMP-2) and Ag nanoparticle contained hydroxyapatite(HA) coatings were prepared on Ti surfaces by combining electrochemical deposition (ED)of Ag and electrostatic immobilization of BMP-2. In the first step of ED process, chitosan(CS) was selected as the stabilizing agent to chelate Ag ions and generate Ag nanoparticles that are uniformly distributed in the coatings. CS also reduces Ag toxicity while retaining its antibacterial activity. In the second step, a BMP/heparin solution was absorbed on the CS/Ag/HA coatings. Consequently, BMP-2 was immobilized on the coatings by the electrostatic attraction between CS, heparin, and BMP-2. Sustained release of BMP-2 and Ag ions from HA coatings was successfully demonstrated for a long period. Results of antibacterial tests indicated that the CS/Ag/HA coatings have high antibacterial properties Osteoblasts (OB) culture revealed that the CS/Ag/HA coatings exhibit good biocompatibility.Bone marrow stromal cells (BMSCs) culture indicated that the BMP/CS/Ag/HA coatings have good osteoinductivity and promote the differentiation of BMSCs. Results of in vivo study indicated that BMP/CS/Ag/HA coatings favor bone formation.(2) Pulse electrochemical deposition was utilized to co-synthesize hydroxyapatite (HA)and silver (Ag) nanoparticles (NPs) with the mediation of polypyrrole (PPy) on Ti surfaces.PPy-NPs formation is critical for the success of spherical HA-NPs and uniform distribution of Ag-NPs. The electrochemical process includes two pulses. In the first pulse, Py is polymerized to PPy-NPs under electrochemical oxidative condition and PO43- ions are doped into the backbone of PPy. Then, PPy-NPs are further polymerized to form PPy coatings on the surfaces of cathode. In the second pulse, HA-NPs are formed by the mediation of PPy-NPs. PPy-NPs with doped PO43- work as a "spherical template" to attract Ca2+ to the surfaces. The PPy-NPs with PO4 3- and Ca2+ move to the working electrode driven by the electric field and form HA-NPs with the addition of OH-, which is supplied by the electrolysis of H2O. Ag-NPs also form because PPy works a stabilizing agent to control Ag reduction and nucleation. The HA-Ag-PPy coatings exhibit both good osteoconductivity and high antibacterial activity. Moreover, Ag-NPs do not affect the biocompatibility of HA-Ag-PPy coatings.(3) A pulse electrochemical driven Layer-by-Layer (PED-LbL) assembly process was developed to rapidly deposit HA-NPs and PDA (HA-PDA) multilayer nanofilms. In this process, PDA and HA-NPs are in situ synthesized in two sequential oxidative and reductive pulses in each electrochemical deposition cycle and alternatively deposited on the substrates.PDA assists the in situ synthesis of HA-NPs by working as a template, which avoids the non-controllable HA nucleation and aggregation. The HA-PDA multilayer nanofilms serve as a tunable reservoir to deliver bone morphogenetic protein-2 and exhibit high osteoinductivity both in vitro and in vivo. This PED-LbL assembly process breaks the limitation of traditional LbL assembly, allowing not only the rapid assembly of polyelectrolytes, but also in situ synthesis of organic/inorganic NPs that are uniformly incorporated in the nanofilm. It has broad applications in the preparation of versatile surface coatings on various biomedical devices.(4) Electroresponsive and conductive poly dopamine-polypyrrole microcapsules(PDA-PPy-MCs) were deposited on the surface of medical devices by the electrochemical deposition. First, sulfonated polystyrene microspheres (SPS-MS) as the sacrificial template were assembled on the surfaces of Ti. Then, PDA-PPy composite coatings were polymerized on the surfaces of the SPS-MS through electrochemical oxidative polymerization. Finally,PDA-PPy-MCs were obtained after removing the SPS-MS template. PDA-PPy-MCs have the cell affinity of PDA, the microporous structures of MCs and the electroresponsive capability of PPy. Incorporation of PDA promoted the conductivity and the adhesive strength of PDA-PPy-MCs. PDA-PPy-MCs could respond to electrical signals so as to on-demand release DEX. During the electrochemical oxidative process, dexamethasone (DEX) as the anion could be doped into the backbone of PPy with PDA to neutralize its positive charges.The microporous structures and PDA improved the drug loading capability of PDA-PPy-MCs. A high throughput BMSCs culture system was designed to study the synergistic effect of compositions, microstructures and electrical stimulation on cell behavior.The results indicated that the cell affinity of PDA and the microporous structures not only enhance the biocompatibility of PDA-PPy-MCs, but also strengthen the effect of electrical stimulation with PDA-PPy-MCs. In vivo study showed that PDA-PPy-MCs have good biocompatibility. The super cell affinity, controllable microstructures, good conductivity and ability to on demand deliver drugs make it a promising candidate for electrical therapy.(5) Biomimetic octacalcuim phosphate mineralized graphene oxide/chitosan(OCP-GO/CS) scaffolds with hierarchical structures were developed, which have good mechanical property and adsorbability. First, GO/CS scaffolds with large micropores (～ 300μm) showed high mechanical strength due to the electrostatic interaction between the oxygen-containing functional groups of GO and the amine groups of CS. Secondly, OCP with porous structures (～1 μm) was biomimetically mineralized on the surfaces of the GO/CS scaffolds (OCP-GO/CS). The hierarchical microporous structures of OCP-GO/CS scaffolds provide a suitable environment for cell adhesion and growth. The scaffolds have exceptional adsorbability of nanoparticles. Bone morphogenetic protein-2 (BMP-2)encapsulated bovine serum albumin (BSA) nanoparticles and Ag nanoparticles (Ag-NPs)were adsorbed in the scaffolds for enhancement of osteoinductivity and antibacterial properties, respectively. Antibacterial tests showed that the scaffolds exhibited high antibacterial properties against both Escherichia coli and Staphylococcus epidermidis. In vitro and in vivo experiments revealed that the scaffolds have good biocompatibility,enhanced BMSCs proliferation and differentiation,and induced bone tissue regeneration.Thus, the biomimetic OCP-GO/CS scaffolds with immobilized growth factors and antibacterial agents might be an excellent candidate for bone tissue engineering.(6) Graphene oxide (GO) were adsorbed on the surfaces of calcium phosphate (CaPs)scaffolds (GO-CaPs) to endow the CaPs scaffolds with the capability of loading BMP-2,immobilizing BMP-2 encapsulated BSA nanoparticles (BSA-NPs) and gelatin microparticles(Gel-MPs). The BMP-2 release study revealed that GO realized the sustain release of BMP-2 from the CaPs scaffolds. The immobilized NPs and MPs on the GO-CaPs scaffolds have the slower BMP-2 release rates than that of BMP-2 directly absorbed on the CaPs and GO-CaPs scaffolds. BMP-2 released from the NPs-immobilized GO-CaPs scaffolds slower than that of MPs-immobilized GO-CaPs scaffolds due to the different BMP-2 loading ability of NPs and MPs. BMP-2 was encapsulated into NPs during the preparation process of NPs, and then the NPs were further stabilized by chitosan layers. In contrast, BMP-2 was adsorbed into MPs by swelling after the MPs formation. In vitro study indicated GO does not affect the biocompatibility of the CaPs scaffolds. However, the immobilization of NPs and MPs through GO introduce the micro/nano structures on the surfaces of the GO-CaPs scaffolds,and the micro/nano structures and BMP-2 synergistically enhance the proliferation and differentiation of BMSCs. In vivo study revealed that the NPs and MPs-immobilization through GO significantly promoted the osteoinductivity of the CaPs scaffolds. These results suggested that the GO modification has great potential for enhancement of osteoinductivity of the CaPs scaffolds through immobilizing drug-loaded NPs/MPs.