Applications Of Layer-by-layer Assembly Technique On Surface Engineering Of Poly(D, L-lactic Acid) And Titanium

Controlled surface property of a biomaterial plays a crucial role in the regulation of interactions between cells and biomaterials, including cell adhesion, migration and differentiation. Biomaterial surface, to some extent, is the link between biaomaterial and life. How to functionalize biomaterial surface, in turn, for regulating the biological functions of cells draws higher demand on the design/research of biomaterials. With the development of biomaterials, the design of new generation of biomaterial is expected to stimulate specific cell/gene responses at molecular level, achieving at directing the cell fate via materials interfaces.In the past decades, poly（D,L-lactic acid）（PDLLA） and titanium were widely used as implants in clinical applications. However, PDLLA and titanium themselves are lack of biocompatibility and tissue inducing property, which are the common challenges for clinical applications of PDLLA and titanium. Failure of implantation is often caused by several factors, such as infection, displacement, allogeneic reaction, fibrous capsulation and cell death at the interface between implant and tissue. Considering the exisiting problems, to regulate cell behaviors and to induce tissue rehabiltation via material surface, it is urgently necessary to develop new technique for surface modification of biomaterial. Layer-by-Layer self-assembly technique （LBL technique）, based on electrostatic absorption, is a simple and effective method to construct polyelectrolyte multilayer films on materials surfaces.In this study, we constructed bioactive interface and cell responsive nanoreservoir system on the surfaces of PDLLA and titanium to improve the cellular inducing capacity of materials interfaces via combined techniques of LBL and gene release/therapy. The study lays basis for developing novel PDLLA- and titanium -based implants. Main contents and conclusions of this study are listed as follows:1. Construction of multilayered thin films with chitosan （Chi）/DNA pairs on poly（D,L-lactic acid） films via layer-by-layer technique, and establishment of the model of DNA release.Fourier transform infrared spectroscopy （FTIR）, X-ray photoelectron spectroscopy （XPS）, atomic force microscopy （AFM） and water contact angle measurement, were used to monitored multilayers formation, respectively. A full coverage of Chi/DNA pair film was formed only after the fifth sequential deposition （PEI/（DNA/Chi）2）, which was revealed by contact-angle measurement. Surface chemistry and topography of multilayered films were directly related to the corresponding outmost layer component by confirms of XPS and AFM. Lysozyme-mediated multilayer degradation and DNA-releasing measurement suggested that DNA was gradually released into the incubation medium over a period of up to 32 h.2. Cell specific/nonspecific gene transfection from gene-functionalized poly（D,L-lactic acid） substrate fabricated by layer-by-layer assembly technique and its mechanism.Three different polymers, including galactosylated chitosan（GC）,galactosylated polyethylenimine（GP） and cyclodextrin grafted polyethylenimine（PEI-CD）, were synthesized in this study. GC/plasmid DNA （pDNA）, GP/ pDNA and PEI-CD/ pDNA multilayered films were fabricated onto poly（D,L-lactic acid） substrates via layer-by-layer assembly technique, respectively. Contact angle measurement and atomic force microscopy （AFM）were employed to monitor assembly process of multilayered films. Results indicated that a full coverage of GC/pDNA pair films was formed only after the fourth sequential deposition.The release profile of pDNA revealed that pDNA gradually released into the incubation medium over a period of up to 42 h. PDLLA surface modified with GC/pDNA multilayered film was benefit to cell growth. Moreover, GC or GP could enhance transfection efficiency via receptor-mediated endocytosis, achieving surface mediated cell-specific gene transfection in situ. Meanwhile, cell transfection on PDLLA/（PEI-CD/pDNA）6 was also confirmed that the feasibility of suface mediated cell-nonspecific gene transfection in situ. The mechanism of surface-mediated gene transfection in situ was revealed by TEM observation and anti-DNase study. The underlying mechanism is that plasmid DNA complexes were formed in situ along the degradation of multilayered films and were subsequently uptaken in situ by those adhered cells on multilayered films, achieving gene transfection in situ.3. Surface mediated in situ differentiation of mesenchymal stem cells on gene-functionalized titanium films fabricated by layer-by-layer assembly techniqueTo further investigate the mechanism of surface mediated gene transfection, LBL technique was employed to construct chitosan/pDNA （pEGFP–hBMP2） multilayered films on titanium surface and to investigate the differentiation behavior of mesenchymal stem cell （MSCs）. Plasmid pEGFP–hBMP2 can be transfected into cells to express green ?uorescence protein （GFP） and bone morphogenetic protein 2 （BMP2）. MSCs were transfected in situ by plasmid DNA complexes, which were formed along degradation of multilayered structures in a sustained manner. The successful transfection to MSCs and differentiation of MSCs into osteoblasts were re?ected by the expression of hBMP2 mRNA, GFP, as well as the production of ALP and osteocalcin. Thus, we achieved the MSCs differentiation in situ via materials surface.4. Surface engineering of titanium thin films with chitosan and silk fibroin via layer-by-layer technique and its effects on osteoblast growth behavior. Layer-by-layer technique was employed to fabricate Chi/SF or Chi/PSS multilayed films as extracellular mixtrix analogues on titanium surface. Contact angle measurement result indicated that a full Chi/SF pair or Chi/PSS film was formed after the fifth sequential deposition layer on the titanium film surfaces. XPS and AFM confirmed that chemical properties and surface topographies of multilayered films were directly related to the corresponding outmost layer components. Chi/SF and Chi/PSS multilayered films with good biocompatibility could promote osteogenesis, which reflected by the results of higher osteoblast prolification, cell viability, the production of ALP and DNA synthesis.5. Construction of nano-reservior structure on titanium surface for mediating bone homeostasis implication via layer-by-layer assembly technique Mesoporous silica nanoparticles （MSN） loaded withβ-estradiol （E2） were assemblyed onto titanium surface via LBL technique. Mesoporous silica nanoparticles were exploited as nano-reservoir to storeβ-estradiol （E2）. We investigated the assembly processes of nano-resoviors on titanium surface and osteoblasts responses on such nano-reservoir structure. TGA analysis revealed thatβ-estradiol could load into mesoporous silica nanoparticles via simple diffusion equilibrium. Zeta potential assay indicated that chitosan/gelatin multilayered film was successful formed on MSN surface, namely E2-MSN@PEM. SEM observation reflected E2-MSN@PEM nanoparticles were assembled very well onto titanium surface, namely estrogen-functionalized titanium or hybrid multilayered film-coated titanium. The new system showed favorable cytocompatiblity and osseogenesis properties, which was reflected by ALP production level, mineralization capability and expression of OPG and OPN mRNA from osteoblasts. Meanwhile, this system implied its potential to inhibit the activities of osteoclasts, i, e, to regulate the dynamic blance of osteoblasts/osteoclasts （bone homeostasis）, which provides new approach for implantation of titanium based implants to ones suffer from osteoporosis in clinical application.6. Surface mediated cells mobility on cytokine-activated TiO₂ nanotube via spin assisted layer-by-layer assembly techniqueCytokine （BMP2）-activated TiO₂ nanotubes were fabricated via spin assisted layer-by-layer assembly technique. TiO₂ nanotubes were firstly synthesized by anodic oxidation method and explored as nano-reservior for storage of BMP2. Chitosan （Chi）/gelatin （Gel） multilayered films were then constructed onto surface of BMP2-laoded TiO₂ nanotubes via layer-by-layer assembly technique, namely TiO₂/BMP2/LBL. SEM observation and contact angle measurement demonstrated the successful construction of Chi/Gel multilayered films on surface of BMP2-laoded TiO₂ nanotube. SOD activity assay confirmed Chi/Gel multilayered film could protect the activity of SOD and prolong its life span. Compared to controls, TiO₂/BMP2/LBL was beneficial for the migration and differentiation of mesenchymal stem cells in vitro.7. Construction of microenvironment onto titanium alloy surface to regulate differentiation of mesenchymal stem cells and osseogenesis in vitro and in vivo. Biofunctionalized, multilayer coated Ti6Al4V （TC4） implants with osteoinductive potential were successfully fabricated via LbL technique. BMP2 released along with the degradation of multilayered structure in a sustained manner. Multilayered structure stimulated the adhesion and differentiation of MSCs in vitro, which were reflected by the production of ALP, mineralization and expressions osteoblasts-related genes of Runx2, Osterix, OCN, OPN, ALP and ColⅠ. Furthermore, MicroCT analysis and histological studies in vivo confirmed that TC4/LbL/BMP2/FN implants could efficiently induce new bone formation at the interface of implant/bone.