ABSTRACT
BACKGROUND: Simple nanotube surface modification of titanium implant has been shown to promote adhesion, proliferation and differentiation of osteoblasts. Collagen coating can promote osteoblast adhesion and osseointegration in vivo. OBJECTⅠVE: To observe the effects of type collagen combined titanium dioxide nanotube composite coating modified titanium surface on osteoblast adhesion in vitro and osseointegration in vivo. METHODS: The titanium dioxide nanotube was fabricated on the pure titanium surface, then type Ⅰ collagen was combined with the nanotube structure to form composite coating. Scanning electron microscope observation was used to characterize the surface topography of the pure titanium, titanium dioxide nanotube and type Ⅰ collagen combined titanium dioxide nanotube surfaces. Contact angle test was employed to evaluate the hydrophilicity of different samples. MC3 T3-E1 murine preosteoblasts were seeded on the three kinds of materials for 4 hours. Cell adhesion morphology was examined by scanning electron microscope. Adherent cell counting was detected under inverted fluorescence microscope. Expression of actin cytoskeleton and vinculin was observed under laser scanning confocal microscope. The gene expression of vinculin and osteoprotegerin mRNA was detected by real-time quantitative PCR. The three kinds of samples were implanted into the tibia of Sprague-Dawley rats (provided by Laboratory Animal Center, Ⅰnstitute of Radiation Medicine, Chinese Academy of Medical Sciences) , and tibia samples were removed after 4 weeks of implantation for biological push-out test and histological observation. RESULTS AND CONCLUSⅠON: (1) Scanning electron microscope: There was mechanical scratch on the pure titanium surface. There was controllable, and uniform vertical arrangement of nanotubular structures with a diameter of approximately 70 nm on the titanium dioxide nanotube surface, and collagen adhered surrounding the nanotubular structures on the type Ⅰ collagen combined titanium dioxide nanotube substrate, and partial tubule orifices were closed. (2) The hydrophicility of type Ⅰ collagen combined titanium dioxide nanotube was significantly larger than those of the other two materials (P < 0.05) . (3) Compared with the pure titanium and titanium dioxide nanotube surfaces, the type Ⅰ collagen combined titanium dioxide nanotube substrate displayed increased adherent cell number, much well-organized cytoskeleton, enhanced immunofluorescence intensity of vinculin protein staining, and increased expression levels of vinculin and osteoprotegerin mRNA levels (all P < 0.05) . (4) Ⅰn vivo test revealed that the maximum push-out force in the type Ⅰ collagen combined titanium dioxide nanotube group was significantly higher than that in the pure titanium and titanium dioxide nanotube groups (P < 0.05) . Hematoxylin-eosin staining results showed that there were few bones, but many fibrous connective tissue surrounding the implant in the pure titanium group; there were more newly-born bones, and less fibrous connective tissue surrounding the implant in the titanium dioxide nanotube group; there were dense newly-born bones, and few thin fibrous connective tissue surrounding the implant in the type Ⅰ collagen combined titanium dioxide nanotube group. (5) These results indicate that type Ⅰ collagen combined titanium dioxide nanotube surface can facilitate osteoblast cell adhesion and promote osseointegration in vivo.
ABSTRACT
BACKGROUND:Nanostructure formation on titanium surface by anodic oxidation has good biocompatibility with bone tissue. OBJECTIVE:To observe the surface morphology and crystal ine constitution of nanopores microstructure on titanium surface formed by anodic oxidation and to further observe its influence on the MC3T3-E1 osteoblast cells’ biological behavior and the gene expression of osteoprotegerin. METHODS:Nanopores forming on titanium surface by anodic oxidation was prepared as experimental group and polished titanium as control group (12 samples for each group). Mouse MC3T3-E1 osteoblasts were co-cultured with polished pure titanium plate group and anodic oxidation nanopores group. After 7 days of inoculation, cellmorphology was observed using scanning electron microscopy, MTT method was used for the cellproliferation test and the growth curve was made. Gene expression of osteoprotegerin was also detected. RESULTS AND CONCLUSION:After anodic oxidation, a homogeneous and uniform array of nanopores formed;however it had no significant influence on the crystal ine phase of the titanium sample surfaces. Titanium surface with nanopore structure was more favorable than polished titanium surface for cellattachment and spreading. cells on the anodized surface with nanopores had higher celldensity and bigger metal coverage area. cells on the nanopores surface also exhibited a polygonal shape with many filopodia extending in al directions. MTT method showed that the anodized nanopore surface had higher cellamount than the as-polished titanium, and the former was about 1.4 times of the latter group after 7 days of culture. The gene expression level of osteoprotegerin in the MC3T3-E1 cells cultured on anodized titanium surface with nanopores was significantly higher than that on the as-polished titanium (P<0.01). The anodic oxidation treatment is more advantageous for the osteoblasts adhesion and gene expression of osteoprotegerin, thereby promoting the growth of osteoblasts.