RESUMEN
Titanium surfaces with micro-nano hybrid topography (nanoscale nodules in microscale pits) have been recently demonstrated to show higher biological capability than those with microtopography alone. On the other hand, UV treatment of titanium surfaces, which is called UV photofunctionalization, has recently been introduced to substantially increase the biological capability and osteoconductivity of titanium surfaces. However, synergistic effects of these two advanced surface modification technologies and regulatory factors to potentially modulate the mutual effects have never been addressed. In this study, utilization of a recently discovered controllable self-assembly of TiO(2) nanonodules has enabled the exploration of the relative contribution of different sizes of nanostructures to determine the biological capability of titanium surfaces and their relative responsiveness to UV photofunctionalization. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules, with or without UV treatment. Although UV treatment increased the attachment, spread, proliferation, and mineralization of these cells on all titanium surfaces, these effects were more accentuated (3-5 times) on nanonodular surfaces than on surfaces with micropits alone and were disproportionate depending on nanonodule sizes. For instance, on UV-treated micro-nano hybrid surfaces, cell attachment correlated with nanonodule sizes in a quadratic approximation with its peak for 300-nm nodules followed by a decline for 500-nm nodules, while cell attachment exponentially correlated with surface roughness with its plateau achieved for 300-nm nodules without a subsequent decline. Moreover, cell attachment increased in a linear correlation with the surface area, while no significant effect of the inter-irregularities space or degree of hydrophilicity was observed on cell attachment. These results suggest that the effect of UV photofunctionalization can be multiplied on micro-nano hybrid titanium surfaces compared with the surfaces with micropits alone. This multiplication is disproportionately regulated by a selected set of topographical parameters of the titanium surfaces. Among the nanonodules tested in this study, 300-nm nodules seemed to create the most effective morphological environment for responding to UV photofunctionalization. The data provide a systematic platform to effectively optimize nanostructures on titanium surfaces in order to enhance their biological capability as well as their susceptibility to UV photofunctionalization.
Asunto(s)
Nanoestructuras/química , Fotoquímica/métodos , Titanio/química , Rayos Ultravioleta , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Masculino , Ensayo de Materiales , Nanoestructuras/ultraestructura , Ratas , Ratas Sprague-Dawley , Propiedades de SuperficieRESUMEN
Biological tissues involve hierarchical organizations of structures and components. We created a micropit-and-nanonodule hybrid topography of TiO(2) by applying a recently reported nanonodular self-assembly technique on acid-etch-created micropit titanium surfaces. The size of the nanonodules was controllable by changing the assembly time. The created micro-nano-hybrid surface rendered a greater surface area and roughness, and extensive geographical undercut on the existing micropit surface and resembled the surface morphology of biomineralized matrices. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules. The addition of nanonodules to micropits selectively promoted osteoblast but not fibroblast function. Unlike the reported advantages of microfeatures that promote osteoblast differentiation but inhibit its proliferation, micro-nano-hybrid topography substantially enhanced both. We also demonstrated that these biological effects were most pronounced when the nanonodules were tailored to a diameter of 300nm within the micropits. An implant biomechanical test in a rat femur model revealed that the strength of bone-titanium integration was more than three times greater for the implants with micropits and 300-nm nanonodules than the implants with micropits alone. These results suggest the establishment of functionalized nano-in-microtitanium surfaces for improved osteoconductivity, and may provide a biomimetic micro-to-nanoscale hierarchical model to study the nanofeatures of biomaterials.
Asunto(s)
Materiales Biocompatibles/química , Células Madre Mesenquimatosas/citología , Nanoestructuras/química , Nanoestructuras/ultraestructura , Oseointegración/fisiología , Osteoblastos/citología , Titanio/química , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Masculino , Células Madre Mesenquimatosas/fisiología , Osteoblastos/fisiología , Ratas , Ratas Sprague-Dawley , Propiedades de SuperficieRESUMEN
UNLABELLED: This study revealed that osteoblasts generate harder, stiffer, and more delamination-resistant mineralized tissue on titanium than on the tissue culture polystyrene, associated with modulated gene expression, uniform mineralization, well-crystallized interfacial calcium-phosphate layer, and intensive collagen deposition. Knowledge of this titanium-induced alteration of osteogenic potential leading to enhanced intrinsic biomechanical properties of mineralized tissue provides novel opportunities and implications for understanding and improving bone-titanium integration and engineering physiomechanically tolerant bone. INTRODUCTION: Bone-titanium integration is a biological phenomenon characterized by continuous generation and preservation of peri-implant bone and serves as endosseous anchors against endogenous and exogenous loading, of which mechanisms are poorly understood. This study determines the intrinsic biomechanical properties and interfacial strength of cultured mineralized tissue on titanium and characterizes the tissue structure as possible contributing factors in biomechanical modulation. MATERIALS AND METHODS: Rat bone marrow-derived osteoblastic cells were cultured either on a tissue culture-grade polystyrene dish or titanium-coated polystyrene dish having comparable surface topography. Nano-indentation and nano-scratch tests were undertaken on mineralized tissues cultured for 28 days to evaluate its hardness, elastic modulus, and critical load (force required to delaminate tissue). Gene expression was analyzed using RT-PCR. The tissue structural properties were examined by scanning electron microscopy (SEM), collagen colorimetry and localization with Sirius red stain, mineral quantification, and localization with von Kossa stain and transmission electron microscopy (TEM). RESULTS: Hardness and elastic modulus of mineralized tissue on titanium were three and two times greater, respectively, than those on the polystyrene. Three times greater force was required to delaminate the tissue on titanium than that on the polystyrene. SEM of the polystyrene culture displayed a porous structure consisting of fibrous and globular components, whereas the titanium tissue culture appeared to be uniformly solid. Cell proliferation was remarkably reduced on titanium. Microscopic observations revealed that the mineralized tissue on titanium was composed of uniform collagen-supported mineralization from the titanium interface to the outer surface, with intensive collagen deposition at tissue-titanium interface. In contrast, tissue on the polystyrene was characterized by collagen-deficient mineralization at the polystyrene interface and calcium-free collagenous matrix formation in the outer tissue area. Such characteristic microstructure of titanium-associated tissue was corresponded with upregulated gene expression of collagen I and III, osteopontin, and osteocalcin mRNA. Cross-sectional TEM revealed the apposition of a high-contrast and well-crystallized calcium phosphate layer at the titanium interface but not at the polystyrene interface. CONCLUSIONS: Culturing osteoblasts on titanium, compared with polystyrene, enhances the hardness, elastic modulus, and interfacial strength of mineralized tissue to a higher degree. Titanium per se possesses an ability to alter cellular phenotypes and tissue micro- and ultrastructure that result in enhanced intrinsic biomechanical properties of mineralized tissue.
Asunto(s)
Calcificación Fisiológica/fisiología , Oseointegración/fisiología , Osteoblastos/citología , Poliestirenos/química , Titanio/química , Animales , Fenómenos Biomecánicos , Huesos/química , Huesos/citología , Huesos/fisiología , Calcio/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Colágeno/genética , Colágeno/metabolismo , Elasticidad , Microanálisis por Sonda Electrónica , Matriz Extracelular/ultraestructura , Expresión Génica/genética , Dureza , Masculino , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Nanotecnología , Osteoblastos/metabolismo , Prótesis e Implantes , Ratas , Ratas Sprague-Dawley , Propiedades de SuperficieRESUMEN
The biological mechanisms underlying bone-titanium integration and biomechanical properties of the integrated bone are poorly understood. This study assesses intrinsic biomechanical properties of mineralized tissue cultured on titanium having different surface topographies. The osteoblastic phenotypes associated with mineral deposition and collagen synthesis underlying the biomechanical modulation are also reported. Rat bone marrow-derived osteoblastic cells were cultured either on the machined titanium disc or acid-etched titanium disc. Nano-indentation study of day 28 culture revealed that the mineralized tissue on the acid-etched surface shows 3-3.5 times greater hardness than that on the machined surface (p < 0.01). Elastic modulus of the mineralized tissue was also 2.5-3 times greater on the acid-etched surface than on the machined surface (p < 0.01). After 28 days of culture, mineralized nodule area was significantly lower on the acid-etched surface than on the machined surface (p = 0.0105), while total calcium deposition did not differ between the two surfaces, indicating denser mineral deposition on the acid-etched surface. Osteopontin and osteocalcin gene expressions assayed by the reverse transcriptase-polymerase chain reaction were upregulated in the acid-etched titanium culture. Collagen synthesis measured by Sirius red stain-based colorimetry was 1.5-10 times higher on the acid-etched surface than on the machined surface in the initial culture period of day 1 to day 14 (p < 0.0001). The amount of collagen synthesis corresponded with the enhanced gene expression of prolyl 4-hydroxylase, a key enzyme for post-translational modification of collagen chains. Scanning electron microscopic images revealed that tissue cultured on the acid-etched titanium exhibited plate-like, compact surface morphology, while the tissue on the machined titanium appeared porous and was covered by fibrous and punctate structures. We conclude that culturing osteoblasts on rougher titanium surfaces enhances hardness and elastic modulus of the mineralized tissue, associated with condensed mineralization, accelerated collagen synthesis, and upregulated expression of selected bone-related genes.