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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.

Reported bone-implant contact percentages are far below the ideal 100%. We tested a hypothesis that the protein adsorption capability of titanium, which is critical to the process of osseointegration, changes over time before its use. Machined, acid-etched, and sandblasted surfaces were prepared and stored under dark ambient conditions for 3 days, 1 week, or 4 weeks. For all surfaces, protein adsorption decreased as the storage time increased, and their decreasing rates were dependent on titanium topography. After 4 weeks, the amounts of albumin and fibronectin adsorbed by the acid-etched surface were only 20% and 35%, respectively, of that adsorbed by the fresh surface after 2 hours of incubation, and remained substantially low even after 24 hours. This time-dependent degradation in protein adsorption of titanium correlated with its naturally decreasing hydrophilicity, which was not observed for the nickel and chromium surfaces, indicating a titanium-specific biological aging.

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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 culturegrade polystyrene dish or titanium-coated polystyrene dish having comparable surface topography. Nanoindentation 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 wellcrystallized calcium phosphate layer at the titanium interface but not ...

Nanostructuring technology has been proven to create unique biological properties in various biomaterials. Here we present a discovered phenomenon of titanium nano-nodular self-assembly that occurs during physical vapor depositions of titanium (Ti) onto specifically conditioned micro-textured titanium surfaces, and test a hypothesis that the Ti nanostructure has the potential to enhance bone-titanium integration. The nanostructure creation effectively provided geometrical undercut and increased the surface area by up to 40% compared with the acid-etched surface with microtopography. Depending on the size control, the nano-nodules can be added without smearing the existing micro-texture, creating a nano-micro-hybrid architecture. Titanium implants with 560-nm nano-nodules produced 3.1 times greater strength of osseointegration than those with an acid-etched surface in a rat femur model. The discovered titanium nano-nodular self-structuring has been proven feasible on biocompatible materials other than titanium, offering new avenues for the development of implant surfaces and other implantable materials for better bone-generative and -regenerative potential.

The application of implant therapy is still limited, because of various risk factors and the long healing time required for bone-titanium integration. This study explores the potential for osseointegration engineering with dental pulp cells (DPCs) by testing a hypothesis that DPCs generate mineralized tissue on titanium. DPCs extracted from rat incisors positive for CD44, alkaline phosphatase activity, and mineralizing capability were cultured on polystyrene and on machined and dual-acid-etched (DAE) titanium. Tissue cultured on titanium with a Ca/P ratio of 1.4 exhibited plate-like morphology, while that on the polystyrene exhibited fibrous and punctate structures. Tissues cultured on titanium were harder than those on polystyrene, 1.5 times on the machined and 3 times on the DAE. Collagen I, osteopontin, and osteocalcin genes were up-regulated on titanium, especially the DAE surface. In conclusion, DPCs showing some characteristics of the previously identified dental pulp stem cells can generate mineralized tissue on titanium via the osteoblastic phenotype, which can be enhanced by titanium surface roughness.

JC virus (JCV) strains worldwide can be classified into various genotypes based on DNA sequence variations. To define the domains of the four major JCV genotypes in Asia, we collected urine samples at six unstudied sites: three in southeastern Asia, two in the central highlands and one in central Asia. DNA was extracted from urine samples, and used to amplify a 610-bp region of the viral genome. For each geographical site, we determined 16 to 31 sequences, from which a phylogenetic tree was constructed to unambiguously classify detected JCV isolates into distinct genotypes. From JCV genotype profiles at the sites studied here and elsewhere, the following conclusions were drawn. Although Af2 is the major genotype in Africa, this genotype also occurs in western and central Asia. B1-b mainly occurs in western and central Asia, including the central highlands. CY occurs in northeastern Asia with the southern boundary between China and southeast Asian countries. Although SC predominates in southeastern Asia, it also occurs in northern and central Asia at lower frequencies. In addition, a few minor JCV genotypes (B1-a, B2 and B3) occur at many sites. We discuss here the anthropological and medical significance of the present findings.