Osteoblasts Generate Harder, Stiffer, and More Delamination-Resistant Mineralized Tissue on Titanium Than on Polystyrene, Associated With Distinct Tissue Micro- and Ultrastructure

Abstract

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 ...

Integrin α2β1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates

“…ECM production is increased on rougher Ti surfaces, and at least some of this increase is caused by an increase in collagen synthesis (51). Others have shown that ECM organization and adhesion strength of osteoblast colonies to their substrate are increased when cells are grown on grit-blasted Ti surfaces (35,37,57). Thus, the cells may use ␣2␤1 to anchor to the collagen-rich matrix, resulting in a more stable construct in vitro and in vivo.…”

“…Although initial attachment can influence the number of cells that can occupy a given surface, it does not appear to be correlated with the long-term adhesion of osteoblasts to the surface once they produce their ECM (35). Moreover, there appear to be surface-specific differences in ECM organization and mineralization (36,37), suggesting that different properties mediate initial attachment and adhesion, proliferation, and ultimately, differentiation.…”

“…However, integrin expression is substratesensitive (40,41); thus assumptions about cell behavior based on TCPS may not be relevant for cells on implant materials (37,42). Osteoblasts express primarily ␣5␤1 when grown on TCPS, but they shift to expression of ␣2␤1 when grown on Ti and Ti-6Al-4V (43,44).…”

TiO2 micro-nano-hybrid surface to alleviate biological aging of UV-photofunctionalized titanium

“…As established previously, 5,11 bone marrow cells isolated from the femurs of 8-week-old male Sprague-Dawley rats were placed into alpha-modified Eagle's medium supplemented with 15% fetal bovine serum, 50 µg/mL ascorbic acid, 10 mM Na-β-glycerophosphate, 10 -8 M dexamethasone, and antibiotic/antimycotic solution. Cells were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37°C.…”

Enhancement of adhesion strength and cellular stiffness of osteoblasts on mirror-polished titanium surface by UV-photofunctionalization

“…Bone marrow cells isolated from the femurs of 8-week-old, male, Sprague-Dawley rats were grown in alpha-modified Eagle's medium supplemented with 15% fetal bovine serum, 50 μg/ml ascorbic acid, 10 mM Na-β-glycerophosphate, 10 -8 M dexamethasone and antibiotic-antimycotic solution according to a previously established method [30]. Cells were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37°C.…”