Titanium nanotopography enhances mechano-response of osteocyte three-dimensional network toward osteoblast activation.

The bone turnover capability influences the acquisition and maintenance of osseointegration. The architectures of osteocyte three-dimensional (3D) networks determine the direction and activity of bone turnover through osteocyte intercellular crosstalk, which exchanges prostaglandins through gap junctions in response to mechanical loading. Titanium nanosurfaces with anisotropically patterned dense nanospikes promote the development of osteocyte lacunar-canalicular networks. We investigated the effects of titanium nanosurfaces on intercellular network development and regulatory capabilities of bone turnover in osteocytes under cyclic compressive loading. MLO-Y4 mouse osteocyte-like cell lines embedded in type I collagen 3D gels on titanium nanosurfaces promoted the formation of intercellular networks and gap junctions even under static culture conditions, in contrast to the poor intercellular connectivity in machined titanium surfaces. The osteocyte 3D network on the titanium nanosurfaces further enhanced gap junction formation after additional culturing under cyclic compressive loading simulating masticatory loading, beyond the degree observed on machined titanium surfaces. A prostaglandin synthesis inhibitor cancelled the dual effects of titanium nanosurfaces and cyclic compressive loading on the upregulation of gap junction-related genes in the osteocyte 3D culture. Supernatants from osteocyte monolayer culture on titanium nanosurfaces promoted osteocyte maturation and intercellular connections with gap junctions. With cyclic loading, titanium nanosurfaces induced expression of the regulatory factors of bone turnover in osteocyte 3D cultures, toward higher osteoblast activation than that observed on machined surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes promoted intercellular 3D network development and regulatory function toward osteoblast activation in osteocytes activated by cyclic compressive loading, through intercellular crosstalk by prostaglandin.

Titanium nanotopography induces osteocyte lacunar-canalicular networks to strengthen osseointegration.

Osteocyte network architecture is closely associated with bone turnover. The cellular mechanosensing system regulates osteocyte dendrite formation by enhancing focal adhesion. Therefore, titanium surface nanotopography might affect osteocyte network architecture and improve the peri-implant bone tissue quality, leading to strengthened osseointegration of bone-anchored implants. We aimed to investigate the effects of titanium nanosurfaces on the development of osteocyte lacunar-canalicular networks and osseointegration of dental implants. Alkaline etching created titanium nanosurfaces with anisotropically patterned dense nanospikes, superhydrophilicity, and hydroxyl groups. MLO-Y4 mouse osteocyte-like cells cultured on titanium nanosurfaces developed neuron-like dendrites with increased focal adhesion assembly and gap junctions. Maturation was promoted in osteocytes cultured on titanium nanosurfaces compared to cells cultured on machined or acid-etched micro-roughened titanium surfaces. Osteocytes cultured in type I three-dimensional collagen gels for seven days on nano-roughened titanium surfaces displayed well-developed interconnectivity with highly developed dendrites and gap junctions compared to the poor interconnectivity observed on the other titanium surfaces. Even if superhydrophilicity and hydroxyl groups were maintained, the loss of anisotropy-patterned nanospikes reduced expression of gap junction in osteocytes cultured on alkaline-etched titanium nanosurfaces. Four weeks after placing the titanium nanosurface implants in the upper jawbone of wild-type rats, osteocytes with numerous dendrites were found directly attached to the implant surface, forming well-developed lacunar-canalicular networks around the nano-roughened titanium implants. The osseointegration strength of the nano-roughened titanium implants was significantly higher than that of the micro-roughened titanium implants. These data indicate that titanium nanosurfaces promote osteocyte lacunar-canalicular network development via nanotopographical cues and strengthen osseointegration. STATEMENT OF SIGNIFICANCE: The clinical stability of bone-anchoring implant devices is influenced by the bone quality. The osteocyte network potentially affects bone quality and is established by the three-dimensional (3D) connection of neuron-like dendrites of well-matured osteocytes within the bone matrix. No biomaterials are known to regulate formation of the osteocyte network. The present study provides the first demonstration that titanium nanosurfaces with nanospikes created by alkali-etching treatment enhance the 3D formation of osteocyte networks by promoting osteocyte dendrite formation and maturation by nanotopographic cues, leading to strengthened osseointegration of titanium implants. Osteocytes attached to the titanium nanosurfaces via numerous cellular projections. The success of osteocyte regulation by nanotechnology paves the way for development of epoch-making technologies to control bone quality.

Titania nanospikes activate macrophage phagocytosis by ligand-independent contact stimulation.

Macrophage phagocytosis is an important research target to combat various inflammatory or autoimmune diseases; however, the phenomenon has never been controlled by artificial means. Titania nanospikes created by alkaline etching treatment can tune macrophage polarization toward a M1-like type and might regulate macrophage phagocytosis. This in vitro study aimed to determine whether the two-dimensional titania nanosurfaces created by alkaline etching treatment activated the macrophage phagocytosis by nanospike-mediated contact stimulation. On two-dimensional pure titanium sheets, alkaline etching treatments with different protocols created superhydrophilic nanosurfaces with hydroxyl function groups and moderate or dense nanospikes. Both types of titania nanosurfaces promoted the phagocytic activity of the mouse macrophage-like cell line, J774A.1, through upregulation of M1 polarization markers and phagocytosis-related receptors, such as toll-like receptors (TLR2 and 4). In contrast, the hydrophobic smooth or micro-roughened titanium surfaces did not activate macrophage phagocytosis or the expression of related receptors. These phenomena remained unchanged even under the antibody blockade of macrophage TLR2 but were either suppressed or augmented for each surface excited by ultraviolet irradiation. Titania nanospikes induced paxillin expression and provided physical stimuli to macrophages, the extent of which was positively correlated with TLR expression levels. Ligand stimulation with lipopolysaccharide did not upregulate macrophage TLR expression but further enhanced M1 marker expression by titania nanosurfaces. These results showed that the two-dimensional titania nanosurfaces activated macrophage phagocytosis by enhancing expression of phagocytosis-related receptors through nanospike-mediated contact stimulation, in assistance with physical surface properties, in a ligand-independent manner.

Titanium Nanosurface with a Biomimetic Physical Microenvironment to Induce Endogenous Regeneration of the Periodontium.

The periodontium supports the teeth by dentoalveolar fibrous joints that serve unique oral functions. Endogenous regeneration of the periodontium around artificial teeth (dental implants) provides a cost-effective solution for the extension of healthy life expectancy but remains a challenge in regenerative medicine. Biomimetics can create smart biomaterials that tune endogenous cells at a tissue-material interface. Here, we created a smart titanium nanosurface mimicking the surface nanotopography and micromechanical properties of the tooth root cementum (TRC), which is essential for the induction of dentoalveolar fibrous joints to regenerate the periodontium. After transplantation into the rat renal capsule, only the titanium artificial tooth with the TRC-mimetic nanosurface formed a complex dentoalveolar fibrous joint structure, with bone tissue, periodontal ligament (PDL), and TRC, in the decellularized jawbone matrix. TRC-mimetic titanium implants induce the formation of functional periodontium, even in a jawbone implantation model, which generally causes osseointegration (ankyloses). In human PDL cells, TRC analogousness in the surface mechanical microenvironment regulates matrix mineralization through bone sialoprotein expression and phosphorus metabolism, which are critical for cementogenesis. Therefore, the titanium nanosurfaces with nanotopographical and mechanical microenvironments mimicking the TRC surface induce dentoalveolar fibrous joints for periodontal regeneration by interfacial tuning of endogenous cells.

Titanium surface with nanospikes tunes macrophage polarization to produce inhibitory factors for osteoclastogenesis through nanotopographic cues.

Definitive prevention of inflammatory osteolysis around peri-implant bone tissue remains unestablished. M1 macrophages play a key role in the host defense against inflammatory osteolysis, and their polarization depends on cell shape. Macrophage polarization is controlled by environmental stimuli, particularly physicochemical cues and hence titanium nanosurface might tune macrophage polarization and function. This study determined whether titanium nanosurfaces with anisotropically patterned nanospikes regulates macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors. Alkaline-etching treatment with different protocols created two types of titanium nanosurfaces that had anisotropically patterned nanospikes with high or low distribution density, together with superhydrophilicity and the presence of hydroxyl groups. J774A.1 cells (mouse macrophage-like cell line), cultured on both titanium nanosurfaces, exhibited truly circulated shapes and highly expressed M1, but less M2, markers, without loss of viability. M1-like polarization of macrophages on both titanium nanosurfaces was independent of protein-mediated ligand stimulation or titanium surface hydrophilic or chemical status. In contrast, other smooth or micro-roughened titanium surfaces with little or no nanospikes did not activate macrophages under any culture conditions. Macrophage culture supernatants on both titanium nanosurfaces inhibited osteoclast differentiation of RAW264.7 cells (mouse osteoclast precursor cell line), even when co-incubated with osteoclast differentiation factors. The inhibitory effects on osteoclast differentiation tended to be higher in macrophages cultured on titanium nanosurfaces with denser nanospikes. These results showed that titanium nanosurfaces with anisotropically patterned nanospikes tune macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors, with nanotopographic cues rather than other physicochemical properties. STATEMENT OF SIGNIFICANCE: Peri-implant inflammatory osteolysis is one of the serious issues for dental and orthopedic implants. Macrophage polarization and function are key for prevention of peri-implant inflammatory osteolysis. Macrophage polarization can be regulated by the biomaterial's surface physicochemical properties such as hydrophilicity or topography. However, there was no titanium surface modification to prevent inflammatory osteolysis through immunomodulation. The present study showed for the first time that the titanium nanosurfaces with anisotropically patterned nanospikes, created by the simple alkali-etching treatment polarized macrophages into M1-like type producing the inhibitory factor on osteoclast differentiation. This phenomenon attributed to nanotopographic cues, but not hydrophilicity on the titanium nanosurfaces. This nanotechnology might pave the way to develop the smart implant surface preventing peri-implant inflammatory osteolysis through immunomodulation.