Mechanoregulation of Osteoclastogenesis-Inducing Potentials of Fibrosarcoma Cell Line by Substrate Stiffness.

A micro-physiological system is generally fabricated using soft materials, such as polydimethylsiloxane silicone (PDMS), and seeks an inflammatory osteolysis model for osteoimmunological research as one of the development needs. Microenvironmental stiffness regulates various cellular functions via mechanotransduction. Controlling culture substrate stiffness may help spatially coordinate the supply of osteoclastogenesis-inducing factors from immortalized cell lines, such as mouse fibrosarcoma L929 cells, within the system. Herein, we aimed to determine the effects of substrate stiffness on the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction. L929 cells showed increased expression of osteoclastogenesis-inducing factors when cultured on type I collagen-coated PDMS substrates with soft stiffness, approximating that of soft tissue sarcomas, regardless of the addition of lipopolysaccharide to augment proinflammatory reactions. Supernatants of L929 cells cultured on soft PDMS substrates promoted osteoclast differentiation of the mouse osteoclast precursor RAW 264.7 by stimulating the expression of osteoclastogenesis-related gene markers and tartrate-resistant acid phosphatase activity. The soft PDMS substrate inhibited the nuclear translocation of YES-associated proteins in L929 cells without reducing cell attachment. However, the hard PDMS substrate hardly affected the cellular response of the L929 cells. Our results showed that PDMS substrate stiffness tuned the osteoclastogenesis-inducing potential of L929 cells via cellular mechanotransduction.

Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation.

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell (iPSC)- derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, a 3D hydrogel-sandwich culture (HGSC) system is developed that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs are seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1 kPa [hard], 28.1 ± 2.3 kPa [moderate], and 5.1 ± 0.1 kPa [soft]) and cultured for 2 days. HGSC induces stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulates the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promotes cardiomyocyte (CM) differentiation and structural maturation of myofibrils. The proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that can be beneficial for research into tissue regeneration and engineering.

Substrate stiffness controls proinflammatory responses in human gingival fibroblasts.

Soft gingiva is often compromised in gingival health; however, the underlying biological mechanisms remain unknown. Extracellular matrix (ECM) stiffness is involved in the progression of various fibroblast-related inflammatory disorders via cellular mechanotransduction. Gingival stiffness might regulate cellular mechanotransduction-mediated proinflammatory responses in gingival fibroblasts. This in vitro study aims to investigate the effects of substrate stiffness on proinflammatory responses in human gingival fibroblasts (hGFs). The hGFs isolated from two healthy donors cultured on type I collagen-coated polydimethylsiloxane substrates with different stiffnesses, representing soft (5 kPa) or hard (25 kPa) gingiva. Expression levels of proinflammatory mediators, prostaglandin E2 or interleukin-1ß, in hGFs were significantly higher with the soft substrate than with the hard substrate, even without and with lipopolysaccharide (LPS) to induce inflammation. Expression levels of gingival ECM and collagen cross-linking agents in hGFs were downregulated more with the soft substrate than with the hard substrate through 14 days of culture. The soft substrate suppressed the expression of mechanotransduction-related transcriptional factors and activated the expression of inflammation-related factors, whereas the hard substrate demonstrated the opposite effects. Soft substrate induced proinflammatory responses and inhibition of ECM synthesis in hGFs by inactivating cellular mechanotransduction. This supports the importance of ECM stiffness in gingival health.

Application of shear stress for enhanced osteogenic differentiation of mouse induced pluripotent stem cells.

The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.

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.