TiO₂ nanostructures and more specifically nanotubes have gained significant attention in biomedical applications, due to their controlled nanoscale topography in the sub-100 nm range, high surface area, chemical resistance, and biocompatibility. Here we review the crucial aspects related to morphology and properties of TiO₂ nanotubes obtained by electrochemical anodization of titanium for the biomedical field. Following the discussion of TiO₂ nanotopographical characterization, the advantages of anodic TiO₂ nanotubes will be introduced, such as their high surface area controlled by the morphological parameters (diameter and length), which provides better adsorption/linkage of bioactive molecules. We further discuss the key interactions with bone-related cells including osteoblast and stem cells in in vitro cell culture conditions, thus evaluating the cell response on various nanotubular structures. In addition, the synergistic effects of electrical stimulation on cells for enhancing bone formation combining with the nanoscale environmental cues from nanotopography will be further discussed. The present review also overviews the current state of drug delivery applications using TiO₂ nanotubes for increased osseointegration and discusses the advantages, drawbacks, and prospects of drug delivery applications via these anodic TiO₂ nanotubes.

Titanium dioxide (TiO₂) nanotube coated substrates have revolutionized the concept of implant in a number of ways, being endowed with superior osseointegration properties and local drug delivery capacity. While accumulating reports describe the influence of nanotube diameter on cell behavior, little is known about the effects of nanotube lateral spacing on cells involved in bone regeneration. In this context, in the present study the MC3T3-E1 murine pre-osteoblast cells behavior has been investigated by using TiO₂ nanotubes of ~78 nm diameter and lateral spacing of 18 nm and 80 nm, respectively. Both nanostructured surfaces supported cell viability and proliferation in approximately equal extent. However, obvious differences in the cell spreading areas, morphologies, the organization of the actin cytoskeleton and the pattern of the focal adhesions were noticed. Furthermore, investigation of the pre-osteoblast differentiation potential indicated a higher capacity of larger spacing nanostructure to enhance the expression of the alkaline phosphatase, osteopontin and osteocalcin osteoblast specific markers inducing osteogenic differentiation. These findings provide the proof that lateral spacing of the TiO₂ nanotube coated titanium (Ti) surfaces has to be considered in designing bone implants with improved biological performance.

With the introduction of a new interdisciplinary field, osteoimmunology, today, it is well acknowledged that biomaterial-induced inflammation is modulated by immune cells, primarily macrophages, and can be controlled by nanotopographical cues. Recent studies have investigated the effect of surface properties in modulating the immune reaction, and literature data indicate that various surface cues can dictate both the immune response and bone tissue repair. In this context, the purpose of the present study was to investigate the effects of titanium dioxide nanotube (TNT) interspacing on the response of the macrophage-like cell line RAW 264.7. The cells were maintained in contact with the surfaces of flat titanium (Ti) and anodic TNTs with an intertube spacing of 20 nm (TNT20) and 80 nm (TNT80), under standard or pro-inflammatory conditions. The results revealed that nanotube interspacing can influence macrophage response in terms of cell survival and proliferation, cellular morphology and polarization, cytokine/chemokine expression, and foreign body reaction. While the nanostructured topography did not tune the macrophages’ differentiation into osteoclasts, this behavior was significantly reduced as compared to flat Ti surface. Overall, this study provides a new insight into how nanotubes’ morphological features, particularly intertube spacing, could affect macrophage behavior.

The functional role of the cholinergic nervous system in regulating gastrin release was investigated using enriched canine antral G cells. Gastrin content was 30.1 ± 2.9 pmol per well and basal gastrin release was 900 ± 27 fmol per well (n = 45). Carbachol (10-8 to 10-5M) dose-dependently stimulated gastrin release with a maximal stimulatory response achieved at a concentration of 10-5M (330% over basal). To characterize the muscarinic receptor which mediates gastrin release from antral G cells, we examined the effect of three muscarinic receptor antagonists on carbachol-stimulated gastrm release; atropine (nonselective muscarinic receptor antagonist), pirenzepine (M1 muscarinic receptor antagonist) and 4-DAMP (M3 muscarinic receptor antagonist). Atropine (10-9 to 10-6M), pirenzepine (10-8 to 10-5M) and 4-DAMP (10-9 to 10-6 M) had no effect on the basal gastrin release. However, carbachol (10-5 M)-stimulated gastrin release was effectively inhibited by atropine and 4-DAMP with Ki values of 0.48 and 0.66 nM, respectively. Pirenzepine at a high concentration (10-5M) also inhibited carbachol-stimulated gastrin release with a Ki value of 46.3 nM. These results suggest that the cholinergic nervous system directly stimulates gastrin release via M3 muscarinic receptors located on antral G cells.

The four and a half LIM domains protein 2 (Fhl2) is an adaptor protein capable of mediating protein–protein interactions. Here, we report for the first time phenotypic changes in the brain of Fhl2-deficient mice. We showed that Fhl2 is expressed in neural stem cells, precursors and mature cells of neuronal lineage. Moreover, Fhl2 deficiency leads to delayed neuroblast migration in vivo, premature astroglial differentiation of neural stem cells (NSCs) in vitro, and a gliosis-like accumulation of glial fibrillary acidic protein (GFAP)-positive astrocytes in vivo that substantially increases with age. Collectively, Fhl2-deficiency in the brain interrupts the maintenance and the balanced differentiation of adult NSCs, resulting in preferentially glial differentiation and early exhaustion of the NSC pool required for adult neurogenesis.

Pathogenic variants of the gene Eda cause X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by structural abnormalities or lack of ectodermal appendages. Signs of dysplasia are not restricted to derivatives of the ectodermal layer, but mesodermal abnormalities, such as craniofacial dysmorphism, are also frequently observed, suggesting close reciprocal interactions between the ectoderm and mesoderm; however, a causal link has remained unsubstantiated. We investigated the functional impact of defective ectodysplasin A1 (Eda1) signaling on postnatal bone homeostasis in Eda1-deficient Tabby mice. Interestingly, Eda1 was detected in wild-type mouse calvariae throughout postnatal lifetime. In calvariae, bone-lining Osterix (Osx)+ osteoblasts stained positive for Eda1, and osteoclasts were revealed as Eda receptor (Edar)-positive. Moreover, adult Eda1-deficient calvarial bone showed osteopetrosis-like changes with significantly diminished marrow space, which was maintained during adulthood. Concomitantly with osteopetrosis-like changes, Tabby calvarial bone and Tabby bone marrow-derived osteoclasts had far less osteoclastic activity-associated co-enzymes including cathepsin K, Mmp9, Trap, and Tcirg1 (V-type proton ATPase a3 subunit) compared with wild-type calvariae in vivo or osteoclasts in vitro, indicating that Eda1 deficiency may affect the activity of osteoclasts. Finally, we confirmed that nuclear Nfatc1-positive osteoclasts were strongly diminished during mature osteoclastic differentiation under M-CSF and RANKL in the Tabby model, while Fc-EDA treatment of Tabby-derived osteoclasts significantly increased nuclear translocation of Nfatc1. Furthermore, we identified enhanced Nfatc1 and NF-κB transcriptional activity following Fc-EDA treatment in vitro using luciferase assays. Overall, the results indicate that diminished expressions of osteoclastic activity-associated co-enzymes may lead to disturbed bone homeostasis in Tabby calvariae postnatally.