Enhanced osteogenic and antibacterial properties of titanium implant surface modified with Zn-incorporated nanowires: Preclinical in vitro and in vivo investigations.

OBJECTIVE: This study aimed to synthesize zinc-incorporated nanowires structure modified titanium implant surface (Zn-NW-Ti) and explore its superior osteogenic and antibacterial properties in vitro and in vivo. MATERIALS AND METHODS: Zn-NW-Ti was synthesized via displacement reactions between zinc sulfate solutions and the titanium (Ti) surface, which was pretreated by hydrofluoric acid etching and hyperthermal alkalinization. The physicochemical properties of the Zn-NW-Ti surface were examined. Moreover, the biological effects of Zn-NW-Ti on MC3T3-E1 cells and its antibacterial property against oral pathogenic bacteria (Staphylococcus aureus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) compared with sandblasted and acid-etched Ti (SLA-Ti) and nanowires modified Ti (NW-Ti) surface were assessed. Zn-NW-Ti and SLA-Ti modified implants were inserted into the anterior extraction socket of the rabbit mandible with or without exposure to the mixed bacterial solution (S. aureus, P. gingivalis, and A. actinomycetemcomitans) to investigate the osteointegration and antibacterial performance via radiographic and histomorphometric analysis. RESULTS: The Zn-NW-Ti surface was successfully prepared. The resultant titanium surface appeared as a nanowires structure with hydrophilicity, from which zinc ions were released in an effective concentration range. The Zn-NW-Ti surface performed better in facilitating the adhesion, proliferation, and differentiation of MC3T3-E1 cells while inhibiting the colonization of bacteria compared with SLA-Ti and NW-Ti surface. The Zn-NW-Ti implant exhibited enhanced osseointegration in vivo, which was attributed to increased osteogenic activity and reduced bacterial-induced inflammation compared with the SLA-Ti implant. CONCLUSIONS: The Zn-incorporated nanowires structure modified titanium implant surface exhibited improvements in osteogenic and antibacterial properties, which optimized osteointegration in comparison with SLA titanium implant surface.

Surface characteristics and in vitro biocompatibility of titanium preserved in a vitamin C-containing saline storage solution.

The purpose of this study is to explore a storage solution for titanium implants and investigate its osteogenic properties. The commercial pure titanium (cp-Ti) surface and double-etched (SLA) titanium surface specimens were preserved in air, saline, 10 mM Vitamin C (VitC)-containing saline and 100 mM VitC-containing saline storage solutions for 2 weeks. The surface microtopography of titanium was observed by scanning electron microscopy (SEM), the surface elemental compositions of the specimens were analyzed by Raman and X-ray photoelectron spectroscopy (XPS), and water contact angle and surface roughness of the specimens were tested. The protein adsorption capacity of two titanium surfaces after storage in different media was examined by BCA kit. The MC3T3-E1 osteoblasts were cultured on two titanium surfaces after storage in different media, and the proliferation, adhesion and osteogenic differentiation activity of osteoblasts were detected by CCK-8, laser confocal microscope (CLSM) and Western blot. The SEM results indicated that the titanium surfaces of the air group were relatively clean while scattered sodium chloride or VitC crystals were seen on the titanium surfaces of the other three groups. There were no significant differences in the micromorphology of the titanium surfaces among the four groups. Raman spectroscopy detected VitC crystals on the titanium surfaces of two experimental groups. The XPS, water contact angle and surface roughness results suggested that cp-Ti and SLA-Ti stored in 0.9% NaCl and two VitC-containing saline storage solutions possessed less carbon contamination and higher surface hydrophilicity. Moreover, the protein adsorption potentials of cp-Ti and SLA-Ti surfaces were significantly improved under preservation in two VitC-containing saline storage solutions. The results of in vitro study showed that the preservation of two titanium surfaces in 100 mM VitC-containing saline storage solution upregulated the cell adhesion, proliferation, osteogenic related protein expressions of MC3T3-E1 osteoblasts. In conclusion, preservation of cp-Ti and SLA-Ti in 100 mM VitC-containing saline storage solution could effectively reduce carbon contamination and enhance surface hydrophilicity, which was conducive to osteogenic differentiation of osteoblasts.

The potential influence of high uric acid exposure on surface and corrosion susceptibility of pure titanium.

This study investigated the corrosion susceptibility of pure titanium under uric acid exposure for 7 days based on surface analysis. The prepared pure titanium specimens, exposed to different concentrations of uric acid, were examined for surface microstructure, surface element composition and surface wettability using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and static contact angle measurement, respectively. The corrosion behaviors of titanium specimens were measured by open-circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization. The titanium ion release from the prepared specimens, which were immersed in Hank's balanced salt solution (HBSS) containing different amount of uric acid, was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). More irregular pitting holes were observed on titanium surfaces exposed to a high concentration of uric acid, and XPS analyses revealed that the amount of titanium dioxide (TiO2) decreased. Titanium surfaces pre-treated with high uric acid became more hydrophobic. Furthermore, the results of OCP and potentiodynamic polarization tests showed increased corrosion susceptibility of titanium samples, while EIS data indicated more active corrosion behavior of titanium materials. The high concentration of uric acid also induced titanium ion release. High concentration of uric acid negatively influenced the surface characteristics and corrosion properties of titanium materials, which destroyed the titanium oxide film barrier. High uric acid exposure increased corrosion susceptibility of pure titanium specimens and accelerated titanium ion release. Graphical abstract.

Anti-inflammatory effects of the immobilization of SEMA4D on titanium surfaces in an endothelial cell/macrophage indirect coculture model.

In this study, we established a procedure to prepare a Semaphorin4D (SEMA4D)-immobilized titanium surface and explored its effects on macrophage behaviors in an endothelial cell/macrophage indirect coculture model. The SEMA4D-bovine serum albumin complex was immobilized onto a preprocessed poly L-lysine titanium surface through NaOH hydrothermal treatment and self-assembly technology. All titanium specimens were examined for surface microstructure, surface element composition, and surface wettability by field emission scanning electron microscopy, x-ray photoelectron spectroscopy (XPS), and water contact angle measurement, respectively. Subsequently, we constructed an endothelial cell/macrophage indirect coculture model and evaluated the activation of NF-κB signaling pathway and the expression of proinflammatory cytokines (TNFα, IL-6, and IL-1ß) in macrophages. In XPS analysis, the SEMA4D-immobilized titanium surface appeared as a loose porous structure covered with uniform film, which exhibited better hydrophilicity than the control smooth titanium surface. In the indirect coculture model, SEMA4D attenuated the activation of NF-κB signaling pathway of lipopolysaccharide-stimulated THP-1 macrophages, thereby downregulating the expression of proinflammatory cytokines in macrophages. In conclusion, SEMA4D could be immobilized on titanium surfaces through NaOH hydrothermal treatment and self-assembly technology. Meanwhile, SEMA4D immobilization altered the characteristics of the titanium surfaces, which negatively regulated macrophage behaviors in the endothelial cell/macrophage indirect coculture model.

Targeting NF-κB c-Rel in regulatory T cells to treat corneal transplantation rejection.

The relevance of Tregs in the induction of tolerance against corneal allografts has been well established. Although it is well known that the conversion of Tregs into effector-like cells contributes to the loss of corneal immune privilege, the underlying mechanism is still not fully understood. Using heterologous penetrating keratoplasty model, we found that Tregs from corneal allograft rejected mice (inflam-Tregs) exhibit impaired function and characteristics of effector T cells. Further study showed that the expression of NF-κB c-Rel, a key mediator of effector T cell function, was significantly increased in inflam-Tregs. Mechanistic study revealed that elevated NF-κB c-Rel level in inflam-Tregs impaired Treg function through the promotion of inflammatory cytokine production and glycolysis. More importantly, we demonstrated that targeting NF-κB c-Rel was able to improve the immune suppressive function of inflam-Tregs in vitro and enhance the potential of them to suppress corneal transplantation rejection. Therefore, our current study identified NF-κB c-Rel as a key mediator of the conversion of Tregs into effector-like cells when under inflammatory environment.

6-Bromoindirubin-3'-oxime Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells and Facilitates Bone Regeneration in a Mouse Periodontitis Model.

Effective bone tissue engineering is important to overcome the unmet clinical challenges of periodontal tissue regeneration. Successful bone tissue engineering comprises three key factors: stem cells, growth factors, and scaffolds. 6-Bromoindirubin-3'-oxime (BIO) is an inhibitor of glycogen synthase kinase-3 (GSK-3) that can activate the Wnt signaling pathway by enhancing ß-catenin activity. In this study, the effects of BIO on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) were investigated. Poly(lactic-co-glycolic acid) (PLGA) and hyaluronic acid (HA) emerged as promising biomaterials; thus, we developed a novel HA hydrogel embedded with BIO-encapsulated PLGA microspheres and injected the formulation into the gingival sulcus of mice with experimental periodontitis. The release speed of this system was fast in the first week and followed a sustained release phase until week 4. In vivo experiments showed that this PLGA-BIO-HA hydrogel system can inhibit periodontal inflammation, promote bone regeneration, and induce the expression of bone-forming markers alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN) in a mouse periodontitis model. Therefore, this PLGA-BIO-HA hydrogel system provides a promising therapeutic strategy for periodontal bone regeneration.

Osteoclast-mediated biocorrosion of pure titanium in an inflammatory microenvironment.

Titanium (Ti) and alloys thereof are commonly utilized in biomedical settings owing to their desirable mechanical properties and good biocompatibility. However, when exposed to biological systems for extended periods of time, Ti still undergoes corrosion. In the present study, we therefore explore the impact of osteoclasts (OC) on the surface characteristics and corrosion of commercially pure Titanium (cpTi) in the context of lipopolysaccharide (LPS)-induced inflammation. We utilized tartrate resistant acidic phosphatase (TRAP) and fluorescence staining to assess OC properties, while scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), optical profilometer, electrochemical impedance spectroscopy (EIS), potentiodynamic polarization tests, and inductively coupled plasma atomic emission spectrometry (ICP-AES) were used to evaluate metal microstructure, surface composition and roughness, electrochemical corrosion properties, and metal ion release. SEM findings demonstrated that the surface of cpTi exhibited micro-pitting as well as the presence of viable OCs. Correspondingly, cpTi that had been exposed to OCs exhibited reduced levels of Ti, oxygen, and oxides within the corroded regions relative to smooth Ti as measured via EDS and XPS. OC exposure was also associated with significant changes in cpTi surface roughness, a significant decrease in corrosion resistance, and a significant increase in the release of Ti ions into the surrounding medium. In summary, these findings indicate that OC culture on the surface of cpTi can directly corrode titanium and lead to the release of Ti ions.

Role of the Hippo-YAP/NF-κB signaling pathway crosstalk in regulating biological behaviors of macrophages under titanium ion exposure.

The presence of metal ions, such as titanium (Ti) ions, is toxic to adjacent tissues of implants. Indeed, Ti ions may induce an inflammatory response through the NF-κB pathway, thus causing damage to soft and hard tissues. The involvement of Yes-associated protein (YAP), a key factor of the Hippo pathway, in an immuno-inflammatory response has been confirmed, whereas its role in Ti ion-mediated inflammation has not been elucidated. Therefore, this study aimed to investigate the role of signal crosstalk between the Hippo/YAP and NF-κB signaling pathways in the pro-inflammatory effect of Ti ions on macrophages. In our work, RAW264.7 cells were cocultured with Ti ions. The migration capacity of macrophages under Ti ion exposure was measured by transwell assay. Western blot analysis was used to detect the expressions of related proteins. Polymerase chain reaction was used to evaluate the expression of pro-inflammatory cytokines. The nucleus translocation of YAP and P65 was visualized and analyzed via immunofluorescence staining. The results showed that the migration of macrophages was promoted under Ti ion exposure. Ten parts per million Ti ions induced nuclear expression of YAP and activated the NF-κB pathway, which finally upregulated the expression of pro-inflammatory cytokines in macrophages. Moreover, the inhibition of the NF-κB pathway rescued the reduction of YAP expression under Ti ion exposure. Most importantly, the overexpression of YAP exacerbated the inflammatory response mediated by Ti ions through the NF-κB pathway. In summary, this study explored the mechanism of Hippo-YAP/NF-κB pathway crosstalk involved in the regulation of macrophage behaviors under Ti ion exposure.

Role of MAPK/JNK signaling pathway on the regulation of biological behaviors of MC3T3­E1 osteoblasts under titanium ion exposure.

The oral cavity is a complex environment that is constantly undergoing remodeling. This provides a favorable electrolytic aqueous condition, which causes the corrosion of titanium implants and the release of titanium (Ti) ions. The accumulation of Ti ions in the peri­implant tissues may affect the osteogenesis process. Therefore, the present study aimed to investigate the possible effects of Ti ions on osteoblast physiology and its underlying mechanism, specifically the MAPK/JNK signaling pathway. In the present study, MC3T3­E1 osteoblasts were cultured the medium containing 10 ppm Ti ions. Confocal laser scanning microscopy was used to analyze cell morphology and adhesion. Alkaline phosphatase (ALP) activity assay and western blotting were performed to evaluate the expression of proteins associated with osteogenesis such as Runx2 and Osterix. Nuclear translocation of JNK, a key factor of the MAPK signaling pathway, was visualized and analyzed using immunofluorescence staining. The results showed that 10 ppm Ti ions exerted negative effects on the biological behaviors of MC3T3­E1 cells, which exhibited reduced adhesion, ALP activity and osteogenic differentiation. It was also found that 10 ppm Ti ions activated the MAPK/JNK signaling pathway by promoting the nuclear translocation of JNK via phosphorylation. In addition, the inhibitory effects of 10 ppm Ti ions on MC3T3­E1 cells was found to be reversed by the JNK inhibitor SP600125. In conclusion, the preset study suggests that the MAPK/JNK signaling pathway serves a key role in the molecular mechanism underlying the changes in osteoblast behavior following Ti ion exposure. These findings may serve as a valuable reference point for the further in­depth exploration of peri­implant bone loss.

The proinflammatory cytokines IL-1ß and TNF-α modulate corneal epithelial wound healing through p16Ink4a suppressing STAT3 activity.

The proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) are involved in the corneal inflammatory response and wound healing following corneal injuries. However, the mechanism by which proinflammatory cytokines modulate corneal epithelial wound healing remains unclear. In this study, we found that IL-1ß or TNF-α was transiently elevated during corneal epithelial wound healing in mice. After corneal epithelial debridement, persistent treatment with IL-1ß or TNF-α restrained the level of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and boosted the level of cell cycle inhibitor p16Ink4a , resulting in impaired corneal epithelial repair. When p16Ink4a was deleted, the p-STAT3 level in corneal epithelium was enhanced and corneal epithelial wound healing was clearly accelerated. In diabetic mice, IL-1ß, TNF-α, and p16Ink4a appeared a sustained and strong expression in the corneal epithelium, and p16Ink4a knockdown partially reverted the defective diabetic corneal epithelial repair. Furthermore, immunoprecipitation proved that p16Ink4a interacted with p-STAT3 and thus possibly suppressed the STAT3 activity. Our findings revealed a novel mechanism that the proinflammatory cytokines modulate corneal epithelial wound healing via the p16Ink4a -STAT3 signaling.

Surface analysis and corrosion behavior of pure titanium under fluoride exposure.

STATEMENT OF PROBLEM: The corrosive effects of oral fluoride products on titanium have been reported, and chronic fluorosis, which causes hyperfluoemia, is one of the world's health problems. Nevertheless, the relationship between high serum fluoride and corrosion on the titanium surface, which might have adverse effects on titanium implant osseointegration, has not been elucidated. PURPOSE: The purpose of this in vitro study was to investigate the corrosion behavior of pure titanium exposed to high serum fluoride with different pH values based on surface analysis. MATERIAL AND METHODS: Pure titanium specimens, exposed to different electrolytes with 0.04 and 0.4 ppm NaF at pH 7.3 and 5.0 values, were examined for surface microstructure by using scanning electron microscopy (SEM) and for surface element composition with X-ray photoelectron spectroscopy (XPS). The corrosion behavior and metal ion release of specimens immersed in the Hanks' balanced salt solution (HBSS) containing 0.04 and 0.4 ppm serum fluoride concentrations (NaF) at 7.3 and 5.0 pH values were measured by electrochemical impedance spectroscopy (EIS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). RESULTS: Pitting holes were observed on pure titanium surfaces exposed to high serum fluoride. The surfaces became rougher with the increase of serum fluoride concentration, especially under acidic conditions. XPS analysis revealed a reduction of dominant titanium dioxide (TiO2) on the pure titanium surface under serum fluoride exposure, corresponding to an increase in the relative level of F. EIS data showed an active corrosion behavior of pure titanium exposed to high serum fluoride and gradually decreased corrosion resistance with increasing concentration of serum fluoride, which was more severe under acidic conditions. The release of titanium ions was also induced by high serum fluoride and acidic conditions. CONCLUSIONS: High serum fluoride had a negative influence on the corrosion behavior of pure titanium. The titanium oxide film barrier could be broken down in the fluoride ions condition, and the corrosion resistance of pure titanium decreased with the increasing concentration of serum fluoride. The increased corrosion susceptibility of pure titanium accelerated the release of titanium ions after exposure to high serum fluoride; this was more pronounced in an acidic environment.

Elimination of Cr(VI) from chromium slag with poplar lignin by electrochemical treatment in sulfuric acid solution.

In this paper, we proposed a novel method to eliminate nocuous Cr(VI) from chromium slag with poplar lignin by electrochemical treatment in sulfuric acid solution. In this electrochemical process, self-made Ti/SnO2-Sb anode and graphite cathode were applied, and the oxidative degradation of lignin proceeded simultaneously with the reduction of Cr(VI) in one pot. The influences of pivotal factors on electrocatalytic redox efficiency were investigated, such as chromium slag concentration, lignin concentration, current density, sulfuric acid concentration, and reaction time. The results showed that the elimination rate of Cr(VI) in chromium slag was 97.16 ± 1.13% and the total yield of lignin degradation products reached 93.78 g/kg lignin under the optimal conditions. X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDS), and UV-visible spectrophotometer studies confirmed that most of the Cr(VI) ions were reduced to Cr(III) ions with the aid of lignin, and a small amount of Cr(VI) ions were adsorbed by lignin residue. Importantly, this method provides a typical example of "waste control by waste", which is treating waste chromium slag with waste lignin that can be an effective way to eliminate Cr(VI).

NAD+ precursors protect corneal endothelial cells from UVB-induced apoptosis.

Excessive exposure of the eye to ultraviolet B light (UVB) leads to corneal edema and opacification because of the apoptosis of the corneal endothelium. Our previous study found that nicotinamide (NIC), the precursor of nicotinamide adenine dinucleotide (NAD), could inhibit the endothelial-mesenchymal transition and accelerate healing the wound to the corneal endothelium in the rabbit. Here we hypothesize that NIC may possess the capacity to protect the cornea from UVB-induced endothelial apoptosis. Therefore, a mouse model and a cultured cell model were used to examine the effect of NAD+ precursors, including NIC, nicotinamide mononucleotide (NMN), and NAD, on the UVB-induced apoptosis of corneal endothelial cells (CECs). The results showed that UVB irradiation caused apparent corneal edema and cell apoptosis in mice, accompanied by reduced levels of NAD+ and its key biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), in the corneal endothelium. However, the subconjunctival injection of NIC, NMN, or NAD+ effectively prevented UVB-induced tissue damage and endothelial cell apoptosis in the mouse cornea. Moreover, pretreatment using NIC, NMN, and NAD+ increased the survival rate and inhibited the apoptosis of cultured human CECs irradiated by UVB. Mechanistically, pretreatment using nicotinamide (NIC) recovered the AKT activation level and decreased the BAX/BCL-2 ratio. In addition, the capacity of NIC to protect CECs was fully reversed in the presence of the AKT inhibitor LY294002. Therefore, we conclude that NAD+ precursors can effectively prevent the apoptosis of the corneal endothelium through reactivating AKT signaling; this represents a potential therapeutic approach for preventing UVB-induced corneal damage.

Biocorrosion of pure and SLA titanium surfaces in the presence of Porphyromonas gingivalis and its effects on osteoblast behavior.

Objective: The study aims to investigate the biocorrosion behavior of Porphyromonas gingivalis on pure and SLA titanium surfaces and its effects on surface characteristics and osteoblast behavior. Methods: Pure and SLA titanium specimens were immersed in culture medium with P. gingivalis and incubated for 7 days. P. gingivalis colonization on the pure and SLA titanium surfaces was observed by scanning electron microscopy (SEM). The pure and SLA titanium surface characteristics were analyzed via X-ray photoelectron spectroscopy (XPS), surface roughness and surface wettability. The corrosion behaviors of pure and SLA titanium specimens were evaluated by electrochemical corrosion test. The osteoblast behavior of MC3T3-E1 cells on the pure and SLA titanium surfaces after P. gingivalis colonization was investigated by cell adhesion and western blot assays. Results: P. gingivalis colonized on the pure and SLA titanium surfaces was observed by SEM. The XPS analysis demonstrated reductions in the relative levels of titanium and oxygen and obvious reductions of dominant titanium dioxide (TiO2) on both titanium surfaces after immersing the metal in P. gingivalis culture. In addition, their roughness and wettability were changed. Correspondingly, the electrochemical corrosion test results revealed significant decreases in the corrosion resistance and increases in the corrosion rate of the pure and SLA titanium specimens after immersion in P. gingivalis culture. The results of the in vitro study showed that the pre-corroded pure and SLA titanium surfaces by P. gingivalis exhibited lower osteocompatibility and down-regulated the adhesion, spreading and osteogenic differentiation abilities of MC3T3-E1 cells. Conclusions: P. gingivalis was able to colonize on the pure and SLA titanium surfaces and weaken their surface properties, especially a decrease in the protective TiO2 film, which induced the biocorrosion and further negatively affected the osteoblast behavior.

Ocular surface repair using decellularized porcine conjunctiva.

The primary functions of the conjunctiva embody ocular surface protection and the maintenance of the tear film equilibrium. Severe conjunctival defects such as symblepharon may impair the integrity of ocular surface and cause loss of visual functions. Here we report the use of a decellularized porcine conjunctiva (DPC) for conjunctival reconstruction in rabbit models and in clinic. Our results show that the major xenoantigens are efficiently removed, while abundant matrix components and integrated microstructures are well preserved in the DPC. These characteristics provide mechanical support and favorable histocompatibility for repairing damaged conjunctiva. The DPC application has demonstrated enhanced transplant stability and improved epithelial regeneration in severe ocular surface damage comparing to those of amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction nowadays. In order to test the DPC performance in clinic, three patients with pterygium and one patient with symblepharon underwent transplant with DPC. The grafts in all cases were completely re-epithelized and no graft melt or fibroplasia were observed. These results suggest that the strategy we developed is feasible and effective for conjunctival reconstruction and ocular surface repair. STATEMENT OF SIGNIFICANCE: In this study, we adopted an innovative approach to prepare decellularized porcine conjunctiva (DPC). The intricate conjunctiva-specific structures and abundant matrix components were preserved in DPC, which offers favorable mechanical properties for graft. DPC has shown positive effects in ocular surface repair, which has been proven particularly in a rabbit model with severe symblepharon. Reconstructed conjunctiva by DPC exhibited epithelial heterogeneity, extremely resembling that of native conjunctiva. In addition, results from clinical studies were encouraging for pterygium and symblepharon and clinical application of DPC is promising.

Cigarette Smoke Extract Exposure: Effects on the Interactions between Titanium Surface and Osteoblasts.

The aim of this study was to explore the changes in the characteristics of titanium surface and the osteoblast-titanium interactions under cigarette smoke extract (CSE) exposure. In this study, CSE was used to simulate the oral liquid environment around the implant under cigarette smoke exposure. Titanium samples were immersed in CSE to explore the changes in the characteristics of titanium surface. The physical properties of titanium surface were measured, including surface micromorphology, surface elemental composition, roughness, and surface hydrophilicity. MC3T3-E1 cells were cultured on the titanium surface in vitro under different concentrations of CSE exposure, and cell adhesion, cell proliferation, and osteogenic differentiation were observed. The surface micromorphology and elemental composition of titanium surface changed under CSE exposure. No obvious changes were found in the surface roughness and the hydrophilicity of titanium samples. Moreover, the results of in vitro study showed that CSE exposure downregulated the cell spreading, proliferation, and osteogenic differentiation of MC3T3-E1 cells on the titanium surface. It could be speculated that some carbon-containing compounds from CSE adsorbed on the titanium surface and the osteoblast-titanium interactions were influenced under CSE exposure. It is hoped that these results could provide valuable information for further studies on smoking-mediated inhibition of implants osseointegration.

Nicotinamide inhibits corneal endothelial mesenchymal transition and accelerates wound healing.

Corneal endothelial cells (CECs) maintain the clarity of the cornea through the barrier and pump function. Ex vivo culture or injury may cause corneal endothelial-mesenchymal transition (EnMT) and lead to loss of function. In this study, we explored the effects of nicotinamide (NIC) on the wound healing of rabbit corneal endothelium and the proliferation, migration, and EnMT of cultured human CEC lines. The animal results showed that corneal clarity was rapidly recovered within seven days through topical application of NIC in the rabbits with mechanical injury of the corneal endothelium, while the control corneas remained edematous and cloudy. Whole-mounted corneal staining found the expressions of Na+/K+-ATPase, aquaporin-1, and zonula occludens-1 were mainly localized to the boundaries of regenerated endothelium in NIC-treated eyes, in contrast to the scattered staining in vehicle-treated eyes. Interestingly, we found that NIC application inhibited the expression of typical EnMT marker alpha-smooth muscle actin, which appeared in the rabbit corneal endothelial wound healing. In vitro, NIC promoted the proliferation, but not the migration, of cultured human CECs. Moreover, NIC effectively inhibited transforming growth factor beta-1-induced corneal EnMT and decreased the levels of EnMT regulators snail and slug. Therefore, our study indicates that NIC enhances corneal endothelial wound healing through the promotion of proliferation and the inhibition of EnMT, which may provide a potential pharmaceutical agent for treating corneal endothelial dysfunction.

Rapid Differentiation of Multi-Zone Ocular Cells from Human Induced Pluripotent Stem Cells and Generation of Corneal Epithelial and Endothelial Cells.

Eye is a complex organ with a highly specialized tissue structure. The establishment of human pluripotent stem cells (hPSCs) has allowed the simulation of eye development in vitro. Most differentiation works of hPSC-derived ocular cells focus on a single, tissue-specific lineage, however, that faces difficulty in reflecting the complexity of eye development. Recently, the generation of a self-formed ectodermal autonomous multi-zone of ocular cells availably mimics the process of whole-eye development. In this study, we developed a rapid defined method to induce the differentiation of multi-zone ocular cells (MZOCs) from human induced pluripotent stem cells, which specifically experienced the key progenitor stages of anterior neuroectoderm and eye field stem cells by a 2.5-dimensional culture. These differentiated cell types spanned neural retina, retinal pigment epithelium, surface ectoderm, and neural crest and lens cells. In addition, the surface ectoderm zone of MZOCs could be mechanically isolated and induced into corneal epithelial cells, and the isolated neural crest zone could be directed into corneal endothelial cells. This in vitro differentiation process vividly mimics the development of vertebrate eye, and it provides a promising model for the study of ocular morphogenesis, as well as an ideal resource of seed cells for corneal regenerative medicine.

Low density lipoprotein adsorption on a titanium surface and its effect on osteoblast behaviors.

Objective: This study aims to investigate the adsorption of low density lipoprotein (LDL) on a titanium surface and to explore its effect on osteoblast behaviors. Materials and methods: LDL adsorption on a titanium surface was analyzed using LDL assay and X-ray photoelectron spectroscopy (XPS). Physical properties, including topography, surface roughness and wettability of a control smooth titanium surface and a LDL pre-adsorbed titanium surface, were assessed. Subsequently, the adhesion, proliferation and differentiation abilities of MC3T3-E1 cells (an osteoblast-like cell line) on the surfaces of control titanium and LDL pre-adsorbed titanium were investigated. Results: LDL assay and XPS confirmed LDL adsorption on the titanium surface. The maximum adsorption of LDL on the titanium surfaces was observed after 150 minutes of incubation. In comparison with the control smooth titanium surface, the roughness and hydrophilicity of the LDL pre-adsorbed titanium surface were significantly altered. Furthermore, in vitro studies demonstrated that LDL adsorption obviously attenuated the adhesion, proliferation and differentiation of MC3T3-E1 cells on the titanium surface. Conclusion: LDL could adsorb on a titanium surface. Meanwhile, LDL adsorption changed the characteristics of the titanium surface, which, in turn, negatively regulated osteoblast behaviors.

Different Effects of Pro-Inflammatory Factors and Hyperosmotic Stress on Corneal Epithelial Stem/Progenitor Cells and Wound Healing in Mice.

Chronic inflammation and severe dry eye are two important adverse factors for the successful transplant of cultured limbal stem cells. The aim of this study was to investigate the effects of inflammation and hyperosmotic stress (a key pathological factor in dry eye) on corneal epithelial stem cells (CESCs) and corneal epithelial wound healing. We observed that the CESCs exhibited significant morphological changes when treated with interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), or hyperosmotic stress. Colony-forming efficiency or colony-forming size was decreased with the increasing concentrations of IL-1ß, TNF-α, or hyperosmotic stress, which was exacerbated when treated simultaneously with pro-inflammatory factors and hyperosmotic stress. However, the colony-forming capacity of CESCs recovered more easily from pro-inflammatory factor treatment than from hyperosmotic stress treatment. Moreover, when compared with pro-inflammatory factors treatment, hyperosmotic stress treatment caused a more significant increase of apoptotic and necrotic cell numbers and cell cycle arrest in the G2/M phase. Furthermore, the normal ability of corneal epithelial wound healing in the mice model was suppressed by both pro-inflammatory factors and hyperosmotic stress treatment, and especially severely by hyperosmotic stress treatment. In addition, inflammation combined with hyperosmotic stress treatment induced more serious epithelial repair delays and apoptosis in corneal epithelium. Elevated levels of inflammatory factors were found in hyperosmotic stress-treated cells and mice corneas, which persisted even during the recovery period. The results suggested that pro-inflammatory factors cause transient inhibition, while hyperosmotic stress causes severe apoptosis and necrosis, persistent cell cycle arrest of CESCs, and severe corneal wound healing delay. Stem Cells Translational Medicine 2019;8:46-57.