Biosynthesis and Function of VIP and Oxytocin: Mechanisms of C-terminal Amidation, Oxytocin Secretion and Transport.

In this review, we provide the status of research on vasoactive intestinal peptide (VIP) and oxytocin, typical C-terminal α-amidated peptide hormones, including their precursor protein structures, processing and C-terminal α-amidation, and the recently identified mechanisms of regulation of oxytocin secretion and its transportation through the blood brain barrier. More than half of neural and endocrine peptides, such as VIP and oxytocin, have the α-amide structure at their C-terminus, which is essential for biological activities. We have studied the synthesis and function of C-terminal α-amidated peptides, including VIP and oxytocin, since the 1980s. Human VIP mRNA encoded not only VIP but also another related C-terminal α-amidated peptide, PHM-27 (peptide having amino-terminal histidine, carboxy-terminal methionine amide, and 27 amino acid residues). The human VIP/PHM-27 gene is composed of 7 exons and regulated synergistically by cyclic AMP and protein kinase C pathways. VIP has an essential role in glycemic control using transgenic mouse technology. The peptide C-terminal α-amidation proceeded through a 2-step mechanism catalyzed by 2 different enzymes encoded in a single mRNA. In the oxytocin secretion from the hypothalamus/the posterior pituitary, the CD38-cyclic ADP-ribose signal system, which was first established in the insulin secretion from pancreatic ß cells of the islets of Langerhans, was found to be essential. A possible mechanism involving RAGE (receptor for advanced glycation end-products) of the oxytocin transportation from the blood stream into the brain through the blood-brain barrier has also been suggested.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

Human Gingival Fibroblast Attachment to Smooth Titanium Disks with Different Surface Roughnesses.

Peri-implantitis is a significant problem associated with dental implants. It has been hypothesized that creating a soft-tissue seal around the implant neck prevents peri-implantitis. This study aims to clarify the effects of the surface smoothness of titanium disks on soft tissues. Thus, titanium disks were prepared through electrolytic composite polishing (ECP), sisal buffing (SB), hairline polishing (HP), and laser cutting (LC). The surface roughness values of seven items was measured. For ECP, SB, HP, and LC samples, the Ra values were 0.075, 0.217, 0.671, and 1.024 µm and the Sa values were 0.005, 0.115, 0.500, and 0.676, respectively, indicating that the surface roughness was remarkably lower with ECP. Moreover, the Wsk values for ECP, SB, HP, and LC were 0.521, 1.018, -0.678, and -0.558, respectively. The smooth surfaces produced by ECP and SB were biased toward the concave surface, whereas those produced by HP and LC were biased toward the convex surface. The Rku values for ECP, SB, HP, and LC were 2.984, 11.774, 14.182, and 26.232, respectively. Only the ECP exhibited a moderate bias peak and produced an extremely smooth surface. The contact angles in the cases of ECP, SB, HP, and LC were 60.1°, 66.3°, 68.4°, and 79.3°, respectively, indicating the hydrophobicity of the titanium disks. Human oral fibroblasts were then incubated on each disk for 24 and 48 h to measure cell attachment, and no significant differences were observed. The differences in Ra and Sa did not affect cell attachment. Therefore, by applying ECP to the abutment or implant neck, the cell attachment required for soft-tissue formation while preventing bacterial adhesion can be achieved.

A Novel Cell Delivery System Exploiting Synergy between Fresh Titanium and Fibronectin.

Delivering and retaining cells in areas of interest is an ongoing challenge in tissue engineering. Here we introduce a novel approach to fabricate osteoblast-loaded titanium suitable for cell delivery for bone integration, regeneration, and engineering. We hypothesized that titanium age influences the efficiency of protein adsorption and cell loading onto titanium surfaces. Fresh (newly machined) and 1-month-old (aged) commercial grade 4 titanium disks were prepared. Fresh titanium surfaces were hydrophilic, whereas aged surfaces were hydrophobic. Twice the amount of type 1 collagen and fibronectin adsorbed to fresh titanium surfaces than aged titanium surfaces after a short incubation period of three hours, and 2.5-times more fibronectin than collagen adsorbed regardless of titanium age. Rat bone marrow-derived osteoblasts were incubated on protein-adsorbed titanium surfaces for three hours, and osteoblast loading was most efficient on fresh titanium adsorbed with fibronectin. The number of osteoblasts loaded using this synergy between fresh titanium and fibronectin was nine times greater than that on aged titanium with no protein adsorption. The loaded cells were confirmed to be firmly attached and functional. The number of loaded cells was strongly correlated with the amount of protein adsorbed regardless of the protein type, with fibronectin simply more efficiently adsorbed on titanium surfaces than collagen. The role of surface hydrophilicity of fresh titanium surfaces in increasing protein adsorption or cell loading was unclear. The hydrophilicity of protein-adsorbed titanium increased with the amount of protein but was not the primary determinant of cell loading. In conclusion, the osteoblast loading efficiency was dependent on the age of the titanium and the amount of protein adsorption. In addition, the efficiency of protein adsorption was specific to the protein, with fibronectin being much more efficient than collagen. This is a novel strategy to effectively deliver osteoblasts ex vivo and in vivo using titanium as a vehicle.

Correlations between implant success rate and personality types in the older people: A preliminary case control study.

Background/ purpose: Older patients inevitably have a higher need for implant treatment, it is unknown how mental changes or psychological aspects affect the outcome of implant treatment. This study evaluated the success rate of implants and the influence of personality traits in the older people. The goal was to provide evidence for predictable implant treatment while taking into account the unique psychological changes of elders. Materials and methods: Participants were patients who were able to independently visit our hospital between March 2004 and May 2021. Inclusion criteria were patients aged 65 years or older at the time of implant placement with regular follow-up for at least 1 year. The implant success rate was calculated by counting peri-implantitis and implant loss as failures. Multivariate analysis was used to examine the effect of patient personality characteristics on the success rate. Results: Fifty-six implants were included in 23 patients (12 men, 11 women), with a mean age of 68.5 years (65-76) and mean maintenance duration of 9 years and 2 months. The cumulative survival rate was 87% at the patient level (94.6% at the implant level). Statistically significant differences were found for adaptive traits (odds ratio [OR] = 0.04) and non-adaptive traits (OR = 6.38); however, no significant differences were found for the other independent variables. Conclusion: The overall implant success rate was 69.6% at the patient level (82.1% at the implant level). The personality traits in older people had a significant effect on the implant failure rate.

Ultraviolet Treatment of Titanium to Enhance Adhesion and Retention of Oral Mucosa Connective Tissue and Fibroblasts.

Peri-implantitis is an unsolved but critical problem with dental implants. It is postulated that creating a seal of gingival soft tissue around the implant neck is key to preventing peri-implantitis. The objective of this study was to determine the effect of UV surface treatment of titanium disks on the adhesion strength and retention time of oral connective tissues as well as on the adherence of mucosal fibroblasts. Titanium disks with a smooth machined surface were prepared and treated with UV light for 15 min. Keratinized mucosal tissue sections (3 × 3 mm) from rat palates were incubated for 24 h on the titanium disks. The adhered tissue sections were then mechanically detached by agitating the culture dishes. The tissue sections remained adherent for significantly longer (15.5 h) on the UV-treated disks than on the untreated control disks (7.5 h). A total of 94% of the tissue sections were adherent for 5 h or longer on the UV-treated disks, whereas only 50% of the sections remained on the control disks for 5 h. The adhesion strength of the tissue sections to the titanium disks, as measured by tensile testing, was six times greater after UV treatment. In the culture studies, mucosal fibroblasts extracted from rat palates were attached to titanium disks by incubating for 24, 48, or 96 h. The number of attached cells was consistently 15-30% greater on the UV-treated disks than on the control disks. The cells were then subjected to mechanical or chemical (trypsinization) detachment. After mechanical detachment, the residual cell rates on the UV-treated surfaces after 24 and 48 h of incubation were 35% and 25% higher, respectively, than those on the control surfaces. The remaining rate after chemical detachment was 74% on the control surface and 88% on the UV-treated surface for the cells cultured for 48 h. These trends were also confirmed in mouse embryonic fibroblasts, with an intense expression of vinculin, a focal adhesion protein, on the UV-treated disks even after detachment. The UV-treated titanium was superhydrophilic, whereas the control titanium was hydrophobic. X-ray photoelectron spectroscopy (XPS) chemical analysis revealed that the amount of carbon at the surface was significantly reduced after UV treatment, while the amount of TiOH molecules was increased. These ex vivo and in vitro results indicate that the UV treatment of titanium increases the adhesion and retention of oral mucosa connective tissue as a result of increased resistance of constituent fibroblasts against exogenous detachment, both mechanically and chemically, as well as UV-induced physicochemical changes of the titanium surface.

EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells.

Angiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

Osteoblast Attachment Compromised by High and Low Temperature of Titanium and Its Restoration by UV Photofunctionalization.

Titanium implants undergo temperature fluctuations during manufacturing, transport, and storage. However, it is unknown how this affects their bioactivity. Herein, we explored how storage (six months, dark conditions) and temperature fluctuations (5-50 °C) affected the bioactivity of titanium implants. Stored and fresh acid-etched titanium disks were exposed to different temperatures for 30 min under wet or dry conditions, and their hydrophilicity/hydrophobicity and bioactivity (using osteoblasts derived from rat bone marrow) were evaluated. Ultraviolet (UV) treatment was evaluated as a method of restoring the bioactivity. The fresh samples were superhydrophilic after holding at 5 or 25 °C under wet or dry conditions, and hydrophilic after holding at 50 °C. In contrast, all the stored samples were hydrophobic. For both fresh and stored samples, exposure to 5 or 50 °C reduced osteoblast attachment compared to holding at 25 °C under both wet and dry conditions. Regression analysis indicated that holding at 31 °C would maximize cell attachment (p < 0.05). After UV treatment, cell attachment was the same or better than that before temperature fluctuations. Overall, titanium surfaces may have lower bioactivity when the temperature fluctuates by ≥20 °C (particularly toward lower temperatures), independent of the hydrophilicity/hydrophobicity. UV treatment was effective in restoring the temperature-compromised bioactivity.

Protective Effects of Collagen Tripeptides in Human Aortic Endothelial Cells by Restoring ROS-Induced Transcriptional Repression.

Collagen tripeptide (CTP) is defined as a functional food material derived from collagenase digests of type I collagen and contains a high concentration of tripeptides with a Gly-X-Y sequence. CTP has several biological effects, including the acceleration of fracture healing, ameliorating osteoarthritis, and improving dryness and photoaging of the skin. Recently, an antiatherosclerotic effect of CTP has been reported, although its molecular mechanism is yet to be determined. In this study, we examined the effects of CTP on primary cultured human aortic endothelial cells (HAECs) under oxidative stress, because oxidative endothelial dysfunction is a trigger of atherosclerosis. DNA microarray and RT-qPCR analyses showed that CTP treatment recovered the downregulated expression of several genes, including the interleukin-3 receptor subunit alpha (IL3RA), which were suppressed by reactive oxygen species (ROS) treatment in HAECs. Furthermore, IL3RA knockdown significantly decreased the viability of HAECs compared with control cells. RT-qPCR analysis also showed that solute carrier 15 family peptide transporters, which are involved in CTP absorption into cells, were expressed in HAECs at levels more than comparable to those of a CTP-responsive human osteoblastic cell line. These results indicated that CTP exerts a protective effect for HAECs, at least in part, by regulating the recovery of ROS-induced transcriptional repression.

Dental implant status in elderly individuals requiring domiciliary dental care in Japan.

BACKGROUND: The presence of implants is a significant burden not only for dentists but also for caregivers and families of elderly individuals requiring nursing and domiciliary dental care. However, few reports have assessed the status of domiciliary dental care or measures employed to deal with related issues. Hence, we aimed to evaluate the dental implant status in elderly patients requiring nursing and domiciliary dental care and to determine the suitable measures for overcoming the associated limitations. A questionnaire was mailed to 1000 dentists who provided domiciliary dental care in the Tokyo metropolitan area of Japan. The questions were classified into three categories: basic information of the dentists, actual implant status of patients requiring domiciliary dental care, and implants in an aging society. RESULTS: The response rate was 36.5%. Approximately 2% of patients requiring domiciliary dental care were implant patients. Many implant-related problems were associated with insufficiency or difficulty in cleaning around the implant, resulting in peri-implantitis. Prosthetic and more serious complications such as implant body fracture or loss were reported and frequently managed by routine follow-ups, cleaning the area around the implant, scaling and polishing, and/or pharmacological modalities. Oral care mainly involved simple toothbrushing instructions, which was not adequate. CONCLUSIONS: Our findings suggest the necessity of simplifying the oral environment and making oral care a simple task before aging individuals require nursing and domiciliary dental care.

Emerging Role of AP-1 Transcription Factor JunB in Angiogenesis and Vascular Development.

Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation.

Metformin Mitigates DPP-4 Inhibitor-Induced Breast Cancer Metastasis via Suppression of mTOR Signaling.

The biological influence of antidiabetic drugs on cancer cells and diabetic cancer patients has not yet been completely elucidated. We reported that a dipeptidyl peptidase (DPP)-4 inhibitor accelerates mammary cancer metastasis by inducing epithelial-mesenchymal transition (EMT) through the CXCL12/CXCR4/mTOR axis. Metformin has been shown to inhibit the mTOR signaling pathway. In this study, we investigated whether metformin mitigates breast cancer metastasis induced by a DPP-4 inhibitor via suppression of mTOR signaling. In cultured mouse mammary and human breast cancer cells, metformin suppressed DPP-4 inhibitor KR62436 (KR)-induced EMT and cell migration via suppression of the mTOR pathway associated with AMPK activation. For the in vivo study, metformin intervention was performed in an allograft 4T1 breast cancer model mouse with or without KR. We also analyzed mice transplanted with shRNA-mediated DPP-4 knockdown 4T1 cells. Treatment with metformin inhibited the lung metastasis of DPP-4-deficient 4T1 mammary tumor cells generated by either KR administration or DPP-4 knockdown. Immunostaining of primary tumors indicated that DPP-4 suppression promoted the expression of EMT-inducing transcription factor Snail through activation of the CXCR4-mediated mTOR/p70S6K pathway in an allograft breast cancer model; metformin abolished this alteration. Metformin treatment did not alter DPP-4-deficiency-induced expression of CXCL12 in either plasma or primary tumors. Our findings suggest that metformin may serve as an antimetastatic agent by mitigating the undesirable effects of DPP-4 inhibitors in patients with certain cancers. IMPLICATIONS: Metformin could combat the detrimental effects of DPP-4 inhibitor on breast cancer metastasis via mTOR suppression, suggesting the potential clinical relevance. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/61/F1.large.jpg.

Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Compromised Epithelial Cell Attachment after Polishing Titanium Surface and Its Restoration by UV Treatment.

Titanium-based implant abutments and tissue bars are polished during the finalization. We hypothesized that polishing degrades the bioactivity of titanium, and, if this is the case, photofunctionalization-grade UV treatment can alleviate the adverse effect. Three groups of titanium disks were prepared; machined surface, polished surface and polished surface followed by UV treatment (polished/UV surface). Polishing was performed by the sequential use of greenstone and silicon rubber burs. UV treatment was performed using a UV device for 12 min. Hydrophobicity/hydrophilicity was examined by the contact angle of ddH2O. The surface morphology and chemistry of titanium were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), respectively. Human epithelium cells were seeded on titanium disks. The number of cells attached, the spreading behavior of cells and the retention on titanium surfaces were examined. The polished surfaces were smooth with only minor scratches, while the machined surfaces showed traces and metal flashes made by machine-turning. The polished surfaces showed a significantly increased percentage of surface carbon compared to machined surfaces. The carbon percentage on polished/UV surfaces was even lower than that on machined surfaces. A silicon element was detected on polished surfaces but not on polished/UV surfaces. Both machined and polished surfaces were hydrophobic, whereas polished/UV surfaces were hydrophilic. The number of attached cells after 24 h of incubation was 60% lower on polished surfaces than on machined surfaces. The number of attached cells on polished/UV surfaces was even higher than that on machined surfaces. The size and perimeter of cells, which was significantly reduced on polished surfaces, were fully restored on polished/UV surfaces. The number of cells remained adherent after mechanical detachment was reduced to half on polished surfaces compared to machined surfaces. The number of adherent cells on polished/UV surfaces was two times higher than on machined surfaces. In conclusion, polishing titanium causes chemical contamination, while smoothing its surface significantly compromised the attachment and retention of human epithelial cells. The UV treatment of polished titanium surfaces reversed these adverse effects and even outperformed the inherent bioactivity of the original titanium.

Role of Arginine Methylation in Alternative Polyadenylation of VEGFR-1 (Flt-1) pre-mRNA.

Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3'-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.

UV-Pre-Treated and Protein-Adsorbed Titanium Implants Exhibit Enhanced Osteoconductivity.

Titanium materials are essential treatment modalities in the medical field and serve as a tissue engineering scaffold and coating material for medical devices. Thus, there is a significant demand to improve the bioactivity of titanium for therapeutic and experimental purposes. We showed that ultraviolet light (UV)-pre-treatment changed the protein-adsorption ability and subsequent osteoconductivity of titanium. Fibronectin (FN) adsorption on UV-treated titanium was 20% and 30% greater after 1-min and 1-h incubation, respectively, than that of control titanium. After 3-h incubation, FN adsorption on UV-treated titanium remained 30% higher than that on the control. Osteoblasts were cultured on titanium disks after 1-h FN adsorption with or without UV-pre-treatment and on titanium disks without FN adsorption. The number of attached osteoblasts during the early stage of culture was 80% greater on UV-treated and FN-adsorbed (UV/FN) titanium than on FN-adsorbed (FN) titanium; osteoblasts attachment on UV/FN titanium was 2.6- and 2.1-fold greater than that on control- and UV-treated titanium, respectively. The alkaline phosphatase activity of osteoblasts on UV/FN titanium was increased 1.8-, 1.8-, and 2.4-fold compared with that on FN-adsorbed, UV-treated, and control titanium, respectively. The UV/FN implants exhibited 25% and 150% greater in vivo biomechanical strength of bone integration than the FN- and control implants, respectively. Bone morphogenetic protein-2 (BMP-2) adsorption on UV-treated titanium was 4.5-fold greater than that on control titanium after 1-min incubation, resulting in a 4-fold increase in osteoblast attachment. Thus, UV-pre-treatment of titanium accelerated its protein adsorptivity and osteoconductivity, providing a novel strategy for enhancing its bioactivity.

UV-Photofunctionalization of Titanium Promotes Mechanical Anchorage in A Rat Osteoporosis Model.

Effects of UV-photofunctionalization on bone-to-titanium integration under challenging systemic conditions remain unclear. We examined the behavior and response of osteoblasts from sham-operated and ovariectomized (OVX) rats on titanium surfaces with or without UV light pre-treatment and the strength of bone-implant integration. Osteoblasts from OVX rats showed significantly lower alkaline phosphatase, osteogenic gene expression, and mineralization activities than those from sham rats. Bone density variables in the spine were consistently lower in OVX rats. UV-treated titanium was superhydrophilic and the contact angle of ddH2O was ≤5°. Titanium without UV treatment was hydrophobic with a contact angle of ≥80°. Initial attachment to titanium, proliferation, alkaline phosphatase activity, and gene expression were significantly increased on UV-treated titanium compared to that on control titanium in osteoblasts from sham and OVX rats. Osteoblastic functions compromised by OVX were elevated to levels equivalent to or higher than those of sham-operated osteoblasts following culture on UV-treated titanium. The strength of in vivo bone-implant integration for UV-treated titanium was 80% higher than that of control titanium in OVX rats and even higher than that of control implants in sham-operated rats. Thus, UV-photofunctionalization effectively enhanced bone-implant integration in OVX rats to overcome post-menopausal osteoporosis-like conditions.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

A Newly Created Meso-, Micro-, and Nano-Scale Rough Titanium Surface Promotes Bone-Implant Integration.

Titanium implants are the standard therapeutic option when restoring missing teeth and reconstructing fractured and/or diseased bone. However, in the 30 years since the advent of micro-rough surfaces, titanium's ability to integrate with bone has not improved significantly. We developed a method to create a unique titanium surface with distinct roughness features at meso-, micro-, and nano-scales. We sought to determine the biological ability of the surface and optimize it for better osseointegration. Commercially pure titanium was acid-etched with sulfuric acid at different temperatures (120, 130, 140, and 150 °C). Although only the typical micro-scale compartmental structure was formed during acid-etching at 120 and 130 °C, meso-scale spikes (20-50 µm wide) and nano-scale polymorphic structures as well as micro-scale compartmental structures formed exclusively at 140 and 150 °C. The average surface roughness (Ra) of the three-scale rough surface was 6-12 times greater than that with micro-roughness only, and did not compromise the initial attachment and spreading of osteoblasts despite its considerably increased surface roughness. The new surface promoted osteoblast differentiation and in vivo osseointegration significantly; regression analysis between osteoconductivity and surface variables revealed these effects were highly correlated with the size and density of meso-scale spikes. The overall strength of osseointegration was the greatest when the acid-etching was performed at 140 °C. Thus, we demonstrated that our meso-, micro-, and nano-scale rough titanium surface generates substantially increased osteoconductive and osseointegrative ability over the well-established micro-rough titanium surface. This novel surface is expected to be utilized in dental and various types of orthopedic surgical implants, as well as titanium-based bone engineering scaffolds.