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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 effectiv

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.

It is a significant challenge for a titanium implant, which is a bio-inert material, to recruit osteogenic factors, such as osteoblasts, proteins and blood effectively when these are contained in a biomaterial. The objective of this study was to examine the effect of ultraviolet (UV)-treatment of titanium on surface wettability and the recruitment of osteogenic factors when they are contained in an atelocollagen sponge. UV treatment of a dental implant made of commercially pure titanium was performed with UV-light for 12 min immediately prior to the experiments. Superhydrophilicity on dental implant surfaces was generated with UV-treatment. The collagen sponge containing blood, osteoblasts, or albumin was directly placed on the dental implant. Untreated implants absorbed only a little blood from the collagen sponge, while the UV-treated implants absorbed blood rapidly and allowed it to spread widely, almost over the entire implant surface. Blood coverage was 3.5 times greater for the UV-treated implants (p < 0.001). Only 6% of the osteoblasts transferred from the collagen sponge to the untreated implants, whereas 16% of the osteoblasts transferred to the UV-treated implants (p < 0.001). In addition, a weight ratio between transferred albumin on the implant and measured albumin adsorbed on the implant was 17.3% in untreated implants and 38.5% in UV-treated implants (p < 0.05). These results indicated that UV treatment converts a titanium surface into a superhydrophilic and bio-active material, which could recruite osteogenic factors even when they were contained in a collagen sponge. The transfer and subsequent diffusion and adsorption efficacy of UV-treated titanium surfaces could be useful for bone formation when titanium surfaces and osteogenic factors are intervened with a biomaterial.

Ultraviolet treatment of titanium implants makes their surfaces hydrophilic and enhances osseointegration. However, the mechanism is not fully understood. This study hypothesizes that the recruitment of fibrinogen, a critical molecule for blood clot formation and wound healing, is influenced by the degrees of hydrophilicity/hydrophobicity of the implant surfaces. Computational fluid dynamics (CFD) implant models were created for fluid flow simulation. The hydrophilicity level was expressed by the contact angle between the implant surface and blood plasma, ranging from 5° (superhydrophilic), 30° (hydrophilic) to 50° and 70° (hydrophobic), and 100° (hydrorepellent). The mass of fibrinogen flowing into the implant interfacial zone (fibrinogen infiltration) increased in a time dependent manner, with a steeper slope for surfaces with greater hydrophilicity. The mass of blood plasma absorbed into the interfacial zone (blood plasma infiltration) was also promoted by the hydrophilic surfaces but it was rapid and non-time-dependent. There was no linear correlation between the fibrinogen infiltration rate and the blood plasma infiltration rate. These results suggest that hydrophilic implant surfaces promote both fibrinogen and blood plasma infiltration to their interface. However, the infiltration of the two components were not proportional, implying a selectively enhanced recruitment of fibrinogen by hydrophilic implant surfaces.

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.

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

Poly(methyl methacrylate) (PMMA)-based bone cement, which is widely used to affix orthopedic metallic implants, is considered bio-tolerant but lacks osteoconductivity and is cytotoxic. Implant loosening and toxic complications are significant and recognized problems. Here we devised two strategies to improve PMMA-based bone cement: (1) adding 4-methacryloyloxylethyl trimellitate anhydride (4-META) to MMA monomer to render it hydrophilic; and (2) using tri-n-butyl borane (TBB) as a polymerization initiator instead of benzoyl peroxide (BPO) to reduce free radical production. Rat bone marrow-derived osteoblasts were cultured on PMMA-BPO, common bone cement ingredients, and 4-META/MMA-TBB, newly formulated ingredients. After 24 h of incubation, more cells survived on 4-META/MMA-TBB than on PMMA-BPO. The mineralized area was 20-times greater on 4-META/MMA-TBB than PMMA-BPO at the later culture stage and was accompanied by upregulated osteogenic gene expression. The strength of bone-to-cement integration in rat femurs was 4- and 7-times greater for 4-META/MMA-TBB than PMMA-BPO during early- and late-stage healing, respectively. MicroCT and histomorphometric analyses revealed contact osteogenesis exclusively around 4-META/MMA-TBB, with minimal soft tissue interposition. Hydrophilicity of 4-META/MMA-TBB was sustained for 24 h, particularly under wet conditions, whereas PMMA-BPO was hydrophobic immediately after mixing and was unaffected by time or condition. Electron spin resonance (ESR) spectroscopy revealed that the free radical production for 4-META/MMA-TBB was 1/10 to 1/20 that of PMMA-BPO within 24 h, and the substantial difference persisted for at least 10 days. The compromised ability of PMMA-BPO in recruiting cells was substantially alleviated by adding free radical-scavenging amino-acid N-acetyl cysteine (NAC) into the material, whereas adding NAC did not affect the ability of 4-META/MMA-TBB. These results suggest that 4-META/MMA-TBB shows significantly reduced cyt

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.

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 adhere

Titanium micro-scale topography offers excellent osteoconductivity and bone-implant integration. However, the biological effects of sub-micron topography are unknown. We compared osteoblastic phenotypes and in vivo bone and implant integration abilities between titanium surfaces with micro- (1-5 µm) and sub-micro-scale (0.1-0.5 µm) compartmental structures and machined titanium. The calculated average roughness was 12.5 ± 0.65, 123 ± 6.15, and 24 ± 1.2 nm for machined, micro-rough, and sub-micro-rough surfaces, respectively. In culture studies using bone marrow-derived osteoblasts, the micro-rough surface showed the lowest proliferation and fewest cells attaching during the initial stage. Calcium deposition and expression of osteoblastic genes were highest on the sub-micro-rough surface. The bone-implant integration in the Sprague-Dawley male rat femur model was the strongest on the micro-rough surface. Thus, the biological effects of titanium surfaces are not necessarily proportional to the degree of roughness in osteoblastic cultures or in vivo. Sub-micro-rough titanium ameliorates the disadvantage of micro-rough titanium by restoring cell attachment and proliferation. However, bone integration and the ability to retain cells are compromised due to its lower interfacial mechanical locking. This is the first report on sub-micron topography on a titanium surface promoting osteoblast function with minimal osseointegration.

PURPOSE: Zirconia is a potential alternative to titanium for dental and orthopedic implants. Here we report the biological and bone integration capabilities of a new zirconia surface with distinct morphology at the meso-, micro-, and nano-scales. METHODS: Machine-smooth and roughened zirconia disks were prepared from yttria-stabilized tetragonal zirconia polycrystal (Y-TZP), with rough zirconia created by solid-state laser sculpting. Morphology of the surfaces was analyzed by three-dimensional imaging and profiling. Rat femur-derived bone marrow cells were cultured on zirconia disks. Zirconia implants were placed in rat femurs and the strength of osseointegration was evaluated by biomechanical push-in test. RESULTS: The rough zirconia surface was characterized by meso-scale (50 µm wide, 6-8 µm deep) grooves, micro-scale (1-10 µm wide, 0.1-3 µm deep) valleys, and nano-scale (10-400 nm wide, 10-300 nm high) nodules, whereas the machined surface was flat and uniform. The average roughness (Ra) of rough zirconia was five times greater than that of machined zirconia. The expression of bone-related genes such as collagen I, osteopontin, osteocalcin, and BMP-2 was 7-25 times upregulated in osteoblasts on rough zirconia at the early stage of culture. The number of attached cells and rate of proliferation were similar between machined and rough zirconia. The strength of osseointegration for rough zirconia was twice that of machined zirconia at weeks two and four of healing, with evidence of mineralized tissue persisting around rough zirconia implants as visualized by electron microscopy and elemental analysis. CONCLUSION: This unique meso-/micro-/nano-scale rough zirconia showed a remarkable increase in osseointegration compared to machine-smooth zirconia associated with accelerated differentiation of osteoblasts. Cell attachment and proliferation were not compromised on rough zirconia unlike on rough titanium. This is the first report introducing a rough zirconia surface wi

Naser Mohammadzadeh Rezaei, Masakazu Hasegawa, Manabu Ishijima, Kourosh Nakhaei, Takahisa Okubo, Takashi Taniyama, Amirreza Ghassemi, Tania Tahsili, Wonhee Park, Makoto Hirota, Takahiro Ogawa Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, UCLA School of Dentistry, Los Angeles, CA, USA Purpose: Zirconia is a potential alternative to titanium for dental and orthopedic implants. Here we report the biological and bone integration capabilities of a new zirconia surface with distinct morphology at the meso-, micro-, and nano-scales.Methods: Machine-smooth and roughened zirconia disks were prepared from yttria-stabilized tetragonal zirconia polycrystal (Y-TZP), with rough zirconia created by solid-state laser sculpting. Morphology of the surfaces was analyzed by three-dimensional imaging and profiling. Rat femur-derived bone marrow cells were cultured on zirconia disks. Zirconia implants were placed in rat femurs and the strength of osseointegration was evaluated by biomechanical push-in test.Results: The rough zirconia surface was characterized by meso-scale (50 µm wide, 6–8 µm deep) grooves, micro-scale (1–10 µm wide, 0.1–3 µm deep) valleys, and nano-scale (10–400 nm wide, 10–300 nm high) nodules, whereas the machined surface was flat and uniform. The average roughness (Ra) of rough zirconia was five times greater than that of machined zirconia. The expression of bone-related genes such as collagen I, osteopontin, osteocalcin, and BMP-2 was 7–25 times upregulated in osteoblasts on rough zirconia at the early stage of culture. The number of attached cells and rate of proliferation were similar between machined and rough zirconia. The strength of osseointegration for rough zirconia was twice that of machined zirconia at weeks two and four of healing, with evidence of mineralized tissue persisting around rough zirconia implants as visu

Naser Mohammadzadeh Rezaei, Masakazu Hasegawa, Manabu Ishijima, Kourosh Nakhaei, Takahisa Okubo, Takashi Taniyama, Amirreza Ghassemi, Tania Tahsili, Wonhee Park, Makoto Hirota, Takahiro Ogawa Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, UCLA School of Dentistry, Los Angeles, CA, USA Purpose: Zirconia is a potential alternative to titanium for dental and orthopedic implants. Here we report the biological and bone integration capabilities of a new zirconia surface with distinct morphology at the meso-, micro-, and nano-scales.Methods: Machine-smooth and roughened zirconia disks were prepared from yttria-stabilized tetragonal zirconia polycrystal (Y-TZP), with rough zirconia created by solid-state laser sculpting. Morphology of the surfaces was analyzed by three-dimensional imaging and profiling. Rat femur-derived bone marrow cells were cultured on zirconia disks. Zirconia implants were placed in rat femurs and the strength of osseointegration was evaluated by biomechanical push-in test.Results: The rough zirconia surface was characterized by meso-scale (50 µm wide, 6–8 µm deep) grooves, micro-scale (1–10 µm wide, 0.1–3 µm deep) valleys, and nano-scale (10–400 nm wide, 10–300 nm high) nodules, whereas the machined surface was flat and uniform. The average roughness (Ra) of rough zirconia was five times greater than that of machined zirconia. The expression of bone-related genes such as collagen I, osteopontin, osteocalcin, and BMP-2 was 7–25 times upregulated in osteoblasts on rough zirconia at the early stage of culture. The number of attached cells and rate of proliferation were similar between machined and rough zirconia. The strength of osseointegration for rough zirconia was twice that of machined zirconia at weeks two and four of healing, with evidence of mineralized tissue persisting around rough zirconia implants as visu

Satomi Sugiyama, Toshinori Iwai, Nobuhide Ohashi, Senri Oguri, Makoto Hirota, Kenji Mitsudo Department of Oral and Maxillofacial Surgery/Orthodontics, Yokohama City University Hospital, Yokohama, Japan Abstract: Intraoral removal of a submandibular gland stone is less invasive than submandibulectomy, with no life-threatening airway complications reported until now. We report a case involving airway obstruction caused by pharyngolaryngeal swelling after intraoral removal of a submandibular gland stone. A 31-year-old man with a left submandibular gland stone underwent intraoral removal of the stone under general anesthesia and was discharged 1 day after surgery. That night, he was transported back to our hospital by ambulance for dyspnea. Because computed tomography showed pharyngolaryngeal swelling, he was intubated and managed by a ventilator. Four days later, the pharyngolaryngeal swelling had mostly resolved and he was extubated and discharged. Surgeons should be vigilant of the potential risk of airway complications following an intraoral approach for submandibular gland stone removal. Keywords: airway obstruction, intraoral removal, sialolithiasis, submandibular gland stone, complication

Titanium undergoes time-dependent degradation in biological capability, or "biological aging". It is unknown whether the biological aging of titanium occurs beyond four weeks and whether age-related changes are definitely associated with surface hydrophilicity. We therefore measured multiple biological parameters of bone marrow-derived osteoblasts cultured on newly prepared, one-month-old, three-month-old, and six-month-old acid-etched titanium surfaces, as well as the hydrophilicity of these surfaces. New surfaces were superhydrophilic with a contact angle of ddH₂O of 0°, whereas old surfaces were all hydrophobic with the contact angle of around 90°. Cell attachment, cell spread, cell density, and alkaline phosphatase activity were highest on new surfaces and decreased in a time-dependent manner. These decreases persisted and remained significant for most of the biological parameters up to six-months. While the number of attached cells was negatively correlated with hydrophilicity, the other measured parameters were not. The biological capability of titanium continues to degrade up to six months of aging, but these effects are not directly associated with time-dependent reductions in hydrophilicity. A full understanding of the biological aging will help guide regulatory improvements in implant device manufacturing and develop countermeasures against this phenomenon in order to improve clinical outcomes.

Titanium undergoes time-dependent degradation in biological capability, or "biological aging". It is unknown whether the biological aging of titanium occurs beyond four weeks and whether age-related changes are definitely associated with surface hydrophilicity. We therefore measured multiple biological parameters of bone marrow-derived osteoblasts cultured on newly prepared, one-month-old, three-month-old, and six-month-old acid-etched titanium surfaces, as well as the hydrophilicity of these surfaces. New surfaces were superhydrophilic with a contact angle of ddH₂O of 0°, whereas old surfaces were all hydrophobic with the contact angle of around 90°. Cell attachment, cell spread, cell density, and alkaline phosphatase activity were highest on new surfaces and decreased in a time-dependent manner. These decreases persisted and remained significant for most of the biological parameters up to six-months. While the number of attached cells was negatively correlated with hydrophilicity, the other measured parameters were not. The biological capability of titanium continues to degrade up to six months of aging, but these effects are not directly associated with time-dependent reductions in hydrophilicity. A full understanding of the biological aging will help guide regulatory improvements in implant device manufacturing and develop countermeasures against this phenomenon in order to improve clinical outcomes.

Pollination of Japanese pear 'Chojuro', self-incompatible cultivar, at bud stages increased persisting fruit on a tree, even though selfing. Buds, selfed during 6 and 3 days before anthesis, produced fruit and they were persisted on a tree till harvest time. After bud pollination, the levels of gibberellin-like substances (GAs) extracted from pistils were assayed by elongation of dwarf rice seedling. Although the amounts of GAs in non-pollinated pistils decreased gradually till 16 days after castration, they increased in the pistils cross-pollinated. In the self-pollinated pistils, relatively high amounts of GAs were observed, especially the activitiy 4 days after pollination was stronger than that cross-pollinated one. These activities were assumed to be gibberellic acid (GA_3) judged from Rf value of paper chromatogram. The overall levels of GAs in self-pollinated pistils were intermediate between non- and cross-pollinated. Exogeneous application of GA_3 and GA_<4+7> had almost no effect on the protrusion of pollen tubes from the excised-styles cultured on an agar medium. From these results and other informations, increasing fruit set when selfed at bud stage was considered to be not only due to overcoming self-incompatibility, but also due to parthenocarpy induced by GAs in pistils produced after pollination.