Transplantation of Mature Adipocyte-Derived Dedifferentiated Fat Cells Facilitates Periodontal Tissue Regeneration of Class II Furcation Defects in Miniature Pigs.

Adipose tissue is composed mostly of adipocytes that are in contact with capillaries. By using a ceiling culture method based on buoyancy, lipid-free fibroblast-like cells, also known as dedifferentiated fat (DFAT) cells, can be separated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and transdifferentiate into various cell types under appropriate culture conditions. Herein, we sought to compare the regenerative potential of collagen matrix alone (control) with autologous DFAT cell-loaded collagen matrix transplantation in adult miniature pigs (microminipigs; MMPs). We established and transplanted DFAT cells into inflammation-inducing periodontal class II furcation defects. At 12 weeks after cell transplantation, a marked attachment gain was observed based on the clinical parameters of probing depth (PD) and clinical attachment level (CAL). Additionally, micro computed tomography (CT) revealed hard tissue formation in furcation defects of the second premolar. The cemento-enamel junction and alveolar bone crest distance was significantly shorter following transplantation. Moreover, newly formed cellular cementum, well-oriented periodontal ligament-like fibers, and alveolar bone formation were observed via histological analysis. No teratomas were found in the internal organs of recipient MMPs. Taken together, these findings suggest that DFAT cells can safely enhance periodontal tissue regeneration.

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.

Ultraviolet treatment restores bioactivity of titanium mesh plate degraded by contact with medical gloves.

Titanium mesh plate (Ti mesh) used for bone augmentation inadvertently comes into contact with medical gloves during trimming and bending. We tested the hypotheses that glove contact degrades the biological capability of Ti mesh and that ultraviolet treatment (UV) can restore this capability. Three groups of Ti mesh specimens were prepared: as-received (AR), after glove contact (GC), and after glove contact followed by UV treatment. The AR and GC meshes were hydrophobic, but GC mesh was more hydrophobic. AR and GC meshes had significant amounts of surface carbon, and Si content was higher for GC mesh than for AR mesh. UV mesh was hydrophilic, and carbon and silicon content values were significantly lower in this group than in the AR and GC groups. The number, alkaline phosphatase activity, and mineralization ability of attached osteoblasts were significantly lower in the GC group than in the AR group and markedly higher in the UV group than in the AR group. In conclusion, glove contact caused chemical contamination of Ti mesh, which significantly reduced its bioactivity. UV treatment restored bioactivity in contaminated Ti mesh, which outperformed even the baseline Ti mesh.

Mouthguards and their use in sports: Report of the 1st International Sports Dentistry Workshop, 2016.

There appears to be much confusion or misinformation worldwide regarding mouthguards and their use in sports. In an effort to clarify where the international dental community stands on mouthguards and mouthguard research, the workshop looked at some important questions. The goal was to one day formulate consensus statements related to these questions, which will be based on current scientific evidence-based research, to motivate the international community of the importance of dentally fitted laminated mouthguards and the wearing of them by athletes of all sports. There are only five sports in the United States that require the use of mouthguards. If, through workshops such as this, the importance of wearing dentally fitted laminated mouthguards can be demonstrated, then more sports may require their athletes to wear them.

Novel antioxidant capability of titanium induced by UV light treatment.

The intracellular production of reactive oxygen species (ROS) is a representative form of cellular oxidative stress and plays an important role in triggering adverse cellular events, such as the inflammatory reaction and delayed or compromised differentiation. Osteoblastic reaction to titanium with particular focus on ROS production remains unknown. Ultraviolet (UV) light treatment improves the physicochemical properties of titanium, specifically the induction of super hydrophilicity and removal of hydrocarbon, and eventually enhances its osteoconductivity. We hypothesized that there is a favorable regulatory change of ROS production within osteoblasts in contact with UV-treated titanium. Osteoblasts were cultured on titanium disks with or without UV-pretreatment. The intracellular production of ROS was higher on acid-etch-created rough titanium surfaces than on machine-prepared smooth ones. The ROS production was reduced by 40-50% by UV pretreatment of titanium regardless of the surface roughness. Oxidative DNA damage, as detected by 8-OHdG expression, was alleviated by 50% on UV-treated titanium surfaces. The expression of inflammatory cytokines was consistently lower in osteoblasts cultured on UV-treated titanium. ROS scavenger, glutathione, remained more without being depleted in osteoblasts on UV-treated titanium. Bio-burden test further showed that culturing osteoblasts on UV-treated titanium can significantly reduce the ROS production even with the presence of hydrogen peroxide, an oxidative stress inducer. These data suggest that the intracellular production of ROS and relevant inflammatory reaction, which unavoidably occurs in osteoblasts in contact with titanium, can be significantly reduced by UV pretreatment of titanium, implying a novel antioxidant capability of the particular titanium.

Effect of UV Photofunctionalization on Osseointegration in Aged Rats.

OBJECTIVES: This study evaluated the effect of photofunctionalization on osseointegration under the biologically adverse conditions of aging. MATERIALS: First of all, bone marrow-derived osteoblastic cells from young (8 weeks old) and aged (15 months old) rats were biologically characterized. Then, the osteoblasts from aged rats were seeded on titanium discs with and without photofunctionalization, and assessed for initial cell attachment and osteoblastic functions. Titanium mini-implants, with and without photofunctionalization, were placed in the femur of aged rats, and the strength of osseointegration was measured at week 2 of healing. Periimplant tissue was examined morphologically and chemically using scanning electron microscopy and energy dispersive x-ray spectroscopy, respectively. RESULTS: Cells from the aged rats showed substantially reduced biological capabilities compared with those derived from young rats. The cells from aged rats showed significantly increased cell attachment and the expression of osteoblastic function on photofunctionalized titanium than on untreated titanium. In addition, the strength of osseointegration was increased by 40% in aged rats carrying the photofunctionalized implants. Robust bone formation was observed around the photofunctionalized implants with strong elemental peaks of calcium and phosphorus, whereas the tissue around untreated implants showed weaker calcium and phosphate signals than titanium ones. CONCLUSION: These in vivo and in vitro results corroboratively demonstrate that photofunctionalization is effective for enhancing osseointegration in aged rats.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

Bone Generation Profiling Around Photofunctionalized Titanium Mesh.

PURPOSE: The aim of this study was to evaluate whether photofunctionalization of titanium mesh enhances its osteoconductive capability. MATERIALS AND METHODS: The titanium mesh (0.2 mm thickness) used in this study was made of commercially pure grade-2 titanium and had hexagonal apertures (2 mm width). Photofunctionalization was performed by treating titanium mesh with UV light for 12 minutes using a photo device immediately before use. Untreated or photofunctionalized titanium mesh was placed into rat femurs, and bone generation around titanium mesh was profiled using three-dimensional (3D) microcomputed tomography (micro-CT). A set of in vitro experiments was conducted using bone marrow-derived osteoblasts. RESULTS: Photofunctionalized titanium mesh surfaces were characterized by the regenerated hydrophilicity and significantly reduced surface carbon. Bone generation profiling at week 3 of healing showed that the hexagonal apertures in photofunctionalized mesh were 95% filled, but they were only 57% filled in untreated mesh, particularly with the center zone remaining as a gap. Bone profiling in slices parallel to the titanium surface showed that photofunctionalized titanium mesh achieved 90% bone occupancy 0 to 400 µm from the surface, compared with only 35% for untreated mesh. Bone occupancy remained as high as 55% 800 to 1,200 µm from photofunctionalized titanium mesh surfaces, compared with less than 20% for untreated mesh. In vitro, photofunctionalized titanium mesh expedited and enhanced attachment and spread of osteoblasts, and increased ALP activity and the rate of mineralization. CONCLUSION: This study may provide novel and advanced metrics describing the osteoconductive property of photofunctionalized titanium mesh. Specifically, photofunctionalization not only increased the breadth, but also the 3D range, of osteoconductivity of titanium mesh, enabling space-filling and far-reaching osteoconductivity. Further translational and clinical studies are warranted to establish photofunctionalized titanium mesh as a novel clinical tool for better bone regeneration and augmentation.

Enhancing osteoblast-affinity of titanium scaffolds for bone engineering by use of ultraviolet light treatment.

Ultraviolet (UV) treatment immediately prior to use is attracting attention as an effective surface conditioning method for titanium to improve osteoblast-affinity. The affinity of titanium to osteoblasts in two-dimensional plate culture has been well studied, but that in three-dimensional cultures remains unclear. Here, we examined the effect of UV treatment on titanium scaffolds, comprising micro-thin titanium fibers, used in bone engineering. Titanium scaffolds, with and without UV treatment, were seeded with rat bone marrow derived osteoblasts, and the number of cells attached to scaffolds and osteoblastic phenotype in the cultures were examined. UV treatment improved the wettability of scaffolds and significantly reduced the percentage of surface carbon. Along with these physicochemical changes in the scaffolds, cell attachment increased by a factor of 1.3 as compared to that of the untreated control. In addition, alkaline phosphatase activity and calcium deposition significantly increased by a factor of 2.3 and 2.0, respectively. Robust formation of mineralized structures consisting of clear peaks of calcium and phosphorus was observed in the UV-treated scaffolds. The observed increase in osteoblast affinity and capability of mineralized matrix formation indicates the potential use of UV-treated titanium scaffolds for bone engineering.

Osteogenic cell sheets reinforced with photofunctionalized micro-thin titanium.

Cell sheet technology has been used to deliver cells in single-sheet form with an intact extracellular matrix for soft tissue repair and regeneration. Here, we hypothesized that titanium-reinforced cell sheets could be constructed for bone tissue engineering and regeneration. Fifty-µm-thick titanium plates containing apertures were prepared and roughened by acid etching, some of which were photofunctionalized with 12 min of UV light treatment. Cell sheets were prepared by culturing rat calvarial periosteum-derived cells on temperature-responsive culture dishes and attached to titanium plates. Titanium-reinforced osteogenic cell sheet construction was conditional on various technical and material factors: cell sheets needed to be double-sided and sandwich the titanium plate, and the titanium plates needed to be micro thin and contain apertures to allow close apposition of the two cell sheets. Critically, titanium plates needed to be UV-photofunctionalized to ensure adherence and retention of cell sheets. Single-sided cell sheets or double-sided cell sheets on as-made titanium contracted and deformed within 4 days of incubation. Titanium-reinforced cell sheets on photofunctionalized titanium were structurally stable at least up to 14 days, developed the expected osteogenic phenotypes (ALP production and mineralization), and maintained structural integrity without functional degradation. Successful construction of titanium-reinforced osteogenic cell sheets was associated with increased cell attachment, retention, and expression of vinculin, an adhesion protein by photofunctionalization. This study identified the technical and material requirements for constructing titanium-reinforced osteogenic cell sheets. Future in vivo studies are warranted to test these titanium-reinforced cell sheets as stably transplantable, mechanically durable, and shape controllable osteogenic devices.

Periodontal tissue regeneration by transplantation of rat adipose-derived stromal cells in combination with PLGA-based solid scaffolds.

Regeneration of damaged periodontium is challenging due to its multi-tissue composition. Mesenchymalstem cell-based approaches using adipose-derived stromal cells (ASCs) may contribute to periodontal reconstruction, particularly when combined with the use of scaffolds to maintain a space for new tissue growth. The aim of this study was to assess the regenerative potential of ASCs derived from inbred or outbred rats in combination with novel solid scaffolds composed of PLGA (Poly D,L-lactic-co-glycolic acid) (PLGA-scaffolds). Cultured ASCs seeded onto PLGA scaffolds (ASCs/PLGA) or PLGA-scaffolds (PLGA) alone were transplanted into periodontal fenestration defects created in F344 or Sprague Dawley (SD) rats. Micro-CT analysis showed a significantly higher percentage of bone growth in the ASCs/PLGA groups compared with the PLGA-alone groups at five weeks after surgery. Similarly, histomorphometric analysis demonstrated thicker growth of periodontal ligament and cementum layers in the ASCs/PLGA-groups compared with the PLGA-alone groups. In addition, transplanted DiI-labeled ASCs were observed in the periodontal regenerative sites. The present investigation demonstrated the marked ability of ASCs in combination with PLGA scaffolds to repair periodontal defects.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

Bone integration capability of nanopolymorphic crystalline hydroxyapatite coated on titanium implants.

The mechanism by which hydroxyapatite (HA)-coated titanium promotes bone-implant integration is largely unknown. Furthermore, refining the fabrication of nano-structured HA to the level applicable to the mass production process for titanium implants is challenging. This study reports successful creation of nanopolymorphic crystalline HA on microroughened titanium surfaces using a combination of flame spray and low-temperature calcination and tests its biological capability to enhance bone-implant integration. Sandblasted microroughened titanium implants and sandblasted + HA-coated titanium implants were subjected to biomechanical and histomorphometric analyses in a rat model. The HA was 55% crystallized and consisted of nanoscale needle-like architectures developed in various diameters, lengths, and orientations, which resulted in a 70% increase in surface area compared to noncoated microroughened surfaces. The HA was free from impurity contaminants, with a calcium/phosphorus ratio of 1.66 being equivalent to that of stoichiometric HA. As compared to microroughened implants, HA-coated implants increased the strength of bone-implant integration consistently at both early and late stages of healing. HA-coated implants showed an increased percentage of bone-implant contact and bone volume within 50 µm proximity of the implant surface, as well as a remarkably reduced percentage of soft tissue intervention between bone and the implant surface. In contrast, bone volume outside the 50 µm border was lower around HA-coated implants. Thus, this study demonstrated that the addition of pure nanopolymorphic crystalline HA to microroughened titanium not only accelerates but also enhances the level of bone-implant integration and identified the specific tissue morphogenesis parameters modulated by HA coating. In particular, the nanocrystalline HA was proven to be drastic in increasing osteoconductivity and inhibiting soft tissue infiltration, but the effect was limited to the immediate microenvironment surrounding the implant.

Bone integration capability of alkali- and heat-treated nanobimorphic Ti-15Mo-5Zr-3Al.

The role of nanofeatured titanium surfaces in a number of aspects of in vivo bone-implant integration, and, in particular, their potential advantages over microfeatured titanium surfaces, as well as their specific contribution to osteoconductivity, is largely unknown. This study reports the creation of a unique nanobimorphic titanium surface comprised of nanotrabecular and nanotuft-like structures and determines how the addition of this nanofeature to a microroughened surface affects bone-implant integration. Machined surfaces without microroughness, sandblasted microroughened surfaces, and micro-nano hybrid surfaces created by sandblasting and alkali and heat treatment of Ti-15Mo-5Zr-3Al alloy were subjected to biomechanical, interfacial and histological analyses in a rat model. The presence of microroughness enabled accelerated establishment of biomechanical implant fixation in the early stages of healing compared to the non-microroughened surfaces; however, it did not increase the implant fixation at the late stages of healing. The addition of nanobimorphic features to the microroughened surfaces further increased the implant fixation by as much as 60-100% over the healing time. Bone area within 50 µm of the implant surface, but not beyond this distance, was significantly increased by the presence of nanobimorphic features. Although the percentage of bone-implant contact was also significantly increased by the addition of nanobimorphic features, the greatest improvement was found in the soft tissue intervention between the bone and the implant, which was reduced from >30% to <5%. Mineralized tissue densely deposited with calcium-binding globular proteins was observed in an extensive area of nanobimorphic surfaces after biomechanical testing. This study clearly demonstrates the nanofeature-enhanced osteoconductivity of titanium by an alkali- and heat-treated nanobimorphic surface compared to that by microfeatured surfaces, which results not only in an acceleration but also an improvement of bone-implant integration. The identified biological parameters that successfully detect the advantages of nanofeatures over microfeatures will be useful in evaluating new implant surfaces in future studies.

Effects of UV photofunctionalization on the nanotopography enhanced initial bioactivity of titanium.

This study addresses the control of the biological capabilities of titanium through specific nanosurface features and its potential modulation by UV photofunctionalization. Rat bone marrow derived osteoblasts were cultured on titanium disks with micropits alone, micropits with 100 nm nodules, micropits with 300 nm nodules, or micropits with 500 nm nodules, with or without UV treatment. After a 24 h incubation protein adsorption, as well as the attachment, retention, and spread of osteoblasts were examined in correlation with the topographical parameters of the titanium substrates. Each of the biological events was governed by a different set of multiple surface topographical factors with a distinctive pattern of regulation. For instance, without UV treatment the protein adsorption and cell attachment capability of titanium substrates increased linearly with increasing average roughness (Ra) and surface area of titanium disks, but increased polynomially with increasing nanonodule diameter. The cell retention capability increased polynomially with increasing nanonodular diameter and Ra, but increased linearly with increasing surface area. Consequently, the micropits with 300 nm nodules created the most favorable environment for this initial osteoblast behavior and response. UV treatment of the nanonodular titanium surfaces resulted in considerable enhancement of all biological events. However, the pattern of UV-mediated enhancement was disproportionate; exponential and overriding effects were observed depending upon the biological event and topographical parameter. As an example of overriding enhancement, the cell retention capability, which fluctuated with changes in various topographical parameters, became invariably high after UV treatment. The present data provide a basis for understanding how to optimize nanostructures to create titanium surfaces with increased biological capabilities and uncover a novel advantage of UV photofunctionalization of titanium substrates that synergistically increases its nanotopography enhanced biological capabilities whereby most of the initial biological events of osteoblasts were overwhelmingly enhanced beyond a simple proportional increase.

Enhanced bone-integration capability of alkali- and heat-treated nanopolymorphic titanium in micro-to-nanoscale hierarchy.

This study introduces nanopolymorphic features of alkali- and heat-treated titanium surfaces, comprising of tuft-like, plate-like, and nodular structures that are smaller than 100 nm and determines whether and how the addition of these nanofeatures to a microroughened titanium surface affects bone-implant integration. A comprehensive assessment of biomechanical, interfacial, and histological analyses in a rat model was performed for machined surfaces without microroughness, sandblasted-microroughened surfaces, and micro-nano hybrid surfaces created by sandblasting and alkali and heat treatment. The microroughened surface accelerated the establishment of implant biomechanical fixation at the early healing stage compared with the non-microroughened surface but did not increase the implant fixation at the late healing stage. The addition of the nanopolymorphic features to the microroughened surface further increased implant fixation throughout the healing time. The area of the new bone within 50 µm proximity of the implant surfaces, which was increased 2-3-fold using microroughened surfaces, was further increased 2-fold using nanopolymorphic surfaces. In contrast, the bone area in a 50-200 µm zone was not influenced by either microroughened or nanopolymorphic surfaces. The percentage of bone-implant contact, which was increased 4-5-fold, using microroughened surfaces, was further increased substantially by over 2-fold throughout the healing period. The percentage of soft tissue intervention between bone and implant surfaces, which was reduced to half by microroughened surfaces, was additionally reduced by the nanopolymorphic surfaces to between one-third and one-fourth, resulting in only 5-7% soft tissue intervention compared with 60-75% for the non-microroughened surface. Thus, using an exemplary alkali- and heat-treated nanopolymorphic surface, this study identified critical parameters necessary to describe the process and consequences of bone-implant integration, for which nanofeatures have specific and substantial roles beyond those of microfeatures. Nanofeature-enhanced osteoconductivity, which resulted in both the acceleration and elevation of bone-implant integration, has clearly been demonstrated.

TiO2 micro-nano-hybrid surface to alleviate biological aging of UV-photofunctionalized titanium.

Bioactivity and osteoconductivity of titanium degrade over time after surface processing. This time-dependent degradation is substantial and defined as the biological aging of titanium. UV treatment has shown to reactivate the aged surfaces, a process known as photofunctionalization. This study determined whether there is a difference in the behavior of biological aging for titanium with micro-nano-hybrid topography and titanium with microtopography alone, following functionalization. Titanium disks were acid etched to create micropits on the surface. Micro-nano-hybrid surfaces were created by depositioning 300-nm diameter TiO(2) nodules onto the micropits using a previously established self-assembly protocol. These disks were stored for 8 weeks in the dark to allow sufficient aging, then treated with UV light for 48 hours. Rat bone marrow-derived osteoblasts were cultured on fresh disks (immediately after UV treatment), 3-day-old disks (disks stored for 3 days after UV treatment), and 7-day- old disks. The rates of cell attachment, spread, proliferation, and levels of alkaline phosphatase activity, and calcium deposition were reduced by 30%-50% on micropit surfaces, depending on the age of the titanium. In contrast, 7-day-old hybrid surfaces maintained equivalent levels of bioactivity compared with the fresh surfaces. Both micropit and micro-nano-hybrid surfaces were superhydrophilic immediately after UV treatment. However, after 7 days, the micro-nano- hybrid surfaces became hydrorepellent, while the micropit surfaces remained hydrophilic. The sustained bioactivity levels of the micro-nano-hybrid surfaces were nullified by treating these surfaces with Cl(-)anions. A thin TiO(2) coating on the micropit surface without the formation of nanonodules did not result in the prevention or alleviation of the time-dependent decrease in biological activity. In conclusion, the micro-nano-hybrid titanium surfaces may slow the rate of time-dependent degradation of titanium bioactivity after UV photofunctionalization compared with titanium surfaces with microtopography alone. This antibiological aging effect was largely regulated by its sustained electropositivity uniquely conferred in TiO(2) nanonodules, and was independent of the degree of hydrophilicity. These results demonstrate the potential usefulness of these hybrid surfaces to effectively utilize the benefits of UV photofunctionalization and provide a model to explore the mechanisms underlying antibiological aging properties.

Synergistic effects of UV photofunctionalization and micro-nano hybrid topography on the biological properties of titanium.

Titanium surfaces with micro-nano hybrid topography (nanoscale nodules in microscale pits) have been recently demonstrated to show higher biological capability than those with microtopography alone. On the other hand, UV treatment of titanium surfaces, which is called UV photofunctionalization, has recently been introduced to substantially increase the biological capability and osteoconductivity of titanium surfaces. However, synergistic effects of these two advanced surface modification technologies and regulatory factors to potentially modulate the mutual effects have never been addressed. In this study, utilization of a recently discovered controllable self-assembly of TiO(2) nanonodules has enabled the exploration of the relative contribution of different sizes of nanostructures to determine the biological capability of titanium surfaces and their relative responsiveness to UV photofunctionalization. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules, with or without UV treatment. Although UV treatment increased the attachment, spread, proliferation, and mineralization of these cells on all titanium surfaces, these effects were more accentuated (3-5 times) on nanonodular surfaces than on surfaces with micropits alone and were disproportionate depending on nanonodule sizes. For instance, on UV-treated micro-nano hybrid surfaces, cell attachment correlated with nanonodule sizes in a quadratic approximation with its peak for 300-nm nodules followed by a decline for 500-nm nodules, while cell attachment exponentially correlated with surface roughness with its plateau achieved for 300-nm nodules without a subsequent decline. Moreover, cell attachment increased in a linear correlation with the surface area, while no significant effect of the inter-irregularities space or degree of hydrophilicity was observed on cell attachment. These results suggest that the effect of UV photofunctionalization can be multiplied on micro-nano hybrid titanium surfaces compared with the surfaces with micropits alone. This multiplication is disproportionately regulated by a selected set of topographical parameters of the titanium surfaces. Among the nanonodules tested in this study, 300-nm nodules seemed to create the most effective morphological environment for responding to UV photofunctionalization. The data provide a systematic platform to effectively optimize nanostructures on titanium surfaces in order to enhance their biological capability as well as their susceptibility to UV photofunctionalization.

Selective cell affinity of biomimetic micro-nano-hybrid structured TiO2 overcomes the biological dilemma of osteoblasts.

OBJECTIVE: There is a great demand for dental implant surfaces to accelerate the process of peri-implant bone generation to reduce its healing time and enable early loading. To this end, an inverse correlation between the proliferation and functional maturation (differentiation) in osteoblasts presents a challenge for the rapid generation of greater amounts of bone. For instance, osteoblasts exhibit faster differentiation but slower proliferation on micro-roughened titanium surfaces. Using a unique micro-nano-hierarchical topography of TiO(2) that mimics biomineralized matrices, this study demonstrates that this challenge can be overcome without the use of biological agents. METHODS: Titanium disks of grade 2 commercially pure titanium were prepared by machining (smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium disks were acid-etched. To create 200-nm TiO(2) nanonodules within the micropits (nanonodule-in-micropit surface), TiO(2) was sputter-deposited onto the acid-etched surface. Rat bone marrow-derived osteoblasts and NIH3T3 fibroblasts were cultured on machined smooth, micropit, and nanonodule-in-micropit surfaces. RESULTS: Despite the substantially increased surface roughness, the addition of 200-nm nanonodules to micropits increased osteoblast proliferation while enhancing their functional differentiation. In contrast, this nanonodule-in-micropit surface decreased proliferation and function in fibroblasts. SIGNIFICANCE: The data suggest the establishment of cell-selectively functionalized nano-in-micro smart titanium surfaces that involve a regulatory effect on osteoblast proliferation, abrogating the inhibitory mechanism on the micropitted surface, while enhancing their functional differentiation. Biomimetic and controllable nature of this nanonodules-in-micropits surface may offer a novel micro-to-nanoscale hierarchical platform to biologically optimize nanofeatures of biomaterials. Particularly, this micro-nano-hybrid surface may be an effective approach to improve current dental implant surfaces for accelerated bone integration.

N-acetyl cysteine prevents polymethyl methacrylate bone cement extract-induced cell death and functional suppression of rat primary osteoblasts.

This study examines the cytotoxicity of bone cement extract to osteoblasts and the potential detoxification and restoration of osteoblastic function by an antioxidant amino acid, N-acetyl cysteine (NAC). The osteoblastic cells derived from rat femurs were cultured with extract from polymethyl methacrylate (PMMA)-based bone cement. The calcein and ethidium homodimer staining of the cells after 24-h incubation showed that 23.0% of the cells were dead in the culture with bone cement extract, while the addition of 5 mM NAC into the culture reduced the percentage to 4.3%. Annexin V and propidium iodide-based flow cytometric analysis also revealed that the apoptotic cells present at 15.8% in the culture with bone cement extract was reduced to 2.4% in the culture cotreated with bone cement extract and NAC. Severely suppressed alkaline phosphatase activity and matrix mineralization in the culture with bone cement extract (reduced to 10% and 5%, respectively, compared with the control culture) were restored to a normal level when treated with 5 mM NAC. The bone cement extract-induced, downregulated expression of osteoblastic genes, such as alkaline phosphatase, collagen I, and osteocalcin, was also restored to the baseline level by cotreatment with NAC. The data indicated that the addition of NAC into acrylic bone cement extract remarkably ameliorated the cytotoxicity to osteoblasts and restored their phenotype and function to a biologically significant degree, suggesting the potential usefulness of NAC in developing more biocompatible acrylic bone cement.