Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola.

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process. KEY POINTS: • An easy and detailed component analysis method for sophorolipids (SLs) is introduced. • Multiple SL derivatives were identified different from known SLs. • A novel hydrophobic acidic SL was isolated and characterized.

Synergistic Enhancement of Protein Recruitment and Retention via Implant Surface Microtopography and Superhydrophilicity in a Computational Fluid Dynamics Model.

The exact mechanisms by which implant surface properties govern osseointegration are incompletely understood. To gain insights into this process, we examined alterations in protein and blood recruitment around screw implants with different surface topographies and wettability using a computational fluid dynamics (CFD) model. Compared with a smooth surface, a microrough implant surface reduced protein infiltration from the outer zone to the implant thread and interface zones by over two-fold. However, the microrough implant surface slowed blood flow in the interface zone by four-fold. As a result, compared with the smooth surface, the microrough surface doubled the protein recruitment/retention index, defined as the mass of proteins present in the area per unit time. Converting implant surfaces from hydrophobic to superhydrophilic increased the mass of protein infiltration 2-3 times and slowed down blood flow by up to two-fold in the implant vicinity for both smooth and microrough surfaces. The protein recruitment/retention index was highest at the implant interface when the implant surface was superhydrophilic and microrough. Thus, this study demonstrates distinct control of the mass and speed of protein and blood flow through implant surface topography, wettability, and their combination, significantly altering the efficiency of protein recruitment. Although microrough surfaces showed both positive and negative impacts on protein recruitment over smooth surfaces, superhydrophilicity was consistently positive regardless of surface topography.

Accuracy and influencing factors of maxillary and mandibular repositioning using pre-bent locking plates: a prospective study.

In-house repositioning methods based on three-dimensional (3D)-printing technology and the use of pre-bent plates has been gaining popularity in orthognathic surgery. However, there remains room for further improvement in methods and investigations on clinical factors that affect accuracy. This single-centre, prospective study included 34 patients and aimed to evaluate the accuracy and factors influencing maxillary and mandibular repositioning using pre-bent locking plates. The plates were manually pre-bent on the 3D-printed models of the planned position, and their hole positions were scanned and reproduced intraoperatively with osteotomy guides. The accuracy of repositioning and plate-hole positioning was calculated in three axes with the set landmarks. The following clinical factors that affect repositioning accuracy were also verified: deviation of the plate-hole positioning, amount of planned movement, and amount of simulated bony interference. The median deviations of the repositioning and hole positioning between the preoperative plan and postoperative results were 0.26 mm and 0.23 mm, respectively, in the maxilla, and 0.69 mm and 0.36 mm, respectively, in the mandible, suggesting that the method was highly accurate, and the repositioning concept based on the plate hole and form matching was more effective in the maxilla. Results of the correlation test suggest that large amounts of bony interference and plate-hole positioning errors in the up/down direction could reduce mandibular repositioning accuracy.

The Effects of a Biomimetic Hybrid Meso- and Nano-Scale Surface Topography on Blood and Protein Recruitment in a Computational Fluid Dynamics Implant Model.

The mechanisms underlying bone-implant integration, or osseointegration, are still incompletely understood, in particular how blood and proteins are recruited to implant surfaces. The objective of this study was to visualize and quantify the flow of blood and the model protein fibrinogen using a computational fluid dynamics (CFD) implant model. Implants with screws were designed with three different surface topographies: (1) amorphous, (2) nano-trabecular, and (3) hybrid meso-spikes and nano-trabeculae. The implant with nano-topography recruited more blood and fibrinogen to the implant interface than the amorphous implant. Implants with hybrid topography further increased recruitment, with particularly efficient recruitment from the thread area to the interface. Blood movement significantly slowed at the implant interface compared with the thread area for all implants. The blood velocity at the interface was 3- and 4-fold lower for the hybrid topography compared with the nano-topography and amorphous surfaces, respectively. Thus, this study for the first time provides insights into how different implant surfaces regulate blood dynamics and the potential advantages of surface texturization in blood and protein recruitment and retention. In particular, co-texturization with a hybrid meso- and nano-topography created the most favorable microenvironment. The established CFD model is simple, low-cost, and expected to be useful for a wide range of studies designing and optimizing implants at the macro and micro levels.

A Novel Orthognathic Surgery With a Half-Millimeter Accuracy for the Maxillary Positioning Using Prebent Plates and Computer-Aided Design and Manufacturing Osteotomy Guide.

This retrospective study aimed to assess the accuracy of prebent plates and computer-aided design and manufacturing osteotomy guide for orthognathic surgery. The prebent plates correspondent to the planning model were scanned with a 3-dimensional printed model for guide design and used for fixation. Forty-two patients who underwent bimaxillary orthognathic surgery using computer-aided design and manufacturing intermediate splint with the guide (guided group: 20 patients) or with conventional fixation under straight locking miniplates (SLMs) technique (SLM group: 20 patients) were analyzed. A deviation of the maxilla between the planned and postoperative positions was evaluated using computed tomography, which was taken 2 weeks before and 4 days after the surgery. The surgery time and the infraorbital nerve paranesthesia were also evaluated. The mean deviations in the mediolateral ( x ), anteroposterior ( y ), and vertical directions ( z ) were 0.25, 0.50, and 0.37 mm, respectively, in the guided group, while that in the SLM group were 0.57, 0.52, and 0.82 mm, respectively. There were significant differences in x and z coordinates ( P <0.001). No significant difference in the surgery duration and paranesthesia was seen, suggesting the present method offers a half-millimeter accuracy for the maxillary repositioning without increasing the risk of extending surgery duration and nerve complication.

Optimization of blood and protein flow around superhydrophilic implant surfaces by promoting contact hemodynamics.

PURPOSE: We examined blood and protein dynamics potentially influenced by implant threads and hydrophilic/hydrophobic states of implant surfaces. METHODS: A computational fluid dynamics model was created for a screw-shaped implant with a water contact angle of 70° (hydrophobic surface) and 0° (superhydrophilic surface). Movements and density of blood and fibrinogen as a representative wound healing protein were visualized and quantified during constant blood inflow. RESULTS: Blood plasma did not occupy 40-50% of the implant interface or the inside of threads around hydrophobic implants, whereas such blood voids were nearly completely eliminated around superhydrophilic implants. Whole blood field vectors were disorganized and random within hydrophobic threads but formed vortex nodes surrounded by stable blood streams along the superhydrophilic implant surface. The averaged vector within threads was away from the implant surface for the hydrophobic implant and towards the implant surface for the superhydrophilic implant. Rapid and massive whole blood influx into the thread zone was only seen for the superhydrophilic implant, whereas a line of conflicting vectors formed at the entrance of the thread area of the hydrophobic implant to prevent blood influx. The fibrinogen density was up to 20-times greater at the superhydrophilic implant interface than the hydrophobic one. Fibrinogen density was higher at the interface than outside the threads only for the superhydrophilic implant. CONCLUSIONS: Implant threads and surface hydrophilicity have profound effects on vector and distribution of blood and proteins. Critically, implant threads formed significant biological voids at the interface that were negated by superhydrophilicity-induced contact hemodynamics.

Bone beads enveloped with vascular endothelial cells for bone regenerative medicine.

The transplantation of pre-vascularized bone grafts is a promising strategy to improve the efficacy of engraftment and bone regeneration. We propose a hydrogel microbead-based approach for preparing vascularized and high-density tissue grafts. Mesenchymal stem cell-encapsulated collagen microgels (2 µL), termed bone beads, were prepared through spontaneous constriction, which improved the density of the mesenchymal stem cells and collagen molecules by more than 15-fold from the initial day of culture. Constriction was attributed to cell-attractive forces and involved better osteogenic differentiation of mesenchymal stem cells than that of spheroids. This approach was scalable, and ∼2000 bone beads were prepared semi-automatically using a liquid dispenser and spinner flask. The mechanical stimuli in the spinner flask further improved the osteogenic differentiation of the mesenchymal stem cells in the bone beads compared with that in static culture. Vascular endothelial cells readily attach to and cover the surface of bone beads. The in vitro assembly of the endothelial cell-enveloped bone beads resulted in microchannel formation in the interspaces between the bone beads. Significant effects of endothelialization on in vivo bone regeneration were shown in rats with cranial bone defects. The use of endothelialized bone beads may be a scalable and robust approach for treating large bone defects. STATEMENT OF SIGNIFICANCE: A unique aspect of this study is that the hMSC-encapsulated collagen microgels were prepared through spontaneous constriction, leading to the enrichment of collagen and cell density. This constriction resulted in favorable microenvironments for the osteogenic differentiation of hMSCs, which is superior to conventional spheroid culture. The microgel beads were then enveloped with vascular endothelial cells and assembled to fabricate a tissue graft with vasculature in the interspaces among the beads. The significant effects of endothelialization on in vivo bone regeneration were clearly demonstrated in rats with cranial bone defects. We believe that microgel beads covered with vascular endothelial cells provide a promising approach for engineering better tissue grafts for bone-regenerative medicine.

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.

A 3-Dimensional Finite Element Analysis of Resected Mandibular Bone to Determine the Most Stable Implant Positions for a Fixed Prosthesis.

This study was conducted to determine the most secure implant positioning on the marginally resected mandible to support a fixed complete denture through finite element analysis. Three or 4 implants were placed at near, middle, or far positions from the resected margin in a simulation model with a symmetrical marginal defect in the mandibular symphysis. The height of the residual bone was 5, 10, or 15 mm. The 4 possible implant patterns for 3 or 4 implants were defined as (1) asymmetrically isolated position 1 to position 2, (2) asymmetrically isolated position 1 to position 3, (3) asymmetrically isolated with greater-length position 1 to position 2, and (4) 2 implants symmetrically positioned on each side of the defect. The von Mises stress in the resected and peri-implant bone with respect to the occlusal force was calculated. Initially, because the peri-implant bone stress around the isolated implant at the near position was greater than at the middle and far positions regardless of the residual bone height, the near position was excluded. Second, the von Mises stress in the resected bone region was >10 MPa when the isolated implant was at the far position, and it increased inversely depending on the bone height. However, the stress was <10 MPa when the isolated implant was placed at the middle position regardless of the bone height, and it was significantly lower compared with the far position and equivalent to the symmetrically positioned implants. Furthermore, the use of a greater-length implant reduced peri-implant bone stress, which was even lower than that of the symmetrically positioned implants. These results suggest that the asymmetrically positioned 3-implant-supported fixed denture, using a greater-length isolated implant, placed neither too close to nor too far from the resected margin, can be an effective alternative to the symmetrically positioned 4-implant-supported fixed denture.

Ultraviolet Light Treatment of Titanium Microfiber Scaffolds Enhances Osteoblast Recruitment and Osteoconductivity in a Vertical Bone Augmentation Model: 3D UV Photofunctionalization.

Vertical bone augmentation to create host bone prior to implant placement is one of the most challenging regenerative procedures. The objective of this study is to evaluate the capacity of a UV-photofunctionalized titanium microfiber scaffold to recruit osteoblasts, generate intra-scaffold bone, and integrate with host bone in a vertical augmentation model with unidirectional, limited blood supply. Scaffolds were fabricated by molding and sintering grade 1 commercially pure titanium microfibers (20 µm diameter) and treated with UVC light (200-280 nm wavelength) emitted from a low-pressure mercury lamp for 20 min immediately before experiments. The scaffolds had an even and dense fiber network with 87% porosity and 20-50 mm inter-fiber distance. Surface carbon reduced from 30% on untreated scaffold to 10% after UV treatment, which corresponded to hydro-repellent to superhydrophilic conversion. Vertical infiltration testing revealed that UV-treated scaffolds absorbed 4-, 14-, and 15-times more blood, water, and glycerol than untreated scaffolds, respectively. In vitro, four-times more osteoblasts attached to UV-treated scaffolds than untreated scaffolds three hours after seeding. On day 2, there were 70% more osteoblasts on UV-treated scaffolds. Fluorescent microscopy visualized confluent osteoblasts on UV-treated microfibers two days after seeding but sparse and separated cells on untreated microfibers. Alkaline phosphatase activity and osteocalcin gene expression were significantly greater in osteoblasts grown on UV-treated microfiber scaffolds. In an in vivo model of vertical augmentation on rat femoral cortical bone, the interfacial strength between innate cortical bone and UV-treated microfiber scaffold after two weeks of healing was double that observed between bone and untreated scaffold. Morphological and chemical analysis confirmed seamless integration of the innate cortical and regenerated bone within microfiber networks for UV-treated scaffolds. These results indicate synergy between titanium microfiber scaffolds and UV photofunctionalization to provide a novel and effective strategy for vertical bone augmentation.

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.

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.

Biomimetic Zirconia with Cactus-Inspired Meso-Scale Spikes and Nano-Trabeculae for Enhanced Bone Integration.

Biomimetic design provides novel opportunities for enhancing and functionalizing biomaterials. Here we created a zirconia surface with cactus-inspired meso-scale spikes and bone-inspired nano-scale trabecular architecture and examined its biological activity in bone generation and integration. Crisscrossing laser etching successfully engraved 60 µm wide, cactus-inspired spikes on yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) with 200-300 nm trabecular bone-inspired interwoven structures on the entire surface. The height of the spikes was varied from 20 to 80 µm for optimization. Average roughness (Sa) increased from 0.10 µm (polished smooth surface) to 18.14 µm (80 µm-high spikes), while the surface area increased by up to 4.43 times. The measured dimensions of the spikes almost perfectly correlated with their estimated dimensions (R2 = 0.998). The dimensional error of forming the architecture was 1% as a coefficient of variation. Bone marrow-derived osteoblasts were cultured on a polished surface and on meso- and nano-scale hybrid textured surfaces with different spike heights. The osteoblastic differentiation was significantly promoted on the hybrid-textured surfaces compared with the polished surface, and among them the hybrid-textured surface with 40 µm-high spikes showed unparalleled performance. In vivo bone-implant integration also peaked when the hybrid-textured surface had 40 µm-high spikes. The relationships between the spike height and measures of osteoblast differentiation and the strength of bone and implant integration were non-linear. The controllable creation of meso- and nano-scale hybrid biomimetic surfaces established in this study may provide a novel technological platform and design strategy for future development of biomaterial surfaces to improve bone integration and regeneration.