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.

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.

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.

Novel Osteogenic Behaviors around Hydrophilic and Radical-Free 4-META/MMA-TBB: Implications of an Osseointegrating Bone Cement.

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 cytotoxicity compared to PMMA-BPO and induces osteoconductivity due to uniquely created hydrophilic and radical-free interface. Further pre-clinical and clinical validations are warranted.

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

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

Disproportionate Effect of Sub-Micron Topography on Osteoconductive Capability of Titanium.

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.

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.

Biological and osseointegration capabilities of hierarchically (meso-/micro-/nano-scale) roughened zirconia.

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 with distinct hierarchical morphology and providing an effective strategy to improve and develop zirconia implants.