Impact of nano-scale trabecula size on osteoblastic behavior and function in a meso-nano hybrid rough biomimetic zirconia model.

PURPOSE: A novel implant model consisting of meso-scale cactus-inspired spikes and nano-scale bone-inspired trabeculae was recently developed to optimize meso-scale roughness on zirconia. In this model, the meso-spike dimension had a significant impact on osteoblast function. To explore how different nano-textures impact this model, here we examined the effect of different nano-trabecula sizes on osteoblast function while maintaining the same meso-spike conformation. METHODS: Zirconia disks with meso-nano hybrid surfaces were created by laser etching. The meso-spikes were fixed to 40 µm high, whereas the nano-texture was etched as large and small trabeculae of average Feret diameter 237.0 and 134.1 nm, respectively. A polished surface was also prepared. Rat bone marrow-derived and human mesenchymal stromal cell-induced osteoblasts were cultured on these disks. RESULTS: Hybrid rough surfaces, regardless of nano-trabecula dimension, robustly promoted the osteoblastic differentiation of both rat and human osteoblasts compared to those on polished surfaces. Hybrid surfaces with small nano-trabeculae further enhanced osteoblastic differentiation compared with large nano-trabeculae. However, the difference in osteoblastic differentiation between small and large nano-trabeculae was much smaller than the difference between the polished and hybrid rough surfaces. The nano-trabecula size did not influence osteoblast attachment and proliferation, or protein adsorption. Both hybrid surfaces were hydro-repellent. The atomic percentage of surface carbon was lower on the hybrid surface with small nano-trabeculae. CONCLUSIONS: Small nano-trabeculae promoted osteoblastic differentiation more than large nano-trabeculae when combined with meso-scale spikes. However, the biological impact of different nano-trabeculae was relatively small compared with that of different dimensions of meso-spikes.

Novel Tuning of PMMA Orthopedic Bone Cement Using TBB Initiator: Effect of Bone Cement Extracts on Bioactivity of Osteoblasts and Osteoclasts.

Bone cement containing benzoyl peroxide (BPO) as a polymerization initiator are commonly used to fix orthopedic metal implants. However, toxic complications caused by bone cement are a clinically significant problem. Poly (methyl methacrylate) tri-n-butylborane (PMMA-TBB), a newly developed material containing TBB as a polymerization initiator, was found to be more biocompatible than conventional PMMA-BPO bone cements due to reduced free radical generation during polymerization. However, free radicals might not be the only determinant of cytotoxicity. Here, we evaluated the response and functional phenotypes of cells exposed to extracts derived from different bone cements. Bone cement extracts were prepared from two commercial PMMA-BPO cements and an experimental PMMA-TBB. Rat bone marrow-derived osteoblasts and osteoclasts were cultured in a medium supplemented with bone cement extracts. More osteoblasts survived and attached to the culture dish with PMMA-TBB extract than in the culture with PMMA-BPO extracts. Osteoblast proliferation and differentiation were higher in the culture with PMMA-TBB extract. The number of TRAP-positive multinucleated cells was significantly lower in the culture with PMMA-TBB extract. There was no difference in osteoclast-related gene expression in response to different bone cement extracts. In conclusion, PMMA-TBB extract was less toxic to osteoblasts than PMMA-BPO extracts. Although extracts from the different cement types did not affect osteoclast function, PMMA-TBB extract seemed to reduce osteoclastogenesis, a possible further advantage of PMMA-TBB cement. These implied that the reduced radical generation during polymerization is not the only determinant for the improved biocompatibility of PMMA-TBB and that the post-polymerization chemical elution may also be important.

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.

Prolonged Post-Polymerization Biocompatibility of Polymethylmethacrylate-Tri-n-Butylborane (PMMA-TBB) Bone Cement.

Polymethylmethacrylate (PMMA)-based acrylic bone cement is commonly used to fix bone and metallic implants in orthopedic procedures. The polymerization initiator tri-n-butylborane (TBB) has been reported to significantly reduce the cytotoxicity of PMMA-based bone cement compared to benzoyl peroxide (BPO). However, it is unknown whether this benefit is temporary or long-lasting, which is important to establish given that bone cement is expected to remain in situ permanently. Here, we compared the biocompatibility of PMMA-TBB and PMMA-BPO bone cements over several days. Rat femur-derived osteoblasts were seeded onto two commercially-available PMMA-BPO bone cements and experimental PMMA-TBB polymerized for one day, three days, or seven days. Significantly more cells attached to PMMA-TBB bone cement during the initial stages of culture than on both PMMA-BPO cements, regardless of the age of the materials. Proliferative activity and differentiation markers including alkaline phosphatase production, calcium deposition, and osteogenic gene expression were consistently and considerably higher in cells grown on PMMA-TBB than on PMMA-BPO, regardless of cement age. Although osteoblastic phenotypes were more favorable on older specimens for all three cement types, biocompatibility increased between three-day-old and seven-day-old PMMA-BPO specimens, and between one-day-old and three-day-old PMMA-TBB specimens. PMMA-BPO materials produced more free radicals than PMMA-TBB regardless of the age of the material. These data suggest that PMMA-TBB maintains superior biocompatibility over PMMA-BPO bone cements over prolonged periods of at least seven days post-polymerization. This superior biocompatibility can be ascribed to both low baseline cytotoxicity and a further rapid reduction in cytotoxicity, representing a new biological advantage of PMMA-TBB as a novel bone cement material.

The Effect of TBB, as an Initiator, on the Biological Compatibility of PMMA/MMA Bone Cement.

Acrylic bone cement is widely used in orthopedic surgery for treating various conditions of the bone and joints. Bone cement consists of methyl methacrylate (MMA), polymethyl methacrylate (PMMA), and benzoyl peroxide (BPO), functioning as a liquid monomer, solid phase, and polymerization initiator, respectively. However, cell and tissue toxicity caused by bone cement has been a concern. This study aimed to determine the effect of tri-n-butyl borane (TBB) as an initiator on the biocompatibility of bone cement. Rat spine bone marrow-derived osteoblasts were cultured on two commercially available PMMA-BPO bone cements and a PMMA-TBB experimental material. After a 24-h incubation, more cells survived on PMMA-TBB than on PMMA-BPO. Cytomorphometry showed that the area of cell spread was greater on PMMA-TBB than on PMMA-BPO. Analysis of alkaline phosphatase activity, gene expression, and matrix mineralization showed that the osteoblastic differentiation was substantially advanced on the PMMA-TBB. Electron spin resonance (ESR) spectroscopy revealed that polymerization radical production within the PMMA-TBB was 1/15-1/20 of that within the PMMA-BPO. Thus, the use of TBB as an initiator, improved the biocompatibility and physicochemical properties of the PMMA-based material.

Redox injectable gel protects osteoblastic function against oxidative stress and suppresses alveolar bone loss in a rat peri-implantitis model.

Dental implant surgery is a routine treatment in clinical dentistry. However, implant surgery is associated with an increased risk of bacterially induced peri-implantitis and the production of reactive oxygen species (ROS), with no established treatment. We recently designed a new redox injectable gel (RIG) containing nitroxide radicals for the treatment of peri-implantitis. Here, we investigated the antioxidative effect of RIG as a preventive therapy for ROS-associated peri-implantitis in a rat model of alveolar bone resorption and in vitro. In each rat, the maxillary first molar tooth was replaced with a screw-type implant, and rats were assigned to one of four groups: an implant alone, an implant with infection, implant with infection and treatment with nRIG (a non-nitroxide radical-containing injectable hydrogel) or RIG. We confirmed the long-term retention of RIG in the peri-implant region and found that RIG significantly protected the alveolar bone volume and decreased lipid peroxidation. In culture, we found that RIG restored osteoblast proliferation and differentiation in the presence of hydrogen peroxide (H2O2)-induced oxidative stress. Moreover, using a malondialdehyde assay of lipid peroxidation, we found that RIG suppressed oxidative stress in H2O2-treated rat osteoblasts. Overall, RIG is anticipated as a prophylactic treatment for peri-implantitis and may help preserve oral function. Statement of Significance 1. Implant surgery is associated with an increased risk of bacterially induced peri-implantitis and the production of reactive oxygen species (ROS). We designed a novel redox injectable gel (RIG) containing nitroxide radicals for the treatment of peri-implantitis. In this study, we investigated the antioxidative effect of RIG as a preventive therapy for ROS-associated peri-implantitis in a rat model and in vitro. 2. We showed that treatment with RIG reduces oxidative damage in a rat peri-implantitis model, protecting against bone resorption and a loss of bone density. We showed that RIG inhibits H2O2-mediated decreases in proliferation, osteoblast differentiation, and mineralization, and also against lipid peroxidation in vitro. Our results indicate that RIG has an antioxidative effect of peri-implantitis.

Computational Fluid Simulation of Fibrinogen around Dental Implant Surfaces.

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.

Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits.

In the present study, we compared bone regeneration ability in sinus floor elevation between a tissue engineering method using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP), and a promising new method using particulate cancellous bone and marrow (PCBM) and PRP. Bilateral sinus floor elevation procedures were performed in 18 adult Japanese white rabbits. MSCs/PRP or PCBM/PRP complexes were grafted to each maxillary sinus in the same rabbits. The MSCs were isolated from rabbit iliac crest marrow, and PRP was obtained from peripheral blood. PCBM were collected from the rabbit iliac crest and mixed with PRP. The animals were sacrificed at 2, 4, and 8 weeks after transplantation, and the bone formation ability of each implant was evaluated histologically and histometrically. According to the histological observations, both sites (MSCs/PRP and PCBM/PRP) showed well newly formed bone and neovascularization at 2 and 4 weeks. However, at 8 weeks, the lamellar bone was observed to be occupied by fatty marrow in large areas in both sites. There was no significant difference in bone volume or augmented height between MSCs/PRP and PCBM/PRP groups each week, but there were significant differences in bone volume and augmented height between 2 and 8 weeks in PCBM/PRP or MSCs/PRP groups and in bone volume between 4 and 8 weeks in the PCBM/PRP group (P<0.05). These results suggest that the MSCs/PRP complex may well be used for bone regeneration in sinus floor elevation, compared with the PCBM/PRP complex.

Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneous implant placement.

This clinical study was undertaken to evaluate the use of tissue-engineered bone, mesenchymal stem cells, platelet-rich plasma, and beta-tricalcium phosphate as grafting materials for maxillary sinus floor augmentation or onlay plasty with simultaneous implant placement in six patients with 3- to 5-mm alveolar crestal bone height. All 20 implants were clinically stable at second-stage surgery and 12 months postloading. A mean increase in mineralized tissue height of 7.3+/-4.6 mm was evident when comparing the pre- and postsurgical radiographs. Injectable tissue-engineered bone provided stable and predictable results in terms of implant success.

Bone regeneration following injection of mesenchymal stem cells and fibrin glue with a biodegradable scaffold.

AIM: The purpose of this study was to determine whether a combination of fibrin glue, beta-tricalcium phosphate as a biodegradable (beta-TCP) and mesenchymal stem cells would provide three-dimensional templates for bone growth resulting in new bone formation at heterotopic sites in the rat with plasticity. MATERIAL AND METHODS: Growing stem cells and developing matrices, explanted from the rat femur, were fragmented and mixed with fibrin glue in a syringe. The cells/beta-TCP fibrin glue admixtures were injected into the subcutaneous space on the dorsum of the rat. RESULTS: Eight weeks after implantation, gross morphology revealed a pearly opalescence and firm consistency. Histological inspections showed newly formed bone structures in all admixtures, but none in the control groups when only fibrin glue and beta-TCP were injected. Osteopontin, a protein important in bone development, was identified by using antibodies in all cells/beta-TCP fibrin glue admixtures. CONCLUSION: Mesenchymal stem cells/beta-TCP fibrin glue admixtures can result in successful bone formation. This technique holds the promise of a minimally invasive means of generating autogenous bone to correct or reconstruct bony defects.