Kosuke Hamajima

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.

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

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.

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.

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.

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-μCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-μCT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.