Effect of Er: YAG Laser Irradiation on Bone Metabolism-Related Factors Using Cultured Human Osteoblasts.

Introduction: A variety of laser treatments have been applied in numerous medical fields. In dentistry, laser treatments are used for caries, root canals, and periodontal disease, as well as surgical resection. Numerous reports have recently been published on the use of lasers for bone regeneration. If laser irradiation is found to promote the activation of bone metabolism, it might also be effective for periodontal treatment, peri-implantitis, and bone regeneration. Therefore, the present in vitro study aimed to elucidate the mechanisms underlying the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on the bone using osteoblast-like cells. Methods: Osteoblast-like Saos 2 cells (5.0×104 cells) were seeded in 24-well plates. 24 hours after being seeded, the cells were subjected to 0.3 W, 0.6 W, and 2.0 W Er: YAG laser irradiation and then allowed to recover for 48 hours. The expression levels of bone metabolism-related factors alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteoprotegerin (OPG) were then evaluated using reverse transcription-quantitative polymerase chain reaction and western blot analyses. Results: Saos 2 cells subjected to Er: YAG laser irradiation at 0.3 W, 0.6 W, and 2.0 W showed normal growth. When the Er: YAG laser irradiation and control groups were compared after 48 hours, increases were observed in ALP, BSP, and OPG gene and protein expression in the 2.0 W group. Similar results were obtained in the western blot analysis. Conclusion: These findings suggest that the Er: YAG laser irradiation of osteoblast-like cells is effective for activating bone metabolism factors.

Fabrication of Gentamicin-Loaded Hydroxyapatite/Collagen Bone-Like Nanocomposite for Anti-Infection Bone Void Fillers.

A gentamicin-loaded hydroxyapatite/collagen bone-like nanocomposite (GNT-HAp/Col) was fabricated and evaluated for its absorption-desorption properties, antibacterial efficacy, and cytotoxicity. The hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) powder was mixed with gentamicin sulfate (GNT) in phosphate-buffered saline (PBS) at room temperature. After 6 h mixing, the GNT adsorption in all conditions reached plateau by Langmuir's isotherm, and maximum GNT adsorption amount was 34 ± 7 µg in 250 µg/mL GNT solution. Saturated GNT-loaded HAp/Col powder of 100 mg was soaked in 10 mL of PBS at 37 °C and released all GNT in 3 days. A shaking culture method for a GNT extraction from the GNT-HAp/Col and an inhibition zone assay for the GNT-HAp/Col compact showed antibacterial efficacy to Escherichia coli (E. coli) at least for 2 days. From the release profile of the GNT from the GNT-HAp/Col powder, antibacterial efficacy would affect E. coli at least for 3 days. Further, no cytotoxicities were observed on MG-63 cells. Thus, the GNT-HAp/Col is a good candidate of bioresorbable anti-infection bone void fillers by prevention initial infections, which is the primary cause of implant-associated infection even for rapid bioresorbable materials.

Enhanced bone formation onto the bone surface using a hydroxyapatite/collagen bone-like nanocomposite.

The process of bone formation onto the bone surface using a hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) was investigated. Immersion tests were performed to evaluate the impact of pH on the degradation of the specimens in an aqueous environment. The specimens were soaked in aqueous solutions of pH 4.0, 5.0, and 7.0. Using standardized images, the top-view areas of the specimens were measured. Animal experiments were performed to investigate the bone formation process onto the bone surface. The specimens were placed under the rat calvarial periosteum, and µCT image analysis and histological observation were performed on samples harvested on postoperative Days 3, 5, and 7. In all experiments, ß-tricalciumphosphate (ß-TCP) was adopted as the control. HAp/Col turned to gel in acidic environments below pH 5.0. In contrast to the ß-TCP, the HAp/Col specimens placed under the periosteum expanded and attained a hollow structure with a gel-filled center, accompanied by larger volume of new bone and appearance of TRAP-positive multinucleated cells on postoperative Day 5. Therefore, HAp/Col can enhance bone formation onto the bone surface via induction of TRAP-positive multinucleated cells, and may have clinical applications.

Preparation of flexible bone tissue scaffold utilizing sea urchin test and collagen.

Gonads of sea urchin are consumed in Japan and some countries as food and most parts including its tests are discarded as marine wastes. Therefore, utilization of them as functional materials would reduce the waste as well as encourage Japanese fishery. In this study, magnesium containing calcite granules collected from sea urchin tests were hydrothermally phosphatized and the obtained granules were identified as approximately 82% in mass of magnesium containing ß-tricalcium phosphate and 18% in mass of nonstoichiometric hydroxyapatite, i.e., a biphasic calcium phosphate, maintaining the original porous network. Shape-controlled scaffolds were fabricated with the obtained biphasic calcium phosphate granules and collagen. The scaffolds showed good open porosity (83.84%) and adequate mechanical properties for handling during cell culture and subsequent operations. The MG-63 cells showed higher proliferation and osteogenic differentiation in comparison to a control material, the collagen sponge with the same size. Furthermore, cell viability assay proved that the scaffolds were not cytotoxic. These results suggest that scaffold prepared using sea urchin test derived calcium phosphate and collagen could be a potential candidate of bone void fillers for non-load bearing defects in bone reconstruction as well as scaffolds for bone tissue engineering.

Substitutions at residues 300 and 389 of the VP2 capsid protein serve as the minimal determinant of attenuation for canine parvovirus vaccine strain 9985-46.

Identifying molecular determinants of virulence attenuation in live attenuated canine parvovirus (CPV) vaccines is important for assuring their safety. To this end, we identified mutations in the attenuated CPV 9985-46 vaccine strain that arose during serial passage in Crandell-Rees feline kidney cells by comparison with the wild-type counterpart, as well as minimal determinants of the loss of virulence. Four amino acid substitutions (N93K, G300V, T389N and V562L) in VP2 of strain 9985-46 significantly restricted infection in canine A72 cells. Using an infectious molecular clone system, we constructed isogenic CPVs of the parental virulent 9985 strain carrying single or double mutations. We observed that only a single amino acid substitution in VP2, G300V or T389N, attenuated the virulent parental virus. Combinations of these mutations further attenuated CPV to a level comparable to that of 9985-46. Strains with G300V/T389N substitutions did not induce clinical symptoms in experimentally infected pups, and their ability to infect canine cells was highly restricted. We found that another G300V/V562L double mutation decreased affinity of the virus for canine cells, although its pathogenicity to dogs was maintained. These results indicate that mutation of residue 300, which plays a critical role in host tropism, is not sufficient for viral attenuation in vivo, and that attenuation of 9985-46 strain is defined by at least two mutations in residues 300 and 389 of the VP2 capsid protein. This finding is relevant for quality control of the vaccine and provides insight into the rational design of second-generation live attenuated vaccine candidates.