Effects of Irradiation by Carbon Dioxide Laser Equipped With a Water Spray Function on Bone Formation in Rat Tibiae.

BACKGROUND/AIM: Irradiation of tissue with carbon dioxide (CO2) laser shows a characteristic thermal effect that causes vaporization of tissue in the target region. However, the thermal effect in places other than the target region induces tissue damage. Two methods are used: high reactive-level laser therapy (HLLT), aimed at surgical treatment, and low reactive-level laser therapy (LLLT), aimed at cell and tissue activation. In both, vaporization of tissue is induced by thermal damage. A water spray function may ameliorate thermal damage from CO2 laser irradiation. In this study, we irradiated CO2 laser on rat tibiae with or without a water spray function and examined the effects of this technique on bone metabolism. MATERIALS AND METHODS: Bone defects were created in rat tibiae by dental bur in a Bur group and by laser in laser irradiation groups with (Spray group) and without (Air group) water spray function. At 1 week postoperatively, histological analyses of tibiae were performed using hematoxylin and eosin staining, immunohistochemical staining (IHC) with anti-sclerostin antibody, and 3-dimensional (3D) observation using micro-computed tomography. RESULTS: Histological findings and 3D observation confirmed induction of new bone formation following laser irradiation in both the Air and Spray groups. No bone formation was seen in the Bur group. IHC revealed that the activity of osteocytes in the region of irradiated cortical bone was markedly impaired in the Air group, but osteocyte impairment was ameliorated in the Spray group and absent in the Bur group. CONCLUSION: The water spray function appears effective in reducing thermal damage to tissues irradiated by CO2 laser. CO2 lasers with water spray function may be useful in bone regeneration therapy.

Diversity of cortical bone morphology in anuran amphibians.

The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.

Positive and Negative Regulators of Sclerostin Expression.

Sclerostin is secreted from osteocytes, binds to the Wnt co-receptor Lrp5/6, and affects the interaction between Wnt ligands and Lrp5/6, which inhibits Wnt/ß-catenin signals and suppresses bone formation. Sclerostin plays an important role in the preservation of bone mass by functioning as a negative regulator of bone formation. A sclerostin deficiency causes sclerosteosis, which is characterized by an excess bone mass with enhanced bone formation in humans and mice. The expression of sclerostin is positively and negatively regulated by many factors, which also govern bone metabolism. Positive and negative regulators of sclerostin expression and their effects are introduced and discussed herein based on recent and previous findings, including our research.

Chemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities.

INTRODUCTION: Bone metabolism imbalances cause bone metabolism diseases, like osteoporosis, through aging. Although some chemokines are known to be involved in bone mass regulation, many have not been investigated. Thus, the present study aimed to investigate the role of chemokine ligand 28 (CCL28) on bone metabolism. MATERIALS AND METHODS: To investigate the role of CCL28 on bone metabolism, 10-week-old male wild-type and Ccl28 knockout (Ccl28 KO) mice were analyzed. Microcomputed tomography analysis and bone tissue morphometry were used to investigate the effect of Ccl28 deficiency on the bone. CCL28 localization in bone tissue was assumed by immunohistochemistry. Osteoblast and osteoclast markers were evaluated by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. Finally, in vitro experiments using MC3T3-E1 and bone marrow macrophages revealed the direct effect of CCL28 on osteoblast and osteoclast. RESULTS: This study showed that Ccl28 deficiency significantly increased bone mass and the number of mature osteoblasts. Immunoreactivity for CCL28 was observed in osteoblasts and osteoclasts on bone tissue. Additionally, Ccl28 deficiency promoted osteoblast and osteoclast maturation. Moreover, CCL28 treatment decreased osteoblast and osteoclast activities but did not affect differentiation. CONCLUSION: In summary, this study indicated that CCL28 is one of the negative regulators of bone mass by suppressing osteoblast and osteoclast activities. These results provide important insights into bone immunology and the selection of new osteoporosis treatments.

A human epidermal growth factor receptor 3/heregulin interaction inhibitor aptamer discovered using SELEX.

The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (KD) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications.

In vitro display evolution of the PURE system-expressed TNFα-binding unnatural cyclic peptide containing an N-methyl-d-amino acid.

Tumor necrosis factor-alpha (TNFα) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNFα from mRNA-displayed peptide library prepared with Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) and cyclized by N-chloroacetyl-N-methyl-d-phenylalanine incorporated by genetic code expansion (sense suppression). We identified a novel TNFα-binding thioether-cyclized peptide that contains an N-methyl-d-phenylalanine. Since cyclic structure and presence of an N-methyl-d-amino acid can increase proteolytic stability, the TNFα binding peptide has potential to be used for therapeutic, research and diagnostic applications.

Figure-eight thermal hysteresis of aminomethylenehelicene oligomers with terminal C16 alkyl groups during hetero-double-helix formation.

1 : 1 mixtures of aminomethylenehelicene (P)-tetramer and (M)-pentamer with terminal C16 alkyl groups in fluorobenzene showed structural changes between hetero-double-helices B and C and random-coils 2A. Figure-eight thermal hysteresis appeared when the solution was cooled and heated at a constant rate and involved the crossing of cooling and heating curves in Δε/temperature profiles. This unusual thermal hysteresis emerged in the intermediate state between counterclockwise and clockwise thermal hystereses. This phenomenon arose from the competition between self-catalytic reactions to form B and C from 2A. Significant effects of terminal C16 alkyl groups on the thermodynamic and kinetic phenomena are also described.