Effects of acidic calcium phosphate concentration on setting reaction and tissue response to β-tricalcium phosphate granular cement

Beta-tricalcium phosphate granular cement (β-TCP GC), consisting of β-TCP granules and an acidic calcium phosphate (Ca-P) solution, shows promise in the reconstruction of bone defects as it sets to form interconnected porous structures, i.e., β-TCP granules are bridged with dicalcium phosphate dihydrate (DCPD) crystals. In this study, the effects of acidic Ca-P solution concentration (0–600 mmol/L) on the setting reaction and tissue response to β-TCP GC were investigated. The β-TCP GC set upon mixing with its liquid phase, based on the formation of DCPD crystals, which bridged β-TCP granules to one another. Diametral tensile strength of the set β-TCP GC was relatively the same, at approximately 0.6 MPa, when the Ca-P concentration was 20–600 mmol/L. Due to the setting ability, reconstruction of the rat’s calvarial bone defect using β-TCP GC with 20, 200, and 600 mmol/L Ca-P solution was much easier compared to that with β-TCP granules without setting ability. Four weeks after the reconstruction, the amount of new bone was the same, approximately 17% in both β-TCP GC and β-TCP granules groups. Cellular response to β-TCP granules and β-TCP GC using the 20 mmol/L acidic Ca-P solution was almost the same. However, β-TCP GC using the 200 and 600 mmol/L acidic Ca-P solution showed a more severe inflammatory reaction. It is concluded, therefore, that β-TCP GC, using the 20 mmol/L acidic Ca-P solution, is recommended as this concentration allows surgical techniques to be performed easily and provides good mechanical strength, and the similar cellular response to β-TCP granules

Histological comparison of three apatitic bone substitutes with different carbonate contents in alveolar bone defects in a beagle mandible with simultaneous implant installation

Since bone apatite is a carbonate apatite containing carbonate in an apatitic structure, carbonate content may be one of the factors governing the osteoconductivity of apatitic bone substitutes. The aim of this study was to evaluate the effects of carbonate content on the osteoconductivity of apatitic bone substitutes using three commercially available bone substitutes for the reconstruction of alveolar bone defects of a beagle mandible with simultaneous dental implant installation. NEOBONE®, Bio-Oss®, and Cytrans® that contain 0.1 mass%, 5.5 mass%, and 12.0 mass% of carbonate, respectively, were used in this study. The amount of newly formed bone in the upper portion of the alveolar bone defect of the beagle’s mandible was 0.7%, 6.6%, and 39.4% at 4 weeks after surgery and 4.7%, 39.5% and 75.2% at 12 weeks after surgery for NEOBONE®, Bio-Oss®, and Cytrans®, respectively. The results indicate that bone-to-implant contact ratio was the largest for Cytrans®. Additionally, the continuity of the alveolar ridge was restored in the case of Cytrans®, whereas the continuity of the alveolar ridge was not sufficient when using NEOBONE® and Bio-Oss®. Both Cytrans® and Bio-Oss® that has a relatively larger carbonate content in their apatitic structure was resorbed with time. We concluded that carbonate content is one of important factors governing the osteoconductivity of apatitic bone substitutes

The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway

OK-432 is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously reported that OK-432 treatment can induce the production of high levels of IFN-γ from peripheral blood mononuclear cells (PBMCs). Moreover, the IFN-γ production from PBMCs by OK-432 is impaired by conditioned media (CM) from oral cancer cells. To determine the inhibitory mechanism of IFN-γ production by CM, the genes involved in IFN-γ production was retrieved by cDNA microarray analysis. We found that CD40 played a key role in IFN-γ production via IL-12 production. Although the expression levels of CD40 were upregulated by OK-432 treatment in PBMCs, CM inhibited OK-432-induced CD40 expression. These findings suggest that uncertain soluble factor(s) in CM may suppress IFN-γ production via the CD40/CD40L–IL-12 axis in PBMCs.(1) Background: OK-432 is a penicillin-killed, lyophilized formulation of a low-toxicity strain (Su) of Streptococcus pyogenes (Group A). It is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously showed that (i) OK-432 treatment induces a high amount of IFN-γ production from peripheral blood mononuclear cells (PBMCs), and (ii) conditioned medium (CM) from oral cancer cells suppresses both the IFN-γ production and cytotoxic activity of PBMCs driven by OK-432. The aim of this study was to determine the inhibitory mechanism of OK-432-induced IFN-γ production from PBMCs by CM. (2) Methods: We performed cDNA microarray analysis, quantitative RT-PCR, and ELISA to reveal the inhibitory mechanism of CM. (3) Results: We found that CD40 plays a key role in IFN-γ production via IL-12 production. Although OK-432 treatment upregulated the expression levels of the IL-12p40, p35, and CD40 genes, CM from oral cancer cells downregulate these genes. The amount of IFN-γ production by OK-432 treatment was decreased by an anti-CD40 neutralizing antibody. (4) Conclusions: Our study suggests that uncertain soluble factor(s) produced from oral cancer cells may inhibit IFN-γ production from PBMCs via suppressing the CD40/CD40L–IL-12 axis

Current status and future development of carbonate apatite as a bone substitute

A main inorganic component of human bone is not hydroxyapatite but carbonate apatite (CQ3Ap). Hydroxyapatite is not resorbed in the body but C03Ap can be resorbed and replaced with bone. We have succeeded in fabricating low crystalline CQ3Ap without sintering by dissolution-precipitation reaction using calcium hydroxide as a precursor. C03Ap showed excellent biocompatibility and faster bone formation compared to other bone substitutes (anorganic bovine bone and hydroxyapatite) in rabbit femur and dog mandible. Clinical study on effectiveness and safety of C03Ap granules in sinus floor augmentation was successfully concluded in 2016. CQ3Ap granules were approved by Japanese government in 2017, and marketed in 2018 as Cytrans® Granules. This comprehensive review explains the clinical cases of sinus lift and alveolar ridge augmentation of Cytrans® and its recommended clinical usage. Furthermore, we have succeeded in developing porous C03Ap and showed it was useful for reconstruction of mandibular bone defect in rabbit model and also aim to use it as a scaffold for bone regenerative medicine.ヒトの骨の無機主成分はハイドロキシアパタイトではなく，炭酸アパタイト (CO3Ap) である．ハイドロキシアパタイトは体内で吸収されないが，CO3Apは吸収されて骨と置換する．われわれは水酸化カルシウムを前駆物質として，焼結操作を用いずに溶解析出反応によって低結晶性の炭酸アパタイトを人工合成することに成功した．炭酸アパタイ卜顆粒は，ウサギ大腿骨とイヌ顎骨における実験で，他の骨補填材よりも骨が速く形成すること，優れた生体親和性を示すことを明らかにした．2016年に上顎洞底挙上術での臨床治験を成功裏に終え，2017年に炭酸アパタイト顆粒は薬事承認され，2018年からサイトランス® グラニュールとして市販された本総説では，サイトランス® による上顎洞底挙上症例と歯槽堤造成術症例を紹介すると共に，その臨床的推奨使用法を説明した．さらに，著者らは多孔質の炭酸アパタイトの作製に成功し，炭酸アパタイト多孔体がウサギの下顎骨欠損モデルにおいて骨再建に有用であることを示した．現在，炭酸アパタイト多孔体の骨再生医療用スキャフォールドヘの応用を目指している

マイクロファイバーを用いた多孔質炭酸アパタイト顆粒の開発とウサギ頭蓋骨における組織学的評価

Carbonate apatite (CO3Ap) granules are known to show good osteoconductivity and replaced to new bone. On the other hand, it is well known that a porous structure allows bone tissue to penetrate its pores, and the optimal pore size for bone ingrowth is dependent on the composition and structure of the scaffold material. Therefore, the aim of this study was to fabricate various porous CO3Ap granules through a two-step dissolution-precipitation reaction using CaSO4 as a precursor and 30-, 50-, 120-, and 205-μm diameter microfibers as porogen and to find the optimal pore size of CO3Ap. Porous CO3Ap granules were successfully fabricated with pore size 8.2-18.7% smaller than the size of the original fiber porogen. Two weeks after the reconstruction of rabbit calvarial bone defects using porous CO3Ap granules, the largest amount of mature bone was seen to be formed inside the pores of CO3Ap (120) [porous CO3Ap granules made using 120-μm microfiber] followed by CO3Ap (50) and CO3Ap (30). At 4 and 8 weeks, no statistically significant difference was observed based on the pore size, even though largest amount of mature bone was formed in case of CO3Ap (120). It is concluded, therefore, that the optimal pore size of the CO3Ap is that of CO3Ap (120), which is 85 μm

Reconstruction of rabbit mandibular bone defects using carbonate apatite honeycomb blocks with an interconnected porous structure

Carbonate apatite (CO3Ap) granules are useful as a bone substitute because they can be remodeled to new natural bone in a manner that conforms to the bone remodeling process. However, reconstructing large bone defects using CO3Ap granules is difficult because of their granular shape. Therefore, we fabricated CO3Ap honeycomb blocks (HCBs) with continuous unidirectional pores. We aimed to elucidate the tissue response and availability of CO3Ap HCBs in the reconstruction of rabbit mandibular bone defects after marginal mandibulectomy. The percentages of the remaining CO3Ap area and calcified bone area (newly formed bone) were estimated from the histological images. CO3Ap area was 49.1 ± 4.9%, 30.3 ± 3.5%, and 25.5 ± 8.8%, whereas newly formed bone area was 3.0 ± 0.6%, 24.3 ± 3.3%, and 34.7 ± 4.8% at 4, 8, and 12 weeks, respectively, after implantation. Thus, CO3Ap HCBs were gradually resorbed and replaced by new bone. The newly formed bone penetrated most of the pores in the CO3Ap HCBs at 12 weeks after implantation. By contrast, the granulation tissue scarcely invaded the CO3Ap HCBs. Some osteoclasts invaded the wall of CO3Ap HCBs, making resorption pits. Furthermore, many osteoblasts were found on the newly formed bone, indicating ongoing bone remodeling. Blood vessels were also formed inside most of the pores in the CO3Ap HCBs. These findings suggest that CO3Ap HCBs have good osteoconductivity and can be used for the reconstruction of large mandibular bone defects

Expression and Role of IL-1β Signaling in Chondrocytes Associated with Retinoid Signaling during Fracture Healing

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1β (IL-1β) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1β and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1β in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1β and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1β decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1β-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1β treatment, and the IL-1β-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1β-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1β via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b+ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment

A Case of Ameloblastoma Removed Using a Three-dimensional Transparent Plastic Jaw Model Which Can Visualize Internal Jawbone Structures

Construction of three-dimensional (3D) models of various organs from CT images using 3D printers has recently become possible. In oral surgery, surgical simulation using a 3D printed jaw model is being widely used. With the conventional use of plaster for jaw modeling, the structures in the jawbone cannot be clearly visualized. Here, we report a case of a mandibular ameloblastoma extirpated using a 3D transparent plastic jaw model which can visualize the structures in the jawbone and assist in recognition of the anatomical position of a tumor. The patient was a 23-year-old woman who visited our hospital in 2016 with a complaint of swelling of the right mandible. Following the diagnosis of ameloblastoma, the tumor was removed. However, after 1 year and 7 months, tumor recurrence was suspected under the mandibular canal. Using a 3D jaw model for visualizing the inside of the jawbone, the tumor was removed with curettage of the surrounding tissue with minimal surgical invasion under general anesthesia. No recurrence has been observed 8 months post-surgery. Therefore, the use of the 3D jaw model facilitated recognition of the anatomical position of the tumor, which resulted in safe and reliable tumor removal