CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development.

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth.

Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is an iatrogenic immunodeficiency-associated lymphoproliferative disorder that occurs mainly with MTX use. This disorder has been associated with Epstein-Barr virus (EBV) infection. In 2017, the WHO newly defined the disease concept of EBV-positive mucocutaneous ulcer (EBV-MCU) as a good-prognosis EBV-related disease. Here, we report 10 cases of MTX-LPD or EBV-MCU in the oral mucosa. This retrospective, observational study was conducted with MTX-LPD or EBV-MCU in the oral mucosa patients who visited us during the nine year period from 2012 to 2021. We gathered the basic information, underlying disease, histopathological evaluation, treatment and prognosis for the subjects. All were being treated with MTX for rheumatoid arthritis. EBV infection was positive in all cases by immunohistochemistry. A complete or partial response was obtained in all cases with the withdrawal of MTX. Our results suggests that the most common risk factor for developing EBV-MCU is the use of immunosuppressive drugs. The most common site of onset is the oral mucosa, which may be attributed to the mode of EBV infection and the high incidence of chronic irritation of the oral mucosa. A small number of patients had been diagnosed with MTX-LPD, but we consider that these cases were EBV-MCU based on our study.

Abstract：Actinomycosis is a specific inflammatory disease that shows typical symptoms of trismus, plate-like induration, and multiple abscess formation. However, it has recently become difficult to diagnose because of a decreased number of cases show typical symptoms. A 85-year-old woman was referred to our clinic because of swelling with left molar region and trismus. Radiographic examination revealed bone destruction and lysis of the mandible. Although a malignant tumor was suspected, the histopathological diagnosis was actinomycosis. Surgical curettage was performed, and Benzylpenicillin and Amoxicillin was administerated.

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