Masakazu Matsubara

Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo

Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry, alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor (bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical purpose.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption

SummaryThe main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506.IntroductionGrowth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling.MethodsFK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments.ResultsCompared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA.ConclusionThese results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Localization of antimicrobial peptides human β‐defensins in minor salivary glands with Sjögren’s syndrome

Sjögrens syndrome is a common systemic autoimmune disease associated with inflammatory cells that infiltrate exocrine glands. The antimicrobial peptides human beta-defensin-1, human beta-defensin-2, and human beta-defensin-3 are expressed in various human epithelial cells and in normal salivary glands. Antimicrobial peptides provide local protection against infection and participate in inflammatory responses. Because of the presence of inflammation, we hypothesized that human beta-defensin expression in minor salivary glands may be increased in subjects with Sjögrens syndrome. However, the expression of human beta-defensins 1 and 2 was decreased in salivary glands affected by Sjögrens syndrome in comparison with the human beta-defensin expression patterns in salivary glands from normal subjects. In addition, the reduction in expression of human beta-defensin-2 was greater than the reduction in expression of human beta-defensin-1. The aforementioned result suggests that the reduction in expression of human beta-defensin-2 may occur earlier than the reduction in expression of human beta-defensin-1, which may lead to a greater decrease in human beta-defensin-2 than in human beta-defensin-1 during disease progression.

Treatment of Osteonecrosis of the Jaw.

The definition of bisphosphonate-related osteonecrosis of the jaw (BRONJ) was recently broadened and it is now known as medication-related osteonecrosis of the jaw (MRONJ). To date, the management of MRONJ is controversial. Conservative treatment is recommended, but it is difficult to successfully treat stage 3 MRONJ. Administration of teriparatide for the MRONJ treatment has only been documented in independent case reports and there are few reports on men with MRONJ treated with teriparatide. An 81-year-old man was referred in May 2014 for treatment of an unhealed tooth extraction wound in the mandible. He took minodronic acid hydrate (1 mg/d orally) for 2 years because of osteoporosis cure. On clinical examination, soft tissue swelling in the left mandibular first molar region extended to the inferior border of the mandible with extraoral fistula. Computed tomography (CT) revealed osteolysis extending to the inferior border resulting in pathologic fracture of mandibular bone. Based on these findings, a diagnosis of stage 3 MRONJ was made. We performed conservative treatment, including amoxicillin, but his symptoms did not improve. He was then treated with once-weekly subcutaneous injection of teriparatide. Although teriparatide injections were started without antibiotics, after 1 week, swelling, erythema, and purulent discharge from the extraoral fistula increased rapidly. Therefore, we combined the once-weekly teriparatide injection with amoxicillin administration. Three months later, the osteonecrosis had healed and CT showed significant bone regeneration and healing of the mandibular pathologic fracture. In addition, the mandibular fistula showed healing and the intraoral fistula was covered with normal mucosa.

Molecular alterations of newly formed mandibular bone caused by zoledronate

Bone quality is defined by structural and material characteristics. Most studies on the mandible have focused on the analysis of structural characteristics, with insufficient investigation of material characteristics. This study tested whether zoledronate affects the material characteristics of newly formed mandibular bone. Thirty-six female Wistar rats were assigned to three groups: sham-ovariectomized rats (SHAM, n=12), ovariectomized rats (OVX, n=12), and ovariectomized rats treated with zoledronate (ZOL, n=12). The left side of the mandibular ramus of all rats was drilled bicortically. Twenty-eight days after surgery, all surviving rats were euthanized and all mandibles were removed. Raman microspectroscopy was performed, and five spectra per specimen of newly formed mandibular bone were analysed. Compared with OVX rats, the mineral/matrix ratio in ZOL rats was significantly increased (5.43±1.88 vs. 7.86±2.05), while crystallinity (0.055±0.002 vs. 0.050±0.002), relative proteoglycan content (0.43±0.10 vs. 0.31±0.05), and collagen structural integrity (1.16±0.21 vs. 0.72±0.06) were significantly decreased. These changes in material characteristics may explain why rats that received zoledronate exhibited peculiar biological phenomena such as bisphosphonate-related osteonecrosis of the jaw.

Rapid occurrence of left ventricular thrombus associated with platinum‑based chemotherapy plus cetuximab for the treatment of metastatic squamous cell carcinoma of the head and neck: A case report

Platinum-based chemotherapy plus cetuximab represents the first-line treatment for recurrent or metastatic squamous cell carcinoma of the head and neck. The most common adverse events associated with cetuximab are infusion reactions and skin reactions, and a risk of venous thromboembolic events has also recently been reported in association with cetuximab. It is well known that thrombosis is a common complication of malignancy, and represents the second most frequent cause of mortality in cancer patients. The present study reports the case of a 79-year-old man who presented with lung and liver metastases from tongue squamous cell carcinoma, for which platinum-based chemotherapy plus cetuximab was administered. After 1 cycle, the patient showed rapid growth of a left ventricular (LV) thrombus, despite ongoing antiplatelet therapy for an old myocardial infarction. Anticoagulant therapy was administered to treat the LV thrombus, which resolved within 1 week. To the best of our knowledge, this represents the first reported case of rapidly occurring LV thrombus associated with platinum-based chemotherapy plus cetuximab. Platinum-based chemotherapy plus cetuximab may be associated with a higher risk of embolic thrombus.

The influence of zoledronate and teriparatide on gamma delta T cells in mice

Background/purpose Few studies have investigated the possibility that bisphosphonate-related osteonecrosis of the jaw (BRONJ) might reflect an immune response; however, gamma delta T cells have been shown to significantly decline in the blood of BRONJ patients. Additionally, there have been some reports of teriparatide usage for the treatment of BRONJ. In this study, we compared the effects of zoledronate and teriparatide on lymphocyte populations and inflammatory cytokine production in mice. Materials and methods Thirty female ICR mice were divided into three groups (n = 10 each): a vehicle, a zoledronate, and a teriparatide group. Drugs were administered for 8 weeks in each group. Lymphocytes in the blood and thymus were analyzed and femurs were used for histological observation and lymphocytes analysis of bone marrow. Cytokines were measured in separated serum using Milliplex® multiplex immunoassay analysis. Results Zoledronate decreased the T cell number in the bone marrow. Additionally, serum levels of interleukin (IL)-2, IL-7, IL-12, IL-15 and RANTES, which are cytokines that affect T cell activation, differentiation and/or proliferation, were significantly lower in zoledronate treated mice. Conversely, teriparatide treatment induced an increase in gamma delta T cells in peripheral blood. Conclusion Gamma delta T cells in the bone marrow are expected to decrease with zoledronate treatment and increase with teriparatide treatment. If BRONJ involves a loss of gamma delta T cells in the circulation or bone marrow, then the increase in gamma delta T cells that is induced by teriparatide may account for its ability to resolve BRONJ.

Stage IV sporadic Burkitt's leukaemia with osteolysis in the maxillary sinuses

We present a case of paediatric Stage IV sporadic Burkitts leukaemia presenting as cheek enlargement with osteolysis of the maxilla. An 8-year-old boy was referred to our department with diffuse swelling of both cheeks. Head and neck examination revealed bilateral diffuse nontender swelling, non-fluctuant but slightly compressible. Computed tomography imaging showed enhancing bilateral bulky lesions expanding the maxillary sinuses, with associated osteolysis in the posterior walls of both sinuses. Laboratory results included blast cells in the peripheral blood, suggesting a haematopoietic tumour. We referred the patient to the Department of Paediatric Haematology and Oncology. Additional examinations eventually led to the diagnosis of Stage IV sporadic Burkitts leukaemia.

Activating stimulatory G protein alpha mutations do not occur in odontogenic myxoma and monostotic fibrous dysplasia of bone in the jaw

Abstract Objective: Activating mutations within the stimulatory G protein alpha gene are typically associated with the McCune-Albright Syndrome. These mutations have been found in endocrine tumours, intramuscular myxomas, and polyostotic fibrous dysplasias of bone in patients with McCune-Albright syndrome. We hypothesised that the same mutations might occur in odontogenic myxoma and monostotic fibrous dysplasia of bone in the jaw. In this study, we aimed to clarify the association between these tumours and mutations in the stimulatory G protein alpha gene. Patients and Methods: Paraffin-embedded specimens of 5 cases each of odontogenic myxoma and monostotic fibrous dysplasia of bone in the jaw were examined. Exon 8 and exon 9 of the stimulatory G protein alpha gene were screened by polymerase chain reaction-single-stranded conformation polymorphism analysis, and the polymerase chain reaction products were then sequenced using the dideoxy method. Result: No activating mutations of the stimulatory G protein alpha gene were found in any of the cases of odontogenic myxoma or monostotic fibrous dysplasia of bone in the jaw. Conclusion: Odontogenic myxoma and monostotic fibrous dysplasia of bone in the jaw are most likely not associated with activating mutations of the stimulatory G protein alpha gene.