Issues with the surgical treatment of antiresorptive agent-related osteonecrosis of the jaws.

Antiresorptive agent-related osteonecrosis of the jaw is a rare but significant complication in patients using antiresorptive agents such as bisphosphonates and denosumab. Although the disease is well recognized, and many studies have been performed on the management of this condition, the treatment of severe osteonecrosis is still a challenge. Most recent studies have shown an advantage of surgical treatment over conservative treatment for stage 2/3 patients, but there is no consensus on the appropriate surgical procedures for antiresorptive agent-related osteonecrosis of the jaw. Furthermore, patients with severe systemic conditions may not be appropriate for extensive surgical treatment, and the treatment protocol for such patients has not been established. In this review, issues regarding the current surgical treatment for antiresorptive agent-related osteonecrosis of the jaws are discussed, with an emphasis on the clinical aspects.

Comparing immunocompetent and immunodeficient mice as animal models for bone tissue engineering.

OBJECTIVES: To understand the differences and similarities between immunocompetent and immunodeficient mice as ectopic transplantation animal models for bone tissue engineering. MATERIALS AND METHODS: Osteogenic cells from mouse leg bones were cultured, seeded on ß-TCP granules, and transplanted onto the backs of either immunocompetent or immunodeficient nude mice. At 1, 2, 4, and 8 weeks postoperatively, samples were harvested and evaluated by hematoxylin-eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining and quantitative PCR. RESULTS: In immunocompetent mice, inflammatory cell infiltration was evident at 1 week postoperatively and relatively higher expression of TNF-α and IL-4 was observed. In immunodeficient mice, new bone area and the number of TRAP-positive cells were larger at 4 weeks than in immunocompetent mice. The volume of new bone area in immunodeficient mice was reduced by 8 weeks. CONCLUSIONS: Bone regeneration was feasible in immunocompetent mice. However, some differences were observed between immunocompetent and immunodeficient mice in the bone regeneration process possibly due to different cytokine expression, which should be considered when utilizing in vivo animal models.

The potential use of cell-based therapies in the treatment of oral diseases.

A recent and exciting development in medicine is the use of living cells for patient treatment. The utility of living cells to treat diseases was first proved in hematopoietic stem cell transplantation for leukemia patients. This approach has been expanded for other diseases such as islet transplantation for diabetic patients. In those cases, the cells were isolated from donors or the patient and used without complex manipulation. Since the 1980s, cells have been expanded outside of the body for the treatment of burn patients' skin. This novel treatment strategy is designated 'tissue engineering' and has been successfully applied for treatment of skin, cartilage, and also bone defects. Due to the recent developments in stem cell science, this area has attracted much attention and the application has been expanding. In this review, the potential of cell-based therapy for oral diseases is discussed with a concise review of recent developments in this field.

Panoramic radiography measurements, osteoporosis diagnoses and fractures in Japanese men and women.

OBJECTIVES: The purpose of this study was to investigate the association of the shape of the mandibular cortex on panoramic radiographs with the risk of an osteoporosis diagnosis without prevalent fractures and with the risk of osteoporotic fractures in Japanese men and women. SUBJECTS AND METHODS: One thousand and twenty-one subjects aged 40-89 years, who visited our university hospital and underwent panoramic radiography between 2007 and 2013, participated in this study. Eighty-eight patients received a diagnosis of osteoporosis without prevalent fractures, and 55 were diagnosed with osteoporotic fractures. Blinded to the groupings, we classified the shape of the mandibular cortex on panoramic radiographs as normal, moderately eroded or severely eroded. RESULTS: After adjustment for confounding factors, the odds ratios for an osteoporosis diagnosis associated with moderately eroded and severely eroded mandibular cortices were 1.4 (95% CI, 0.8-2.6) and 2.6 (95% CI, 1.4-5.0), respectively. The odds ratios for an osteoporotic fracture associated with moderately eroded and severely eroded cortices were 0.8 (95% CI, 0.4-1.7) and 1.1 (95% CI, 0.5-2.5), respectively. CONCLUSIONS: Subjects in Japan with eroded mandibular cortices tended to be at increased risk of osteoporosis diagnoses but not of fractures.

Effect of GDF-5 and BMP-2 on the expression of tendo/ligamentogenesis-related markers in human PDL-derived cells.

OBJECTIVES: The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers. MATERIALS AND METHODS: Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control. RESULTS: Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal ligament-derived cells. CONCLUSION: Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament-derived cells. The expression of scleraxis was higher in STRO-1-negative fraction, suggesting more differentiated state of the cells.

Characteristic changes of periodontal ligament-derived cells during passage.

BACKGROUND AND OBJECTIVE: Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage. MATERIAL AND METHODS: Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat. RESULTS: Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue. CONCLUSION: Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes.

Cell therapy for facial anti-aging.

Two types of cell therapy for facial anti-aging in my clinical experience are introduced in this presentation. One therapy is cultured gingival fibroblasts injection. This procedure lasts for at least one year, making it a good option for patients. The other is platelet rich plasma injection. The results of the preliminary data are promising, but not yet well understood. More clinical data and long-term follow-up is needed.

Bone marrow stromal cell therapy improves femoral bone mineral density and mechanical strength in ovariectomized rats.

BACKGROUND: This study aimed to determine the influence of bone marrow stromal cells (BMSC) on the degree and sustainability of ovariectomy-induced bone loss. METHODS: Allogenic BMSC were injected into either the left or right femur of 15 ovariectomized rats (OVX). Saline was injected into the contralateral femur as a vehicle control. Five rats were killed at 8 weeks and 5 rats at 24 weeks. The other five OVX rats received serial injections 4 weeks after the first injection and were killed 24 weeks after the first injection. To confirm osteoporotic model, five rats received sham operation. Bone mineral density (BMD) was measured using dual-energy X-ray absorptometry. Mechanical properties were evaluated by three-point bending. RESULTS: The OVX rats showed significantly lower BMD compared with that of the sham operated rats. BMD at the femoral mid-shaft was significantly greater in the BMSC-injected bones compared with the control bones. At week 8, ultimate load and stiffness were also improved in the BMSC-injected bones compared with controls. At 24 weeks, the stiffness of control and BMSC-injected bones was statistically indistinguishable. The additional injection aided preservation of both BMD and mechanical properties. DISCUSSION: The present study suggests that bone strength may be improved by direct BMSC injection.

Restoring the function of salivary glands.

Salivary gland destruction occurs as a result of various pathological conditions such as radiation therapy for head and neck cancer and Sjögren's syndrome. As saliva possesses self-cleaning and antibacterial capability, hyposalivation is known to deteriorate dental caries and periodontal disease. Furthermore, hyposalivation causes mastication and swallowing problems, burning sensation of the mouth and dysgeusia. Currently available treatments for dry mouth are prescription for artificial saliva, moisturizers and medications which induce salivation from the residual tissue. Unfortunately, these treatments cannot restore the acini functions. This review focuses on various efforts to restore the function of damaged salivary gland. First, the possibility of salivary gland regeneration and tissue engineering is discussed with reference to stem cells, growth factors and scaffold materials. Second, the current status of gene transfer to salivary glands is discussed.

Migration and proliferation of primordial germ cells in the early chicken embryo.

In avian species, primordial germ cells (PGC) use the vascular system as a vehicle to transport them to the future gonadal region. The aim of this study was to elucidate the details of migration system and size of the PGC population in the early chicken embryo. We analyzed whole chicken embryos during stages X and 2 to 17 by immunohistochemical staining using specific antibody raised against chicken vasa homolog. At stage X, PGC were dense in the central zone of the area pellucida. Following the formation of the primitive streak, PGC moved anteriorly to the edge of the extraembryonic region. The size of the PGC population increased gradually during stages X (130.4 +/- 31.9) to 10 (439.3 +/- 93.6). At stage 10, PGC began to accumulate in the region anterior to the head, and then we could observe that PGC invaded into the vascular system in this region. At stage 11, the number of PGC decreased in the region anterior to the head (129.8 +/- 42.5 to 46.7 +/- 4.2) and increased in the blood vessels (194.0 +/- 41.6 to 285.0 +/- 7.5). No PGC could be recognized in the intermediate mesoderm, the future gonadal region, until stage 14, but they first appeared there at stage 15. The number of PGC recognized in the intermediate mesoderm increased from stage 15 to 17. Interestingly, the number of PGC between the left and right sides of this region was consistently and significantly different (P < 0.05) in females and males. The present study mainly clarified that chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively left the blood vessels and migrated to the intermediate mesoderm from stage 15.

Odontoblast marker gene expression is enhanced by a CC-chemokine family protein MIP-3alpha in human mesenchymal stem cells.

OBJECTIVE: Macrophage inflammatory protein-3 alpha (MIP-3alpha) is a major CC-chemokine family protein, which serves as a differentiation factor for mesenchymal cells, including osteoblasts and dental pulp cells. The purpose of this study was to investigate the influence of MIP-3alpha on human mesenchymal stem cell differentiation in vitro. DESIGN: Human mesenchymal stem cells were maintained in Dulbecco's modified Eagle's medium in the presence or absence of MIP-3alpha and the presence or absence of osteogenic factors (dexamethasone, beta-glycerophoshate and ascorbic acid). Alkaline phosphatase (ALP) activity was measured, and expression of odontoblast and osteoblast markers were examined by RT-PCR and Western blotting. RESULTS: MIP-3alpha alone did not increase ALP activity, as compared to controls. The combination of MIP-3alpha and osteogenic factors increased ALP activity beyond increases observed with osteogenic factors alone. mRNA expression of the odontoblast marker dspp was only detectable when MIP-3alpha was added together with osteogenic factors at day 7 in three out of four samples. DSP protein level was increased only in the samples treated with both MIP-3alpha and osteogenic factors until day 5. In contrast, MIP-3alpha did not influence levels of the osteoblast markers CBFA1 or BSP. CONCLUSIONS: The present study demonstrated that MIP-3alpha enhanced gene expression and protein levels of odontoblast-related genes, without affecting levels of the osteogenic proteins CBFA1 or BSP.

Effects of preceding sialadenitis on the development of autoimmunity against salivary gland.

OBJECTIVE: The mechanism underlying the onset and development of autoimmune diseases such as Sjogren's syndrome is not well understood. Here, we examined the effects of preceding inflammation of the salivary gland at the onset of autoimmunity against the salivary gland. MATERIALS AND METHODS: One side of the submandibular gland duct was ligated in mice and the effect on the contralateral gland was investigated. After histological evaluation with hematoxylin and eosin staining, the presence of autoantibodies and immune compounds was examined. RESULTS: In all five strains of mice that were used, the salivary gland of the ligated side showed severe inflammation and atrophic change. In two mouse strains (SJL/J and PL/J), mild sialadenitis was observed on the non-ligated side 8 weeks after ligation. Autoantibodies reacting to the salivary gland were detected in three mouse strains (C3H/He, SJL/J, and PL/J). Immune complex was also detected in the duct basement membrane. CONCLUSION: The results indicate that the autoimmune mechanism is activated by the transient inflammation in the salivary gland under a specific genetic background.

Effective bone engineering with periosteum-derived cells.

Bone augmentation via tissue engineering has generated significant interest. We hypothesized that periosteum-derived cells could be used in place of bone marrow stromal cells (which are widely used) in bone engineering, but the differences in osteogenic potential between these 2 cell types are unclear. Here, we compared the osteogenic potential of these cells, and investigated the optimal osteoinductive conditions for periosteum-derived cells. Both cell types were induced, via bFGF and BMP-2, to differentiate into osteoblasts. Periosteal cells proliferated faster than marrow stromal cells, and osteogenic markers indicated that bone marrow stromal cells were more osteogenic than periosteal cells. However, pre-treatment with bFGF made periosteal cells more sensitive to BMP-2 and more osteogenic. Transplants of periosteal cells treated with BMP-2 after pre-treatment with bFGF formed more new bone than did marrow stromal cells. Analysis of these data suggests that combined treatment with bFGF and BMP-2 can make periosteum a highly useful source of bone regeneration.

Shear stress facilitates tissue-engineered odontogenesis.

Numerous studies have demonstrated the effect of shear stress on osteoblasts, but its effect on odontogenic cells has never been reported. In this study, we focused on the effect of shear stress on facilitating tissue-engineered odontogenesis by dissociated single cells. Cells were harvested from the porcine third molar tooth at the early stage of crown formation, and the isolated heterogeneous cells were seeded on a biodegradable polyglycolic acid fiber mesh. Then, cell-polymer constructs with and without exposure to shear stress were evaluated by in vitro and in vivo studies. In in vitro studies, the expression of both epithelial and mesenchymal odontogenic-related mRNAs was significantly enhanced by shear stress for 2 h. At 12 h after exposure to shear stress, the expression of amelogenin, bone sialoprotein and vimentin protein was significantly enhanced compared with that of control. Moreover, after 7 days, alkaline phosphatase activity exhibited a significant increase without any significant effect on cell proliferation in vitro. In vivo, enamel and dentin tissues formed after 15 weeks of in vivo implantation in constructs exposure to in vitro shear stress for 12 h. Such was not the case in controls. We concluded that shear stress facilitates odontogenic cell differentiation in vitro as well as the process of tooth tissue engineering in vivo.

Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit.

BACKGROUND: Although accumulating evidence shows that mesenchymal stem cells (MSC) are a promising cell source for articular cartilage repair, the fate of transplanted MSC has not been extensively studied. METHODS: To monitor their persistence and differentiation, we labeled uninduced MSC with a fluorescent dye, PKH26, and transplanted them, in a poly-glycolic-acid scaffold, to full-thickness defects made in the weight-bearing area of rabbit femoral trochleae with hyaluronate sheets. The fate of the labeled cells was monitored for up to 8 weeks. RESULTS: Two weeks after transplantation, immature cartilage containing collagen type II had formed. By 8 weeks, this cartilage had thinned and immunolabeling for collagen type II gradually disappeared from the basal region, which became positive for collagen type I. Most chondrocytes within the regenerated cartilage were PKH26-positive and, therefore, derived from transplanted MSC, whereas osteoblasts within the regenerated bone were a mixture of donor- and host-derived cells. The thickness of the cartilage became thinner up to 8 weeks and then remained stable up to 42 weeks after surgery. DISCUSSION: These results showed that uninduced MSC were able to survive osteochondral defects and differentiated according to the environment, making a major contribution to initial cartilage formation and a partial contribution to bone regeneration.

Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer.

BACKGROUND: The techniques to isolate and purify retinal pigment epithelial (RPE) cells from small piece of autologous tissues are extremely difficult, and it is important to develop an efficient cell culture technique for RPE cells. The purpose of this study was to investigate the effect of 3T3-J2 cells and conditioned medium from 3T3-J2 cells on the proliferation of cultured RPE cells. METHODS: RPE cells from pigmented rabbits and a human RPE-derived cell line, ARPE-19, were used. First, the effects of co-culturing RPE cells with 3T3-J2 cells on the growth of the cells were analyzed. Second, the effects of the conditioned medium from 3T3-J2 cells on the proliferation of both types of cells were investigated. And third, the effects of the conditioned medium on RPE cell culture from a surgically removed choroidal neovascular (CNV) membrane were investigated. RESULTS: The 3T3-J2 cells increased the proliferation of both rabbit RPE cells and ARPE-19 cells. The number of rabbit RPE cells cultured in a mixture of the conditioned medium from 3T3-J2 cells was significantly higher than that in the reported optimal condition, and a similar tendency was observed for ARPE-19 cells. The results from enzyme-linked immunosorbent assay showed the presence of PDGF-AB, VEGF and IGF-I in the conditioned medium. The conditioned medium also promoted selective growth of human RPE cells from CNV. DISCUSSION: The results from this study present the conditions for efficient and selective culture of primary RPE cells.

Transplantation of cultured salivary gland cells into an atrophic salivary gland.

Patients with dry mouth have been treated with salivary substitutes and/or medications such as pilocarpine or cevimeline hydrochloride. These treatments temporarily relieve their symptoms and induce salivation from residual tissue. However, no treatment is available for the purpose of regenerating an atrophic gland. In this study, the feasibility of a cell transplantation therapy for the atrophic submandibular glands was investigated in rats. Further, the potential of cell differentiation into a useful phenotype was assessed by immunohistochemistry together with cell tracking with the fluorescent dye PKH 26. Rat submandibular glands were excised, and the salivary gland epithelial cells were cultured for 3 weeks with 3T3 cells as a feeder layer. Ductal ligation of the submandibular gland was employed to generate an atrophic gland. One week after the operation, the ligation was removed, and the cultured cells labeled with PKH 26 were injected into the atrophic submandibular glands. As a control, the cultured cells were also injected into normal submandibular glands. Two weeks after cell transplantation, the transplanted cells were detectable in both the experimental and control groups. The cells were clustered in the connective tissue between the lobules. Four weeks after transplantation, the labeled cells were detectable in the experimental group but not in the control group. In the atrophic glands, the scattered transplanted cells were observed over a broad area of the gland but localized mainly around the acini and ductal region. Immunostaining results showed a possible involvement of the transplanted cells in ductal regeneration, while neither myoepithelial nor acinar differentiations were observed within the 4 weeks since transplantation. This study demonstrated that cell transplantation to the salivary gland is feasible, and that the transplanted cells were selectively attracted to and remained in the damaged area without affecting normal tissue.

Small intestinal metastasis from renal cell carcinoma exhibiting rare findings.

Small intestinal metastasis from renal cell carcinoma (RCC) has only rarely been described. We report two patients who developed small bowel metastases from RCC showing different clinicopathological characteristics. Both patients underwent hemilateral nephrectomy for RCC and developed lung metastases metachronously or simultaneously. One patient developed occlusive ileus caused by multiple polypoid tumours composed of sarcomatoid tissue in the jejunum shortly after nephrectomy. The other patient presented melaena due to bleeding from a Borrmann 2-like tumour in the jejunum six years after nephrectomy. Clinically, his disease was slow-growing. Sarcomatoid histology and Borrmann 2-like tumour in this report are rare findings in metastatic tumour of RCC in the small bowel.

Small bowel bacterial overgrowth and rice malabsorption in healthy and physically disabled older adults.

AIM: To determine whether there are differences in small bowel bacterial overgrowth (SBBO) and rice digestion between healthy and disabled older adults and to estimate the influence of physical activity on these nutritional statuses. METHOD: Fifteen disabled adults who commute to a day-care centre and 11 healthy older adults participated in this study. SBBO and rice absorption were judged using a breath hydrogen test. Physical activity was estimated using a pedometer. RESULTS: The average number of steps taken per day by the disabled was 1056 +/- 243, which was statistically lower than that of the healthy, 6904 +/- 782 (P < 0.001). No SBBO-positive subject was seen in the healthy group, whereas five (33.3%) of 15 disabled older adults were SBBO-positive. After ingesting glucose solution, the triangle up H2 of disabled subjects was higher than that of the healthy subjects (7.6 +/- 2.7 versus 0.5 +/- 0.3 p.p.m., P < 0.05). Rice malabsorption was seen in one (9.1%) of 11 in the healthy and two (14.3%) of 14 in the disabled groups, which was not statistically significant. CONCLUSIONS: Disabled older people who have a physically inactive lifestyle are at risk of SBBO, probably because of a reduction in their intestinal motility. SBBO has no influence on absorption of rice, and some older adults, independent of physical condition, can not absorb rice adequately.