Hiroaki Tsuno

Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation.

Background Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. Methods In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. Results We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAM&agr; cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen–DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor &bgr;3, transforming growth factor &bgr;3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAM&agr; cells achieved 8-week survival in vivo. Conclusions These results suggest that HAM&agr; cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAM&agr; cells could be an allotransplantable cell resource for cartilage repair.

CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics

BACKGROUND Cancer may be derived from cancer stem-like cells (CSCs), which are tumor-initiating cells that have properties similar to those of stem cells. Identification and isolation of CSCs needs to be improved further. MATERIALS AND METHODS CSCs markers were examined in human oral cancer cell lines by flow cytometry. The stem cell properties of subpopulations expressing different markers were assessed further by in vitro sphere formation assays, expression of stemness genes, drug resistance assays, and the ability to form tumors in nude mice. RESULTS We demonstrated that CSCs could be isolated by the cell surface markers CD44 and stage-specific embryonic antigen-4 (SSEA-4). CD44+SSEA-4+ cells exhibited cancer stem-like properties, including extensive self-renewal into the bulk of cancer cells. In vivo xenograft experiments indicated that CD44+SSEA-4+ cells exhibit the highest tumorigenic capacity compared with the remaining subpopulations and parental cells. Double-positive cells for CD44 and SSEA-4 exhibited preferential expression of some stemness genes and were more resistant to the anticancer drugs, cisplatin and 5-fluorouracil (5-FU). In addition, cells expressing CD44 and SSEA-4 were detected in all tumor specimens analyzed, while coexpression of CD44 and SSEA-4 was not detectable in normal oral mucosa. CONCLUSION Our findings suggest that CD44+SSEA-4+ cells exhibit the characteristics of CSCs in oral squamous cell carcinoma and provide a target for the development of more effective therapies.

Clinical application of a hyperdry amniotic membrane on surgical defects of the oral mucosa.

PURPOSE The aim of this study was to evaluate the usefulness of a hyperdry amniotic membrane (AM), a novel preservable human amnion, as a wound-dressing material for surgical defects of the oral mucosa. MATERIALS AND METHODS A hyperdry AM was used in the treatment of 10 patients who had developed secondary defects in the tongue and buccal mucosa after the surgical removal of cancerous or precancerous lesions. The effectiveness of the hyperdry AM was assessed by scoring its operability during the surgical procedure and by the hemostatic status, pain relief, feeding situation, epithelialization, and scar contracture in the postoperative period. Its usefulness was evaluated by considering its effectiveness and safety based on the absence of wound infection and graft rejection. RESULTS The membrane was found to be easy to handle as an oral-dressing material. It adhered well to the bare connective and muscular tissues. One lingual case showed slight postoperative bleeding, which astriction then stopped. No remarkable adverse effects were observed in the process of wound healing. The average score of the patients was 11.2 points (10 to 13 points) in the present evaluation, with 14 being the highest possible score. CONCLUSIONS This study showed the clinical usefulness of the hyperdry AM as an intraoral wound-dressing material. Although the number of cases was small, the results suggested that the hyperdry AM is biologically acceptable to oral wounds and could be a suitable clinical alternative for the repair of the oral mucosa.

Intraoral application of hyperdry amniotic membrane to surgically exposed bone surface

Hyperdry amniotic membrane, a novel preservable material derived from the human amnion, has been introduced clinically in ophthalmology and other fields. This membrane is available as a wound dressing material for surgical wounds of the tongue and buccal mucosa but has not been used on wounds of the alveolar mucosa. This paper reports 2 cases in which intraoral alveolar wounds with bone exposure were successfully treated with the use of hyperdry amniotic membrane: a 74-year-old woman with gingival leukoplakia of the edentulous mandible, and a 43-year-old man who underwent vestibuloplasty of the reconstructed mandible. The results indicate that the hyperdry amniotic membrane is a useful dressing material, not only for soft tissue wounds, but also for exposed bone in the oral cavity.

Use of hyperdry amniotic membrane in operations for cleft palate: a study in rats

The growth of maxillary bone and the development of dentition are often impaired in patients who have had pushback operations for repair of a cleft palate. There has been considerable discussion about the most suitable technique or material used in such repairs to resolve the problem. Hyperdry amniotic membrane, a new preservable material derived from human amnion, has recently been introduced in several procedures. We have evaluated its use during pushback surgery in animal studies to try to correct the inhibition of growth and development of the maxilla. Mucosal defects were created in 3-week-old rats, and then covered with hyperdry amniotic membrane or not. Healing was assessed by histological and morphological examination at 1 week and 7 weeks postoperatively. In the group treated with hyperdry amniotic membrane, submucosal tissue was reconstructed successfully during the early postoperative period. Lateral palatal growth was not inhibited as much, and medial inclination of the teeth was less, after a period of growth using this material. The results suggest that hyperdry amniotic membrane is a suitable new dressing material for use in the treatment of cleft palate.

In vitro synergistic effects of zoledronic acid and calcium on viability of human epithelial cells

OBJECTIVE Bisphosphonate-related osteonecrosis of the jaw is a common complication with defective wound healing of oral mucosa and frequently occurs in patients receiving zoledronic acid (ZA). The aim of this in vitro study was to investigate whether ZA has a cytotoxic effect at clinically relevant concentrations on epithelial cells when calcium conditions are altered. METHODS HaCaT human keratinocyte cells were treated with ZA in the presence of various concentrations of calcium. The concentrations of ZA included submicromolar ones, which are comparable with those found in the plasma of patients. Cell viability and apoptosis were assessed using MTT assay and annexin V flow cytometry. RESULTS Under standard culture conditions, cell growth was inhibited at 1 μM of ZA or above, but was unaffected by lower concentrations. However, when calcium concentrations were moderately increased, cell viability was decreased and apoptosis was induced at 0.2-0.3 μM of ZA. Moreover, a 50% reduction in serum in the hypercalcemic medium resulted in a significant decrease in cell viability at a much lower concentration (0.05 μM). CONCLUSION These results suggest that clinically relevant concentrations of ZA, which alone have little effects, can be toxic to the epithelial cells depending on the conditions of extracellular calcium.

Mucosa-associated Lymphoid Tissue Lymphoma of the Palate

Abstract This report is of a 60-year-old woman with mucosa-associated lymphoid tissue lymphoma of the palate. A moderate dose of radiotherapy and anti-CD20 monoclonal antibody chemotherapy were administered after tumour resection. There was no sign of recurrence 14 months after treatment.

Frey's Syndrome after Surgery for Fracture of the Mandibular Condyle

Abstract This report is of a 55-year-old woman presenting with Freys syndrome 6 months after surgical fixation of a fracture of the mandibular condyle. Treatment consisted of a fascia lata graft placed under the involved skin. No recurrence of gustatory sweating was observed.