Kazuhide Mineda

Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers

Stage‐specific embryonic antigen‐3 (SSEA‐3)‐positive multipotent mesenchymal cells (multilineage differentiating stress‐enduring [Muse] cells) were isolated from cultured human adipose tissue‐derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic‐activated cell sorting into positive and negative fractions, a SSEA‐3+ cell‐enriched fraction (Muse‐rich) and the remaining fraction (Muse‐poor). Muse‐rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse‐poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse‐poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse‐rich cells significantly accelerated wound healing compared with treatment with Muse‐poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell‐depleted or ischemic conditions of various organs and tissues.

Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel

Three‐dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose‐derived stem/stromal cells (hASCs) in a non‐cross‐linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20–50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX‐2), and 40% of the cells were SSEA‐3‐positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia‐reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate‐buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage.

Normobaric hyperoxygenation enhances initial survival, regeneration, and final retention in fat grafting.

Background: Fat grafting is a promising modality for soft-tissue augmentation/reconstruction. However, grafted fat tissue is not initially perfused and relies on plasmatic diffusion from the recipient bed until revascularization occurs. The authors evaluated the therapeutic effects of normobaric hyperoxygenation for enhancing fat graft retention. Methods: Aspirated human fat tissue was cultured under tissue hypoxia (1% oxygen), normoxia (6%), and hyperoxia (20%) levels, and evaluated for adipocyte viability. Inguinal fat pads were autografted under mouse scalps (n = 36), and mice were housed in either 20% (control) or 60% (normobaric hyperoxygenation) atmospheric oxygen for the first 3 days, and then returned to normoxia. Samples harvested at 0, 1, 2, 4, 8, and 12 weeks were analyzed immunohistochemically for adipocyte viability and regeneration. Results: Organ culture adipocytes died more quickly under lower oxygen tensions; thus, hyperoxygenation of recipient tissues may delay adipocyte death after fat grafting. Autografted mouse adipose tissue underwent dynamic remodeling, from ischemic degeneration to partial regeneration, over 12 weeks. Normobaric hyperoxygenation grafted samples showed significantly larger survival zones and engraftment scores (calculated using sample weight and adipocyte viability) at 1 and 12 weeks, respectively, than control samples. In addition, adipocyte regeneration (number of perilipin-positive preadipocytes), which peaked at 4 weeks, was significantly increased in normobaric hyperoxygenation samples. Conclusion: The normobaric hyperoxygenation protocol using 60% oxygen can be safely applied to enhance adipocyte survival, regeneration, and final engraftment after fat grafting.

An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20–100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.

Micronized cellular adipose matrix as a therapeutic injectable for diabetic ulcer

BACKGROUND Despite the clinical potential of adipose-derived stem/stromal cells (ASCs), there are some clinical difficulties due to the regulation of cell therapies. MATERIALS & METHODS Micronized cellular adipose matrix (MCAM) injectable was prepared through selective extraction of connective tissue fractions in fat tissue only through mechanical minimal manipulation procedures. RESULTS It retained some capillaries and ASCs, but most adipocytes were removed. The presence of viable ASCs, vascular endothelial cells was confirmed and ASCs of MCAM kept intact mesenchymal differentiation capacity. In diabetic mice, skin wounds treated with MCAM showed significantly accelerated healing compared with phosphate-buffered saline-treated ones. CONCLUSION The proven potential of MCAM to accelerate healing in ischemic diabetic ulcers may offer a simple, safe and minimally invasive means for tissue repair and revitalization.

Application of normobaric hyperoxygenation to an ischemic flap and a composite skin graft.

Background: Hyperbaric oxygenation has been used for various purposes, but its clinical application is limited due to its pulmonary toxicity. We evaluated the therapeutic value of normobaric hyperoxygenation (NBO) for vascularized and nonvascularized tissue transplantation. Methods: Tissue oxygen partial pressure (PtO2) was measured for various organs in mice under inspiratory oxygen of 20%, 60%, or 100%. A rectangular skin flap (1 × 4 cm) or a composite skin graft (2 × 2 cm) was made on the back of mice, which were housed under 20% or 60% oxygen for the first 3 days after surgery. Cell survival was also examined in organ culture skin samples. Results: PtO2 varied among tissues/organs, but increased depending on inspiratory oxygen concentration in all tissues/organs. Although NBO with 100% O2 was toxic, NBO with 60% O2 was safe even when used continuously for a long period. NBO did not significantly improve survival of the rectangular skin flap. On the other hand, in the composite skin graft model, the engraftment area increased significantly (52 ± 10 at 20% vs 68 ± 5.1 at 60%) and contraction decreased significantly (42 ± 8.0 at 20% vs 27 ± 5.7 at 60%). Organ culture of a composite skin sample showed significant cell death under lower oxygen concentrations, supporting the data in vivo. Conclusions: The composite graft was maintained until revascularization by plasmatic diffusion from surrounding tissues, in which PtO2 was improved by NBO. NBO may be an effective adjunct therapy that can be performed readily after nonvascularized tissue grafting.

Blood Congestion Can Be Rescued by Hemodilution in a Random-Pattern Skin Flap

Background: There is no standard method to ensure survival of random-pattern skin flaps. The authors developed a rat anemia model to observe survival of random-pattern skin flaps after blood transfusion and hemodilution. Methods: Anemia was induced by withdrawal of 35 percent blood volume followed by compensation with the same amount of blood (blood transfusion model) or plasma equivalent (normovolemic hemodilution). Control rats were subjected to a sham procedure. Subsequently, a random-pattern skin flap (1.5 × 6 cm) was elevated on the back of each rat. Physiologic assessments of flap vascularity/viability were performed using laser Doppler spectrophotometry before and after flap elevation. Results: The normovolemic hemodilution group showed anemia (hemoglobin, 9.5 ± 0.8 g/dl) but less flow occlusion and greater flap survival (72.8 ± 8.6 percent) compared with control (57.4 ± 9.6 percent; p < 0.01) and blood transfusion (62.1 ± 6.5 percent; p < 0.089) groups. In control and blood transfusion groups but not the normovolemic hemodilution group, blood flow was decreased and relative quantity of hemoglobin was increased toward the flap tip, indicating congestion. In control and blood transfusion groups, blood flow and tissue oxygen saturation dropped after flap elevation, but recovered by day 7; congestion gradually improved by day 7. Conclusions: The authors determined that congestion promoted necrosis and hemodilution reduced microcirculatory occlusion and increased blood flow and oxygenation in skin flaps. It was suggested that perioperative hemodilution is superior to blood transfusion in any flap operations unless there is a critical systemic need for blood transfusion.

Cell and Tissue Damage after Skin Exposure to Ionizing Radiation: Short- and Long-Term Effects after a Single and Fractional Doses

Ionizing radiation is often used to treat progressive neoplasms. However, the consequences of long-term radiation exposure to healthy skin tissue are poorly understood. We aimed to evaluate the short- and long-term radiation damage to healthy skin of the same irradiation given either as single or fractional doses. C57BL/J6 mice were randomly assigned to one of three groups: a control and two exposure groups (5 Gy ×2 or 10 Gy ×1). The inguinal area was irradiated (6-MeV beam) 1 week after depilation in the treatment groups. Skin samples were evaluated macroscopically and histologically for up to 6 months after the final exposure. After anagen hair follicle injury by irradiation, hair cycling resumed in both groups, but hair graying was observed in the 10 Gy ×1 group but not in the 5 Gy ×2 group, suggesting the dose of each fractional exposure is more relevant to melanocyte stem cell damage than the total dose. On the other hand, in the long term, the fractional double exposures induced more severe atrophy and capillary reduction in the dermis and subcutis, suggesting fractional exposure may cause more depletion of tissue stem cells and endothelial cells in the tissue. Thus, our results indicated that there were differences between the degrees of damage that occurred as a result of a single exposure compared with fractional exposures to ionizing radiation: the former induces more severe acute injury to the skin with irreversible depigmentation of hairs, while the latter induces long-term damage to the dermis and subcutis.

Clinicopathologic Assessment of Myositis Ossificans Circumscripta of the Masseter Muscles.

and there was no abnormality in intraoral examination. The patient had no history of dentomaxillofacial trauma, craniofacial anomalies, syndromes, or infection. A computerized tomography (CT) scan was performed, which revealed a supernumerary intranasal tooth in the floor of left nasal cavity, 2 cm posterior to the anterior end of the inferior turbinate, with the same attenuation as that of the oral teeth (Fig. 1). The intranasal tooth was removed by forceps with endoscopic guidance under general anesthesia. The postoperative course was uneventful, except for a few days of serious discharge, which diminished. Ectopic teeth is a rare disease, which may be supernumerary or may result from abnormal dentition. The supernumerary intranasal teeth may be observed at various ages, ranging from 6 to 61 years. It is reported to be more common in male patients (62%) and in the left side. The etiology of intranasal tooth eruption has not been clarified. Abnormal tissue interactions during development may potentially lead to ectopic tooth development and eruption. A total of 84.6% of patients are symptomathic. Nasal teeth can cause nasal discharge, foul smell, nasal obstruction, nasal discomfort, headache, facial pain, epistaxis, epiphora, sinusitis, rhinitis, nasal septal deviations, nasal septal abscess, and oronasal fistula. Sometimes an intranasal tooth may be asymptomatic as in the current case. The diagnosis of an intranasal tooth can be confirmed clinically and radiologically. An intranasal tooth is often a hard white mass and can sometimes be covered completely by nasal mucosa and surrounded by granulation tissue and necrotic debris. Radiologic examinations, especially CT, are useful to identify the exact position and helps to decide the surgical approach. The differential diagnosis of intranasal white mass includes nasal foreign body, rhinolith, exocytosis, odontomas, osteomas, malignant tumors. The general opinion on the treatment of nasal teeth is the extraction when diagnosed because of potential morbidity, even if, asymptomatic. It may be extracted with either transnasal or transpalatal approach. Endoscopic removal of intranasal teeth can provide good illumination, better visualization, and has less morbidity compared with conventional approach. As the diagnosis of the disease is easy with a simple endoscopic examination, all of the patients referred to an otorhinolaryngology clinic should be completely examined. The supernumerary intranasal teeth are rare cases. Early extraction is advocated because of potential morbidity.

Heterotopic ossification in cauliflower ear

The cauliflower ear is a thickened deformity of the external ear seen after repeated traumatic hematoma formation in the auricle, and most commonly caused by sporting activity, such as rugby, boxing, wrestling and judo or injury from a fall or motor vehicle accident. This hypertrophied tissue is commonly considered to consist of fibrosis and neocartilage, resulting from the scarring and stimulation of mesenchymal stem cells in the perichondrium caused by bleeding between the perichondrium and the cartilage or perichondritis. When performing otoplasty, however, we sometimes experience a bone-like hardness in the hypertrophied tissues of a cauliflower ear. We have previously reported multiple ossifications in the cauliflower ear patient using computed tomographic scan. Herein, we report another case of the cauliflower ear in which detailed histopathological findings showed rigid bone formation in the excised specimen. A 22-year-old male presented with a severe deformity of the right ear as a result of playing rugby for 4 years (Figure 1). The outline of the entire ear had significantly changed with protruding thick tissues, and the structural integrity was poor. Otoplasty was performed from a postauricular incision, and the ear cartilage and hypertrophied tissues were completely exposed. The deformed, hyperplastic tissues were then shaved with a scalpel to restore normal shape, and the skin was closed with application of a bolster suture to the scaphoid fossa. Surgical specimens, grossly ash gray solid and hard masses, were submitted to the pathological department for detailed examination. The wound healed well, and the ear maintained a good contour at 1-year follow-up observation. The decalcified, and azan-stained (which shows collagen fiber as deep blue, cartilage matrix as light blue, and nuclei as bright red) specimens revealed presence of fibrosis, cartilage, and bone structure (Figure 2). As only abnormal tissues were removed, all of these tissues were thought to