Muneo Miyasaka

Induction of vascular endothelial growth factor by fibrin as a dermal substrate for cultured skin substitute.

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human keratinocytes and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after 2 days of incubation. Experiments were performed using 12 cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was 27.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.226, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating keratinocyte layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.

The effects of flap ischemia on normal and diabetic progenitor cell function.

Background: Endothelial progenitor cells play an important role in neovascularization of ischemic flaps, a process that is significantly impaired in diabetes. This is the first investigation into the effects of flap ischemia on circulating and bone marrow–derived endothelial progenitor cells. Potential mechanisms for impaired vasculogenesis in diabetes are also investigated. Methods: Circulating and bone marrow–derived endothelial progenitor cells were isolated from wild-type (n = 24) and diabetic mice (n = 24) with ischemic flaps (days 0, 1, 3, and 7). The number and vasculogenic function of primitive and definitive endothelial progenitor cells were determined by fluorescence-activated cell sorting analysis, culture assay, and vasculogenic colony-forming assay. Results: Ischemia mobilized endothelial progenitor cells (25 ± 0.5 cells per high-power field at day 7 versus 9.0 ± 0.6 cells per high-power field, p < 0.01) and enhanced the vasculogenic potential of circulating primitive endothelial progenitor cells (23 ± 3.2 at day 3 versus 14 ± 0.8, p < 0.01) relative to baseline. In the bone marrow, endothelial progenitor cell number and vasculogenic potential peaked at day 3 (2.1 ± 0.3 × 105 cells versus 1.3 ± 0.1 × 105 cells, p < 0.05; 36 ± 1.9 versus 27 ± 1.6, p < 0.05, respectively). In diabetes, circulating endothelial progenitor cell mobilization (5.8 ± 0.4 cells per high-power field versus 9.0 ± 0.6 cells per high-power field, p < 0.01) and vasculogenic potential (36 ± 1.7 versus 43 ± 2.6, p < 0.05) were impaired relative to the wild-type animals. Bone marrow–derived endothelial progenitor cell number was normal in diabetic animals, but the vasculogenic potential of these cells was significantly impaired (5.7 ± 0.8 day 1 versus 13.4 ± 2.5, p < 0.05). Conclusions: Flap ischemia induces phenotypic changes in bone marrow–derived endothelial progenitor cells that subsequently traffic through the circulation. The vasculogenic potential of endothelial progenitor cells at various stages of differentiation is impaired in diabetes and thus may account for impaired ischemia-induced vasculogenesis observed clinically.

Subcutaneous laser treatment of axillary osmidrosis: a new technique.

Axillary osmidrosis, also referred to as bromidrosis, is a distressing condition that can pose significant social embarrassment, especially in Japan and other Asian countries. Conservative treatments such as topical agents, systemic agents, and botulinum toxin are only temporarily effective and may lead to surgical treatment.1–3 However, surgical interventions using gland excision or liposuction have associated downtimes and complications that may be unacceptable for the treatment of a benign condition.1–3 The following is a report of a new technique for resolving problems in the management of axillary osmidrosis. We describe subcutaneous application of pulsed neodymium:yttriumaluminum-garnet laser for ablation of the sweat glands.

Autologous G-CSF-Mobilized Peripheral Blood CD34 + Cell Therapy for Diabetic Patients With Chronic Nonhealing Ulcer

Recently, animal studies have demonstrated the efficacy of endothelial progenitor cell (EPC) therapy for diabetic wound healing. Based on these preclinical studies, we performed a prospective clinical trial phase I/IIa study of autologous G-CSF-mobilized peripheral blood (PB) CD34+ cell transplantation for nonhealing diabetic foot patients. Diabetic patients with nonhealing foot ulcers were treated with 2 × 107 cells of G-CSF-mobilized PB CD34+ cells as EPC-enriched population. Safety and efficacy (wound closure and vascular perfusion) were evaluated 12 weeks posttherapy and further followed for complete wound closure and recurrence. A total of five patients were enrolled. Although minor amputation and recurrence were seen in three out of five patients, no death, other serious adverse events, or major amputation was seen following transplantation. Complete wound closure was observed at an average of 18 weeks with increased vascular perfusion in all patients. The outcomes of this prospective clinical study indicate the safety and feasibility of CD34+ cell therapy in patients with diabetic nonhealing wounds.

Quality-Control Culture System Restores Diabetic Endothelial Progenitor Cell Vasculogenesis and Accelerates Wound Closure

Delayed diabetic wound healing is, in part, the result of inadequate endothelial progenitor cell (EPC) proliferation, mobilization, and trafficking. Recently, we developed a serum-free functional culture system called the quality and quantity culture (QQc) system that enhances the number and vasculogenic potential of EPCs. We hypothesize that QQc restoration of diabetic EPC function will improve wound closure. To test this hypothesis, we measured diabetic c-kit+Sca-1+lin− (KSL) cell activity in vitro as well as the effect of KSL cell–adoptive transfer on the rate of euglycemic wound closure before and after QQc. KSL cells were magnetically sorted from control and streptozotocin-induced type I diabetic C57BL6J bone marrow. Freshly isolated control and diabetic KSL cells were cultured in QQc for 7 days and pre-QQc and post-QQc KSL function testing. The number of KSL cells significantly increased after QQc for both diabetic subjects and controls, and diabetic KSL increased vasculogenic potential above the fresh control KSL level. Similarly, fresh diabetic cells form fewer tubules, but QQc increases diabetic tubule formation to levels greater than that of fresh control cells (P < 0.05). Adoptive transfer of post-QQc diabetic KSL cells significantly enhances wound closure compared with fresh diabetic KSL cells and equaled wound closure of post-QQc control KSL cells. Post-QQc diabetic KSL enhancement of wound closure is mediated, in part, via a vasculogenic mechanism. This study demonstrates that QQc can reverse diabetic EPC dysfunction and achieve control levels of EPC function. Finally, post-QQc diabetic EPC therapy effectively improved euglycemic wound closure and may improve diabetic wound healing.

The influence of different types of hard-palate closure in two-stage palatoplasty on maxillary growth: cephalometric analyses and long-term follow-up.

Using cephalometric analysis we investigated the influence on maxillary growth of two different types of hard-palate closure in two-stage palatoplasty. In 12 patients with complete unilateral cleft lip, alveolus, and palate, the lip and soft-palate were closed between 3 and 7 months of age. These 12 patients were then assigned to two groups of 6. In one group the hard palate was closed at 1 year 5 to 11 months of age by a vomer flap with a skin graft (VF group, Osadas two-stage palatoplasty) and in the other group it was closed by the mucoperiosteal pushback procedure (PB group). Sella-nasion-point A (SNA) in the VF group at 3 to 4 and after 10 years of age were within normal range and significantly larger than in the PB group. Two patients in the PB group required orthognatic surgery to obtain normal occlusion and a well-balanced profile. We concluded that in two-stage palatoplasty better maxillary growth can be obtained using the vomer flap method than using the pushback procedure.

Liposome-Encapsulated Hemoglobin Accelerates Skin Wound Healing in Mice

Effects of liposome-encapsulated hemoglobin with high O₂ affinity (m-LEH, P₅₀O₂ = 17 mm Hg) on skin wound healing in mice were examined. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in Balb/c mice. Two days later (day 2), the animals randomly received intravenous m-LEH (2 mL/kg, n = 12), homologous blood transfusion (red blood cell [RBC], n = 11), or saline (n = 12). The same treatment was repeated 4 days after wounding (day 4), and the sizes of the skin defects and ulcers were monitored on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. While the size of the skin defect in relation to the stent ring remained the same in all groups, the size of the ulcer compared with the skin defect (or silicone stent) became significantly reduced on days 4 and 7 in mice treated with m-LEH (46 ± 10% of pretreatment size, P < 0.01) compared with mice treated with RBC transfusion (73 ± 6%) or saline (76 ± 7%). m-LEH treatment significantly accelerated granulation, increased epithelial thickness, suppressed early granulocyte infiltration, and increased Ki67 expression in accordance with the ulcer size reduction, while there was no difference in surface blood flow or CD31 expression among the groups. The results suggest that m-LEH (2 mL/kg) may accelerate skin wound healing in Balb/c mice via mechanism(s) involving reduced inflammation and increased metabolism, but not by improved hemodynamics or endothelial regeneration.

Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: In vivo analysis by film model method

Hyaluronan (HA) is known to inhibit neurons from regenerating in the central nervous system. However, hyaluronan tetrasaccharide (HA4) was found in in vitro experiments to promote outgrowth of neurons. To investigate the promotion by HA4 of nerve regeneration in vivo, we analyzed outgrowth of regenerating axons treated with HA4, using a film model method. After the common peroneal nerve in mice was transected, the proximal end of cut nerve was placed on a sheet of thin plastic film, immersed in several drops of HA4 solution, covered with another sheet of film, and then kept in vivo. Six hours after the procedure, terminal sprouts had grown out from ending bulbs formed at the cut end of parent nerve administered with HA4 solution 100 or 1000 μg/mL, while no sprouts were observed in groups treated with 10 μg/mL of HA4 or in controls. On the 2nd day after axotomy (day 2), many regenerating axons in the group treated with 100 μg/mL of HA4 extended onto the flat film for a longer distance than those treated with 1000 μg/mL of HA4 and controls. With the optimal dose of HA4 (100 μg/mL), axonal outgrowth was significantly (p<0.01) greater than that in controls at each time point. Schwann cells appeared migrating from parent nerve onto the film from day 3 as well as in controls. Thus, enhanced outgrowth of regenerating axons and normal behavior of migratory Schwann cells suggested that HA4 promoted regeneration of neurons without the mediation of Schwann cells.

A new objective histological scale for studying human photoaged skin

A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging.

Comparison of mandibular stability after SSRO with surgery-first approach versus conventional ortho-first approach

Abstract Methods: Postoperative mandibular stability in the surgery-first (SF) approach and ortho-first (OF) approach in orthognathic surgery was retrospectively assessed using the lateral cephalo X-P in 38 patients with skeletal Angle Class III malocclusion who underwent sagittal split ramus osteotomy (SSRO). Results: The postoperative mandibular relapse of the two groups observed from T1 (2 weeks after the surgery) to T2 (for the OF group, a year after surgery; for the SF group, the day orthodontic treatment was completed) was compared. The mean (SD) horizontal relapse at pogonion was 0.86 (0.92) mm in the forward direction in the SF group and 0.90 (1.09) mm in the forward direction in the OF group. No significant difference was found in the amount of horizontal movement between the two groups. On the other hand, the mean (SD) vertical relapse at pogonion was 1.59 (2.91) mm in the downward direction in the SF group and 0.14 (1.30) mm in the upward direction in the OF group, showing a significant difference in the amount of movement between the two groups. The degree of completion of the occlusion at T2 in the SF group was compared with that in the OF group by measuring OB, OJ, L1-occlusal plane angle, and interincisal angle. No significant difference was found between the two groups and the post-treatment occlusion was clinically favourable. Conclusion: Although the SF approach has several advantages for patients, the method of operation and fixation should be selected carefully to maintain postoperative mandibular stability.