Eijiro Tokuyama

Mechanical Stretch on Human Skin Equivalents Increases the Epidermal Thickness and Develops the Basement Membrane

All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.

Evaluation of the Microvascular Research Center Training Program for Assessing Microsurgical Skills in Trainee Surgeons

Background We established the Microvascular Research Center Training Program (MRCP) to help trainee surgeons acquire and develop microsurgical skills. Medical students were recruited to undergo the MRCP to assess the effectiveness of the MRCP for trainee surgeons. Methods Twenty-two medical students with no prior microsurgical experience, who completed the course from 2005 to 2012, were included. The MRCP comprises 5 stages of training, each with specific passing requirements. Stages 1 and 2 involve anastomosing silicone tubes and blood vessels of chicken carcasses, respectively, within 20 minutes. Stage 3 involves anastomosing the femoral artery and vein of live rats with a 1-day patency rate of >80%. Stage 4 requires replantation of free superficial inferior epigastric artery flaps in rats with a 7-day success rate of >80%. Stage 5 involves successful completion of one case of rat replantation/transplantation. We calculated the passing rate for each stage and recorded the number of anastomoses required to pass stages 3 and 4. Results The passing rates were 100% (22/22) for stages 1 and 2, 86.4% (19/22) for stage 3, 59.1% (13/22) for stage 4, and 55.0% (11/20) for stage 5. The number of anastomoses performed was 17.2±12.2 in stage 3 and 11.3±8.1 in stage 4. Conclusions Majority of the medical students who undertook the MRCP acquired basic microsurgical skills. Thus, we conclude that the MRCP is an effective microsurgery training program for trainee surgeons.

Histologic Evaluation of Lymphaticovenular Anastomosis Outcomes in the Rat Experimental Model: Comparison of Cases with Patency and Obstruction.

Background: Lymphaticovenular anastomosis plays an important role in the surgical treatment of lymphedema. The outcomes of lymphaticovenular anastomosis are evaluated based on changes in edema; however, isolated assessment of the anastomosis itself is difficult. The authors used an animal experimental model to conduct a detailed examination of histologic changes associated with lymphaticovenular anastomosis and determined the factors important for success. Methods: The experimental lymphaticovenular anastomosis model was created using lumbar lymph ducts and iliolumbar veins of Wistar rats. The authors performed anastomosis under a microscope and reviewed postoperative histologic changes using optical and electron microscopy. In addition, electron microscopy and histology were used for detailed examination of the area in the vicinity of the anastomotic region in cases with patency and obstruction. Results: The patency rates immediately after, 1 week after, and 1 month after lymphaticovenular anastomosis were 100 percent (20 of 20), 70 percent (14 of 20), and 65 percent, respectively. A detailed examination of the anastomotic region with electron microscopy revealed that, in cases with patency, there was no notable transformation of the endothelial cells, which formed a smooth layer. In contrast, in obstruction cases, the corresponding region of the endothelium was irregular in structure. Conclusions: Vessel obstruction after lymphaticovenular anastomosis may be associated with irregular arrangement of the endothelial layer, leading to exposure of subendothelial tissues and platelet formation. One part of the postoperative changes after anastomosis and a cause of obstruction were elucidated in this study. The authors’ results may enable improvements in lymphaticovenular anastomosis by translating back to real clinical operations.

Efficacy of Honeycomb TCP-induced Microenvironment on Bone Tissue Regeneration in Craniofacial Area.

Artificial bone materials that exhibit high biocompatibility have been developed and are being widely used for bone tissue regeneration. However, there are no biomaterials that are minimally invasive and safe. In a previous study, we succeeded in developing honeycomb β-tricalcium phosphate (β-TCP) which has through-and-through holes and is able to mimic the bone microenvironment for bone tissue regeneration. In the present study, we investigated how the difference in hole-diameter of honeycomb β-TCP (hole-diameter: 75, 300, 500, and 1600 μm) influences bone tissue regeneration histologically. Its osteoconductivity was also evaluated by implantation into zygomatic bone defects in rats. The results showed that the maximum bone formation was observed on the β-TCP with hole-diameter 300μm, included bone marrow-like tissue and the pattern of bone tissue formation similar to host bone. Therefore, the results indicated that we could control bone tissue formation by creating a bone microenvironment provided by β-TCP. Also, in zygomatic bone defect model with honeycomb β-TCP, the result showed there was osseous union and the continuity was reproduced between the both edges of resected bone and β-TCP, which indicated the zygomatic bone reproduction fully succeeded. It is thus thought that honeycomb β-TCP may serve as an excellent biomaterial for bone tissue regeneration in the head, neck and face regions, expected in clinical applications.

Determination of reference values for normal cranial morphology by using mid-sagittal vector analysis in Japanese children

Mid-Sagittal Vector Analysis (MSVA) is a method of measuring the distance from a defined central point on the skull surface in the entire mid-sagittal plane and provides a clear description of the lateral view of the skull. We used a series of images of normal skulls of Japanese children to determine normal MSVA values. For this cross-sectional study, we first constructed a database of head CT and MRI images of children aged 0-6 years (41.5 ± 24.9 month (mean ± SD)) who showed no abnormality of cranial development and growth at the time of imaging. Measurement errors due to lateral shifting of the sagittal plane during MSVA were examined, CT and MRI images taken in the same patients at the same time were compared, and measurement differences were examined. Finally, MSVA was carried out, and the mean of the measured values was calculated according to age group. Two hundred ninety-five images were included in the database. When the lateral shifting of the sagittal plane was within 4 mm from the true mid-sagittal plane, the mean errors were less than 1 mm at all measurement points. Between the CT and MRI images from the same patients, most differences in MSVA values were within ±1 mm. These differences were thus acceptable for use in clinical settings. After the above verifications, 220 images were extracted for determination of normal MSVA values. We established a normal dataset of MSVA for Japanese children that can be used effectively for preoperative diagnosis, surgery planning, and postoperative assessment of cranial deformities.