Preparation of virus-like particle mimetic nanovesicles displaying the S protein of Middle East respiratory syndrome coronavirus using insect cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) first emerged in 2012, and over 2000 infections and 800 deaths have been confirmed in 27 countries. However, to date, no commercial vaccine is available. In this study, structural proteins of MERS-CoV were expressed in silkworm larvae and Bm5 cells for the development of vaccine candidates against MERS-CoV and diagnostic methods. The spike (S) protein of MERS-CoV lacking its transmembrane and cytoplasmic domains (SΔTM) was secreted into the hemolymph of silkworm larvae using a bombyxin signal peptide and purified using affinity chromatography. The purified SΔTM forms small nanoparticles as well as the full-length S protein and has the ability to bind human dipeptidyl peptidase 4 (DPP4), which is a receptor of MERS-CoV. These results indicate that bioactive SΔTM was expressed in silkworm larvae. To produce MERS-CoV-like particles (MERS-CoV-LPs), the coexpression of spike proteins was performed in Bm5 cells and envelope (E) and membrane (M) proteins secreted E and M proteins extracellularly, suggesting that MERS-CoV-LPs may be formed. However, this S protein was not displayed on virus-like particles (VLPs) even though E and M proteins were secreted into the culture supernatant. By surfactant treatment and mechanical extrusion using S protein- or three structural protein-expressing Bm5 cells, S protein-displaying nanovesicles with diameters of approximately 100-200 nm were prepared and confirmed by immuno-TEM. The mechanical extrusion method is favorable for obtaining uniform recombinant protein-displaying nanovesicles from cultured cells. The purified SΔTM from silkworm larvae and S protein-displaying nanovesicles from Bm5 cells may lead to the development of nanoparticle-based vaccines against MERS-CoV and the diagnostic detection of MERS-CoV.

Experimental Rat Skin Flap Model That Distinguishes between Venous Congestion and Arterial Ischemia: The Reverse U-Shaped Bipedicled Superficial Inferior Epigastric Artery and Venous System Flap.

BACKGROUND: The commonly used flap models have drawbacks that limit their usefulness. In the random skin flap model, flap necrosis is caused by both arterial and venous insufficiency. In the axial skin flap model, flap viability is easily affected by the pedicle blood flow and can result in complete necrosis. This study aimed to establish a new rat skin flap model that has a consistent flap survival rate and in which venous congestion and arterial ischemia can be readily distinguished macroscopically. METHODS: Rats underwent reverse U-shaped bipedicled superficial epigastric artery flap elevation. The right superficial epigastric vessels formed the pedicle. In the control rats (n = 3), the left superficial epigastric vessels were left intact. In the ischemia group (n = 10), the left superficial epigastric artery was ligated. In the congestion group (n = 10), the left superficial epigastric vein was ligated. The flap was returned to the original site and sutured. The surrounding neovascularization was blocked by polyurethane film. Flap survival rates were evaluated on postoperative day 3. RESULTS: The flaps in the ischemia and congestion groups were noticeably pale and violet, respectively. Flap necrosis was noted in the contralateral distal zone only. It started on postoperative day 2 in the ischemia and congestion groups. The mean flap survival rates of the control, ischemia, and congestion groups were 100 percent, 61.8 percent (range, 56.9 to 67.1 percent), and 42.3 percent (35.7 to 48.7 percent), respectively (all p < 0.001). CONCLUSIONS: The flap facilitated discrimination of the effects of ischemia and congestion. This new rat skin flap model is simple and easy to construct, and has a consistent flap survival rate.

A novel cell-containing device for regenerative medicine: biodegradable nonwoven filters with peripheral blood cells promote wound healing.

The efficacy of skin regeneration devices consisting of nonwoven filters and peripheral blood cells was investigated for wound healing. We previously found that human peripheral blood cells enhanced their production of growth factors, such as transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor, when they were captured on nonwoven filters. Cells on biodegradable filters were expected to serve as a local supply of growth factors and cell sources when they were placed in wounded skin. Nonwoven filters made of biodegradable polylactic acid (PLA) were cut out as 13-mm disks and placed into cell-capturing devices. Mouse peripheral blood was filtered, resulting in PLA filters with mouse peripheral blood cells (m-PBCs) at capture rates of 65.8 ± 5.2%. Then, the filters were attached to full-thickness surgical wounds in a diabetic db/db mouse skin for 14 days as a model of severe chronic wounds. The wound area treated with PLA nonwoven filters with m-PBCs (PLA/B+) was reduced to 8.5 ± 12.2% when compared with day 0, although the non-treated control wounds showed reduction only to 60.6 ± 27.8%. However, the PLA filters without m-PBCs increased the wound area to 162.9 ± 118.7%. By histopathological study, the PLA/B+ groups more effectively accelerated formation of epithelium. The m-PBCs captured on the PLA filters enhanced keratinocyte growth factor (FGF-7) and TGF-ß1 productions in vitro, which may be related to wound healing. This device is useful for regeneration of wounded skin and may be adaptable for another application.

Clinical application and histological properties of autologous tissue-engineered skin equivalents using an acellular dermal matrix.

We developed a transplantable tissue-engineered skin equivalent composed of autologous cultured keratinocytes, fibroblasts, and a decellularized allogeneic dermis (acellular allogeneic dermal matrix; ADM) obtained from cadavers. In a process taking 3 weeks, cultured autologous keratinocytes from burn patients were expanded and then grown on ADMs. The tissue-engineered autologous skin equivalents (TESEs) were then transplanted in a one-stage procedure to the debrided third-degree burn wounds of 4 patients. The mean graft survival rate was 96%. Delayed graft loss and graft fragility were not observed. Histological and immunohistological findings indicated that the transplanted TESE had similar characteristics to normal human split-thickness skin grafts. These results suggest that the TESE using ADM can be used for permanent repair of full-thickness skin defects.

A case of breast reconstruction with bilaterally divided transverse rectus abdominis musculocutaneous flaps after removal of injected silicone and granuloma.

We present a case of breast reconstruction using bilaterally divided transverse rectus abdominis musculocutaneous flaps after the removal of severe siliconoma. In Japan, we have examined many patients who have had foreign substance injected into the breasts for augmentation. Most of these patients have had delayed complications. We believe that autologous tissue transfer is an ideal procedure for breast reaugmentation in patients who want to have artificial materials removed from the breasts but to maintain breast contour.

Perforator-supercharged occipitocervicopectoral flaps for lower face and neck reconstruction.

BACKGROUND: The occipitocervicopectoral flap has a local skin pedicle in the occipital region, with the distal portion of the flap in the pectoral region. One disadvantage of the occipitocervicopectoral flap is its limited flap length. To overcome this disadvantage, a perforator supercharging technique was applied to enlarge the original flap length. The aims of this study are to present a clinical application of the perforator-supercharged occipitocervicopectoral flap for face and neck reconstruction and to discuss a perforator supercharging technique and perforator anatomy. METHODS: All perforator-supercharged occipitocervicopectoral flaps that were used for face and neck reconstructions were analyzed retrospectively. RESULTS: In all nine cases, the second internal mammary artery perforator was attached at the end of the occipitocervicopectoral flap and supercharged with the contralateral recipient facial artery vessels. The average flap size was 22.6 × 6.2 cm, without any flap loss. It was possible to cover a large defect extending to bilateral sides with thin and pliable local skin tissue. All patients were satisfied with functional and aesthetic results achieved postoperatively after 6 months. CONCLUSIONS: The internal mammary artery perforator-supercharged occipitocervicopectoral flap can be considered a type of bipedicle perforator flap and can provide reliable flap vascularity. By using a perforator supercharging technique, we can adjust and enlarge the flap length tailored to the defect. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Acellular dermal matrix seeded with adipose-derived stem cells as a subcutaneous implant.

BACKGROUND: Soft tissue augmentation using autologous materials usually is associated with high resorption rates. To obtain more durable soft tissue filler, acellular dermal matrix (ADM) was seeded with adipose-derived stem cells (ASCs) in this study. METHODS: For ADM preparation, split-thickness skin was obtained from the dorsum of two Fischer rats. Harvested skin was acellularized to obtain ADM and subsequently seeded in vitro with ASCs isolated from the same rats. Subcutaneous soft tissue augmentation was carried out in the dorsal area of 20 Fischer rats. The implant materials were ADM (group 1), ADM with ASCs (group 2), collagen type 1 gel (group 3), and collagen type 1 gel with ASCs (group 4). Each specimen was harvested after 8 weeks for quantitative evaluation of thickness, vascular density, and collagen content. RESULTS: Histologic analysis showed that ASCs were successfully seeded onto ADM. The thickness of the implanted material and the vascular density were highest 8 weeks postoperatively in group 2. The subpanniculus layer became significantly thicker in group 3 than in group 4. The collagen content was significantly higher in group 2 than in the other groups. CONCLUSION: These findings suggest that ADM seeded with ASCs forms the best implant material in terms of volume maintenance, vascular density, and collagen content.

Application of multidetector-row computed tomography in propeller flap planning.

BACKGROUND: The propeller flap is defined as (1) being island-shaped, (2) having an axis that includes the perforators, and (3) having the ability to be rotated around an axis. The advantage of the propeller flap is that it is a pedicle flap that can be applied to cover defects located at the distal ends of the extremities. The specific aims of the authors' study were (1) to evaluate the usefulness of multidetector-row computed tomography in the planning of propeller flaps and (2) to present a clinical case series of propeller flap reconstructions that were planned preoperatively using multidetector-row computed tomography. METHODS: The authors retrospectively analyzed all cases between April of 2007 and April of 2010 at Nippon Medical School Hospital in Tokyo, where multidetector-row computed tomography was used preoperatively to plan surgical reconstructions using propeller flaps. RESULTS: Thirteen patients underwent 16 flaps using the propeller flap technique. The perforators were identified accurately by multidetector-row computed tomography preoperatively in all cases. CONCLUSIONS: This is the first report describing the application of multidetector-row computed tomography in the planning of propeller flaps. Multidetector-row computed tomography is superior to other imaging methods because it demonstrates more precisely the perforator's position and subcutaneous course using high-resolution three-dimensional images. By using multidetector-row computed tomography to preoperatively identify a flap's perforators, the surgeon can better plan the flap design to efficiently conduct the flap surgery.

Multidetector-row computed tomography (MDCT) analysis of the supra-fascial perforator directionality (SPD) of the occipital artery perforator (OAP).

BACKGROUND: The occipital artery (OA) has three major cutaneous branches, all of which can be used to make occipital artery perforator (OAP) flaps on the posterior scalp and nape. To optimise OAP flap design, it is important to determine the supra-fascial perforator directionality (SPD) of the three branches. MATERIALS AND METHODS: We analysed the anatomical characteristics of the OAP by using the stored Digital Imaging and Communications (DICOM) multidetector-row CT (MDCT) data of 10 cases. For all cases, the branches arising from the OAP were counted and their origins and courses were traced on the computer. Branch diameters and lengths, and the distance between branch origin and the occipital protuberance or mastoid process, were measured. Averages were calculated. RESULTS: All cases had three OAP branches, namely an ascending, a transverse and a descending branch (the average branch number was 3.2±0.4). Their average vessel diameters were 16.8, 10.8 and 10.0mm, respectively. Their average SPD lengths were 71.1, 20.8 and 21.6mm, respectively. After the OA penetrated the fascia (thus becoming the OAP), the descending branch arose from the OAP within 10mm of the piercing point and travelled downwards and inwards. Above the descending branch bifurcation point, the OAP divided into the transverse branch, which travelled upwards and inwards, and the ascending branch, which travelled upwards. CONCLUSIONS: The OAP has three branches, all of which can be reliable vascular pedicles for scalp and nape skin flaps. Knowing the SPD of each branch is important for designing flap shape.

Bone marrow-derived myofibroblasts recruited to the upper dermis appear beneath regenerating epidermis after deep dermal burn injury.

Fibroblasts and myofibroblasts migrating to sites of tissue repair after injury may not only be locally recruited but could also be recruited from the bone marrow. However, the characteristics and functional roles, if any, of these cells in wound healing are poorly understood. Here, we show unequivocally that bone marrow-derived fibroblasts do contribute to deep dermal burn wound healing. Bone-marrow stromal cells were collected from femurs of male Lewis rats, cultured for a week, and then the adherent cells were labeled with the fluorescent marker PKH-26. These cells stained positive for alpha-smooth muscle actin and prolyl 4-hydroxylase, but did not express RM-4 (a macrophage marker), CD34, or cytokeratin, characteristic of myofibroblastic differentiation. When injected intravenously into Lewis rats, they homed to the bone marrow. Five days after transplantation, a deep dermal burn was made on the back of the rat, and biopsies were taken 7, 10, and 14 days later. PKH-positive cells were not found at day 7, but by day 10, they were easily detected mainly in the upper dermis close beneath the regenerating epidermis. These PKH-positive cells still stained for alpha-SMA and prolyl 4-hydroxylase, but not RM4. Thus, it is suggested that myofibroblasts originating in the bone marrow contribute not only to promotion of granulation but also enhancement of dermal-epidermal interaction after thermal injury.

Development of a tissue-engineered human oral mucosa equivalent based on an acellular allogeneic dermal matrix: a preliminary report of clinical application to burn wounds.

Tissue-engineered skin equivalents composed of epidermal and dermal components have been widely investigated for coverage of full-thickness skin defects. We developed a tissue-engineered oral mucosa equivalent based on an acellular allogeneic dermal matrix and investigated its characteristics. We also tried and assessed its preliminary clinical application. Human oral mucosal keratinocytes were separated from a piece of oral mucosa and cultured in a chemically-defined medium. The keratinocytes were seeded on to the acellular allogeneic dermal matrix and cultured. Histologically, the mucosa equivalent had a well-stratified epithelial layer. Immunohistochemical study showed that it was similar to normal oral mucosa. We applied this equivalent in one case with an extensive burn wound. The equivalent was transplanted three weeks after the harvest of the patient's oral mucosa and about 30% of the graft finally survived. We conclude that this new oral mucosa equivalent could become a therapeutic option for the treatment of extensive burns.

Protective effects of a free radical scavenger, MCI-186, on high-glucose-induced dysfunction of human dermal microvascular endothelial cells.

Functional damage to microvascular endothelial cells by hyperglycemia is thought to be one of the critical risk factors in the impaired wound healing seen with diabetes mellitus. It is also thought that free radical stress plays a significant role in this endothelial cell dysfunction. In the present study, the effect of a free radical scavenger, MCI 186, on the endothelial cell dysfunction of cultured cells induced by high-glucose conditions was studied. Human dermal microvascular endothelial cells were cultured with high-glucose medium (50 mM) with or without MCI-186 (10 microM) for 7 days. Fifty mM mannitol was used as an osmotic control in this study. After this treatment, cell proliferation, activation of mitogen-activated protein kinase (MAPK), the level of apoptosis, and caspase-3 activation induced by removal of growth factors or tumor necrosis factor-alpha treatment were studied. High-glucose conditions significantly decreased cell proliferation and increased apoptosis levels with the activation of caspase-3 induced by growth factor removal. The high-glucose condition significantly activated MAPK. MCI-186 treatment improved cellular proliferation and reduced apoptosis and caspase-3 activation induced by high-glucose conditions. MCI-186 also inhibited the activation of MAPK. On the other hand, MCI-186 did not alter the level of apoptosis and caspase-3 activation induced by TNF-alpha treatment. In conclusion, we suggest that MCI-186 may be beneficial for improving the endothelial cell dysfunction induced by hyperglycemia.

Frontal sinus repair with free autologous bone grafts and fibrin glue.

BACKGROUND: The authors describe a technique for repairing the frontal sinus with autologous bone grafts removed during craniotomy and fibrin glue. METHODS: This technique was used in 12 patients who underwent craniotomy for aneurysms (n = 9), brain tumors (n = 2), and acute epidural hematoma (n = 1). RESULTS: The repair was successful in all cases. There were no instances of postoperative infection or leakage of cerebrospinal fluid. No complications from the repair have occurred over a mean follow-up of 51 months. CONCLUSION: This technique is simpler than others used to seal a frontal sinus.