Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis.

Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded "dense ER" using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.

Actin Filament in the First Cell Cycle Contributes to the Determination of the Anteroposterior Axis in Ascidian Development.

In many animal species, the body axis is determined by the relocalization of maternal determinants, organelles, or unique cell populations in a cytoskeleton-dependent manner. In the ascidian first cell cycle, the myoplasm, including mitochondria, endoplasmic reticulum (ER), and maternal mRNAs, move to the future posterior side concomitantly (called ooplasmic segregation or cytoplasmic and cortical reorganization). This translocation consists of first and second phases depending on the actin and microtubule, respectively. However, the transition from first to second phase, that is, translocation of myoplasmic components from microfilaments to microtubules, has been poorly investigated. In this study, we analyzed the relationship between these cytoskeletons and myoplasmic components during the first cell cycle and their role in morphogenesis by inhibitor experiments. Owing to our improved visualization techniques, there was unexpected F-actin accumulation at the vegetal pole during this transition period. When this F-actin was depolymerized, the microtubule structure was strongly affected, the myoplasmic components, including maternal mRNA, were mislocalized, and the anteroposterior axis formation was disordered. These results suggested the importance of F-actin during the first cell cycle and the existence of interactions between microfilaments and microtubules, implying the enigmatic mechanism of ooplasmic segregation. Solving this mystery leads us to an improved understanding of ascidian early development.

Omental flap closure of refractory wounds: rat model.

Omental flaps, with their associated rich and pliable vascular arcades, are frequently used in clinical practice for the reconstruction of complex and irregular defects. There is little experimental evidence, however, to prove that omental flaps can be a useful tool for the defects. Using a gastric-wall defect model, we performed histological and immunocytochemical examinations. We created an omental flap lining a 2.0-mm defect perforating the center of the anterior wall of a rat stomach. We examined the tissue response during gastric wall regeneration by H&E and Masson trichrome stains. We also performed immunocytochemical studies for the detection of proliferating cell nuclear antigen (PCNA), factor VIII-related antigen, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). One day after the operation, the omental flap was found to firmly adhere to the gastric serosa surrounding the defect. An extensive inflammatory response occurred from Day 1 to 3 with dilated vessels in the omentum. From Days 3 to 7, a significant number of PCNA-positive cells, FGF-2-positive cells and VEGF-positive cells were observed at the edge of the mucosa and within the granulation tissue. On Day 4, in place of extensive inflammation, an exuberant granulation tissue response was observed from the omentum. The defect had been covered by stratified villi by Day 7. This study demonstrated that an omental flap came to rapidly adhere to the defect serving as a source of extensive inflammation and granulation for the rich and pliable vascular arcades.

Cryptic fragment alpha4 LG4-5 derived from laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of fibroblast growth factor-2.

Cleavage of the extracellular matrix (ECM) by proteolysis unmasks cryptic sites and generates novel fragments with biological activities functionally distinct from those of the intact ECM molecule. The laminin G-like (LG)4-5 fragment has been shown to be excised from the laminin alpha4 chain in various tissues. However, the functional role of this fragment has remained unknown to date. To investigate this, we prepared alpha4 LG1-3 and alpha4 LG4-5 fragments by elastase digestion of recombinant alpha4 LG1-5, and examined their effects on de novo adipogenesis in mice at the site of injection of basement membrane extract (Matrigel) and fibroblast growth factor (FGF)-2. Although the addition of whole alpha4 LG1-5 suppressed adipogenesis to some extent, the alpha4 LG4-5 fragment could strongly suppress adipogenesis at a concentration of less than 20 nm. Addition of the alpha4 LG4 module, which contains a heparin-binding region, had a suppressive effect, but this was lost in mutants with reduced heparin-binding activity. In addition, antibodies against the extracellular domain of syndecan-2 and -4, which are known receptors for the alpha4 LG4 module, suppressed adipogenesis. Thus, these results suggest that the cryptic alpha4 LG4-5 fragment derived from the laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of FGF-2 through syndecans.

Analysis of salvage operation in head and neck microsurgical reconstruction.

In this study, we examined salvage operations after reexploration in head and neck reconstruction and analyzed ways to solve problems. Free flap reconstruction of the head and neck lesion was carried out for 513 cases in our hospital over the past 12 years. Twenty-one cases of reexploration were caused by postoperative thrombosis (4.1%). We could only salvage seven cases (33.3%) of 21 cases from flap thrombosis. All seven cases were included in the category of venous thrombosis, and they were undertaken within 3 days postoperatively. Our results have shown that once thrombosis occurs, there is little possibility of flap salvage, particularly 3 days after operation and in infectious cases. When no flow phenomena are observed and no flap salvage is deemed possible, aggressive treatment such as a second free flap or next pedicle flap should be chosen as soon as possible to avoid any delay in postoperative treatment.

Endogenous adipocyte precursor cells for regenerative soft-tissue engineering.

Subcutaneous injection of reconstituted basement membrane (Matrigel) in combination with basic fibroblast growth factor induces de novo adipogenesis in which endogenous precursor cells invade the artificially formed Matrigel space, proliferate and differentiate to form adipose tissue. Since this adipogenesis offers us a novel approach for soft-tissue reconstruction without transplanting preadipocytes, the early process was examined by optical and electron microscopy. Formation of multiple layers of fibroblast-like cells at the surface of Matrigel implant was the first response of connective tissue. The cells within four to five layers proximal to Matrigel implant acquired a thick cytoplasm and an enlarged nucleus, and they invaded Matrigel space together with endothelial cells which caused neovascularization. Phagocytotic incorporation and digestion of Matrigel components by well-developed lysosomes appeared to be a stimulus of fibroblast-like cells to mature depending on proximity to Matrigel. The fibroblast-like cells often contacted to the outer surface of capillary over a large area and rapidly accumulated lipid droplets. Electron microscopy of the developing adipocytes showed a well-organized smooth endoplasmic reticulum and mitochondria. This investigation thus revealed the characteristics of adipocyte precursor cells, which can be recruited for regenerative engineering of soft tissues.

Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells.

In clinical studies and animal models, low-intensity ultrasound (US) promotes fracture repair and increases mechanical strength. US also promotes cartilage healing by increasing glycosaminoglycan synthesis of chondrocytes. As mesenchymal stem cells (MSCs) have the ability to differentiate into chondrocytes, US may promote their differentiation. Here, we evaluated the effects of US on the differentiation of MSCs toward chondrocytes and cartilage matrix formation. When human MSCs cultured in pellets were treated with transforming growth factor beta (TGF-beta, 10 ng/mL), they differentiated into chondrocytes as assessed by alcian blue staining and immunostaining for aggrecan, but nontreated cell pellets did not. Furthermore, when low-intensity US was applied for 20 min every day to the TGF-beta-treated cell pellets, chondrocyte differentiation was enhanced. Biochemically, aggrecan deposition was increased by 2.9- and 8.7-fold by treatment with TGF-beta alone, and with both TGF-beta and US, respectively. In contrast, cell proliferation and total protein amount appeared unaffected by these treatments. These results indicate that low-intensity US enhances TGF-beta-mediated chondrocyte differentiation of MSCs in pellet culture and that application of US may facilitate larger preparations of chondrocytes and the formation of mature cartilage tissue.

Origin and course of nerves immunoreactive for calcitonin gene-related peptide surrounding the femoral artery in rat.

Appreciation of anatomic relationships between perivascular nerve fibers and blood vessels is essential in reconstructive surgery. We examined the origin and neural connections of perivascular nerve fibers containing calcitonin gene-related peptide surrounding the femoral artery that regulate vascular tone. We used immunohistochemistry, denervation, and retrograde labeling methods. Peptide-immunoreactive fibers surrounding the femoral artery formed a complex network, with numerous small fibers extending from nerve fiber bundles located in the perivascular connective tissue. In middle and distal arterial segments, these fibers originated from the femoral nerve, the artery's main accompanying nerve. More proximally, fibers arose from the genitofemoral nerve and sympathetic nerves. Nerve branches terminating in various arterial segments had origins corresponding to those of somatic sensory nerve fibers, although pathways innervating the femoral artery took different courses.

Usefulness of a first transferred free flap vascular pedicle for secondary microvascular reconstruction in the head and neck.

The authors found that a previously transferred free flap vascular pedicle, distal to the first microvascular anastomosis, can be used as a recipient vessel for an additional free flap transfer. Free flap transfers were performed by using the standard procedure in patients with head and neck cancer. The mean age of the patients was 62 years. Five patients were men and three were women. A second free flap was transferred for secondary primary head and neck cancer in two cases, facial deformity in two cases, osteomyelitis of the skull in two cases, recurrent cancer in one case, and exposure of a mandibular reconstruction plate in one case. The interval between the two operations was from 4 months to 12 years (median, 21 months). All secondary free flaps were performed successfully. In two cases, the external jugular vein proximal to the previously anastomosed site was used for venous drainage. In another case, additional venous anastomosis was performed for flap congestion. It became clear that a previously transferred free flap vascular pedicle could be used as a recipient vessel for microvascular anastomosis. This is an excellent procedure for additional free flap transfers.

Management of postpneumonectomy empyema using free flap and pedicled flap.

Treatment of empyema after pneumonectomy is challenging. We treated 4 patients with postpneumonectomy empyema using a free rectus abdominis myocutaneous flap together with a pedicled pectoralis major muscle flap. The pectoralis major flap was used to fill the apical portion of the cavity, while the rectus abdominis flap was used to fill the caudal portion and occlude any bronchial fistula present. All empyemas were treated successfully without recurrence. This method has the advantage of filling apical and caudal parts of the space at once, making effective use of the combined volume of the two flaps.

Reconstruction of the first and second metatarsals with free vascularised double-barrelled fibular graft after resection of a chondrosarcoma.

We report the case of a 71-year-old woman with a chondrosarcoma originating in the first metatarsal of the left foot. We performed a wide resection and reconstructed the first and second metatarsals with a free vascularised double-barrelled fibular graft. Her limb was successfully salvaged with functional and aesthetic results. Two years after surgery, the patient is able to walk without any ortho-prosthetic devices and has returned to farm work. The details of the technique applied in this case have been described in this article.

Differences in blood flow volume and vascular resistance between free flaps: assessment in 58 cases.

In free-flap transfer, blood flow in the transferred flap contributes to wound healing and to resistance against infection in the recipient site. Successful reconstructions using free tissue transfers may be required to define and choose flaps with abundant blood flow in necessary cases. We investigated blood flow in the flap by transit-time ultrasound flowmeter in 58 free-flap transfers. Flow volume was compared between flap tissues as vascular resistance in the flap was calculated. Fasciocutaneous and osteocutaneous flaps had relatively low blood flow volume, myocutaneous flaps had more, and intraperitoneal flaps had still higher blood flow volume. These differences were statistically significant. Vascular resistance significantly decreased in the same order of comparison. Our findings will help in selecting the most suitable flaps for reconstructive surgery.

Novel culture system of mesenchymal stromal cells from human subcutaneous adipose tissue.

Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and fibroblast growth factor-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) than hASCs cultured in 20% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.

A new design for free flap reconstruction of the tongue and oropharynx.

Radical resection of tongue and oropharyngeal carcinomas often results in complex defects that are challenging to reconstruct. Furthermore, remaining function should not be disturbed by the reconstruction. We introduce a new flap design and monitored swallowing and speech following reconstruction of complex oral defects. The new design consists of four lobes on a single pedicle. The first reconstructs the tongue, the second the tongue base, the third the oral floor, and the fourth the lateral wall. The records of cases performed between July 2002 and December 2006 were reviewed. Twenty-three patients (17 men and 6 women) with an average age of 60 years underwent immediate reconstruction using a free flap after radical resection of a malignant carcinoma of tongue or oropharynx using a free flap. Postoperative swallowing and speech were rated using a self-report questionnaire. Moreover, the functional outcome in patients with and without a tongue base defect was compared. All free flaps took entirely, and postoperative function was largely satisfactory. Swallowing in patients with and without a tongue base defect was similar, but speech was more impaired in patients who had a tongue base defect. Our new design is effective for reconstructing intraoral defects three-dimensionally. However, further refinement is required to preserve speech.

Analysis of 13 cases with gastroepiploic vessels used as grafts.

Free flaps are used increasingly in reconstructive surgery. However, many patients lack a satisfactory recipient vessel near the defect because of radiotherapy, post-traumatic infection, or treatment for recurrent tumor. We usually use a venous graft in such cases, but when both long arterial and venous segments are needed, we use the gastroepiploic vessels because they include extensive, viable arterial and venous segments. Between 1998 and 2006, we used gastroepiploic vessels as grafts in the 13 cases analyzed here. All defects lacked satisfactory recipient vessels nearby. Patients included 10 men and 3 women; mean age was 51.2 years (range, 14 to 82 years). Demographic and clinical background, results, and complications were analyzed retrospectively. Mean length of gastroepiploic vessels was 13.3 cm (range, 10 to 17 cm). All outcomes were satisfactory. A complication, abdominal wound infection secondary to harvesting of the gastroepiploic vessels, occurred in one case. Although the number of gastroepiploic vessels used as grafts analyzed here is relatively small, this experience confirms their clinical usefulness.

Posterolateral cervical vein as a recipient vein in reconstructive microvascular surgery of the head and neck.

Free flaps are becoming the preferred method of choice for head and neck reconstruction. However, many patients who have undergone radiotherapy and radical neck dissection or who require treatment for recurrent tumor, often present difficulty in choosing recipient vessels. The authors have noted a potential recipient vein coursing vertically along the anterior ridge of the trapezius muscle. They used this vein as the recipient vein in two patients; the two free flaps were transferred successfully without complications. This vein, which they provisionally named the posterolateral cervical vein (PLCV), is considered an important option as a recipient vein in head and neck reconstruction when more commonly used recipient cervical veins are unavailable.

Use of the internal mammary vessels as recipient vessels for an omental flap in head and neck reconstruction.

The availability of reliable recipient vessels for free flap transfer in head and neck reconstruction may be limited in cases of prior neck dissection or radiation therapy. One solution is to use the internal mammary vessels as recipients for a free omental flap. Five patients were treated with free omental flap transfer using the internal mammary vessels as recipient vessels during head and neck reconstruction. Two patients presented with a pharyngocutaneous fistula, 1 had mandibular osteomyelitis, 1 had primary esophageal cancer, and 1 had bilateral cervical radiation ulcers. All patients had received radiation therapy previously (average dose, 75.4 Gy), and 4 had undergone neck dissection (3 bilateral and 1 ipsilateral). All patients were reconstructed using a free omental flap. Four patients had a second free flap combined with the free omental flap (3 free jejunal flaps and 1 free fibular osteocutaneous flap). The mean follow-up was 26.4 months. All free flaps took entirely, the only complication ileus requiring reoperation in 1 patient. The internal mammary vessels are reliable recipient vessels for a free omental flap in head and neck reconstruction. This procedure is a good option for patients in whom previous surgery or radiation therapy has compromised local recipient vessels.

A survey of the use of synthetic surgical sutures.

UNLABELLED: We conducted a survey of the use of synthetic surgical sutures for the purpose of making clear the current status of their use. METHOD: We collected the suture packages used from each surgery over a one month period, and counted the number of sutures. RESULT: We found that for aortic aneurysm surgeries, liver surgeries, pancreatic tumor surgeries, etc., over 100,000 yen worth of synthetic surgical sutures were used. Also, for surgeries such as plastic surgery procedures that involve microsurgery, cardiovascular surgery, thyroid surgery, liver surgery, pancreatic tumor surgery, and open gynecological surgeries, the average cost of synthetic sutures is over 10% of the surgical charge. As is seen above, for surgeries which used a great amount of synthetic surgical sutures, the cost of synthetic surgical sutures occupied the surgical technical fee. Based upon the concept of separating the technical fee and the device cost, the cost of synthetic surgical sutures should be excluded from the surgical charge, and a process should be put into place whereby they can be billed to insurance separately.

Management of a total parotidectomy defect with a gastrocnemius muscle transfer and vascularized sural nerve grafting.

Immediate facial nerve reconstruction is very demanding after total parotidectomy. Under such conditions, we reconstructed facial nerves using vascularized sural nerve with free lateral gastrocnemius muscle flap. The patient was a 14-year-old male diagnosed with mucoepidermoid carcinoma of the right parotid gland. We reconstructed zygomatic, buccal, and mandibular branches of facial nerve using 2 vascularized sural nerves, medial sural cutaneous nerve, and the lateral sural cutaneous nerve. The postoperative course was good, and there was no flap trouble. The reinnervated nerve recovered from 3 months after the operation. In 6 months after operation, almost normal animation was recovered, except for the corners of the patient's mouth. The advantages of this flap are feasible harvesting in the supine position, feasible filling of the dead space, possible harvesting of 2 series of lateral sural cutaneous nerve and median sural cutaneous nerve, and less sacrifice of the donor site. The disadvantage of this method is that the diameter of the pedicle is smaller than that of the medial pedicle. But the diameters of the lateral sural artery and vein are suitable to anastomose the cervical artery and vein. This flap is one of the good options for reconstruction of facial nerves after total parotidectomy.