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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.

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.

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.

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.

Background: The free vascularized fibular bone graft, first described in 1975, has become one of the most commonly used bone grafts. It is useful for reconstructing mandibular and traumatic long bone defects and defects following tumor resection. This flap, however, does not have a long pedicle and does not have very much volume. The authors report a solution to these problems through the use of an omental flap as a bridge. Methods: Over a 3-year period, nine patients were treated by means of a free fibular osteocutaneous flap with an omental flap. These cases required bone reconstruction without suitable recipient vessels that existed near the defect. The nine patients had the following disorders: two femoral pseudoarthrosis, two tibial pseudoarthrosis, two malignant femoral tumors, one maxillary defect after tumor resection, and one femoral and tibial osteomyelitis. Results: All flaps were transferred successfully. In one case, an additional skin graft was performed because of partial omental necrosis that occurred within 2 weeks. Fibular synostosis developed in eight cases, but amputation was performed in one case because of failed fibular synostosis. In one case, femoral amputation was performed because the femoral artery became obstructed 3 months postoperatively. Conclusions: An omental flap has a long pedicle for use as a bridge flap. It is useful as a vascular flap. A combined fibular osteocutaneous and omental flap is useful for bone reconstruction without satisfactory recipient vessels that exist near the defect. Furthermore, the flap is useful for infected wounds.

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.

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.

In this study, innervation of dental pulp in the tooth of green iguana (Iguana iguana) was investigated. Cryosections of dissected maxillae and mandibles were examined by free-floating technique with anti-PGP9.5 antibodies and anti-S-100 protein antibodies, and identified by electron microscopy. Immunoreactive fibers were observed in the pulp. They appeared to be a mixture of myelinated and unmyelinated fibers with small varicosities and were distributed in the pulp along the gap between the tooth and the jaw bone. Dental pulp of polyphyodonts has not been investigated much as replacement of teeth occurs in succession. These findings may promote investigations that could improve our understanding of the function of dental pulp in reptiles.

Romberg disease is a progressive hemifacial atrophy of unknown etiology. Several methods have been described for its reconstruction. Microsurgical reconstruction, focusing on the correction of facial asymmetry and restoration of contour, has become the standard. We have succeeded a reconstructive technique for Romberg disease using by omental flap. All patients achieved good healing of the transferred omentum without recurrences and complications with a good cosmetic result on long-term follow-up. Donor-site morbidity resulting from omental free flap harvest was minimal. Based on the results of our patients in this series, the surgical results were favorable. This report describes the successful treatment of 8 patients using this approach.

While the free fibular osteocutaneous flap is indispensable for mandibular reconstruction, reliable setting is often difficult because relative positions of the bone, skin island, and vascular pedicle are critical. We have an algorithm for donor-side selection of free fibular osteocutaneous flap. From July 2002 to March 2004, we performed 15 mandibular reconstructions using free fibular osteocutaneous flaps. We retrospectively classified these procedures as follows. In type I (flap harvested ipsilaterally to defect, n = 5), the skin island was fixed to the oral cavity, and the vascular pedicle emerged from the anterior aspect of the fibula. In type II (flap contralateral to defect, n = 5), the skin island was fixed to the oral cavity, and the vascular pedicle arose from the posterior aspect. In type III (flap contralateral to defect, n = 4), the skin island was fixed to the facial skin and the vascular pedicle arose from the anterior aspect. In type IV (flap ipsilateral to defect, n = 1), the skin island was fixed to the facial skin and the vascular pedicle arose from the posterior aspect. Flaps took completely except in 1 group II case with partial necrosis. Close attention to geometric characteristics of a free fibular osteocutaneous flap facilitates reconstruction of mandibular defects and selection of donor side.

Background: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy. Methods: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2). Results: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG. Conclusion: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.

頭蓋底の再建では，頭蓋と鼻腔との確実な閉鎖が求められる。通常は遊離腹直筋皮弁などの遊離皮弁にて再建することが多い。しかし中頭蓋底のように奥深く複雑な欠損を死腔なく充填することは難しい。われわれは，中頭蓋底再建に遊離大網移植を行い有用であったので報告する。最近の5年間にわれわれの施設で行った中頭蓋底再建は8例で，側頭窩下腫瘍が3例，外耳道腫瘍が2例，上顎癌の頭蓋底侵潤が1例，副咽頭間隙腫瘍が1例，斜台腫瘍が1例であった。再建に用いたのは大網が5例，腹直筋が3例であった。大網移植を行った5例のうち1例で髄液瘻を認めたが，保存的に治癒した。腹直筋皮弁にて再建した3例のうち2例において感染し，1例は保存的に治癒したが，1例では遊離大網移植を行うことで治癒した。大網は，volumeの調節がしやすく，柔軟性があるため，狭い部分にも充填しやすく，複雑な欠損となる中頭蓋底の再建に有用であると考えられた。

The surgical strategy for maxillary reconstruction after maxillectomy has yet to be standardized. The authors developed a technique using a three-dimensional orbitozygomatic skeletal model of a titanium mesh for skeletal reconstruction after maxillectomy. From May of 1996 to September of 2000, 18 patients underwent reconstruction using the titanium mesh model in conjunction with a soft-tissue free flap following total maxillectomy for a maxillary malignancy. The soft-tissue free flap was conventional and consisted of two skin paddles to the maxillary defect. One skin paddle became the lateral nasal wall and the other was used to close the palatal defect. After modeling, the titanium mesh plate was implanted between the orbital contents and the upper edge of the free flap to lie over the front of the flap. The model was fixed to the residual zygoma laterally and to the nasal or frontal bone medially. The palatal skin paddle was anchored by three or four dermal stitches to the bottom edge of the titanium mesh to create a concave neopalate that allowed the patient to wear a denture. Thirteen of 18 patients who underwent implantation had good facial appearance and oral function. This procedure prevented lagophthalmos, facial deformity, and sagging of the palatal skin paddle caused by gravitational force. Five patients (27.8 percent) developed exposure or infection of the implant and lost the benefit of having the prosthesis. However, treatment did not require total removal of the implant. Maintaining adequate tissue volume during soft-tissue transfer on either side of the mesh plate may minimize the complication rate. Titanium mesh implantation for skeletal reconstruction after maxillectomy avoids the need for bone grafting and may be especially beneficial in fragile or aged patients.

Midkine is a heparin-binding cytokine or growth factor and promotes the migration of inflammatory leukocytes. Upon partial hepatectomy, adhesion of the intestine was less severe in midkine-deficient mice than in wild-type mice. In a newly developed assay, in which the omentum adhered to the injured peritoneal wall, the incidence of adhesion in the deficient mice was reduced to 20% of that in the wild-type mice. Administration of midkine to the deficient mice increased the frequency of adhesion. The area of adhesion was also reduced to 8.3% in the deficient mice. The extent of migration of macrophages and neutrophils in the omentum around the adhesive region was reduced in the deficient mice. Therefore, midkine was concluded to play important roles in the formation of intraperitoneal adhesions, at least partly by promoting the migration of macrophages and neutrophils to the omentum.

われわれは側頭骨亜全摘を行った側頭骨悪性腫瘍5例に対し, 顔面神経動的再建を行った。側頭骨亜全摘術を要する悪性腫瘍切除例は, 比較的稀でしかも予後不良であり, 顔面神経動的再建を行いその結果について述べた報告は少ない。神経移植と遊離皮弁移植を用いた再建法と神経血管柄付き筋肉移植を用いた再建法の術式を報告し, 顔面神経機能の回復について述べた。その結果, 顔面神経機能は25～50%程度回復することがわかった。

一般に, 下咽頭腫瘍における遊離空腸移植を除く口腔咽頭再建では, 皮弁, 筋皮弁による再建が多い。今回われわれは, 下咽頭癌における遊離空腸移植を除いた症例で, 皮膚成分を用いない口腔咽頭再建を経験したので報告するとともに考察を加えて報告する。用いた皮弁は, 空腸5例, 胃壁3例, 腹膜3例であった。空腸を用いた再建では, 咽頭皮膚瘻3例, 口腔底再建1例, 頬粘膜の再建1例であった。胃壁は頬粘膜の再建1例, 口腔底再建1例, 口蓋再建1例であった。腹膜は口腔底に2例, 頬粘膜1例に用いた。腹膜を用いた2例で, 腹膜は脱落して瘢痕治癒した。空腸による再建例は全て経過良好であった。胃壁を用いた再建では, 2例において胃酸の分泌により潰瘍ができたが, 放射線照射により1例は治癒した。粘膜を用いた再建は, 工夫次第で皮膚成分を用いた再建より, 有用な再建法になり得ると考えられた。

Skin-flap thickness is an important consideration when choosing a free flap for head and neck reconstruction. The anterolateral thigh flap, the rectus abdominis flap, and the radial forearm flap, which included the epidermis, the dermal, and the subcutaneous layers, were measured using ultrasonography in 31 patients. The mean skin and subcutaneous thickness of the anterolateral thigh flap was 7.1 mm; the rectus abdominis flap was 13.7 mm; and the radial forearm flap was 2.1 mm. Further analysis revealed a statistically significant difference among the skin and subcutaneous thickness of the three flap groups. Of the 44 anterolateral thigh flap transfers done for head and neck reconstruction after cancer ablative surgery, 41 (93.2 percent) were transferred successfully. The anterolateral thigh flap creates a moderately thick skin flap, and is less variable in thickness across its area than is the rectus abdominis flap. The flap is adaptable for reconstruction of head and neck soft-tissue defects.

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

A 72-year-old man presented with a right parapharyngeal mass, 4 cm in diameter, which was subsequently diagnosed as a metastasis originating from papillary carcinoma of the thyroid gland. The parapharyngeal tumor was successfully removed by the mandibular swing-transcervical approach with pharyngeal reconstruction, performed using a buccal mucosal island flap based on the facial artery. His postoperative course was uneventful, and the preoperative clinical symptoms such as dysphagia and head-aches completely resolved after surgery.

Expression of midkine (MK), a retinoic acid-inducible heparin binding growth factor, was examined immunohistochemically in normal and burn sites of rat skin. In the normal skin, MK was localized in the epidermis and dermal appendages such as hair follicles and sebaceous glands. Mast cells in the subdermal connective tissue also accumulated MK. After burn injury, MK-positive cells began to infiltrate into subdermal connective tissue, and the number of MK-positive cells in the region increased to a maximum at postburn day two and then decreased gradually. Western blotting analysis of both normal and postburn skin revealed a 30 kDa band reactive with anti-MK antibody; this band was concluded to be a dimer of MK. These findings were discussed from the viewpoint of the possible role of MK in wound healing.

Adipocytes forming fat pad in vivo are surrounded by well developed basement membranes. Synthesis of basement membrane is enhanced during in vitro differentiation of preadipocyte line. In order to know the role of basement membrane in adipogenesis in vivo, we injected 3T3-F442A preadipocytes subcutaneously into nude mice together with or without the reconstituted basement membrane, Matrigel. Histological sections of the fat pads newly formed by injecting the cell alone showed dense population of immature adipocytes and microvessels within 2 weeks and they matured rapidly. In contrast, injection of the cells together with Matrigel showed sparse adipocytes after 2 weeks and they matured slowly over the period of 6 weeks. Quantification of the process by measuring the weight, DNA content, triglyceride content and glycerophosphate dehydrogenase (GPDH) activity of the fat pads showed that injection of the cell alone resulted in early maturation of adipose tissue with fewer adipocytes while the presence of Matrigel decelerated but potentiated the maturation of adipose tissue with 2 fold contents of DNA, triglyceride and GPDH activity. We thus showed that reconstituted basement membrane (Matrigel) supported the survival and maturation of adipocytes.

Autografting of fat pads has a long history in plastic and reconstructive surgery for augmentation of lost soft tissue. However, the results are disappointing because of absorption of the grafts with time. The fate of transplanted fat is linked to adipose precursor cells distributed widely in connective tissues. Adipocyte precursor cells can proliferate and mature into adipocytes even in the adult body depending on microenvironment. When reconstituted basement membrane, Matrigel, supplemented with more than 1 ng/ml bFGF was injected s.c. into 6-week-old mice, the neovascularization induced within 1 week was followed by migration of endogenous adipose precursor cells, and a clearly visible fat pad was formed. The pad grew until 3 weeks after the injection and persisted for at least 10 weeks. Such de novo adipogenesis was induced reproducibly by s.c. injection of Matrigel and bFGF over the chest, lateral abdomen, or head. Adipogenesis could be induced even in ear cartilage or in muscle. Thus, our results demonstrated that an abundant population of adipose precursor cells is distributed widely in connective tissues of the adult body and that they migrate into the neovascularized plug of Matrigel for proliferation and maturation. These results suggest a technique of augmenting lost soft tissue in plastic and reconstructive surgery.

Through the frontal approach, a skull base tumor in the deep midline region is obscured by the cribriform plate. This paper demonstrates our experience of operating on seven patients with midline skull base tumors posterior to the cribriform plate using a technique of maintaining the olfactory-cribriform anatomy. Following a bifrontal craniotomy and an orbitonaso-glabellar osteotomy, a circumferential osteotomy of the cribriform plate and horizontal incision of the nasal mucosa permitted mobilization of the cribriform plate unit along with the frontal lobe, providing a wide exposure of the posterior midline area. Tumors were resected from the posterior nasopharynx, ethmoid sinus, sphenoid sinus, and upper portion of the clivus. The skull base defects were repaired with the bipedicled temporoparietal galeal flap. Two patients lost olfaction because of poor preservation of the nasal mucosa and intradural dissection of the olfactory bulb. Five patients showed complete recovery of olfaction in 8 weeks. None of the patients developed cerebrospinal fluid leakage, infection, or increased neurologic deficits. En bloc mobilization of the cribriform plate and the bipedicled temporoparietal galeal flap are very useful for the management of midline cranial base tumors posterior to the cribriform plate as long as the olfactory unit is not involved with tumors.

This study was performed to investigate the capability of iontophoretic delivery of prostaglandin E1 (PGE1) and the effect of this treatment on the viability of skin flaps on the rat dorsum model as described by Hammond and Ronald in 1993. The PGE1 level in the tissue sample under the electrode was assessed with radioimmunoassay. The iontophoretic treatment (for 20 minutes at 4 mA) with PGE1 solution (20 micrograms PGE1 in 2 ml saline) showed a significant increase of PGE1 in the flap tissue under the negative electrode (p < 0.05). Very little increase was observed after the iontophoresis of saline alone. This was not statistically significant. Iontophoretic treatment was given for 5 consecutive days following flap elevation. The survival area on the seventh postoperative day was significantly greater in the PGE1-treated flaps than that of either the control or saline-treated flaps. These results confirm the beneficial effect of iontophoretic treatment with PGE1 on the augmentation of skin flap viability.

Cultured epithelium has proven to be a good grafting material for skin defects. In our experience two kinds of epithelial cells, skin keratinocytes and mucosal cells, have been used to fabricate cultured epithelial sheets and autografted to the patients. Traumatic scars of the face were treated by cultured epidermal epithelium (CEE). The skin graft in the oral cavity was replaced by mucosa using cultured mucosal epithelium (CME). Also, the CME was applied to the skin defects at the donor sites of split-thickness skin grafts. Postsurgical follow-up showed good results. As a result, CME was useful in improving the biological environment around the abutments of dental implants, and it also promoted the re-epithelialization of skin defects. From our investigations, CEE/CME are promising treatment modalities which can reduce pain and speed up the healing process in burn patients. Therefore, cultured epithelium banks are worth establishing for auto- and allografting of skin/mucosal defects.

We investigated morphological changes after transplantation of cultured mucosal epithelium using a modified Barrandon's method (1988). Serially cultivated human mucosal epithelium was transplanted onto the reverse side of rectangular dorsal skin flaps in hairless mice. The morphological changes in the epithelium were studied using paraffin sections. The modified Barrandon's method used in this study has advantages such as minimum external trauma and less chance of infection. The cultured epithelium was taken within 1 week and gradually increased its epithelial thickness. Keratinized epithelium arises after 3 weeks. At 4 weeks after grafting, the grafted epithelium comprised 7-10 cell layers. The structure of transplanted tissue, in conjunction with surrounding connective tissues, showed dermis-like features at day 7 after transplantation. From these results, it was confirmed that cultured mucosal epithelium could be successfully transplanted and its morphology was similar to that of normal mucosal tissue.

症例は68歳の男性で, 胃切除術後の食道癌に対して右側結腸による食道再建術を施行したが, 血行障害によって挙上大腸壊死に陥った術後困難例である. 本例に対して早期に壊死腸管切除術を施行し, 2期的に有茎空腸による食道再建術を施行した. 術後縫合不全を回避するためにマイクロサージャリー下に挙上空腸の動静脈を内胸動静脈と吻合する“supercharge法”を付加することで, 術後合併症もなく満足すべき経過を得ることができた. 本法は再建腸管の血流に不安のある症例に対して, 今後積極的に用いられるべき有用な付加術式と思われる.

Cultured epidermal autografting has been employed in a variety of clinical treatments including vitiligo management. In this study, we successfully treated 2 patients with vitiligo using a short-pulsed CO2 laser and by grafting the autologous cultured epidermis. Small pieces of uninvolved skin (2 x 1 cm) were taken for cultivation from a pudendal or axillary area and were expanded into 2 pieces of epidermal sheets 100 cm. Before grafting, the lesions were abraded superficially using a short-pulsed CO2 laser with a computerized pattern generator. After successful grafting, repigmentation was visible within 1 to 2 months. One year after grafting, the skin color was almost the same as that of the surrounding normal skin. Thus, the combination of short-pulsed CO2 laser resurfacing and cultured epidermal grafting is a powerful option for treating an asymmetric and wide vitiliginous lesion.

We have achieved functional reconstruction for a bilateral upper alveolar bone, gingival, and palatal defect that has various problems originating from instability of the prosthesis using the fibula osteocutaneous flap with osseointegrated implants. The flap had three bone segments and two skin paddles. The combined bone segments created the upper alveolar arch, and the skin paddles closed the palatal defect. Nine months later, prosthodontic treatment was performed successfully. Our procedure restored the patient to masticatory function of the upper jaw, intelligible speech, and natural facial appearance. As a result, quality of life of the patient was extremely improved.

The characteristics of cultured mucosal cells from the oral mucosa were investigated and compared with those of cultured epidermal cells. Total cell counts showed that mucosal cells possessed greater proliferating ability than epidermal cells. The results of 3(4,5-dimethyle-thiazoyl-2-yl)2,5 diphenyltetrazolium bromide assay confirmed this observation and also suggested that the mucosal cells maintained biological activity longer than epidermal cells. The most important morphological characteristics of mucosal cells in culture were their low grade of differentiation. Interestingly, the epidermal cells showed enucleation and keratinization progressively during culture, whereas the mucosal cells showed no obvious enucleation when examined by light microscopy. Transmission electron microscopy showed a smaller number of desmosomes in cultured mucosal cells than epidermal cells. The results of this study reveal cultured mucosal cell sheets to be a possible material for grafting in addition to cultured epidermal cell sheets.

Cultured stratified epithelium for grafting was prepared using human keratinocytes. In the field of plastic surgery, many patients who received grafts of cultured epithelium have been reported. However, no case in the maxillofacial region has been reported. Here we report 2 cases of traumatic scars in the maxillofacial region that were treated by cultured epithelium.The patients were a 21-year-old man and a 9-year-old boy who complained of deformity due to a traumatic scar. In both cases, small skin segments, 1-2cm2, were obtained before operation, and epithelial cells were cultured by the methods of Green et al, using 3T3 cells as the feeder layer. After 3 weeks, 175-350cm2 cultured epithelium was prepared. Then, surgical debridment of the scar was carried out, and cultured epithelium was grafted onto the epithelial defects. Cultured epithelium was completely taken within 1 week. Subsequently, that the surface of the graft became smooth and pigmentation faded away.

Many clinical studies have demonstrated the usefwness of cultured epithelial grafts. Recently, cultured epithelium has been preserved by freezing and successfuly grafted after thawing. However, few papers have described morphological changes that occur after freezing cultured epithelium. We studied the morphological changes of cultured mucosal epithelial sheets and cultured epidermal sheets after freezing. In our freezing procedure, glycerin and dimethyl sulfoxide (DMSO) were used as cryoprotectants. Each epithelial sheet was stored for 3 weeks at -80°C or -196°C.The structures of both types of epithelial sheets were generally maintained after freezing storage. However, cultured mucosal epithelium showed mild showing vacuolation around the nuclei after freezing with DMSO.The results indicated that cultured mucosal epithelial sheets can be stored by freezing and that their structure can be maintained by using glycerin.

顎顔面領域の再建術に対する手術支援方法として光硬化樹脂製顔面マスクを製作し, 臨床応用した。顔面マスクの製作は非接触3次元レーザ計測装置による顔表面形状計測, CAD (Computer Aided Design) による仮想対象顔面形状データの生成, 光造形法による3次元モデリングマシンによって行った。顔面変形の把握, 術中, 再建目標としてのオリエンテーション, 術後評価基準として有用なシミュレーションモデルであった。症例を呈示し, その詳細について報告し考察する。

Objective The authors performed a study to see if gastric seromuscular patch elevation is anatomically feasible and to estimate the maximum size of the patch relative to its blood supply. Summary background data A flap composed of greater omentum and a full-thickness segment of the greater curvature of the stomach, based on the right gastroepiploic artery, was first described in 1977. Elevation of the greater omentum along with a seromuscular patch of stomach has not been reported previously. Methods Angiography was performed via the right gastroepiploic artery in a stomach obtained from ten patients who underwent gastrectomy, then india ink was injected. The stomach was then fixed in 10% formalin, and histochemical examination was performed to determine if seromuscular patch elevation was possible. Result Results of the angiography and injection experiment of india ink indicated that the territory of a single gastric ramus was approximately 5 cm x 5 cm in both anterior and posterior walls of the stomach. Histology revealed that the gastric rami lay between the muscular layer and the mucosa. Conclusion The authors believed that a composite gastric seromuscular patch and omental pedicle flap would be clinically applicable. The authors also believed that the maximum size of the patch was 10 cm x 10 cm. They used this flap to successfully treat three patients with chronic soft-tissue defects--two bronchopleural fistulae and a radiation ulcer.

To analyze irradiation effects on wound contraction, fibroblasts harvested from human oral mucosa were irradiated (single 2-, 4-, 6-, 8-, and 10-Gy exposures of x-rays). The irradiated fibroblasts were examined for growth ability, and they were cultured three-dimensionally in hydrated collagen gels. The irradiation inhibited growth of fibroblasts and contraction of collagen gels in a dose-dependent manner. In addition, inhibition depended on the duration after exposure. The organization of actin filaments of fibroblasts in this model was examined with fluorescent dye-conjugated phalloidin. Many elongated cell projections were formed in cells of the control group, but the cell projections were almost disrupted in the irradiated cells. X-ray irradiation is thus shown to inhibit wound contraction by affecting the organization of actin filaments in fibroblasts.

We report a case in which venous occlusion occurred after fibular osteocutaneous flap transfer for open tibial fracture. The flap was salvaged using an external venous shunt. The shunt consisted of a Teflon catheter for intravenous infusion, a three-way stopcock, a urokinase-immobilised catheter and a pressurising bag for arterial-pressure monitoring. This method can be used to establish venous drainage when there is no accessible recipient vein. It may improve the success rate of microvascular surgery and extend the indications for surgery.

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 HE 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.

巨大な皮膚直接浸潤で発見されたびまん浸潤型横行結腸癌を経験した.症例は79歳の女性.左下腹部腫瘤にて来院, 入院後腫瘤の表面が潰瘍形成し, カリフラワー状に腫瘍が露出した.びまん性浸潤型横行結腸癌の皮膚浸潤.S状結腸浸潤と診断し, 結腸左半切除術, 腹壁合併切除術 (肉眼的相対治癒切除) を施行した.広範な腹壁欠損部は下腹壁動静脈と浅腹壁動静脈を血管皮膚茎とした拡大腹直筋皮弁により再建した.びまん浸潤型大腸癌は診断がついた時点で広範な進展をきたしている例が多く, 転移臓器の病変が初期症状を発現することがあるが, 皮膚直接浸潤で発見されたびまん浸潤型大腸癌の報告例は本例が本邦第1例目である.また病理学的にlymphangiosis typeに属し, 元来が分化型腺癌であったため, 比較的膨張性の発育を示し切除が可能であった.さらに広範な腹壁欠損に対し拡大腹直筋皮弁といった形成外科的手技がきわめて有用であった.

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.