Single-stage reconstruction of a full-thickness alar defect using a folded nasolabial flap combined with a redundant skin turnover flap.

BACKGROUND: The reconstructive strategy for full-thickness nasal skin defects should include recreation of a cutaneous cover, support, and internal nasal lining. The most challenging aspect of this procedure is provision of the nasal lining. These reconstructions typically require a 2-step process. Satisfactory nasal skin reconstruction in a single operation is ideal. OBJECTIVE: We used a folded nasolabial flap combined with a turnover flap for reconstruction of full-thickness alar defects. METHODS: The donor material of the lining flap was a combination of the distal portion of the nasolabial flap and redundant skin resected during its transposition. The redundant skin flap was turned upside down, with the skin surface inside the nasal cavity. The remaining portion of the defect was covered with a folded nasolabial flap. RESULTS: This procedure was successful in all 5 patients. All flaps survived completely without evidence of necrosis or narrowing of airways. Aesthetic concerns, including effacement of the nasofacial sulcus, were minor. CONCLUSION: This method has the advantage of providing well-vascularized tissue of appropriate color, texture, and thickness for external coverage, as well as a satisfactory internal lining in a single-stage procedure.

Surgical management of breast deformity in a young patient with localized scleroderma: a case report and literature review.

Localized scleroderma in the chest region of adolescent girls leads to incomplete breast development and breast asymmetry, for which patients may require treatment. The site of localized scleroderma, its activity, the surgeon, and the patient's desires influence the selection of treatment method. There have been few reports on surgical treatment of this disease. In the current report, we present a case in which improved breast asymmetry was achieved through multiphased surgery, and we review treatment methods and indications of this disease.

Osteogenic potential of human bone marrow-derived mesenchymal stromal cells cultured in autologous serum: a preliminary study.

PURPOSE: As part of the authors' research on potential osteogenesis by filling bone defects with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) in patients with cleft lip and palate, they examined the cytoproliferative potential and cytobiological activity of hBM-MSCs in vitro and their osteogenic potential in vivo without performing osteoinduction. MATERIALS AND METHODS: The hBM-MSCs were collected from iliac cancellous bone and then used in primary culture, followed by 2 subcultures using an autologous serum (AS)-containing medium and a fetal bovine serum (FBS)-containing medium. Cytoproliferative potential and cytobiological activity as expressed by bone markers (alkaline phosphatase and osteocalcin) in hBM-MSCs cultured in the AS-containing medium (AS-cultured hBM-MSCs) and the FBS-containing medium (FBS-cultured hBM-MSCs) were examined in vitro, and the osteogenic potential of AS- and FBS-cultured hBM-MSCs was examined in mice. RESULTS: On day 6 of the second subculture, the number of hBM-MSCs per milliliter of specimen from 8 pediatric patients was significantly larger (P < .05) in FBS-cultured compared with AS-cultured hBM-MSCs. The alkaline phosphatase activity of hBM-MSCs was significantly greater (P < .05) when cultured in the AS-containing medium compared with the FBS-containing medium. The in vivo study showed the formation of an osteoid-like matrix rather than definite bone tissue. CONCLUSIONS: 1) FBS is appropriate for the cytoproliferation of hBM-MSCs; 2) the AS-containing medium is likely to have a good possibility of inducing the differentiation of hBM-MSCs; and 3) AS-cultured hBM-MSCs contain a group of cells that spontaneously differentiate into an osteoid-like matrix without performing osteoinduction.

Clinical trial of allogeneic cultured dermal substitutes for intractable skin ulcers.

The effect of allogeneic cultured dermal substitute (CDS) on wound healing was evaluated in 9 intractable skin ulcers in 5 patients who had failed to improve despite conventional topical treatment with basic fibroblast growth factor (bFGF) for more than 2 months. In general, the topical application of bFGF is effective in facilitating wound healing. However, skin regeneration was very slow in the present 9 cases. In this study, to improve the condition of these wounds, allogeneic CDS was applied once a week for 2 months. The wound healing process was evaluated, focusing on the reduction ratio of wound size through the granulation tissue formation associated with epithelialization. In all 9 cases, the wound size was successfully decreased after the application of CDS, and ulcers were completely resurfaced in 2 cases. In all cases, except the 2 cases showing complete wound closure, the mean wound size decreased to 33.3% of the original size, i.e., a mean reduction ratio of 33.3%. The present results indicate that allogeneic CDS can promote wound healing of intractable skin ulcers that fail to improve despite treatment with bFGF.

Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema.

The pathophysiology of secondary lymphedema remains poorly understood. To clarify the roles of cyclooxygenase (COX)-2 in enhancement of lymphangiogenesis during secondary lymphedema, we tested a mouse tail model and evaluated the recurrence of lymph flow. To induce lymphedema, a circumferential incision was made in the tail of anesthetized mice to sever the dermal lymphatic vessels. The maximum diameters of the tails were measured weekly. We found that the diameters of the tails around the wounds were markedly increased after surgery, and reached maximum size 2 weeks after wounding in mice without a COX-2 inhibitor, celecoxib (Celecoxib-). Expression of COX-2 in wound granulation tissues was markedly increased 1 week after surgery compared with unwounded naive control mice. In Celecoxib-, recurrence of lymphatic flow in the wound granulation tissues was detected 3 weeks after surgical treatment. In contrast, lymphatic flow was markedly suppressed in mice treated with celecoxib (Celecoxib+). Newly formed lymphatic structures were identified in the granulation tissues formed at wounded lesions in Celecoxib-, whereas those were markedly suppressed in Celecoxib+. Interstitial tissue pressures in the distal areas of the tail wounds were markedly increased in Celecoxib+ with reduced expression of vascular endothelial cell growth factor (VEGF)-C. F4/80-positive cells were accumulated to the wound granulation tissues in Celecoxib-, and the accumulation of these cells was suppressed in Celecoxib+. Prostaglandin E(2) (PGE(2)) upregulated the expressions of VEGF-A and VEGF-C in cultured macrophages, but not human lymphatic microvascular endothelial cells. The present study therefore suggests that lymphangiogenesis, together with recurrence of lymph flow after surgical induction of lymphedema, is upregulated by COX-2 possibly via generation of PGs.

A patient with macrodystrophia lipomatosa bilaterally affecting the entire upper extremity: reporting of a rare case and literature review.

The patient, a 58-year-old Asian female, had the progressive, bilateral overgrowth of the entire upper extremity since her childhood and has undergone debulking surgery twice in her country. However, overgrowth progressed after surgery. The patient was diagnosed with Macrodystrophia lipomatosa (MDL) by physical and imaging findings in our departments.

Hes1 regulates formations of the hypophyseal pars tuberalis and the hypothalamus.

The hypophyseal pars tuberalis surrounds the median eminence and infundibular stalk of the hypothalamus as thin layers of cells. The pars tuberalis expresses MT1 melatonin receptor and participates in mediating the photoperiodic secretion of pituitary hormones. Both the rostral tip of Rathke's pouch (pars tuberalis primordium) and the pars tuberalis expressed alphaGSU mRNA, and were immunoreactive for LH, chromogranin A, and TSHbeta in mice. Hes genes control progenitor cell differentiation in many embryonic tissues and play a crucial role for neurulation in the central nervous system. We investigated the Hes1 function in outgrowth and differentiation of the pars tuberalis by using the markers for the pars tuberalis. In homozygous Hes1 null mutant embryos, the rostral tip was formed in the basal-ventral part of Rathke's pouch at embryonic day (E)11.5 as well as in wild-type embryos. In contrast to the wild-type, the rostral tip of null mutants could not extend rostrally with age; it remained in the low extremity of Rathke's pouch during E12.5-E13.5 and disappeared at E14.5, resulting in lack of the pars tuberalis. Development of the ventral diencephalon was impaired in the null mutants at early stages. Rathke's pouch, therefore, could not link with the nervous tissue and failed to receive inductive signals from the diencephalon. In a very few mutant mice in which the ventral diencephalon was partially sustained, some pars tuberalis cells were distributed around the hypoplastic infundibulum. Thus, Hes1 is required for development of the pars tuberalis and its growth is dependent on the ventral diencephalon.

Significance of CD271 in bone marrow mesenchymal stem cells--changes by cryopreservation.

In investigating the biological activities of bone marrow mesenchymal stem cells (BMMSCs), an important task is cell sorting of effective BMMSCs. In the present study, we examined the usefulness of CD271 as a cell surface marker of BMMSCs. Specifically, we investigated (1) CD271 expression before and after freezing, (2) difference between the CD271(+) and CD271(-) fractions regarding calcium formation level after bone differentiation, and (3) method of harvesting effective BMMSCs from marrow tissue. CD271 was partially expressed in cryopreserved BMMSCs (CBMMSCs). We used CD271 in separating CBMMSCs into different cell groups and compared the calcium formation levels between the CD271-expressed and CD271-nonexpressed groups. A significant amount of BMMSCs existed even in the CD271(-) fraction, and the calcium formation level was high in 4 of 5 CD271(-) fraction specimens. The same investigation was conducted on nonfrozen BMMSCs. No major difference was found in CD271 expression compared with CBMMSCs. However, the calcium formation level of the CD271(+) fraction was higher in 3 of 5 specimens. We presumed that CD271 expression might have been substantially changed during culture and cryopreservation. We compared the method of directly culturing bone marrow tissue and that of washing the sample before culture and confirmed that the calcium formation level of BMMSCs was higher when the marrow tissue was washed before culture.

Hes1 is required for the development of craniofacial structures derived from ectomesenchymal neural crest cells.

The cranial neural crest cells contribute extensively to the formation of skeletogenic mesenchyme in the head and neck. Hes1 functions as a repressor of basic helix-loop-helix transcription factors and is implicated in controlling the maintenance of undifferentiated cells and the timing of cell differentiation. We show here that Hes1 homozygous null mutant mice exhibit multiple craniofacial malformations including calvaria agenesis, defective anterior cranial base, shortened maxilla and mandible, and abnormal palate and tongue. In the null mutant cranium, the calvarial bones, meninges including the dura mater and skin were not formed, and the brain was therefore exposed without the outer cover. The defective anterior cranial base in the mutants was attributable to the lack of presphenoid bone and the flexed cranial base angle, which was in contrast with the flat cranial base of wild-type mice. Furthermore, in the null mutants, palatal shelf growth was impaired because of the early elevation of the palatal shelves, resulting in a narrow palate and oral cavity, which were consistently associated with a small size of the tongue. These craniofacial anomalies could be the result of the defective development of neural crest cells. Taken together, it is supposed that Hes1 signaling plays an essential role in regulating the development of various craniofacial structures derived from the cranial neural crest cells.

A novel cell-adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds.

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

Comparative evaluation of re-epithelialization promoted by fresh or cryopreserved cultured dermal substitute.

Allogeneic cultured dermal substitute (CDS) was prepared by plating cultured fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen, followed by culturing for 1 week. The resulting fresh CDS was then cryopreserved in a freezer at -152 degrees C in accordance with conventional procedures. Fresh CDS was rinsed thoroughly with lactated Ringer's solution to remove fetal bovine serum (FBS) and was then used in the clinical study, whereas cryopreserved CDS was thawed and then rinsed with lactated Ringer's solution to remove cryoprotectant and FBS. The present study was designed to compare the efficacy of fresh and cryopreserved CDS in promoting reepithelialization at donor sites leaving behind split-thickness skin autografts. Fourteen donor sites were used for this comparative study. There were no differences in the period of complete re-epithelialization between the fresh and cryopreserved CDS. The results of this comparative study thus suggest that cryopreserved CDS is able to maintain the same level of potency to promote re-epithelialization as fresh CDS. This indicates that, although the release of growth factors is suppressed to some extent during the course of cryopreserving, thawing, and rinsing procedures, it is still sufficient to promote re-epithelialization.

Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture.

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.

Wound healing process of a full-thickness skin wound model in rats.

Using a rat full-thickness skin wound model, confocal laser scanning microscopy was carried out for examination of angiogenesis in granulation tissue formation. By semi-quantitation analysis with reverse transcription-polymerase chain reaction (RT-PCR), assessment was made of mRNA expression in the vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). Immunohistological analysis was also made to determine the location of these factors in granulation tissue at the protein level. Comparison was made of the cytokines in granulation and angiogenesis. Chronic granulation tissue with advanced fibrosis could be clearly demonstrated by the inhibition of wound contraction and epithelialization. From the results of the present study, VEGF would appear quite closely involved in angiogenesis in granulation tissue formation and pericytes may possibly give rise to VEGF and have significant roles in angiogenesis even with inhibition of epithelialization. CTGF expression in parenchyma is considerable during granulation and thus possibly may be related to the progress of fibrosis.

Novel mutations of the HOXD13 gene in hand and foot malformations.

Homeobox genes encode a set of transcription factors of fundamental importance for body patterning during embryogenesis. Hoxa9-a13 and Hoxd9-d13 play an especially important part in vertebrate limb development. Synpolydactyly (SPD) is characterized by various malformations of the limbs. The expansion of the polyalanine tract in 1OXD13 is one of its major causes. Recently, there have been many analysis studies of HOXD13 in patients with SPD and limb malformations. We analyzed HOXD13 in 100 patients with limb malformations, which affects the limbs in the distal parts of the metacarpal and/or metatarsal bones. Seven mutations in the coding region and two mutations in the 5'-untranslated region were identified. All were novel mutations. In this study, the mutations were located upstream in the homeobox. Thus, translation of the homeobox was affected by upstream mutations. Consequently, this suggested the possibility that abnormalities in the hands and feet could be caused by novel HOXD13 gene mutations.

Laminin peptide-conjugated chitosan membrane: Application for keratinocyte delivery in wounded skin.

Tissue engineering requires the delivery and survival of cells to organ sites needing repair. Previously, we showed that an active laminin peptide (AG73: RKR-LQVQLSIRT)-conjugated chitosan membrane promoted cell adhesion and spreading in vitro. Here, we seeded human keratinocytes onto AG73-chitosan membranes and found that nearly 80% of the cells were attached to the membranes within 2 h. The membranes carrying the keratinocytes were inverted and placed onto exposed muscle fascia on the backs of nude mice. After 3 days, the keratinocytes had migrated from the membrane and established a stratified epidermis-like structure on the fascia. Cells recognize the AG73 through transmembrane proteoglycan syndecans, which recognition system has not previously been tested in tissue engineering applications. We suggest that the AG73-chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system such as delivering keratinocytes to a wound bed.

Clinical features of primary vein grafts in free tissue transfers.

The outcomes of free tissue transfers combined with vein grafts have been inconsistent, and discussions continue regarding their appropriate use. Of the 142 free tissue transfers that we performed from January 2004 to December 2011, we retrospectively analyzed 15 consecutive patients who underwent free tissue transfers in combination with vein grafts. Etiologies included trauma (8 patients), infection (4), and tumor (3). Types of free tissue transfers were fibula (4), anterolateral thigh (3), groin (3), jejunum (3), latissimus dorsi (1), and dorsal pedis (1). Vein grafts were used for the artery (6), vein (2), or both (7). The donor veins were the saphenous vein (12) and the external jugular vein (3). The mean length of the grafted veins was 10.8 cm (range: 4-18 cm). Even though complications of congestion occurred in 2 patients, these flaps survived by reexploration. The flap success rate was 15 of 15 (100%) of vein grafted free flaps versus 124 of 127 (97.6%) of free flaps not requiring vein grafts. To improve the success rate of free tissue transfers combined with vein grafts, securing healthy recipient vessels, meticulous surgical handling, a reliable vascular anastomosis technique, and strict postoperative monitoring are crucial.

Effects of neuropeptides and their local administration to cutaneous wounds in sensory-impaired areas.

Wound healing in sensory-impaired areas such as diabetic foot and spinal cord injuries is intractable. Previous studies have shown that delayed wound healing both of wound contraction and epithelialization in denervated rat skin. The aim of this study was to investigate whether the wound healing process was affected by the administration of substance P to skin defects and histological analysis in denervated skin. Full thickness circular skin defects 15 mm in diameter were made symmetrically on the denervated area and the normal innervated area, and substance P and vehicle was administered over a period of 3 days by injecting with a syringe. The rate of wound contraction and epithelialization were measured. The wound surface area in saline injections were larger than the group of substance P injections in denervated area and controls (p < 0.05) on day 3. Wound healing in local administration of substance P to denervated skin defect was equal to in normal animals. It seems that the presence of substance P in the wound area positively affects the early stages of wound healing.

Preoperative risk factors of lymph node metastasis in cutaneous squamous cell carcinoma.

Lymph node metastasis of cutaneous squamous cell carcinoma ("SCC") affects the prognosis. A variety of risk factors of lymph node metastasis have been reported. Predicting lymph node metastasis prior to surgery, which is a major treatment method for cutaneous SCC, contributes to the effects of treatment. Factors that can be obtained prior to surgery were weighed between a lymph node metastasis group and a non-metastasis lymph node group. One hundred and sixty-four cutaneous SCC patients were operated on. The following factors, which can be obtained prior to surgery, were compared between the lymph node metastasis group and the non-metastasis lymph node group: age, sex, tumour size, symptom period, lesions, and local recurrence. The detection rate from lymph node metastasis of the sentinel lymph node biopsy using the blue dye technique was studied. Among all subjects, lymph node metastasis was observed in 17 cases (10.4%). Lower lip SCC was observed only in the higher metastasis rate. Significant local recurrence occurred more frequently in the lymph node metastasis group. For other factors, no significant difference was observed between the lymph node metastasis group and the non-metastasis lymph node group. A sentinel lymph node biopsy was given in 21 cases, two false-negative cases were observed, and local recurrence and lymph node metastasis were observed postoperatively. Operation should be given to the lower lip SCC and local recurrence cases considering lymph node metastasis. It is hard to say that the sentinel lymph node biopsy of cutaneous SCC using the blue dye technique has sufficient detection rates.

Free tensor fascia lata flap and synthetic mesh reconstruction for full-thickness chest wall defect.

A large full-thickness chest wall defect over 10 cm in diameter requires skeletal reconstruction and soft tissue coverage. Use of various flaps for soft tissue coverage was previously reported, but en bloc resection in each case affects these flap pedicles and sizes. We present a case of a 74-year-old man with a soft tissue tumor involving the left lateral chest wall. We performed an en block resection and skeletal reconstruction using a mesh, free tensor fascia lata (TFL) flap for soft tissue coverage. This procedure could be performed in one position. A fixed fascia lata of the flap was also useful for tight reconstruction with the mesh. We suggest that free TFL and/or anterior lateral thigh flap is a useful technique to reconstruct anterior to posterior lateral chest wall defects.