吉村 浩太郎

Several putative biomarkers have been suggested for identifying murine follicular stem cells; however, human hair follicles have a different pattern of biomarker expression, and follicular stem cell isolation methods have not been established. To isolate a stem cell population applicable to clinical settings, we conducted a comprehensive survey of the expression of stem-cell-associated (K15, CD200, CD34, and CD271) and other biomarkers (K1, K14, CD29, and CD49f) in immunohistological sections of the human epidermis and follicular outer root sheath (ORS). We also examined freshly isolated and cultured epidermal or follicular cells with single- and multicolor flow cytometry or immunocytochemistry. After sorting cells by CD200, CD34, and forward scatter (FSC) values (cell size), colony-forming assays were performed. We found that biomarkers were differentially expressed in the epidermis and ORS. Basal bulge cells were mainly K15(+)CD200(+)CD34(-)CD271(-), and suprabasal cells were K15(-)CD200(+)CD34(-)CD271(-). We categorized follicular cells into nine subpopulations according to biomarker expression profiles. The CD200(+)CD34(-) bulge cells had much higher colony-forming abilities than the CD34(+) population, and were divided into two subpopulations: a CD200(+)CD34(-)FSC(high) (K15-rich, basal) and a CD200(+)CD34-FSC(low) (K15-poor, suprabasal) population. The former formed fewer but larger-sized colonies than the latter. Follicular epithelial cell cultivation resulted in loss of K15, CD200, CD34, and CD271 expression, but maintenance of K14, CD29, and CD49f expression. We found that the bulge contained two populations with different localizations, cell sizes, and colony-forming abilities. We showed that K15, CD200, CD34, and CD271 were useful biomarkers for characterizing freshly isolated human follicular epithelial cells in diverse stages of differentiation. Laboratory Investigation (2009) 89, 844-856; doi:10.1038/labinvest.2009.48; published online 8 June 2009

Background: The dermal papilla (DP) interacts with epithelial cells for folliculogenesis. For translational research on cell therapies for hair regrowth with cultured human DP cells (hDPCs), a model to evaluate the capacity of hDPCs to induce hair formation is inevitable. Methods: Chamber models were constructed by transplanting 4 different combinations of mouse or human epithelial and mesenchymal cells into a silicone chamber implanted onto the back of nude mice. In parallel, 3 types of sandwich constructs were created by inserting hDPCs or human DP tissue between the epidermis and dermis of isolated rat footpad skin or human facial skin, and subcutaneously transplanting the constructs into the back of nude mice. Four to six weeks later, skin sections of each model were histologically examined. Results: Folliculoneogenesis was detected in both chamber and sandwich models, although the induction rate and maturity of the hair follicles varied among cell combination subgroups in each model. The difference in hair induction rate was not statistically significant between 2 representative chamber and sandwich subgroups using cultured hDPCs. The sandwich model, however, required fewer hDPCs, did not require human keratinocytes, and exhibited a higher rate of successful sample collection. Conclusions: Although there is no significant difference in hair induction rate, the sandwich model using cultured hDPCs and the rat sole skin is more feasible than the chamber model using human cultured keratinocytes and hDPCs as a tool to evaluate the hair-inducing capacity of cultured hDPCs. Copyright (c) 2008 S. Karger AG, Basel

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs. STEM CELLS 2009; 27: 238-249

Background: To avoid potential risks of animal-derived products such as viral transmission and immunologic reactions, usefulness of human-derived products in manipulation of cells for cell-based therapies has been investigated but has not yet been completely clarified. Methods: Three types of human sera-serum from whole blood, serum from platelet-rich plasma, and serum from platelet-poor plasma-were prepared from blood samples obtained from the same four volunteers. The authors investigated the biochemical profiles of the three serum preparations as well as their potential as culture additives using three types of human cells: dermal fibroblasts, adipose-derived stem/stromal cells, and umbilical vein endothelial cells. Results: Platelet counts differed among serum from whole blood (100 percent), platelet-rich plasma (75.1 percent), and platelet-poor plasma (12.6 percent), resulting in differential concentrations of platelet-derived growth factor and epidermal growth factor, although other biochemical values such as total protein and albumin were similar. Serum from whole blood and platelet-rich plasma highly enhanced proliferation of dermal fibroblasts compared with the effects of serum from platelet-poor plasma, but no differences in proliferative efficacy were observed in cultures of adipose-derived stem/stromal cells and vascular endothelial cells. Conclusions: Serum from platelet-rich plasma, which is less invasive to prepare than serum from whole blood, was superior to serum from platelet-poor plasma as a substitute for animal-derived serum in culture expansion of dermal fibroblasts. Although autologous or human-derived serum preparations may be of great use in cell-based therapies, this usefulness strongly depends on the target cell species and the purpose of the cell culture.

Background: A reliable method with which to assay viability and number of adipocytes and other cellular components in adipose tissue remains to be established. Methods: The authors assessed cell viability and number obtained from 1 g of suctioned adipose tissue and respective layers (the top, middle, and bottom layers) before and after digestion and centrifugation, using cell staining with Hoechst 33342 and propidium iodide and the 2,3-bis(2-methoxy-4-vitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazoliumhydroxide (XTT) and glycerol-3-phosphate dehydrogenase assays (n = 10). The correlation between the number of prepared cells (adipocytes, adipose stromal cells, and white blood cells) and the resulting values from the XTT and glycerol-3-phosphate dehydrogenase assays was also examined (n = 5). The cell composition of the stromal vascular fraction isolated from the same adipose tissue was determined by multicolor flow cytometry (n = 5). Results: Hoechst 33342 and propidium iodide staining allowed distinguishing of viable adipocytes from lipid droplets, dead adipocytes, and cells other than adipocytes. The authors obtained 6.9 X 10(5) nonruptured adipocytes from 1 g of suctioned adipose tissue; 30 percent of the original adipocytes appeared to have been ruptured. Both the XTT and glycerol-3-phosphate dehydrogenase assays provided good correlations between the number of viable adipocytes and resulting values, but only the glycerol-3-phosphate dehydrogenase assay was strictly specific for adipocytes. The ratio of adipose stromal cells to adipocytes was found to be much larger than previously described. Conclusion: Single use or a combination of the viability assays used in this study can appropriately determine the number of adipocytes and other cells, although it remains difficult to assess original cells directly without tissue dissociation.

Background: Although injective autologous fat transplantation is one of the most attractive options for soft-tissue augmentation, problems such as unpredictability and fibrosis resulting from fat necrosis limit its universal acceptance. Centrifugation is one of most common methods for overcoming these difficulties. This study was performed to investigate quantitatively the effects of centrifugation on liposuction aspirates to optimize centrifugal conditions for fat transplantation and isolation of adipose-derived stem cells. Methods: Liposuction aspirates, obtained from eight healthy female donors, were either not centrifuged or centrifuged at 400, 700, 1200, 3000, or 4200 g for 3 minutes. The volumes of the oil, adipose, and fluid portions and numbers of blood cells and adipose-derived cells in each portion were examined. The processed adipose tissues (1 ml) were injected into athymic mice, and grafts were harvested and weighed at 4 weeks. Morphologic alterations were observed using light and scanning electron microscopy. Results: Centrifugation concentrated adipose tissues and adipose-derived stem cells in the adipose portion and partly removed red blood cells from the adipose portion. Centrifugation at more than 3000 g significantly damaged adipose-derived stem cells. Centrifugation enhanced graft take per 1 ml centrifuged adipose but reduced calculated graft take per 1 ml adipose before centrifugation. Conclusions: Excessive centrifugation can destroy adipocytes and adipose-derived stem cells, but appropriate centrifugation concentrates them, resulting in enhanced graft take. The authors tentatively recommend 1200 g as an optimized centrifugal force for obtaining good short- and long-term results in adipose transplantation.

Background: Few studies have addressed anti-androgenic therapy using oral spironolactone for acne in Asians. Obtaining this race-specific information is important because Westerners and Asians respond differently to hormone therapy. This study aimed to examine the efficacy and safety of oral spironolactone used to treat acne in Asians. Methods: Spironolactone (initial dose, 200 mg/day) was administered orally to 139 Japanese patients (116 females and 23 males) with acne. Serum laboratory data, including various hormones and electrolytes, were examined for 25 of the subjects. Results: Most of the female patients who completed the 20-week regimen exhibited excellent improvement (evaluated by a photographic grading scale), although some discontinued treatment because of menstrual disturbances or other reasons. The treatment was less efficacious for the males than for the females, and because gynecomastia developed in three male patients, spironolactone treatment for males was stopped. Examination of the serum of 25 patients did not identify any toxicity associated with the treatment. Drug eruptions and edema in the lower extremities were each seen in three patients. Conclusion: Oral spironolactone is effective and safe for the treatment of acne in Asian females, and can be a good option for severe, recurring, and widespread types of the condition.

Sonic hedgehog (Shh) is a well-known morphogen indispensable in facial and nervous development, and recently it has also garnered much attention as a potent angiogenic factor. We previously created an animal model of holoprosencephaly by administration of cyclopamine, a specific inhibitor of hedgehog signaling, to the mouse embryos cultured in vitro, and found several types of angiogenic defects. In this study, we focused on other angiogenic phenotypes in the same model. When cyclopamine was added for embryonic day (E) 8.0-9.5, a pair of immature dorsal aortae, which normally fuse to form the single aorta by E9.5, remained to be separated. Expressions of vascular endothelial growth factor and bone morphogenetic protein 4, putative mediators of aortic fusion, were also reduced around the aorta by blockade of Shh signaling. When cyclopamine was added for E8.5-10.5, vessels on the surface of craniofacial region (possibly external cardinal veins) were extended and malformed. These results suggest that Shh signaling is essential for some aspects of embryonic angiogenesis, and that pathophysiology of holoprosencephaly may involve, at least in part, the Shh-dependent angiogenesis.

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34(+)CD45(-) cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45(+)), ASCs (CD31(-)CD34(+)CD45(-)CD90(+)CD105(-)CD146(-)), endothelial (progenitor) cells (CD31(+)CD34(+)CD45(-) CD90(+)CD105(low)CD146(+)), pericytes (CD31(-)CD34(-)CD45(-)CD90(+)CD105(-)CD146(+)), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.

Background: The mechanisms responsible for the incomplete recovery of muscle function after microneurovascular transfer have not been fully determined. Because fiber degeneration and regeneration can impact muscle mechanical function, the authors tested the null hypothesis that ischeinia-induced fiber degeneration is not responsible for the force deficits observed after neurovascular muscle transfer. Methods: Rats were assigned to one of three groups: orthotopic. nonvascularized grafting of the extensor digitorum longus muscle (STD group); orthotopic, nenrovascular transfer of the extensor digitorum longus muscle With no intra-operative ischemia (NV-0 group); or orthotopic, neurovascular transfer of the extensor digitorum longus muscle with 3 hours of intraoperative ischemia (NV-3 group. At 1 and 2 weeks, extensor digitorum longus muscle cross-sections were labeled For developmental in),myosin heavy chain isoforms, markers of fiber regeneration. Results: In extensor (digitorum longus muscles from animals in the STD group, many small cells strongly), labeled for developmental myosin heavy chain were observed and identified as inyoblasts, indicating recent muscle fiber necrosis With Subsequent regeneration. Extensor digitorum longus muscles from rats in the NV-0 and NV-3 groups contained no cells labeled for developmental myosin heavy chain. Conclusions: After neurovascular muscle transfer (with ischemia times up to 3 hours), ischemia-induced muscle fiber degeneration and regeneration does not Occur. muscle fiber degeneration is not responsible for the force deficits observed after microneurovascular skeletal muscle transfer.

BACKGROUND AND OBJECTIVE Melasma and acquired dermal melanocytosis (ADM; acquired bilateral nevus of Ota-like macules) are both seen most commonly symmetrically on the face of women with darker skin and are also known as difficult conditions to treat. METHODS Our topical bleaching protocol with 0.1 to 0.4% tretinoin gel and 5% hydroquinone was performed repeatedly (1-3 times) for melasma (n=163), and a combination treatment with topical bleaching and Q-switched ruby (QSR) laser was performed repeatedly (1-3 times) for ADM (n = 62). RESULTS There is a significant correlation between clinical results (clearance of pigmentation) and the number of sessions in both melasma (p=.019) and ADM (p <.0001). CONCLUSION The repeated treatment protocol for melasma and ADM showed successful clinical results compared with conventional ones, and they may be applied to other pigment conditions. It may be better that epidermal and dermal pigmentations are treated separately, especially in dark-skinned people who are more likely to suffer postinflammatory hyperpigmentation after inflammation-inducing therapies.

We have constructed a pigmented skin equivalent and used it to study the hyperpigmentation seen in cafe-au-lait macules to elucidate whether the pigmented skin equivalent could be used as a model of congenital hyperpigmentary disorders. When we used fibroblasts derived from cafe-au-lait macules of neurofibromatosis type 1, the amount of pigment was significantly greater than in models using cells derived from normal skin. Quantities of pigment were not seen when keratinocytes derived from solitary cafe-au-lait macules were used, a possible reason being that keratinocytes on the skin equivalent are in a proliferating condition and are not well-differentiated enough to act on other cells. Our results suggested that our pigmented skin equivalent is useful for the study of congenital hyperpigmentary disorders, although insufficient differentiation of keratinocytes might be a disadvantage.

To clarify the mechanism of accentuated melanisation in non-syndromic solitary cafe-au-lait macules we used an enzyme-linked immunosorbent assay (ELISA) to measure the concentration of melanogenic cytokines secreted by cultured keratinocytes and fibroblasts derived from the skins of the macules and compared them with those derived from normal people. Endothelin-1 (ET-1) was significantly increased in cultured keratinocytes in the macules compared with the normals. In contrast, the secretion of other cytokines secreted by keratinocytes or fibroblasts did not differ between the groups. It may be that the increased secretion of ET-1 by epidermal keratinocytes has a role in the accentuated epidermal melanisation seen in non-syndromic macules.

In denervated skeletal muscle, mononuclear interstitial cells accumulate in the perisynaptic regions before fibrotic change occurs. These cells are currently considered to be fibroblasts that originate from muscle tissue. However, when we denervated hind limbs of GFP-bone marrow chimeric mice by excising the sciatic nerve unilaterally, many bone marrow-derived cells (BM-DCs) infiltrated the interstitial spaces and accumulated in the perisynaptic regions, peaking 14 days after denervation. They accounted for nearly one-half of the increase in mononuclear interstitial cells. Although BM-DCs did not incorporate into satellite cells, immunohistochemical and FACS analyses revealed that BM-DCs were both CD45 and CD11b positive, indicating that they were of macrophage/ monocyte lineage. BrdU staining showed inactive proliferation of BM-DCs. Reverse transcriptase-polymerase chain reaction of mononuclear cells isolated by FACS revealed that BM-DCs did not express type I collagen or tenascin-C; however, they did express transforming growth factor-beta 1, suggesting that they regulate the fibrotic process. In contrast, muscle tissue-derived interstitial cells expressed type I collagen and tenascin-C, suggesting that these populations were the final effectors of fibrosis. These findings identify elementary targets that may regulate the migration, homing, differentiation, and function of BM-DCs, leading to amelioration of the excessive fibrosis of denervated skeletal muscle.

The problem of postoperative reduction of projecting reconstructed nipples remains to be resolved. To this end we did a clinical study of reconstructing the nipple at the same time as the breast. A tissue-expander was placed under the skin of the breast at the first operation, and then the breast and nipple were reconstructed at the second. A nipple was reconstructed using a dermal-fat flap harvested from the myocutaneous flaps used for reconstruction of the breast. A small hole was made in the corresponding site of the skin of the breast, and the reconstructed nipple was projected through the hole. This method was used in 8 cases. This method is useful in reconstructing a breast without a pad of skin and a projected nipple simultaneously. Its disadvantages are the relatively weak blood supply of the flaps, and difficulty in calculating the position of the nipple. The procedure may be beneficial for selected cases.

A prominent mandibular angle is a relatively common aesthetic problem among Asians, and the reduction angle-splitting ostectomy is now becoming a very popular procedure in Asian countries. Although this operation is usually performed on young patients, the same aesthetic demands are also seen in the elderly. In this report, the authors describe their experience with angle-splitting ostectomies followed by face lifts in three patients older than 50 years. The angle-splitting procedure was the same as that performed in young patients, and clinical results were assessed with photographs and three-dimensional computed tomographic scans. The facial contours after angle-splitting ostectomy were satisfactory, but the patients showed postoperative redundancy of the skin, especially along the jaw line, because of the loss of bony protrusion laterally. Therefore, the patients underwent subsequent superficial musculoaponeurotic system cheek lifts. The final aesthetic results were satisfactory in all cases. When surgeons want to perform the angle-splitting ostectomy safely and effectively on the elderly, they should be aware of the risks and indications specific for elderly patients, and a multidisciplinary support system should be available. Subsequent face lifts can improve skin redundancy and lead to better cosmetic results.

The pathogenesis of holoprosencephaly is multifactorial, and blockage of Sonic hedgehog signaling is one of the most important causative factors in animal models and human cases. In this study, the authors analyzed facial anomalies of mouse embryos, which were cultured in vitro and exposed to cyclopamine, an alkaloid blocker of Sonic hedgehog signaling. When cultured with cyclopamine for embryonic day 8.5 to 10.5, the whole body size was smaller than normal, and the distance and angle between the nasal placodes were remarkably reduced. Extension of the cranial surface vessels also was noted. No cyclopia was observed. Migration of the cranial neural crest cells seemed to be intact. Expressions of Patched-1 and Gli-1, downstream genes of Sonic hedgehog signaling, also were down-regulated in in situ hybridization and real-time reverse transcriptase-polymerase chain reaction analyses. The authors consider that these facial anomalies represent milder phenotypes of holoprosencephaly.

BACKGROUND AND OBJECTIVE. Acquired dermal melanocytosis (ADM; acquired bilateral nevus of Ota-like macules) is known for its recalcitrance compared with Nevus of Ota, and we assume that one of the reasons is a higher rate and degree of postinflammatory hyperpigmentation (PIH) seen after laser treatments. METHODS. Topical bleaching treatment with 0.1% tretinoin aqueous gel and 5% hydroquinone ointment containing 7% lactic acid was initially performed ( 4 to 6 weeks) to discharge epidermal melanin. Subsequently, Q-switched ruby (QSR) laser was irradiated to eliminate dermal pigmentation. Both steps were repeated two to three times until patient satisfaction was obtained ( usually at a 2-month interval for laser sessions). This treatment was performed in 19 patients with ADM. Skin biopsy was performed in six cases at baseline, after the bleaching pretreatment, and at the end of treatment. RESULTS. All patients showed good to excellent clearing after two to three sessions of QSR laser treatments. The total treatment period ranged from 3 to 13 ( mean of 8.3) months. PIH was observed in 10.5% of the cases. Histologically, epidermal hyperpigmentation was observed in all specimens and was dramatically improved by the topical bleaching pretreatment. CONCLUSION. QSR laser combined with the topical bleaching pretreatment appeared to treat ADM consistently with a low occurrence rate of PIH and lessen the number of laser sessions and total treatment period and may also be applied to any other lesions with both epidermal and dermal pigmentation.

Among concerned nasal appearances, a deformity with supero-lateral displacement of the nostril rim, called retracted nostril rim or elevated nostril rim is commonly seen and is considered one of the most difficult types of cases to treat aesthetically. A new surgical method for treating retracted nostril rim was performed in 10 patients, using the combination of auricular composite graft, internal fixation with a retainer, and external continuing suspension with anchoring sutures. The procedure was successful in maintaining the grafted cartilage in the ideal position and in avoiding recurrence of retraction or elevation of the constructed alar rim. The presented method merits consideration as a standard operative approach for correction of retracted nostril rim.

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 X 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells. may still be stronger than extrinsic regulation by mesenchymal fibroblasts.

Keloids are skin abnormalities that are characterized by excessive deposition of collagen bundles in the dermis. Patients with keloids complain not only about their cosmetic appearance, but also about continuous itching and/or tenderness associated with chronic inflammation. Degradation of extracellular matrix (ECM) may be upregulated, associated with the expansion of keloids into circumferential skin, and high metabolic activity of keloid tissues may be due to increased matrix metalloproteinase (NIMP) activity. Based on these hypotheses, we examined differences in expression of MMP-1, MMP-8, and MMP-13 between keloid-derived and normal dermal fibroblasts. Since retinoids are potent inhibitors of MMPs in the treatment of photoaged skin and cancers, we also examined whether or not tretinoin affects MMP expression of keloid-derived fibroblasts. The results of real-time polymerase chain reaction and ELISA demonstrated significant upregulation of MMP-13 and significant downregulation of MMP-1 and MMP-8 in keloid-derived fibroblasts, at both mRNA and protein levels. MMP-1 mRNA expression in the control group was significantly upregulated after the addition of tretinoin, whereas no significant change was observed in the keloid group. MMP-8 mRNA expression in the control group was significantly upregulated by tretinoin, with the peak at 12 h. while no significant change was observed in the keloid-derived fibroblasts. In contrast, the remarkably elevated MMP-13 mRNA expression in the keloid group was significantly suppressed, with the peak suppression at 12 h after addition of tretinoin, while MMP-13 mRNA expression in the control group was not significantly changed. The decrease in MMP-1 and MMP-8 may contribute to accumulation of type I and type III collagen in keloid tissues, and this mechanism may be modulated by molecular interaction with MMP-13. Tretinoin appeared to reverse the abnormal expression profile of MMPs in keloid-derived fibroblasts, such as markedly elevated expression of MMP-13, partly through inactivation of AP-1 pathway. The present results suggest that tretinoin may be clinically useful to improve the chronic inflammation seen in keloids and prevent expansion of keloid tissues into circumferential normal skin.

It was recently revealed that epidermal growth following topical treatment with all-trans retinoic acid (atRA) was at least partly induced by heparin-binding epidermal growth factor-like growth factor (HB-EGF) released from suprabasal keratinocytes. Since proliferation of keratinocytes appears to be one of the critical roles of atRA in depigmentation treatment and promotion of wound healing, HB-EGF is considered suitable for assessing the therapeutic value of topical retinoids. In this study, HB-EGF mRNA expression in normal human keratinocytes after atRA treatment was examined, and the effects of a variety of natural and synthetic retinoids were compared. The results of reverse transcription polymerase chain reaction (RT-PCR) suggested that induction of differentiation increased HB-EGF mRNA expression in cultured keratinocytes. Real-time PCR analyses revealed that HB-EGF mRNA expression was elevated dose-dependently with atRA, peaking at 12 h. This elevation was more prominent in confluent keratinocytes than in subconfluent cells, suggesting that differentiated keratinocytes are more subject to stimulation of HB-EGF expression by atRA than proliferating keratinocytes. HB-EGF mRNA was upregulated in differentiation-induced keratinocytes by all retinoids used in this study at 1 mumol/l, and marked upregulation was seen when treated with three isotypes of retinoic acid (atRA, and 9-cis and 13-cis retinoic acid). RARalpha-selective agonists (Am80, Am580, ER-38925, and TAC-101) and a panagonist of RARs (Re80) caused relatively low elevation of HB-EGF transcripts, as did all-trans retinol (Rol) and all-trans retinal (Ral). Although another panagonist (Ch55) showed the highest elevation of HB-EGF mRNA, it was relatively cytotoxic at the concentration employed. Ral and Rol were found to upregulate HB-EGF when used at 100 mumol/l to 1 mmol/l, to a similar extent of atRA at 1-10 mumol/l. The capacity of retinoids to upregulate HB-EGF may be an important index for investigation and development of an ideal synthetic retinoid, which has maximum benefits and minimum side-effects.

Epithelium in the nail matrix is different from that at other body sites, in terms of clinical and histological appearance. Hard keratins are exclusively expressed in the nail matrix and bed and the hair apparatus, and hard keratin is considered a differentiation marker of these sites. Whether the expression of hard keratin in non-nail-matrical keratinocytes could be induced by nail-matrical fibroblasts v,,as examined. Skin equivalents were constructed in three ways; ventral keratinocytes (from the ventral side of the digit) were cocultured with ventral fibroblasts (group A), ventral keratinocytes were cocultured with nail-matrical fibroblasts (group B), and nail-matrical keratinocytes were cocultured with ventral Fibroblasts (group C). Immunohistochemical examinations with anti-hard keratin antibody (HKN-7) revealed hard keratin expression in groups B and C. HKN-7-positive cells were distributed continuously in the en tire epithelial strata or in the suprabasal layer in group B, whereas HKN-7-positive cells were distributed spottily in group C This study indicates extrinsic hard keratin induction in non-nail-matrical keratinocytes by nail-matrical fibroblasts and suggests that non-nail-matrical epidermal grafts may be effective in the treatment of deepithelized nail injuries. In addition, it is possible that lost nails could be reconstructed with grafts of "tissue-engineered' nail equivalent.

Oral mucosa heals faster with less scar formation than skin and a hypertrophic scar is very rare in the oral cavity, but its mechanism has not been elucidated enough. To elucidate whether or not there are differences in growth factor expression between fibroblasts derived from buccal mucosal and normal skin, we investigated the expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) by cultured fibroblasts. The semiquantitative RT-PCR revealed that the expression of HGF and KGF transcripts by buccal mucosal fibroblasts was significantly elevated compared with that by dermal fibroblasts. In parallel, ELISA revealed the significant increase of HGF production by buccal mucosal fibroblasts. The level of production of SCF protein did not differ significantly. Our study suggests that increased expression of HGF and KGF by buccal mucosal fibroblasts may partly be responsible for the faster wound healing with less scar formation in the oral cavity compared with normal skin. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Oral mucosa heals faster with less sear formation than skin and a hypertrophic scar is very rare in the oral cavity, but its mechanism has not been elucidated enough. To elucidate whether or not there are differences in growth factor expression between fibroblasts derived from buccal mucosal and normal skin, we investigated the expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) by cultured fibroblasts. The semiquantitative RT-PCR revealed that the expression of HGF and KGF transcripts by buccal mucosal fibroblasts was significantly elevated compared with that by dermal fibroblasts. In parallel, ELISA revealed the significant increase of HGF production by buccal mucosal fibroblasts. The level of production of SCF protein did not differ significantly. Our study suggests that increased expression of HGF and KGF by buccal mucosal fibroblasts may partly be responsible for the faster wound healing with less scar formation in the oral cavity compared with normal skin. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Many methods have been reported for the treatment of ganglions. The authors present their modified technique for ganglion sclerotherapy. Their modification enables them to perform sclerotherapy safely and consistently, and they have treated 10 patients in this manner. The method is described and the cases are illustrated.

Sensory or motor "baby-sitting" has been proposed its a clinical strategy to preserve muscle integrity if motion-specific axons must regenerate over a long distance to reach denervated target muscles. Denervated muscles are innervated temporarily by using axons from nearby sensory or motor nerves. After motion specific motor axons have reached the target, the baby-sitter nerve is severed and motion-specific axons are directed to [lie target. Although this strategy minimizes denervation time, the requisite second episode of denervation and reinnervation might be deleterious to muscle contractile function. This study was designed to test the hypothesis that two sequential episodes of skeletal muscle denervation and reinnervation result in greater force and power deficits than a single peripheral nerve injury and repair. Adult Lewis rats underwent either transection and epineurial repair or sham exposure of the left peroneal nerve. After a 4-month recovery period, the contractile properties of the extensor digitorum longus muscle of the sham exposure group (control, n = 9) and one of the nerve division and repair groups (repair group 1, n = 9) were evaluated with measurements of the maximum tetanic isometric force, peak power, and maximal sustained power. A third group of rats Underwent a second cycle of nerve division and repair (repair group 2, n = 9) at this same time point. Four months postoperatively, contractile properties of the extensor digitorum longus muscles were evaluated. Maximum tetanic isometric force and peak power were significantly reduced in repair group 2 rats as compared with repair group I and control rats. Maximal sustained power was not significantly different between the groups. These data Support our working hypothesis that skeletal muscle contractile function is adversely affected by two cycles of denervation and reinnervation as compared with a single episode of nerve division and repair.

Sensory or motor "baby-sitting" has been proposed its a clinical strategy to preserve muscle integrity if motion-specific axons must regenerate over a long distance to reach denervated target muscles. Denervated muscles are innervated temporarily by using axons from nearby sensory or motor nerves. After motion specific motor axons have reached the target, the baby-sitter nerve is severed and motion-specific axons are directed to [lie target. Although this strategy minimizes denervation time, the requisite second episode of denervation and reinnervation might be deleterious to muscle contractile function. This study was designed to test the hypothesis that two sequential episodes of skeletal muscle denervation and reinnervation result in greater force and power deficits than a single peripheral nerve injury and repair. Adult Lewis rats underwent either transection and epineurial repair or sham exposure of the left peroneal nerve. After a 4-month recovery period, the contractile properties of the extensor digitorum longus muscle of the sham exposure group (control, n = 9) and one of the nerve division and repair groups (repair group 1, n = 9) were evaluated with measurements of the maximum tetanic isometric force, peak power, and maximal sustained power. A third group of rats Underwent a second cycle of nerve division and repair (repair group 2, n = 9) at this same time point. Four months postoperatively, contractile properties of the extensor digitorum longus muscles were evaluated. Maximum tetanic isometric force and peak power were significantly reduced in repair group 2 rats as compared with repair group I and control rats. Maximal sustained power was not significantly different between the groups. These data Support our working hypothesis that skeletal muscle contractile function is adversely affected by two cycles of denervation and reinnervation as compared with a single episode of nerve division and repair.

In vivo gene transfer is a recently developed device for efficient delivery of a therapeutic recombinant protein. We formulated the hypothesis that a high level of expression of bone morphogenetic protein 2 (BMP-2) could be a future therapeutic modality in terms of inducing substantial bone formation in vive. First, to test this hypothesis, adenoviruses carrying BMP-2 gene were directly injected into the soleus muscle of adult rat. The BMP-2 gene was successfully overexpressed in the target muscle by adenovirus-mediated transfer, whereas bone formation in and around the muscle failed to occur in this case. Second, to recruit putative osteoprogenitor cells, we then induced ischemic degeneration of the target muscle by orthotopically grafting it simultaneously with the gene transfer. The combination of BMP-2 gene transfer and orthotopic muscle grafting resulted in successful ossification of almost the whole grafted muscle, whereas neither muscle grafting alone nor the combination of muscle grafting and adenovirus-mediated transfer of reporter gene LacZ induced any bone formation in the muscle. The ossification process was evident by positive von Kossa staining of the histological sections and roentgenographical radio-opacity of the region. It was also found that the BMP-2 transgene overexpressed in grafted muscles inhibited muscle regeneration, which should otherwise follow the muscle degeneration. We further demonstrated an up-regulation of BMP receptor type IA in grafted muscles, suggesting its involvement in the bone-formation process. In conclusion, overexpression of BMP-2 gene induced massive heterotopic ossification in skeletal muscles under graft-induced ischemic degeneration, which possibly up-regulates osteoprogenitor cells in situ.

An approach for the correction of cryptotia using a superiorly based superficial mastoid fascial flap and a skin paddle is introduced. The buried portion of the auricle was exposed through an incision made along the upper part of the helix, followed by an appropriate correction of the deformed cartilage. Protrusion of the upper portion of the auricle was accomplished using anchoring sutures. A small skin paddle was elevated from the caudal portion of the auricular sulcus with the superiorly based superficial mastoid fascia as the nutrient pedicle and transferred to the temporal skin defect. The procedure was performed in eight auricles in a total of seven patients with cryptotia. A satisfactory contour and protrusion of the auricle were maintained postoperatively, leaving the scar within the auricular sulcus.