Preexisting gastric carcinoid in a gastro-omental free flap.

The authors present a 72-year-old man with an extensive medical history including stage III squamous cell carcinoma of the right pyriform sinus diagnosed approximately 10 years before this report. They were asked to evaluate the patient for esophageal reconstruction after local radiation had led to benign stricture of his esophagus and subsequent development of a large, draining esophagocutaneous fistula. A gastro-omental free flap reconstruction of the esophagus and overlying skin defect was complicated by the intraoperative diagnosis of gastric carcinoid obtained from several polyps noticed on the gastric mucosa on routine inspection. This case report signifies the importance of close inspection of all free tissue transfers before interposition. Failure to do so could result in disastrous outcomes.

Preparation and transplantation of a composite graft of epidermal keratinocytes on acellular dermis.

Loss of skin because of burns or ulcers is a major medical problem, and is the impetus for the development of skin substitutes and skin replacement technologies. Efforts in this area have focused on developing suitable substitutes for the epidermis or for the dermis, as well as ways to combine both technologies in a composite skin. In this chapter, we describe the construction of a skin substitute of cultured human keratinocytes on acellular human dermis.

Characterization of a composite tissue model that supports clonal growth of human melanocytes in vitro and in vivo.

To aid in the investigation of factors that control the proliferation and function of melanocytes, we have characterized a skin equivalent model that supports melanocyte growth and function in vitro and in vivo. Passenger melanocytes survive and proliferate at low numbers when keratinocytes of the epidermis are cultured in serum-containing medium using a fibroblast feeder layer. When the surface of de-epidermalized acellular dermis was seeded with these cultured cells, the keratinocytes formed a stratified epithelium in vitro containing rete ridges, and the melanocytes were preferentially located in the bottom of these rete ridges. Melanocyte cell number was much less than in normal skin, but in some areas the melanocytes were in clusters, consistent with clonal growth of the cells. When transplanted to athymic mice, the grafts formed foci of pigmentation at 3 wk that expanded and repigmented the entire graft by 8 wk. Histologic examination of these foci revealed that they corresponded to clusters of melanocytes that proliferated and migrated to eventually repopulate the entire graft. In grafts of mixed cells from light and dark skin donors, distinct foci of pigmentation were obvious at 3 wk and, instead of progressing to complete repigmentation, these foci remained stable for over 6 wk. Histologic examination confirmed that these grafts of mixed cells were entirely repopulated with melanocytes and that the grafts contained distinct zones of melanocytes that were of exclusively dark or light skin origin. This model should be valuable for studying the clonal growth of melanocytes in the context of the epidermis.

Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft.

Skin loss due to burns and ulcers is a major medical problem. Bioengineered skin substitutes that use cultured keratinocytes as an epidermal layer with or without analogues of the dermis are one strategy for skin repair. However, none can achieve definitive wound closure, function, or cosmesis comparable to split-thickness autografts. Moreover, autograft donor sites, which require time to heal, may be limited or have attendant problems such as infection or functional/cosmetic deficiencies. To determine if the performance of composite skin grafts of keratinocytes on a dermal analogue could be enhanced, human keratinocytes were genetically modified to overexpress platelet-derived growth factor A chain (PDGF-A). Composite grafts of modified keratinocytes seeded onto acellular dermis, prepared from cryopreserved cadaver skin, secreted PDGF-AA protein in vitro [90 ng/graft (1.5 x 1.5 cm)/24 hr]. To test their performance in a wound healing model, composite grafts were transplanted to full-thickness excisional wounds on the back of athymic mice. PDGF-A grafts formed a stratified differentiated epidermis similar to control grafts. The acellular dermis was repopulated with host fibrovascular cells and by day 7, the PDGF-A grafts had significantly more cells in the dermis and increased staining for murine collagen types I and IV. At this early time point, wound contraction was also significantly inhibited in PDGF-A grafts versus control grafts. Thus, PDGF-A overexpression improves graft performance during the first critical week after transplantation.

Differences in dermal analogs influence subsequent pigmentation, epidermal differentiation, basement membrane, and rete ridge formation of transplanted composite skin grafts.

This study evaluated the in vitro and in vivo function of composite skin equivalents based on two different dermal analogs. Keratinocytes derived from the same dark-skinned neonatal foreskins were seeded onto both acellular human dermis and fibroblast-contracted collagen gels. Each type of composite graft readily formed an epithelium in vitro. However, the undulating surface of the acellular dermis acted as a template and organized the seeded keratinocytes into a rete ridge-like pattern, whereas the smooth surface of the fibroblast-contracted collagen gels generated an epithelium with a linear basal layer. Moreover, when acellular dermis was used, the composite grafts demonstrated enhanced melanocyte proliferation. When transplanted to athymic mice, both composite grafts formed a fully differentiated human epidermis, but repigmentation of the grafts when acellular dermis was used was more extensive and only the epidermis on the fibroblast-contracted collagen gels showed signs of hyperproliferation at 6 weeks after grafting. These results demonstrate that the type of dermal analog incorporated into a composite skin graft can influence the subsequent functionality of the skin substitute.

Corrective gene transfer in the human skin disorder lamellar ichthyosis.

Lamellar ichthyosis (LI) is a disfiguring skin disease characterized by abnormal epidermal differentiation and defective cutaneous barrier function. LI has been associated with loss of keratinocyte transglutaminase 1 (TGase1), an enzyme believed necessary for normal formation of the cornified epidermal barrier. Using LI as a prototype for therapeutic cutaneous gene delivery, we have used the human skin/immunodeficient mouse xenograft model to correct the molecular, histologic and functional abnormalities of LI patient skin in vivo. We have used TGase1-deficient primary keratinocytes from LI patients combined with high-efficiency transfer of functional TGase1 to regenerate engineered human LI epidermis on immunodeficient mice. Engineered LI epidermis displayed normal TGase1 expression in vivo, unlike unengineered LI epidermis where TGase1 was absent. Epidermal architecture was also normalized by TGase1 restoration, as was expression of the epidermal differentiation marker filaggrin. Engineered LI skin demonstrated restoration of cutaneous barrier function measures to levels seen in epidermis regenerated by keratinocytes from patients with normal skin, indicating functional correction in vivo of the proposed primary pathophysiologic defect in LI. These results confirm a major role for TGase1 in epidermal differentiation and demonstrate a potential future approach to therapeutic gene delivery in human skin.

Evaluation of acellular human dermis as a dermal analog in a composite skin graft.

This study evaluated the structure and function of a dermal substitute composed of de-epidermalized, acellular human dermis, and tested its performance as part of a composite skin graft with cultured human keratinocytes. The acellular dermis retained much of the complex structure of native dermis, as demonstrated by immunostaining for collagen Types IV and VII. Collagen Types IV and VII were found on the papillary surface, and collagen Type IV was found at sites throughout the acellular dermis. To further demonstrate the functionality of the acellular dermis material, we seeded the papillary surface with cultured keratinocytes. In response to the architecture of the dermal papillae, the keratinocytes formed a three-dimensional epidermal structure that was several cell layers thick, and recreated the original skin rete ridges at the interface with the dermis. When these composite grafts were transplanted to athymic mice, host fibrovascular cells repopulated the acellular dermis. Some vessels grew into the acellular dermis along the original pathways of the human blood vessels, as demonstrated by co-localization of human and mouse collagen Type IV. The skin that developed from these grafts repigmented completely via passenger melanocytes from the keratinocyte cultures, was durable, and remained stable for more than 5 months.

Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice.

This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections. Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds.

Effects of luteal estradiol on the secretory transformation of human endometrium and plasma gonadotropins.

To study the role of luteal estradiol (E2), we interrupted the supply of E2 during the luteal phase of E2 and progesterone (P) replacement cycles. Thirty-one women, aged 26-37 yr, with absent or inactive ovaries received three different treatment regimens: group I (n = 11) received transdermal E2 and vaginal P according to a protocol designed to approximate levels of estrone (E1), E2, and P seen during the menstrual cycle. Groups II (n = 11) and III (n = 9) received identical treatments, except that in group II no E2, and in group III no E2 or P, was administered after day 15. Endometrial biopsies were obtained on days 20 and 24 in groups I and II, and on days 14 and 20 in group III. In group I, plasma E1 and E2 reached menstrual cycle levels, whereas in groups II and III, discontinuation of the E2 supply on day 15 resulted in a prompt decrease to castrate levels of plasma E1 and E2. In groups I and II, menopausal FSH and LH levels decreased to 26 +/- 6 and 30 +/- 7 IU/L, respectively, on day 13 (mean +/- SEM). In group I, administration of E2 and P starting on day 15 further lowered plasma gonadotropin levels. In group II, administration of P only failed to induce a similar decrease in plasma FSH and LH. No uterine bleeding occurred before day 25 in women of groups I or II, while women of group III bled within 2 days of E2 withdrawal. Endometrial biopsies were similar in groups I and II. Histological features were characteristic of early and late luteal phases on days 20 and 24, respectively. Endometrial maturation assessed by estrogen and progesterone receptors identified by immunocytochemistry showed the typical distribution seen on day 24 of the menstrual cycle with no difference between groups I and II. We conclude that in women deprived of ovarian function, administration of P only after 14 days of E2 priming prevented uterine bleeding and induced normal secretory transformations of the endometrium, but failed to suppress plasma gonadotropins.

Immunocytochemical localization of oestradiol and progesterone receptors in human endometrium: a tool to assess endometrial maturation.

Uterine oestrogen (ER) and progesterone (PR) receptors are subject to fine hormonal control by oestradiol and progesterone. In order to assess the role of ER and PR measurement in the evaluation of endometrial maturation, both receptors were studied by immunocytochemical techniques using monoclonal antibodies during the menstrual cycle, and in women with inactive ovaries treated by different regimens of hormonal substitution with oestradiol and progesterone. During the normal menstrual cycle, the concentrations and distribution of ER and PR changed markedly. During the mid follicular period (days 7-8), a small proportion of stromal and glandular cells stained positively for PR while staining for ER was more intense and more frequent. During the late follicular phase and early luteal period (days 9-19), the staining for PR increased markedly in glandular cells. During the mid and late luteal phase (days 21-27), ER and PR staining disappeared in glandular cells. Thus, while oestradiol increases the staining for ER and PR in both glands and stroma, progesterone decreases ER and PR staining in the glands in a dramatic fashion. These variations, especially the disappearance of PR under the effect of progesterone, are potentially useful for studying the cumulative effect of progesterone on endometrial maturation. This was confirmed in anovulatory women, where a late luteal phase aspect was observed, i.e. the absence of a reduction in ER and PR in glandular cells. In women with ovarian failure, the disappearance of ER and PR in glandular cells is correlated with the duration of progesterone therapy.(ABSTRACT TRUNCATED AT 250 WORDS)