Clinical Indications of Cultured Epithelial Autografts.

ABSTRACT: Cultured epithelial autografts (CEAs) have been used for decades as a treatment for massive burn injuries. Cultured epithelial autografts allow for wounds to heal by taking a small sample and growing a patient's own epithelium in culture to create large, graftable sheets. This technique is especially useful in large wounds where donor sites are limited compared with conventional skin grafting. However, CEAs have a variety of uses in wound healing and reconstruction and have the potential to aid in the closure of several types of defects. Cultured epithelial autografts have shown applicability in large burns, chronic nonhealing wounds, ulcerating wounds of various etiologies, congenital defects, wounds requiring specialized epithelium to replace like by like, and wounds in critically ill patients. Several factors must be considered when using CEAs, such as time, cost, and outcomes. In this article, we detail the various clinical applications of CEAs and how they can be situationally advantageous outside of their original purpose.

Use of mouse liver mesothelial cells to prevent postoperative adhesion and promote liver regeneration after hepatectomy.

BACKGROUND & AIMS: Repeated hepatectomy is widely accepted as one of the most effective curative treatment for recurrent hepatocellular carcinoma or liver metastasis from colorectal cancer. It has, however, two critical issues; postoperative adhesion and decrease of liver regenerative capacity. Postoperative adhesion makes surgical operations technically more demanding, leading to increased mortality and morbidity rates. Although the liver has a remarkable regenerative ability, volume and functional restoration after multiple repeated hepatectomy is not generally complete. So a new procedure that overcomes these two issues is required. We examined if a fetal liver mesothelial cells (FL-MCs) sheet could solve these two clinical issues simultaneously. METHODS: We established a novel mouse hepatectomy model that reproduces postoperative adhesion on the resected liver surface. We isolated FL-MCs from mouse fetal liver and prepared a cell sheet. The FL-MCs sheet was then transplanted to the resected liver surface. RESULTS: The FL-MCs sheet effectively prevented postoperative adhesion by expressing PCLP1, one of the transmembrane sialomucin family proteins and by activating the fibrinolytic system. Furthermore, the FL-MCs sheet facilitated liver regeneration by providing growth factors for hepatocytes, allowing quick recovery of liver weight and function. Additionally, we showed that an allogeneic FL-MCs sheet was as effective as a syngeneic cell sheet. CONCLUSIONS: We demonstrate that the FL-MCs sheet is able to not only prevent postoperative adhesion but also promote liver regeneration in both syngeneic and allogeneic transplantation, and hence FL-MCs may serve as a potentially useful cell source for regenerative medicine after hepatectomy.

Experience of using cultured epithelial autografts for the extensive burn wounds in eight patients.

UNLABELLED: In Japan, the cultured epithelial autografts "JACE" was accepted as a health insurance adaptation from January 1, 2009. We examined the extensive burn wounds in 8 patients by using a combination of autograft and JACE. After debridement, we managed the wound bed preparation by using artificial dermis. The wound bed was covered with fine tissue 2 weeks after we implanted artificial dermis and trafermin was used every day. Meshed 6:1 split-thickness autografts were placed onto the recipient wound bed under the JACE. The epidermalization was nearly complete within 3 to 4 weeks. RESULTS: A total of 39 patients underwent medical treatment of burns. All patients burned more than 30% total body surface area (TBSA). We divided them into 2 groups. The control group consisted of 31 patient, 23 men and 8 women. They underwent operation not using JACE but only autograft. The average age of the patients was 59.61 (3.85) years. The TBSA burned in this control group was 58.94% (3.89%). Operation times were 2.16 (0.24) hours. The overall survival rate was 35.5%. The study group consisted of 8 patients, 5 men and 3 women. The average age of the patients was 56.38 (7.04) years. The TBSA burned in this study group was 51.63% (4.17%). Operation times were 4.25 (0.59) hours, and the overall survival rate in this study group was 87.5%. The average take rate of JACE was 80.0% (3.09%) 4 weeks postoperatively. CONCLUSIONS: JACE is one of the cultured epithelial autografts. Although we managed the wound bed preparation by using artificial dermis instead of cryopreserved cadaver allograft, we were able to recognize a good result from grafting JACE on meshed 6:1 split-thickness autografts. The study group observed a significant difference in operation times compared with the control group. However, this treatment contributed to reducing the area of the donor site.

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland.

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p<0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.

Repigmentation of poliosis after epithelial grafting for vitiligo.

BACKGROUND: Vitiligo is a disease of color loss from skin and possibly also from hair. The presence of white hair follicles is known to be a bad prognostic sign. OBJECTIVES: To evaluate the possibility of repigmentation of white hair follicles after epithelial grafting. METHODS: Patients with recalcitrant vitiligo with loss of hair pigment were treated using Chinese cupping blisters or ultrathin Thiersch grafting after de-epithelialization of vitiliginous patches by dermabrasion. Phototherapy was used afterward to enhance success. RESULTS: Repigmentation of the skin surface was obtained with as little as 1 to 2 months of phototherapy, as expected, and further follow-up of cases revealed the re-coloring of hair follicles after 4 to 11 months. CONCLUSION: Re-coloring of poliosis with vitiligo is possible but was unexpected because of the difference in mechanism and signaling required between hair bulb melanization and the surface skin. One likely mechanism to explain this change is that melanocyte stem cells are stimulated and migrate to supply hair bulbs with new mature melanocytes. Epithelial grafting of vitiligo with poliosis in hairy areas should be a treatment of choice when white hair tufts cause cosmetic disfigurement.

In vitro characterization of multipotent mesenchymal stromal cells isolated from palatal subepithelial tissue grafts.

The aim of this study was to analyze whether the mesenchymal stromal cells (MSCs) isolated from palatal tissue grafts harvested in order to cover gingival recessions have the basic characteristics of stem cells. The palatal tissue cells were processed using a special culture medium that stimulated the development of only undifferentiated cellular lines. Cells at passage 4 were evaluated by flow cytometry to examine the expression of specific surface markers and were tested for multilineage differentiation capacity. These cells collected at passage 4 were also investigated for the capacity to cluster into embryoid body aggregates. Palatal MSCs displayed positive staining for the mesenchymal markers CD29, CD73, CD105, CD 49e, and CD44, but did not express hematopoietic markers CD34/45. The palatal MSCs successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. When seeded in special conditions, palatal MSCs propagated into unattached spheres resembling embryoid body aggregates consisting both of differentiated and undifferentiated cells as revealed at the ultrastructural evaluation. It is concluded that the isolated palatal MSCs fulfilled the basic criteria defining the stem cells. This new source of stem cells characterized here for the first time opens new perspectives on possible applications in basic research and in regenerative medicine.

Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses.

We describe the use of a human bronchial xenograft model for studying the efficiency and biology of in vivo gene transfer into human bronchial epithelia with recombinant E1 deleted adenoviruses. All cell types in the surface epithelium except basal cells efficiently expressed the adenoviral transduced recombinant genes, lacZ and CFTR, for 3-5 weeks. Stable transgene expression was associated with high level expression of the early adenoviral gene, E2a, in a subset of transgene expressing cells and virtually undetectable expression of the late adenoviral genes encoding the structural proteins, hexon and fiber. These studies begin to address important issues that relate to safety and in vivo efficacy of recombinant adenoviruses for gene delivery into the human airway.

Mechanisms and interventions in peritoneal fibrosis.

Peritoneal dialysis (PD) is an attractive treatment for patients with end-stage kidney disease (ESKD). However, long-term peritoneal dialysis is associated with development of functional and structural alterations of the peritoneal membrane. Several factors are implicated in the development of peritoneal fibrosis in PD patients. Inflammatory cytokines, which are induced in the peritoneal cavity during peritonitis, may also induce chronic inflammation and fibrosis. Transforming growth factor ß1 (TGF-ß1) is generally considered to play an important role in peritoneal fibrosis. The objective of this review is to summarize the mechanisms of peritoneal fibrosis using in vitro and in vivo studies, and the current status and future prospects of interventions in the peritoneal fibrosis.

Profibrotic potential of prominin-1+ epithelial progenitor cells in pulmonary fibrosis.

BACKGROUND: In idiopathic pulmonary fibrosis loss of alveolar epithelium induces inflammation of the pulmonary tissue followed by accumulation of pathogenic myofibroblasts leading eventually to respiratory failures. In animal models inflammatory and resident cells have been demonstrated to contribute to pulmonary fibrosis. Regenerative potential of pulmonary and extra-pulmonary stem and progenitor cells raised the hope for successful treatment option against pulmonary fibrosis. Herein, we addressed the contribution of lung microenvironment and prominin-1(+) bone marrow-derived epithelial progenitor cells in the mouse model of bleomycin-induced experimental pulmonary fibrosis. METHODS: Prominin-1(+) bone marrow-derived epithelial progenitors were expanded from adult mouse lungs and differentiated in vitro by cytokines and growth factors. Pulmonary fibrosis was induced in C57Bl/6 mice by intratracheal instillation of bleomycin. Prominin-1(+) progenitors were administered intratracheally at different time points after bleomycin challenge. Green fluorescence protein-expressing cells were used for cell tracking. Cell phenotypes were characterized by immunohistochemistry, flow cytometry and quantitative reverse transcription-polymerase chain reaction. RESULTS: Prominin-1(+) cells expanded from healthy lung represent common progenitors of alveolar type II epithelial cells, myofibroblasts, and macrophages. Administration of prominin-1(+) cells 2 hours after bleomycin instillation protects from pulmonary fibrosis, and some of progenitors differentiate into alveolar type II epithelial cells. In contrast, prominin-1(+) cells administered at day 7 or 14 lose their protective effects and differentiate into myofibroblasts and macrophages. Bleomycin challenge enhances accumulation of bone marrow-derived prominin-1(+) cells within inflamed lung. In contrast to prominin-1(+) cells from healthy lung, prominin-1(+) precursors isolated from inflamed organ lack regenerative properties but acquire myofibroblast and macrophage phenotypes. CONCLUSION: The microenvironment of inflamed lung impairs the regenerative capacity of bone marrow-derived prominin-1(+) progenitors and promotes their differentiation into pathogenic phenotypes.

Treatment of gingival recession in the anterior mandible using the tunnel technique and a combination epithelialized-subepithelial connective tissue graft-a case series.

Covering exposed roots becomes more and more difficult as the gingiva becomes thinner and the vestibule becomes more shallow. Also, the outcome becomes less predictable. In addition, where there is high frenal attachment or muscle pull, such as the mentalis muscle in the mandibular anterior region, secondary retraction of a coronally advanced flap will likely occur. Therefore, a transplanted connective tissue graft may not completely cover the recession. This case series presents a technique where the roots are covered with a combination epithelialized-subepithelial connective tissue graft. The epithelialized portions of the graft are positioned directly over the exposed roots to aid in resistance to the environment of the mouth, and there is no displacement of the mucogingival junction or flattening of the vestibule.

[Regenerative medicine in cornea].

Cornea is unique organ in its transparency. It consists of three different layers epithelium, stroma, and endothelium. Defect of each layers decrease the transparency resulting in blindness. Corneal transplant from donors is performed for these conditions. However it sometimes does not work because of immuno-rejection and shortage of donors is still problem. Regenerative medicine resolves these problems. According to epithelium, we had succeeded in making epithelial sheet from oral mucosa epithelium. The sheet is clear and very resembles normal corneal epithelium in histology. We have auto transplantation of this epithelial sheet to severe corneal deficiency patients and obtained good clinical results. According to endothelium, we are trying to make the sheet from various stem sells including iPS cells.

Combined Complex Skin Repair in Patient With Extensive Burns: A Case Report.

Auto-skin grafting is the current treatment of choice for extensive burns. Nevertheless, the lack of donor sites for skin grafting remains one of the greatest limiting factors for the treatment of extensively burned patients. We present the case of a 53-year-old male patient with deep and full thickness burns on 91% of the total body surface area. We used the Meek technique for split-thickness skin graft expansion to treat this patient. In order to obtain sufficient skin for grafting, we repeatedly harvested the same anatomical areas. Acceleration of burn wounds, recipient, and donor site healing was achieved by systemic treatment with recombinant human growth hormone and topical recombinant human epidermal growth factors. This combined, complex treatment modality contributed to the successful skin repair in this patient.

Successful Ocular Surface Reconstruction in Complete Ankyloblepharon With the Simple Oral Mucosal Epithelial Transplantation Technique: A Case Report.

PURPOSE: To report an outcome of a patient with complete ankyloblepharon successfully managed with simple oral mucosal epithelial transplantation (SOMET). METHODS: A 55-year-old woman presented with complete adhesion of both lids to the ocular surface as a complication from Stevens-Johnson syndrome. We performed 2-staged reconstructive surgeries: the first stage was to perform ankyloblepharon lysis and surface reconstruction with a mucosal graft on the palpebral area and an amniotic membrane on the bulbar area, and the second stage was to reconstruct the bulbar area with a transplantation of small pieces of oral mucosa (SOMET technique). Postoperatively, the patient was evaluated for ocular surface stability, recurrent symblepharon, in vivo confocal microscopy, and impression cytology with immunofluorescence staining. RESULTS: Complete epithelialization of cornea-like epithelium was observed within 6 weeks after SOMET was performed. The ocular surface was stable over 1 year. Both fornices remained deep. In vivo confocal microscopy showed cornea-like epithelium mixed with conjunctival epithelium, as confirmed with immunofluorescence staining, which revealed cytokeratin 3, cytokeratin 7, and cytokeratin 12 positivity. CONCLUSIONS: SOMET is a simple modified technique using minimal oral mucosal tissue to regenerate epithelialization for complicated ocular surface reconstruction such as a complete ankyloblepharon repair. Although there was evidence of conjunctival invasion, stable ocular surface and deep fornices can be achieved for further visual rehabilitative procedure.

Allogeneic simple limbal epithelial transplantation: an appropriate treatment for bilateral stem cell deficiency.

A 39-year-old man presented with both eyes limbal stem cell deficiency status post chemical injury. He was managed initially with topical medications to subside the ocular surface inflammation. Over the course of subsequent visits, the fibrovascular pannus over the cornea gradually progressed, leading to further diminution of vision in left eye more than right eye. Since, the ocular surface was wet, the patient committed for lifelong immunosuppression and his brother consented to donate healthy limbal tissue; he underwent living-related allogeneic simple limbal epithelial transplantation in the left eye.

The effect of epithelial remnant and whole organ grafts of thymus on the recovery of thymectomized irradiated mice.

Work has been presented which suggests that thymus epithelial reticular cells are not effective in restoring the microscopic morphology of lymphoid tissues and their immunologic capacities. They function in recruiting precursors of thymus lymphocytes from the host animals to produce an organ which, after it becomes architecturally normal, can reconstitute the defective host. Intact thymus grafts in situ from 10-14 days, but not for shorter periods of time, have been shown to result in a return toward normal of these two parameters. Evidence is offered to show that few dividing cellular components in the lymphoid tissue originate from the thymus remnant grafts, and that a minor cellular component is contributed by the intact grafts. These data support the concept that the structural and functional development of the lymphatic tissue in thymectomized animals is dependent on thymus lymphoid cells and/or their products, and that the epithelial-reticular cells do not have a direct action in peripheral lymphoid reconstitution.

Use of a combination epithelized-subepithelial connective tissue graft for closure and soft tissue augmentation of an extraction site following ridge preservation or implant placement: description of a technique.

An esthetic implant-supported rehabilitation continues to be a major challenge in patients with a thin periodontium. Ridge preservation and immediate implant placement are intended to preserve the hard tissue volume and prevent preimplant bone loss following tooth extraction. Since these techniques are almost always combined with bone grafting, primary wound closure is indispensable. Therefore, a technique for reliable wound closure was developed. This technique employs a combined epithelized-subepithelial connective tissue graft, leaves the mucogingival line in its place, and has the added advantage of thickening the buccal soft tissue with the resultant local conversion of a thin marginal gingiva to a thick marginal gingiva.

Cultured Epithelial Autograft Combined with Micropatterned Dermal Template Forms Rete Ridges In Vivo.

For patients with large, full-thickness burn wounds, sufficient donor sites for autografting are not available, and thus, alternate strategies must be used to close these wounds. Cultured epithelial autografts (CEAs) can aid in closing these wounds but are often associated with slow deposition of basement membrane proteins, leading to blistering and graft loss. Rete ridges and dermal papillae present at the dermal-epidermal junction (DEJ) play a key role in epidermal adhesion and skin homeostasis. Promoting the development of an interdigitated DEJ may enhance basement membrane protein deposition and provide enhanced physical interlock of the epidermis and dermis. To develop a dermal template with stable dermal papillae, an electrospun collagen scaffold was seeded with human dermal fibroblasts. Ridged topographies were patterned into the cell-seeded dermal template using laser ablation, creating wide and shallow (ActiveFX) or narrow and deep (DeepFX) wells. Micropatterned or flat (control) dermal templates were combined with CEAs immediately before grafting to full-thickness excisional wounds on immunodeficient mice. CEAs grafted in conjunction with ridged templates showed rete ridge formation at 2 weeks after grafting and led to increased epidermal thickness, proliferation, and stemness compared to templates with a flat DEJ. As this technology is further developed, the dermal papilla-containing dermal templates may be utilized in combination with CEAs to improve adhesion and clinical function. Impact statement Cultured epithelial autografts (CEAs) serve as an adjunct to conventional split-thickness autograft in patients with very large burns, but they are susceptible to blistering that can reduce engraftment. Blistering results, in part, from relatively slow basement membrane deposition after grafting. This study demonstrates that basement membrane deposition and rete ridge formation are enhanced by combination of CEAs with a micropatterned, cell-seeded dermal template. These findings may lead to improved treatment and increased survival in patients with very large burns.

Dual-label centromere and telomere FISH identifies human, rat, and mouse cell contribution to Multispecies recombinant urogenital sinus xenografts.

BACKGROUND: Recombinant xenografts of human cells growing in immunocompromised rodents are widely used for studying stem cell biology, tumor biology, and epithelial to mesenchyme transitions. Of critical importance is the correct interpretation of the cellular composition of such xenografts. METHODS: Here we present a rapid and robust method employing protein nucleic acid (PNA) FISH probes to dual-label centromeres and telomeres (Cen/Tel FISH). Such labeling allows unambiguous discrimination between human, mouse, and rat cells in paraffin-embedded tissue sections, providing significant advantages over current methods used to discern human versus rodent cell types. RESULTS: Using an in vivo prostatic developmental system where rat embryonic urogenital sinus mesenchyme is recombined with human prostate epithelial organoids and grown in an immunocompromised mouse, Cen/Tel FISH documents that all three species contribute to the development of glandular structures. CONCLUSIONS: The method is an indispensable tool to analyze xenograft/host interactions and prevent misinterpretation of data using tissue recombination approaches.

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.

Flap advancement: practical techniques to attain tension-free primary closure.

BACKGROUND: Primary and tension-free closure of a flap is often required after particular surgical procedures (e.g., guided bone regeneration). Other times, flap advancement may be desired for situations such as root coverage. METHODS: The literature was searched for articles that addressed techniques, limitations, and complications associated with flap advancement. These articles were used as background information. In addition, reference information regarding anatomy was cited as necessary to help describe surgical procedures. RESULTS: This article describes techniques to advance mucoperiosteal flaps, which facilitate healing. Methods are presented for a variety of treatment scenarios, ranging from minor to major coronal tissue advancement. Anatomic landmarks are identified that need to be considered during surgery. In addition, management of complications associated with flap advancement is discussed. CONCLUSIONS: Tension-free primary closure is attainable. The technique is dependent on the extent that the flap needs to be advanced.