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.

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.

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.

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.

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.

The Full-Thickness Palatal Graft Technique: Description of an Original Surgical Technique and 15 Case Reports.

Gingival thickness plays a pivotal role in both the etiology and treatment of gingival recessions. When treating gingival recessions by different periodontal plastic surgery techniques, a relationship has been reported between the thickness of the tissue used to cover the exposed root surface and the recession reduction. This case series illustrates a technique making use of a very thick graft, which consists of the entire thickness of the palate in its central part, from the periosteum to the epithelium, to obtain complete root coverage with high predictability. At 12 months, the treatment resulted in 100.22% ± 6.95% root coverage, and 14 of the 15 treated teeth (93.3%) were completely covered.

Wound healing after cultured epithelial autografting in patients with massive burn injury: A cohort study.

BACKGROUND/AIM: Last century, our laboratory produced Cultured Epithelial Autograft (CEA) for clinical use by the affiliated adult burn service and other burn units across the country. Production of CEA for clinical use was discontinued after several years because of a low success rate and subsequent low demand. Recently, at our burns unit, a cell culture program was reintroduced as a direct response to the need for improvement in ongoing deficiencies and clinical requirements in burn wound closure. The aim of this study was to validate the laboratory processes and clinical algorithms established and share our recent clinical experiences involving CEA. METHODS: This observational cohort study recruited patients with burns exceeding 35% TBSA admitted to the Victorian Adult Burns Service at The Alfred (December 2013-December 2016). Autologous keratinocytes were expanded and delivered through sheets of fibrin carrier. RESULTS: Twelve patients were recruited to participate in the study. Thirty-two sites were treated with CEA. CEA applied in combination with widely meshed SSG led to the highest take rate (90.1%) at 7-10 days. Further, debridement and grafting were necessary in sixteen of thirty-two sites treated, all involving wound beds prepared with Cuono method or sites treated with CEA only. CONCLUSION: It is important to address the problem of wound bed contamination, either through increased resistance on the part of the construct or wound bed sterilization. Improved understanding of the relative importance of vascularization, control of cell behavior, the extracellular matrix, immune function, and intrinsic antimicrobial capacity for graft take would then inform a more targeted approach to skin tissue engineering for wound closure in severe burns.

Combination of meshed dermis graft and cultured epithelial autograft for massive burns: Three case reports.

RATIONALE: This study reviewed the use of a combination of meshed dermis graft and cultured epithelial autografts (CEA) made in Japan "JACE" (JACE; Japan Tissue Engineering Co., Ltd. Japan) for the treatment of massively burns. JACE is a Green-type CEA. We recently described a method in which we prepare the wound bed for burned patients by using artificial dermis and graft with JACE on a meshed 6:1 split-thickness autograft. In this report, we used a meshed 3:1 split-thickness dermis graft without epithelial cells. There are several reports of combination of using CEA on meshed split-thickness autograft, however this is the first report of using CEA on meshed split-thickness dermis graft. PATIENT CONCERNS AND DIAGNOSIS: Between March 2015 and August 2017, 3 burn patients were enrolled in this study. The patients ranged in age from 51 to 66 years. All 3 patients suffered severe burn injury that caused by flame. % Total Body Surface Area (TBSA) burned were ranged from 37.5% to 69%. INTERVENTIONS: All patients received surgical treatment with tangential excision within a week from admission. We implanted artificial dermis immediately after debridement. Basically, we applied meshed 6:1 split-thickness autografts to the wound bed and covered with JACE. However, in the absence of split-thickness autografts, we used a meshed 3:1 split-thickness dermis graft instead of a meshed 6:1 split-thickness autograft. OUTCOMES: At 3 weeks after the transplantation of JACE, the take rate for JACE sheets was >60% on the meshed 3:1 split-thickness dermis graft. Furthermore, almost all of the burn wounds had healed at 6 weeks after surgery. LESSONS: We observed good results by grafting JACE on meshed 3:1 dermis graft. With this new method, it is possible to cover a large burn wound by harvesting tissue from only a small site.

Sub-epithelial connective tissue graft for the management of Miller's class I and class II isolated gingival recession defect: A systematic review of the factors influencing the outcome.

The aim of the present systematic review was to analyze the factors that affect the outcome of subepithelial connective tissue graft (SCTG) for managing Miller's class I and class II isolated gingival recession defect. The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for systematic reviews were used. Quality assessments of selected articles were performed. Data on root surface condition, recession type defect, flap thickness, different flap designs, different harvesting techniques, presence/absence of the epithelial collar, graft thickness, flap tension, suturing techniques, and smoking-related outcomes on root coverage were assessed. The SCTG procedure provides the best root coverage outcomes for Miller's class I and class II recession. The critical threshold of flap thickness was found to be 1 mm. Maximum root coverage was achieved by envelope and modified tunnel technique. SCTG with the epithelial collar does not provide additional gains than SCTG without the epithelial collar. The thickness of SCTG for root coverage was found to be 1.5-2 mm. Greater flap tension and smoking adversely affect root coverage outcomes. Analysis of the factors discussed would be of key importance for technique selection, and a combined approach involving factors favoring outcomes of SCTG could be of clinical relevance in recession coverage.

Vermillion Reconstruction With Anal Verge Transitional Epithelium: Turning Things Upside Down.

Postburn lip deformities pose a significant set of challenges to reconstructive burn surgeons because of the complex anatomy, diverse functions, and specialized nature of the lip tissues. There has been a paucity of literature on restoration of the vermillion to date. The authors report on two patients who suffered burn injuries resulting in significant lip deformities who underwent a novel method of vermillion reconstruction with a full thickness anal verge skin graft. Both patients tolerated the procedure well without complications. One patient had slight hyperpigmentation of the graft which was treated with a phenol peel to cause intentional lightening. Overall, both patients had a restored vermillion border and improved color match and contour of the lip. Histologic analysis of the anal verge demonstrates that it has a nonkeratinized, transitional epithelial architecture which is nearly identical to that of the vermillion tissue. Skin grafting remains one of the cornerstones of tissue replacement in acute burn care and burn reconstruction. The vermillion represents an area of specialized tissue that is not well reconstructed with simple skin grafts. Other methods for reconstruction involve lip switch operations or local flaps, like a ventral tongue flap. These procedures are not without limitation and can often cause microstomia among other issues. With no donor site morbidity, a full thickness anal verge skin graft represents the closest approximation of actual vermillion tissue found anywhere else in the body and should be considered a viable option in the reconstruction of these challenging patients.

Depigmentation during vitiligo activity spares epithelial grafted areas.

BACKGROUND: Vitiligo is a depigmenting disease characterized by episodes of stability and activity without a fixed sequence, and active phases demonstrate immune attacks toward vulnerable melanocytes. Epithelial grafts were introduced to treat recalcitrant vitiligo patches aiming to provide new generations of melanocytes. AIMS: The aim of study was to evaluate the possible permanency of succeeded epithelial grafts during a coming activity episode. METHODS: This study included 20 nonsegmental vitiligo cases. They were epithelial grafted, success was maintained with the use of UV sessions for a year, and further follow-up was allowed until new phases of activity developed. RESULTS: The grafted areas, in all cases, were spared during the activity episodes in spite of the development of new depigmented lesions in other parts of skin neighboring the grafted areas or in remote sites. CONCLUSION: The permanency of epithelial grafts in spite of disease reactivity is a good sign and magnifies the value of surgical approaches in management of vitiligo. New genetically different melanocytes should have been provided and were able to resist the new immune attacks in spite of the yielding of other skin melanocytes.

Soft tissue surgery for implants.

Adequate quality and quantity of soft tissue plays an integral part in the esthetic outcome of dental implants. Adequate band of attached tissue decreases the incidence of mucositis and improves hygiene around implants. This article discusses a variety of techniques for soft tissue augmentation. Soft tissue grafting can be achieved at various stages of implant therapy. Epithelial connective tissue grafts are commonly used to increase the band of attached tissue. Subepithelial connective tissue grafts are great for increasing soft tissue thickness and improving the gingival biotype.

Postextraction socket preservation using epithelial connective tissue graft vs porcine collagen matrix. 1-year results of a randomised controlled trial.

PURPOSE: To compare epithelial connective tissue graft vs porcine collagen matrix for sealing postextraction sockets grafted with deproteinised bovine bone. MATERIALS AND METHODS: A total of 30 patients, who needed a maxillary tooth to be extracted between their premolars and required a delayed, fixed, single implant-supported restoration, had their teeth atraumatically extracted and their sockets grafted with deproteinised bovine bone. Patients were randomised according to a parallel group design into two arms: socket sealing with epithelial connective tissue graft (group A) vs porcine collagen matrix (group B). Outcome measures were: implant success and survival rate, complications, horizontal and vertical alveolar bone dimensional changes measured on Cone Beam computed tomography (CBCT) scans at three levels localised 1, 3, and 5 mm below the most coronal aspect of the bone crest (levels A, B, and C); and between the palatal and buccal wall peaks (level D); and peri-implant marginal bone level changes measured on periapical radiographs. RESULTS: 15 patients were randomised to group A and 15 to group B. No patients dropped out. No failed implants or complications were reported 1 year after implant placement. Five months after tooth extraction there were no statistically significant differences between the 2 groups for both horizontal and vertical alveolar bone dimensional changes. At level A the difference was 0.13 ± 0.18; 95% CI 0.04 to 0.26 mm (P = 0.34), at level B it was 0.08 ± 0.23; 95% CI -0.14 to 0.14 (P = 0.61), at level C it was 0.05 ± 0.25; 95% CI -0.01 to 0.31 mm (P = 0.55) and at level D it was 0.13 ± 0.27; 95% CI -0.02 to 0.32 mm (P = 0.67). One year after implant placement there were no statistically significant differences between the 2 groups for peri-implant marginal bone level changes (difference: 0.07 ± 0.11 mm; 95% CI -0.02 to 0.16; P = 0.41). CONCLUSIONS: When teeth extractions were performed atraumatically and sockets were filled with deproteinised bovine bone, sealing the socket with a porcine collagen matrix or a epithelial connective tissue graft showed similar outcomes. The use of porcine collagen matrix allowed simplification of treatment because no palatal donor site was involved.

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.

Luminal epithelium in endometrial fragments affects their vascularization, growth and morphological development into endometriosis-like lesions in mice.

In endometriosis research, endometriosis-like lesions are usually induced in rodents by transplantation of isolated endometrial tissue fragments to ectopic sites. In the present study, we investigated whether this approach is affected by the cellular composition of the grafts. For this purpose, endometrial tissue fragments covered with luminal epithelium (LE(+)) and without luminal epithelium (LE(-)) were transplanted from transgenic green-fluorescent-protein-positive (GFP(+)) donor mice into the dorsal skinfold chamber of GFP(-) wild-type recipient animals to analyze their vascularization, growth and morphology by means of repetitive intravital fluorescence microscopy, histology and immunohistochemistry during a 14-day observation period. LE(-) fragments developed into typical endometriosis-like lesions with cyst-like dilated endometrial glands and a well-vascularized endometrial stroma. In contrast, LE(+) fragments exhibited a polypoid morphology and a significantly reduced blood perfusion after engraftment, because the luminal epithelium prevented the vascular interconnection with the microvasculature of the surrounding host tissue. This was associated with a markedly decreased growth rate of LE(+) lesions compared with LE(-) lesions. In addition, we found that many GFP(+) microvessels grew outside the LE(-) lesions and developed interconnections to the host microvasculature, indicating that inosculation is an important mechanism in the vascularization process of endometriosis-like lesions. Our findings demonstrate that the luminal epithelium crucially affects the vascularization, growth and morphology of endometriosis-like lesions. Therefore, it is of major importance to standardize the cellular composition of endometrial grafts in order to increase the validity and reliability of pre-clinical rodent studies in endometriosis research.

The selection of peritoneal mesothelial cells is important for cell therapy to prevent peritoneal fibrosis.

Long-term peritoneal dialysis (PD) causes chronic peritoneal damage. Peritoneal mesothelial cells (PMCs) play an important role in peritoneal function. We investigated the possibility of cell therapy using the PMCs to prevent peritoneal damage in PD patients. We harvested human PMCs from the PD effluent of PD patients. The PMCs were separated based on morphological characteristics into epithelial-like (Epi) cells and fibroblast-like (Fib) cells by the limiting dilution method. We transplanted these cells into nude mice whose parietal and visceral peritoneum were scratched by mechanical scraping. The transplanted cells were detected at the parietal and visceral peritoneum. Compared with the positive control, the Epi cell therapy group showed very few adhesions and exhibited no thickening of the parietal and visceral peritoneum. However, the group with Fib cell therapy could not inhibit peritoneal adhesion and thickening. In addition, hepatocyte growth factor was expressed by the grafted Epi cells but not Fib cells. Fib cells expressed vascular endothelial growth factor stronger than Epi cells. These two types of cells from the same patient showed different characteristics and effects for cell therapy. These findings suggest that the PMCs from the PD patient showed different characteristics, such as Epi cells and Fib cells, and the selection of PMCs is important for cell therapy on the point of not only the direct cellular interactions but also cytokine secretion from the grafted cells. Furthermore, the differences in the morphological cell characteristics may influence their role in peritoneal regeneration.

Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development.

Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFß, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age- and stress-related thymic decline.