Sheet Grafts Research Articles

A Composite Application Technique of Single-stage Dermal Templates to Improve Handling and Ease of Use.

The "work horse" for the treatment of full-thickness defects of any etiology, including chronic nonhealing wounds and traumatic injuries, is generally autologous split-thickness skin grafts (STSGs), meshed, hand-fenestrated, or a sheet graft. Advancements in skin tissue engineering have allowed for the integration of dermal substitutes to be combined with autologous STSGs, adding valuable options for restoring the skin's complex multilayered structure. Although dermal templates offer a promising avenue for more nuanced reconstruction in certain cases, their application is not without challenges, particularly when they are made from delicate materials. The following technique simplifies the application technique of single-stage dermal templates by using a composite approach. To demonstrate this technique, MatriDerm, a 1-mm-thick bovine collagen-elastin dermal template, is used to enable an STSG to be applied in a one-stage procedure.

Glans Adipodermal Augmentation for Management of Neophallus Fat Atrophy After Penile Implant Insertion: Surgical Technique and Outcomes

To describe our surgical technique and outcomes of glans augmentation with autologous adipodermal or acellular dermal matrix (ADM) interposition grafts for fat atrophy of the neophallus following penile implant insertion. We retrospectively reviewed the outcomes of glans augmentation in phalloplasty patients presenting with fat atrophy following penile prosthesis insertion. Glans augmentation is performed by making a small posterior coronal incision to preserve the shaft-to-glans dermal blood supply. A plane is made between the glans skin and the capsule of the distal penile implant cylinder. An adipodermal graft or ADM sheet graft is then sized to the glans dissection space and inserted, covering the implant capsule and filling the glans. The graft harvest site and posterior coronal incisions are then closed. The primary postoperative outcome was the recurrence of implant glans skin impingement or erosion. From October 2017 through January 2023, 15 patients underwent glans augmentation after penile prosthesis insertion. The mean follow-up was 20 months. Adipodermal grafts were placed in 12 (80%) patients and ADM grafts in 3 (20%) patients. Two patients developed complications requiring surgical revision and 3 patients are considering a secondary glans augmentation, for a potential surgical revision rate of 33% (5/15). There were no wound infections, implant infections, or erosions. Glans augmentation with adipodermal or ADM graft interposition between the glans skin and implant capsule improves the neophallus appearance and may help prevent future implant erosion in phalloplasty patients who develop fat atrophy after penile implant insertion.

Application of a Single T-Shaped Porous High-Density Polyethylene in Oriental Nasal Tip-Plasty.

Porous high-density polyethylene (pHDPE) is an alternative material for septal extension grafts. This study investigated the clinical application of a single T-shaped pHDPE graft combined with auricular cartilage in oriental nasal tip-plasty. A retrospective analysis was conducted of 66 patients who underwent septal extension graft with a T-shaped pHDPE and auricular cartilage graft from December 2018 to November 2021. All patients were female from 19 to 51 years old (average: 28.9 years). Patients were followed up for 5 to 40 months with an average of 23.8 months. The postoperative nasal length (39.8±3.7mm) was significantly improved (35.6±5.7mm preoperatively, P <0.0001) with the tip projection increasing from 23.9±2.3mm preoperatively to 25.3±2.4mm postoperatively ( P =0.0005). There was a significant difference in the nasolabial angle (96.8±14.1 degrees preoperatively versus 101.8±7.2 degrees postoperatively, P =0.0105) but no significant change in the nasofrontal angle (143.3±9.5 degrees preoperatively versus 143.7±5.5 degrees postoperatively, P =0.7546), with an improved tip projection achieved in 93.9% of patients. One patient had an abscess in the nasal septum and a tip graft was performed using auricular cartilage in another patient because of an insufficient postoperative tip projection. There was a slight tip deviation in 2 patients, 1 case of a deviated nasal tip was corrected with a pHDPE sheet graft, while the other was left intact. Two patients had excess cartilaginous graft protruding to the nasal cavity that did not affect nasal ventilation and the shape of the nasal tip was relatively good, so the prominent cartilaginous graft was not addressed. In conclusion, this technique not only improved the bulbous nasal tip, lengthened the short nose, and increase the tip projection, but also prevented absorption of the existing septal cartilage and reduced infection or extrusion of the pHDPE.

The use of human-derived feeder layers for the cultivation of transplantable human epidermal cell sheet to repair second degree burn wounds.

Human epidermal cell sheet (human-ECS) is a feasible treatment option for wound injury. Traditionally, researchers often use murine 3T3 fibroblast cells as feeder layer to support human epidermal cell sheet grafts, thus increase risk to deliver animal-borne infection. To overcome the potential risks involved with xenotransplantation, we develop human foreskin fibroblast cell as feeder layer culture system and investigate the effects of human-ECS on second-degree burn wound healing in mini-pig in order to develop more effective and safer therapies to enhance wound healing in human. Human epidermal keratinocytes and fibroblasts were isolated from foreskin tissue and were co-cultured to manufacture human-ECS. The cell morphology was monitored with phase-contrast microscopy, the stem cell markers were assessed by flow cytometry, and by colony-forming efficiency (CFE) assay. The structure of human-ECS was observed by hematoxylin and eosin staining. Expression of cytokines in human-ECS was confirmed by enzyme-linked immunosorbent assay. Second-degree burn wounds were created on the dorsal of miniature pig to evaluate the effect of oil gauze, oil gauze combined with commercial epidermal growth factor (EGF) cream, and oil gauze combined with human-ECS. Wound healing rate, histological examination, and Masson staining were measured to observe the wound repair efficacy. Real-time PCR and Western blot were utilized to detect the expression level of EGF and interleukin 6 (IL-6). Stratified human-ECS with 6-7 layers of epidermal cells was successfully cultivated with human-derived feeder cells, in which epidermal cell highly expressed CD49f and CFE was 3%±0.45%. Application of human-ECS induced a higher wound healing rate than commerical EGF cream and oil gauze control. The expression of EGF in human-ECS group was higher than those in the other groups; however, the expression of IL-6 was significantly decreased at day14 by human-ECS treatment group. Human-derived feeder cells are suitable for cultivation of human-ECS, avoiding pathogen transmission. Human-ECS could enhance second-degree burn wound healing, and its promoting effect involved secreting a variety of cytokines to regulate tissue reparative process.

86 Outcomes for 43 Hand Burns Treated with 2:1 Meshed and Epidermal Autografts with Abundant Donors

IntroductionOur group has previously reported our experience in treating hand burns with 2:1 meshed autografting and simultaneous application of autologous skin cell suspension (ASCS) even when donor sites are abundant. Here, we sought to expand on this experience. We hypothesized that the use of 2:1 meshed autografting and ASCS (MA/ASCS) would provide comparable outcomes to hand burns treated with sheet graft.MethodsA retrospective review was conducted of all subjects operated on for deep 2nd and 3rd degree hand burns at our ABA-verified burn center from April, 2018 to May, 2021. Exclusion criterion was a burn of >20% TBSA. The cohorts were those subjects treated with MA/ASCS versus those treated with split thickness sheet, pie-crust, or 1:1 meshed autograft alone (STAG). Outcomes included proportion returning to work (RTW), length of time for RTW, and time to wound closure. Mann-Whitney U test was used for comparisons of continuous variables, and Fishers Exact test for categorical variables. Values are reported as median and interquartile range.ResultsSixty-eight subjects fit the study criteria (MA/ASCS n=43, STAG n=25). The MA/ASCS group was significantly older than the STAG cohort (45.5 yrs [32, 59.25] vs 35 [28, 45], p=0.013) with larger %TBSA burns overall (11.5% [7, 16.25] vs 2% [1, 3], p < 0.0001), and larger hand burns (186 cm2 [124.75, 330.5] vs 104 cm2 [56, 164], p=0.001). Comparable results were seen between MA/ASCS and STAG, respectively, for time to wound closure (8 days [7, 13] vs 8 [6, 14], p=0.48), proportion RTW (51% vs 64%, p=0.33), and days for RTW among those returning (38 [29.25, 62.75] vs 34.5 [20.75, 57], p=0.471). Fractional ablations were performed in 14% of the MA/ASCS group and 12% of the STAG group.ConclusionsDespite being significantly older, having larger hand wounds, and larger overall wounds within the parameters of the study criteria, patients with 20% TBSA burns or smaller whose hand burns were treated with 2:1 mesh and ASCS overspray had comparable time to wound closure and return to work as subjects treated with 1:1, pie-crust, or sheet STAG.

797 Single Stage Application of Autologous Skin Cell Suspension in Deep Partial Thickness Pediatric Facial Burns

IntroductionDeep partial thickness facial burns in the pediatric population present a dilemma. Standard burn treatment includes excision and split thickness sheet grafting for the face or closure which often yield sub-optimal results. Point-of-care system for autologous skin cell suspension (ASCS) can be utilized as a stand alone option for partial thickness burns. We sought to evaluate our early results from the treatment of pediatric deep partial thickness facial burns using ASCS.MethodsThis was a retrospective cohort analysis of pediatric deep partial thickness facial burns treated with early excision and application of ASCS. After assessment by two surgeons, patients underwent tangential excision with either standard surgical burn knives or dermabrasion. Depth was assessed and if dermal elements were present, then ASCS was applied. Primary outcome was time to >95% reepithelialization, and secondary outcomes were aesthetics, Vancouver Scar Scale, postoperative complications, number of reoperations for wound closure, and need for burn reconstruction.ResultsFrom September 2020 to July 2021, eight pediatric patients (six male and two female) with deep partial thickness facial burns were treated within one week of injury with excision and ASCS. TBSA ranged from 0.3%-24% with some having additional anatomic areas. The mean age was 2.5 years old, with a range of 1.08-5.0 years old. Burn mechanisms included flame, scald, and contact. The mean time from burn injury to initial excision and ASCS was 4.3 days, with a range of 2-7 days. Average time to complete reepithelialization was 20.3 days, with a range of 4-47 days. Average follow up duration was 218 days with a range of 98-365 days. No patients experienced complete graft loss or infection. One patient required need for reoperation with reapplication of polylactic acid skin substitute placed to a small area on the face during closure for other non-face burns. Of the 8 patients, the two patients with the longest times to complete reepithelialization required laser therapy, of which one patient also needed limited facial scar contracture release.ConclusionsEarly, single-staged ASCS application results in good aesthetic and relatively quick healing times with no complications and limited need for additional burn reconstruction. While the results are promising, there is a need for larger studies with longer term follow up before wider applicability.

Meshed Versus Sheet Skin Graft for Scrotum and Perineal Skin Loss: A Retrospective Comparative Study.

BackgroundA wide array of diseases can lead to skin defects of the male genitalia. Although reconstructive options have been debated in the literature, no study has compared the effectiveness of a meshed split-thickness skin graft (STSG) and a sheet STSG in perineal and scrotal wound coverage. In this study, we report our experience in a tertiary trauma center.MethodologyIn this retrospective study, we included cases with a skin defect of the male genitalia, for which genital reconstruction with a skin graft was performed at our hospital from December 2017 to February 2020. This study was approved by the institutional review board. The analysis was performed at 95% confidence interval using the Statistical Package for Social Science (SPSS) version 23.0 (IBM Corp., Armonk, NY, USA).ResultsA total of 27 patients were included in the study. The most common indication for genital reconstruction was Fournier’s gangrene (59.3%). In 15 (55.6%) patients, a meshed skin graft was utilized to cover the defect, whereas a sheet graft was utilized in 12 (44.4%) patients. Out of the 15 patients who underwent genital reconstruction with a meshed graft, 10 (66.6%) had complete graft take. On the other hand, out of the 12 (44.4%) patients who underwent genital reconstruction using a sheet graft, five (41.6%) had complete graft take. A statistically significant relationship was found between aesthetic and functional outcomes and the type of skin graft used. The satisfaction rate was higher among meshed skin graft recipients (86.2%) compared to sheet skin graft recipients (41.7%) (p = 0.014).ConclusionsBased on our observational experience, we found that meshed STSG to cover male genital skin defects is safe with satisfactory cosmetic outcomes. Further prospective randomized studies are needed.

Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line

ObjectivesCone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. MethodsWe generated OPN1LW-EGFP/NRL−/− mice by crossing NRL−/− and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL−/− mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL−/− mice were transplanted subretinally into immunodeficient Rd1 mice. ResultsOPN1LW-EGFP/NRL−/− retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL−/− compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL−/− were similar as the NRL−/− with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL−/− was thinner than NRL−/− controls. The OPN1LW-EGFP/NRL−/− ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL−/− controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL−/− mice showed greater S-opsin + cone survival than suspension grafts from the same strain. ConclusionsOPN1LW-EGFP/NRL−/− retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Temporary Storage of the Human Nasal Tissue and Cell Sheet for Wound Repair.

Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen–thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank’s balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank’s balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank’s balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.

Outcomes for Hand Burns Treated With Autologous Skin Cell Suspension in 20% TBSA and Smaller Injuries.

In order to address the confounder of TBSA on burn outcomes, we sought to analyze our experience with the use of autologous skin cell suspensions (ASCS) in a cohort of subjects with hand burns whose TBSA totaled 20% or less. We hypothesized that the use of ASCS in conjunction with 2:1 meshed autograft for the treatment of hand burn injuries would provide comparable outcomes to hand burns treated with sheet or minimally meshed autograft alone. A retrospective review was conducted for all deep partial and full-thickness hand burns treated with split-thickness autograft (STAG) at our urban verified burn center between April 2018 and September 2020. The exclusion criterion was a TBSA greater than 20%. The cohorts were those subjects treated with ASCS in combination with STAG (ASCS(+)) vs those treated with STAG alone (ASCS(-)). All ASCS(+) subjects were treated with 2:1 meshed STAG and ASCS overspray while all ASCS(-) subjects had 1:1, piecrust, or unmeshed sheet graft alone. Outcomes measured included demographics, time to wound closure, proportion returning to work (RTW), and length of time to RTW. Mann-Whitney U test was used for comparisons of continuous variables and Fisher's exact test for categorical variables. Values are reported as medians and 25th and 75th interquartile ranges. Fifty-one subjects fit the study criteria (ASCS(+) n = 31, ASCS(-) n = 20). The ASCS(+) group was significantly older than the ASCS(-) cohort (44 [32-54] vs 32 years [27.5-37], P = .009) with larger %TBSA burns (15% [9.5-17] vs 2% [1-4], P < .0001) and larger size hand burns (190 [120-349.5] vs 126 cm2 [73.5-182], P = .015). Comparable results were seen between ASCS(+) and ASCS(-), respectively, for time to wound closure (9 [7-13] vs 11.5 days [6.75-14], P = .63), proportion RTW (61% vs 70%, P = .56), and days for RTW among those returning (35 [28.5-57] vs 33 [20.25-59], P = .52). The ASCS(+) group had two graft infections with no reoperations, while ASCS(-) had one infection with one reoperation. No subjects in either group had a dermal substitute placed. Despite being significantly older, having larger hand wounds, and larger overall wounds within the parameters of the study criteria, patients with 20% TBSA burns or smaller whose hand burns were treated with 2:1 mesh and ASCS overspray had comparable time to wound closure, proportion of RTW, and time to return to work as subjects treated with 1:1 or piecrust meshed STAG. Our group plans to follow this work with scar assessments for a more granular picture of pliability and reconstructive needs.

Multilayer Collagen Sheet Graft for Corneal Perforation with a Corneal Ulcer

Multilayer Collagen Sheet Graft for Corneal Perforation with a Corneal Ulcer

Artificial Dermal Template for Cultured Keratinocyte Sheet Grafting Detached by Temperature Responsive Dish in an Ovine Burn Wound Model

Artificial Dermal Template for Cultured Keratinocyte Sheet Grafting Detached by Temperature Responsive Dish in an Ovine Burn Wound Model

Bladder reconstruction using autologous smooth muscle cell sheets grafted on a pre-vascularized capsule.

Rationale: Construction of functional vascularized three-dimensional tissues has been a longstanding objective in the field of tissue engineering. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate functional vascularized smooth muscle tissue flaps for bladder reconstruction in a rabbit model was tested.Methods: Skin tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Smooth muscle cells and endothelial progenitor cells were harvested and cocultured to form pre-vascularized smooth muscle cell sheet. Then repeated transplantation of triple-layer cell sheet grafts onto the vascularized capsular tissue was performed at 2-day intervals to prefabricate functional vascularized smooth muscle tissue flaps. Bladder muscular wall defects were created and repaired by six-layer cell sheet graft (sheet only), capsule flap (capsule only) and vascularized capsule prelaminated with smooth muscle cell sheet (sheet plus capsule). The animals were followed for 3 months after implantation and their bladders were explanted serially.Results: Bladder capacity and compliance were maintained in sheet plus capsule group throughout the 3 months. Tissue bath stimulation demonstrated that contractile responses to carbachol and KCl among the three groups revealed a significant difference (p < 0.05). Histologically, inflammation was evident in the capsule only group at 1 month and fibrosis was observed in sheet only group at 3 months. The vessel density in capsule only and sheet plus capsule group were significantly higher than in the sheet only group at each time point (p < 0.05). Comparison of the smooth muscle content among the three groups revealed a significant difference (p < 0.05).Conclusion: These results proved that the capsule may serve as an induced vascular bed for vascularized smooth muscle tissue flap prefabrication. The prefabricated functional vascularized smooth muscle tissue flap has the potential for reliable bladder reconstruction and may create new opportunities for vascularization in 3-D tissue engineering.

Keratinocyte Monolayers on Hyaluronic Acid Membranes as "Upside-Down" Grafts Reconstitute Full-Thickness Wounds.

BackgroundSkin replacement by means of cultured epithelial keratinocytes is a well-accepted method. However, several clinical drawbacks of sheet autografts (CEA – cultured epithelial autografts) have stimulated various efforts to optimize cell culture and cell delivery. Recent developments include use of cell monolayers instead of a fully differentiated epithelium, as well as use of various biomaterials to grow and transport the cultured cells.To optimize the transfer of human keratinocytes directly to the recipient wound bed, we used an “upside-down” technique, delivering cultured cells directly to the wound with the carrier material on top.Material/MethodsSubconfluent second-passage human keratinocyte monolayers on esterified hyaluronic acid membranes (KHAMC – Keratinocyte-Hyaluronic-Acid-Membrane-Composites) were transplanted either as upside-down grafts or as upside-up grafts onto standardized full-thickness wounds in athymic nude mice versus controls with the cell-free membrane alone.ResultsIn the upside-down group, 14 days after grafting, a multi-layered, differentiating epidermis was found, whereas the wounds in the upside-up group and in the control group were not completely closed up to day 21. Persistence of human keratinocytes was shown in the upside-down group only, from day 7 until day 35 after grafting.ConclusionsThis study confirms that upside-down grafting of subconfluent monolayers of serum-free cultured human keratinocytes on esterified hyaluronic acid membranes is a suitable means to transfer actively proliferative keratinocytes, and reduces wound contraction. Compared to standard grafting protocols of cultured epithelium, such as CEA sheet grafts, it is easier to apply, does not need enzymatic detachment of cells from the culture dish, and limits the number of production steps required.

Scar Quality of Skin Graft Borders: A Prospective, Randomized, Double-Blinded Evaluation

Prominent scars may remain around the border of a mature skin graft (SG) at the interface of the SG with normal skin. The border of a SG may be constructed by either exactly approximating (A) or slightly overlapping (O) the edge of the SG on the wound margin. The purpose of this study was to evaluate whether Aor O affects the quality of the border scar of SGs applied to burn patients. This prospective study was a within-border design in which adult burn patients requiring SGs served as their own control. Half of each study border was fashioned using O and the immediately adjacent other half was made using A.We randomly assigned O or Ato the proximal or distal halves of vertical borders and the medial and lateral halves of horizontal borders. Both halves of the study border were identically fixated with staples or sutures and were managed in the same fashion postoperatively. Blinded evaluations at 3, 6, and 12months of O and Aborders were performed using the Vancouver Scar Scale (VSS), the observer component of the Patient and Observer Scar Assessment Scale (POSAS), and a global binary assessment of which half of the study border "looked better." Blinded patients also rated each half of the study border with a 10-point Likert scale. Values are reported as the mean ± SD or median (interquartile range), as appropriate. There were 34 borders studied in 15 subjects (46.7% female, age 29 [22,57], % TBSA burn 9.7± 5.3, and no inhalation injuries). Study borders were constructed at 7 (5,11) days postburn, had a total length of 12 (9.3,14.5) cm, and all involved split thickness SGs of thickness 13 (12,14)/1000th of an inch. Sheet grafts were applied in 27% and meshed grafts in 73%. SGs were applied immediately after excision in 75% or after allografting in 25%. Border scars matured between 3 and 12months with reductions in total VSS from 8 (7,8) to 4 (3,6) for O borders (P < .001) and from 8 (7,9) to 4 (1,6) for Aborders (P < .001). However, there were no significant differences between O and Aborders in total VSS at 3months (P=.165), 6months (P=.602), and 12months (P=.358) or in total OSAS at 3months (P=.681), 6months (P=.890), or 12months (P=.601). At 12months, 60% of O borders and 40% of Aborders were globally rated as "better" (P=.258). There were no significant differences in the patients' subjective ratings of the O and Aborders at 3months (P=.920), 6months (P=.960), and 12months (P=.66). The scar quality at the border of a skin graft does not appear to be affected by the surgical technique used to construct the border at the time of grafting.

Preserving the extra cellular matrix is critical for improving efficacy of keratinocyte sheets for treatment of burn wounds

IntroductionCultured autologous keratinocyte sheets (KS) have been proposed for treating burn wounds for over 30 years; however, they have not been translated to clinical practice because of negative effects of enzymes, used for detachment, on KS quality. The aim of this study was to test the hypothesis that non‐enzymatically detached KS exert better effects on grafted burn wound healing compared to enzymatically detached KS.MethodsFor non‐enzymatic detachment, autologous ovine primary keratinocytes were cultured in novel temperature‐responsive dishes (T sheets), and were detached by temperature reduction to 20°C. For comparison, the aliquots of keratinocytes were also cultured in conventional dish to be detached by use of enzyme (Dispase) (D sheets). We have tested the efficacy of these sheets using a well‐characterized, novel and clinically relevant ovine model of grafted burn wounds. Briefly, six full thickness skin burns (5×5cm) were induced in sheep dorsum under anesthesia and analgesia. After 24 hrs, burned skin was excised and wounds were grafted with ovine cadaver skin. After 3 weeks, rejected cadaver skin epidermis was debrided and wounds were covered with T and D sheets. Thereafter, the wounds were assessed for two more weeks. Wound samples were collected postmortem for various assays. The autologous keratinocytes for the sheet culture were isolated at the same day of skin grafting which mimics the clinical situation.ResultsCultured keratinocytes in both dishes formed firm sheets within three weeks, mimicking the time of cadaver skin epidermis rejection. Keratinocyte sheet thickness was significantly higher in T sheets compared to D sheets. In vivo, T sheets were better accepted with significantly higher wound re‐epithelialization percentage than D sheets at days 7 and 14 after sheet grafting. Although the epidermis thickness was comparable in wounds treated with both types of sheets, the dermal‐epidermal junction was well defined in wounds covered with T sheets at 14th day as evidenced by continuous lamina densa and high number of hemi‐desmosomes. Histopathological evaluation for burn wounds revealed that wounds treated with T sheets had less hemorrhage, ulceration incidence and neutrophils infiltration.ConclusionNovel non‐enzymatic method for KS detachment provides better sheet quality and accelerates healing of grafted burn wounds.Support or Funding InformationShriner's Hospital for Children SHC85100 and SHC84050This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Early Versus Delayed Excision and Grafting on Restoring the Functionality of Deep Burn-Injured Hands: A Double-Blind, Randomized Parallel Clinical Trial

Hand burns have major impacts on daily activities. An interdisciplinary and multifaceted approach is necessary to effectively treat these burns. Generally, hands are common sites for burn injuries. It is known that Early Excision and Grafting (EEG) of deep burn wounds can decrease scar and subsequent contracture formation compared with Delayed Excision and Grafting (DEG) method. Accordingly, this study aimed to compare the effects of these procedures on function of hands with deep burns, by using the Michigan Hand Questionnaire (MHQ). In this double-blind randomized clinical trial, we evaluated a total of 50 patients (age range: 15-70 years) with deep second-degree burns of both hands from January 2013 to December 2015. The subjects were randomly divided into two groups, treatment (EEG) and control (DEG) groups (25 patients per group). They followed up postoperatively for 3 months. MHQ was completed for each of them to determine the function of the hands, pain sensation, limitation of daily activities, overall satisfaction, and cosmetic appearance during follow-up. Data were analyzed in the groups using SPSS version 16. Baseline characteristics of the groups were matched. In early postoperation, hand functions and daily activities, gross appearance, and pain sensation were better in EEG group. After 1 and 3 months, no significant difference was detected between the groups except for patient satisfaction which was better in EEG group. Also, EEG group was associated with a significant shorter hospital stay and lower treatment costs compared with DEG group. Graft take rate was similar in both groups. EEG is recognized as the standard treatment for deep skin burns, but we did not find any significant difference between effects of EEG and DEG on hand functions although EEG group had shorter hospital stay and lower treatment costs compared with DEG. The main goal of treatment of hand burns is restoration of the hand functions. Based on our findings, hand function was not related to the type of surgery. Also, as all wounds in both surgical groups healed less than 3 weeks, the authors suggest that the excision time limit could be change in our burn center from 10 days to 2 to 3 weeks when we use sheet graft as for hands.

495 Factors Affecting Optimal Outcome in Pediatric Skin Grafts

The majority of skin grafts done at our pediatric burn center are sheet grafts performed exclusively by two surgeons with similar techniques. In spite of the excellent take and appearance of sheet grafts in the acute and subacute post operative period, a number of grafts developed hypertrophic scarring yielding suboptimal long term outcomes when mature. The aim of this study is to explore contributory factors that may potentially be modified to yield better long term outcomes in our sheet skin grafts and reduce the need for additional scar treatment interventions. Records for all patients who underwent split-thickness skin grafts at our institution between January of 2013 and July of 2018 were reviewed. Data including age, gender, race, Fitzpatrick score, mechanism of injury, burn anatomic location, joint involvement, time to skin graft, time to first pressure garment fitting, percentile weight change during graft healing and maturation, and compliance with garment wear was collected and analyzed. Grafts were scored from most recent medical photographs by a panel of clinicians and non clinicians as optimal or suboptimal. We identified 131 grafts to review. The median follow-up was 249 days (range 23-2455 days). Sixty nine grafts (53%) had a suboptimal long term outcome. Of all factors studied, only joint involvement was significantly associated with suboptimal outcome (p=0.0009). Compliance with pressure garment wear resulted in significantly more optimal outcomes overall (p=0.025). However, in the subgroup of patients that was highly compliant with pressure garments and had skin grafts involving joints, graft outcome was optimal in only 50%, indicating equivocal value of pressure in those areas. Skin grafts involving joints are more likely to have a suboptimal outcome requiring further scar treatment after maturation. This is an injury related factor that cannot be affected. However, patient compliance with pressure therapy yielded more optimal graft outcomes. We recommend pressure garment therapy following skin grafting in pediatric burn patients with an emphasis on outpatient compliance to reduce the risk of long term hypertrophic scarring. The applicability of this research study will eventually be utilizable and adapted to our institute practice, in order to produce the desired outcome over time.

474 Cyanoacrylate Glue for Sheet Graft Fixation in Patients with Burn Injuries

474 Cyanoacrylate Glue for Sheet Graft Fixation in Patients with Burn Injuries

Construction of Pedicled Smooth Muscle Tissues by Combining the Capsule Tissue and Cell Sheet Engineering.

The survival of engineered tissue requires the formation of its own capillary network, which can anastomose with the host vasculature after transplantation. Currently, while many strategies, such as modifying the scaffold material, adding endothelial cells, or angiogenic factors, have been researched, engineered tissue implanted in vivo cannot timely access to sufficient blood supply, leading to ischemic apoptosis or shrinkage. Constructing vascularized engineered tissue with its own axial vessels and subsequent pedicled transfer is promising to solve the problem of vascularization in tissue engineering. In this study, we used the tissue expander capsule as a novel platform for vascularizing autologous smooth muscle cell (SMC) sheets and fabricating vascularized engineered tissue with its own vascular pedicle. First, we verified which time point was the most effective for constructing an axial capsule vascular bed. Second, we compared the outcome of SMC sheet transplantation onto the expander capsule and classical dorsal subcutaneous tissue, which was widely used in other studies for vascularization. Finally, we transplanted multilayered SMC sheets onto the capsule bed twice to verify the feasibility of fabricating thick pedicled engineered smooth muscle tissues. The results indicated that the axial capsule tissue could be successfully induced, and the capsule tissue 1 week after full expansion was the most vascularized. Quantitative comparisons of thickness, vessel density, and apoptosis of cell sheet grafts onto two vascular beds proved that the axial capsule vascular bed was more favorable to the growth and vascularization of transplants than classical subcutaneous tissue. Furthermore, thick vascularized smooth muscle tissues with the vascular pedicle could be constructed by multi-transplanting cell sheets onto the capsule bed. The combination of axial capsule vascular bed and cell sheet engineering may provide an efficient strategy to overcome the problem of slow or insufficient vascularization in tissue engineering.