Translational application of human keratinocyte-fibroblast cell sheets for accelerated wound healing in a clinically relevant type 2 diabetic rat model.

BACKGROUND AIMS: Despite advancements in wound care, wound healing remains a challenge, especially in individuals with type 2 diabetes. Cell sheet technology has emerged as an efficient and promising therapy for tissue regeneration and wound repair. Among these, bilayered human keratinocyte-fibroblast cell sheets constructed using temperature-responsive culture surfaces have been shown to mimic a normal tissue-like structure and secrete essential cytokines and growth factors that regulate the wound healing process. METHODS: This study aimed to evaluate the safety and therapeutic potential of human skin cell sheets to treat full-thickness skin defects in a rat model of type 2 diabetes. RESULTS: Our findings demonstrate that diabetic wounds transplanted with bilayered cell sheets resulted in accelerated re-epithelialization, increased angiogenesis, enhanced macrophage polarization and regeneration of tissue that closely resembled healthy skin. In contrast, the control group that did not receive cell sheet transplantation presented characteristic symptoms of impaired and delayed wound healing associated with type 2 diabetes. CONCLUSIONS: The secretory cytokines and the upregulation of Nrf2 expression in response to cell sheet transplantation are believed to have played a key role in the improved wound healing observed in diabetic rats. Our study suggests that human keratinocyte-fibroblast cell sheets hold great potential as a therapeutic alternative for diabetic ulcers.

Recipient-to-Donor Ratios in the Surgical Treatment of Vitiligo, Leukoderma, and Piebaldism: A Retrospective Review.

BACKGROUND: The autologous noncultured melanocyte keratinocyte transplant procedure (MKTP) has emerged as a popular grafting technique with proven efficacy for achieving repigmentation. However, there remains no consensus regarding the optimal recipient-to-donor (RD) ratio required to achieve acceptable repigmentation. In this retrospective cohort study of 120 patients, we sought to examine whether expansion ratios impact the repigmentation success rates following MKTP. RESULTS: A total of 69 patients (mean [SD] age was 32.4 [14.3] years, mean follow-up was 30.4 [22.5] months, 63.8% were male; 55% were dark-skinned individuals [Fitzpatrick IV-VI]) were included. The mean percent change in the Vitiligo Area Scoring Index (VASI) was 80.2 (±23.7; RD of 7.3) in patients with focal/segmental vitiligo (SV), 58.3 (±33.0; RD of 8.2) in those with non-segmental vitiligo (NSV), and 51.8 (±33.6; RD of 3.7) in those with leukoderma and piebaldism. Focal/SV was positively associated with a higher percent change in VASI (parameter estimate: 22.6, p value <0.005). In the SV/focal group, non-white patients had a higher RD ratio compared to White individuals (8.2 ± 3.4 vs. 6.0 ± 3.1, respectively, p value = 0.035). DISCUSSION: In our study, we found that patients with SV were significantly more likely to achieve higher repigmentation rates compared to those with NSV. Although repigmentation rates were higher in the low expansion ratio group than in the high expansion ratio group, we did not observe a significant difference between the two groups. CONCLUSION: MKTP is an effective therapy for restoring repigmentation in patients with stable vitiligo. Therapeutic response of vitiligo to MKTP appears to be influenced by the type of vitiligo, rather than a specific RD ratio.

Dermabrasion Versus Microneedling in Transplantation of Autologous Noncultured Melanocyte-Keratinocyte Cell Suspension in Patients With Vitiligo.

BACKGROUND: Autologous noncultured melanocyte-keratinocyte transplantation is one of the procedures used to treat stable vitiligo with varying reported results. Recipient site preparation is one of the variables that could affect repigmentation outcomes. OBJECTIVE: To assess the effectiveness of transplanting autologous melanocyte-keratinocyte suspension in patients with stable vitiligo and to compare recipient site preparation using dermabrasion versus microneedling. METHODS: From March 2020 to September 2022, this randomized comparative study included 40 patients with 40 stable vitiligo lesions managed by suspension transplants of melanocytes. Patients were divided into 2 groups: group A, where the recipient site was prepared using dermabrasion, and group B, which was done by microneedling. The assessment was performed 3 months after the treatment based on the degree of repigmentation (excellent, ≥90%; good, 50%-89%; fair, 20%-49%; and poor response, <20%). RESULTS: Both modalities resulted in effective repigmentation, but the dermabrasion group showed a statistically significant improvement and a satisfactory repigmentation rate. CONCLUSION: Autologous melanocyte transplantation is a safe and effective treatment method for stable vitiligo lesions that have not responded to other therapies. When compared with microneedling, dermabrasion produced better outcomes for recipient site preparation.

Transplantation of autologous epidermal melanocyte-keratinocyte cells suspension for cell therapy of vitiligo: A clinical evaluation and biometric assessment.

INTRODUCTION: Among several surgical treatments, the use of transplantation of epidermal cultured melanocytes or melanocytes-keratinocytes cell suspension has gained many researchers and dermatologists' attention as a new technique for the treatment of vitiligo. The present study aimed to transplant autologous epidermal melanocytes-keratinocytes cell suspension for the treatment of vitiligo. METHODS: In this study, 15 volunteer patients aged between 18 and 45 years old were studied. The autologous melanocytes-keratinocytes cell suspension was then transplanted to the region after dermabrasion. The included patients were evaluated by VisioFace, MPA9, and Skin Scanner-DUB once before and 1, 2, and 6 months after the transplantation, while the extents of stainability and changes in the transplanted region were recorded. RESULTS: The color contrast between the lesion and normal skin significantly decreased after 1, 2, and 6 months of the melanocytes transplantation compared with the pre-procedure (13.8 ± 0.45 before vs. 12.9 ± 0.43, 12.2 ± 0.45, and 10.2 ± 0.34 at months 1, 2, and 6, p < 0.001). Furthermore, melanin index significantly increased six months after cell transplantation compared to the pretreatment (168.3 ± 4.22 vs. 130.5 ± 3.98, p < 0.001). CONCLUSION: Transplantation of melanocytes cells with dermabrasion can be effective on vitiligo improvement, so it is recommended.

The influence of CD26+ and CD26- fibroblasts on the regeneration of human dermo-epidermal skin substitutes.

CD26, also known as dipeptidyl peptidase IV (DPPIV), is a multifunctional transmembrane protein playing a significant role in the cutaneous wound healing processes in the mouse skin. However, only scarce data are available regarding the distribution and function of this protein in the human skin. Therefore, the aim of this study was to investigate the impact of CD26 deficiency in human primary fibroblasts on the regeneration of human tissue-engineered skin substitutes in vivo. Dermo-epidermal skin analogs, based on collagen type I hydrogels, were populated either with human CD26+ or CD26knockout fibroblasts and seeded with human epidermal keratinocytes. These skin substitutes were transplanted onto the back of immune-incompetent rodents. Three weeks post-transplantation, the grafts were excised and analyzed with respect to specific epidermal and dermal maturation markers. For the first time, we show here that the expression of CD26 protein in human dermis is age-dependent. Furthermore, we prove that CD26+ fibroblasts are more active in the production of extracellular matrix (ECM) both in vitro and in vivo and are necessary to achieve rapid epidermal and dermal homeostasis after transplantation.

Dried human cultured epidermis accelerates wound healing in diabetic mouse skin defect wounds.

Cryopreserved allogeneic cultured epidermis (CE) is used for treating second-degree burn wounds and diabetic foot ulcers; however, the need for cryopreservation limits its use. We have previously reported that CE accelerates wound healing irrespective of its viability and hypothesized that dehydrated CEs lacking living cells may act as an effective wound dressing. We prepared dried CE and investigated its morphological and physical properties and wound-healing effects and compared them with those of cryopreserved CE. Hematoxylin-eosin staining, immunostaining for basement membrane, and electron microscopy revealed that the morphologies of dried CE and cryopreserved CE were comparable and that the membrane structure was not damaged. The breaking strength, modulus of elasticity, and water permeability of dried CE were comparable with those of the cryopreserved CE. Furthermore, the levels of various active cytokines and chemokines in dried CE were comparable with those in cryopreserved CE. Dried CE applied to skin defect in diabetic mice significantly reduced the wound area and increased the new epithelium length 4 and 7 days after implantation, similar to that observed for cryopreserved CE. Consequently, dried CE had similar morphological and physical properties and wound-healing effects compared with those of cryopreserved CE and can be a physiological and versatile wound-dressing.

New options to manage epidermolysis bullosa.

RDEB is a genetic skin disorder involving COL7A1, the gene encoding C7, which is a component of the fibers anchoring the epidermis to the dermis. Affected patients (about 1.35 per million persons in the United States) experience lifelong painful itching, blistering, fibrosis, impaired healing, and scarring, with increased likelihood of infection and cancer. RCT involving those affected by RDEB are rare with limited sample sizes due to the low prevalence of this genetic disorder. Treatment with topical or systemic agents has not consistently improved patient outcomes. Therapies focused on replacing C7 using autologous bone marrow or keratinocyte grafts have been difficult, with high complication rates, and have been met with mixed success. Recent HSV-1 vector research4 led to development of HSV-1 vectors capable of transferring the COL7A1 coding sequence to keratinocytes and fibroblasts in vitro and to mice deficient in C7, as well as to RDEB human skin xenografts. This research offered new opportunities for gene therapy for patients with RDEB. In this final Evidence Corner, readers are invited to consider the implications of 2 small RCTs that suggest fruitful avenues for RDEB research and practice. The first study describes a pioneering phase 1 and 2 RCT of topical gene therapy for RDEB. The second study describes a small crossover RCT exploring the effect of topical calcipotriol (VD3) ointment on wound healing and pruritis in patients with RDEB.

Effect of Rho-Associated Protein Kinase Inhibitors on Epidermal Keratinocytes: A Proposed Application for Burn Wound Healing.

Rho-associated protein kinases (ROCKs) affect a variety of cellular functions, including cell attachment, migration, and proliferation. ROCK inhibitors therefore have potential as tools for optimizing cell behavior in tissue engineering applications, including the manufacturing of cultivated epithelial autografts (CEAs) used in the treatment of burn patients. For example, ROCK inhibitors may facilitate earlier engraftment of CEA sheets by increasing the proliferation of skin keratinocytes ex vivo. Nevertheless, the current understanding of ROCK inhibitor action on epidermal keratinocytes is unclear owing to multiple drug formulations, drug concentrations, and cellular function assays having been used. The aim of this review article therefore is to identify consistent patterns of ROCK inhibitor action on human keratinocytes, as well as revealing key knowledge gaps. In doing so, we propose a clearer course of action for pursuing the potential benefits of ROCK inhibitors for the future treatment of burn patients. Impact statement The properties of Rho-associated protein kinase (ROCK) inhibitors are already used clinically within the fields of cardiology, neurology, and ophthalmology. These results encourage the broadening of ROCK inhibitor uses for other clinical applications. With respect to burn patients, ROCK inhibitors may facilitate improvements in patient survival and healing by reducing the time required for generating cultivated epithelial autograft (CEA) sheets from patient biopsies. Nevertheless, varying approaches to studying the effects of ROCK inhibitors on skin cells in vitro have complicated the development of improved protocols. Our review aims to clarify a diverse and growing body of literature as to the potential benefits for burn patients.

Fractional versus full ablative CO2 laser in recipient site of non-cultured melanocytes and keratinocyte transplantation in treatment of vitiligo.

BACKGROUND: Melanocyte-keratinocyte transplant procedure (MKTP) or non-cultured epidermal cell suspension transplantation is a very popular surgical modality for treating stable vitiligo. The recipient-site preparation is one potential determinant in the repigmentation outcomes. AIM: To assess the efficacy of fractional CO2 (FCO2 ) laser in recipient-site preparation before MKTP and comparing it to the frequently used full surface laser ablation. METHODS: This randomized comparative trial included 19 patients with 40 stable vitiligo lesions. In each patient, the treated sites were randomly categorized into two groups according to the recipient-site ablation (either fractional or full ablative CO2 laser). Assessment of repigmentation was performed six months after the procedure. RESULTS: Both modalities achieved successful repigmentation of a median of 80% and 77.5% for fractional and full ablation groups, respectively, with a non-statistically significant difference between them. The median of VASI change percent was -73% and -71% with fractional and full surface ablation, respectively. CONCLUSIONS: FCO2 laser ablation is effective for recipient-site preparation before cell suspension transplantation as well as the full ablative CO2 laser.

Transgenic Epidermal Cultures for Junctional Epidermolysis Bullosa - 5-Year Outcomes.

Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014-2020 - Regione Emilia-Romagna and others.).

In vitro cell culture of amniotic fluid keratinocytes on amniotic membrane: the ideal tissue for repairing skin ulcers.

OBJECTIVE: The amniotic fluid contains a large population of stem keratinocytes demonstrating minimal immunological rejection. Recent evidence suggests that stem cells from the amniotic fluid can be employed in the field of tissue engineering. In this work we identified precursors of the epithelial cells and expanded them in vitro. MATERIALS AND METHODS: After collecting samples of amniotic fluid and separating the cells via centrifugation, we seeded a portion of these cells in selection media to analyze the proliferation of epithelial cells. The stem cells precursors of keratinocytes were identified through specific markers. The expression of these markers was evaluated by immunofluorescence and reverse transcription polymerase chain reaction (PCR). RESULTS: The stem cells demonstrated 90% confluence, after undergoing proliferation in the selection medium for 15 days. Most of these cells tested positive for the keratinocyte-specific markers, but negative for stem cell specific markers. Of note, the identity of the keratinocytes was well established even after several subcultures. CONCLUSIONS: These results suggested that it is feasible to isolate and expand differentiated cell populations in the amniotic fluid from precursor cells. Furthermore, amniotic membranes can be utilized as scaffolds to grow keratinocytes, which can be potentially exploited in areas of skin ulcer transplantation and tissue engineering interventions.

RAS induced senescence of skin keratinocytes is mediated through Rho-associated protein kinase (ROCK).

Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated ß-galactosidase (SAßgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.

The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity.

Cells/colony motion of oral keratinocytes determined by non-invasive and quantitative measurement using optical flow predicts epithelial regenerative capacity.

Cells/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cells/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage [passage 1 (p1)] cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 µm/h reflects cellular damages by experimental metabolic challenges although this value shall only apply in case of our culture system. Nonetheless, the motion index can be used as the threshold to determine the quality of cultured cells while it may be affected by any different culture conditions. Because the p1 cells/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

Scale-up of a Composite Cultured Skin Using a Novel Bioreactor Device in a Porcine Wound Model.

Extensive deep-burn management with a two-stage strategy can reduce reliance on skin autografts; a biodegradable polyurethane scaffold to actively temporize the wound and later an autologous composite cultured skin (CCS) for definitive closure. The materials fulfilling each stage have undergone in vitro and in vivo pretesting in "small" large animal wounds. For humans, producing multiple, large CCSs requires a specialized bioreactor. This article reports a system used to close large porcine wounds. Three Large White pigs were used, each with two wounds (24.5 cm × 12 cm) into which biodegradable dermal scaffolds were implanted. A sample from discarded tissue allowed isolation/culture of autologous fibroblasts and keratinocytes. CCS production began by presoaking a 1-mm-thick biodegradable polyurethane foam in autologous plasma. In the bioreactor cassette, fibroblasts were seeded into the matrix with thrombin until established, followed by keratinocytes. The CCSs were applied onto integrated dermal scaffolds on day 35, alongside a sheet skin graft (30% of one wound). Serial punch biopsies, trans-epidermal water loss readings (TEWL), and wound measurements indicated epithelialization. During dermal scaffold integration, negligible wound contraction was observed (average 4.5%). After CCS transplantation, the control skin grafts were "taken" by day 11 when visible islands of epithelium were clinically observed on 2/3 CCSs. Closure was confirmed histologically, with complete epithelialization by day 63 post-CCS transplantation (CCS TEWL ~ normal skin average 11.9 g/m2h). Four of six wounds demonstrated closure with robust, stratified epithelium. Generating large pieces of CCS capable of healing large wounds is thus possible using a specialized designed bioreactor.

A retrospective study of long term follow-up of 2283 vitiligo patients treated by autologous, non-cultured melanocyte-keratinocyte transplantation.

BACKGROUND: Autologous non-cultured melanocyte-keratinocyte transplantation (MKTP) can be used to treat stable vitiligo cases, but there were insufficient clinical data to evaluate its safety and efficacy. OBJECTIVE: To assess the influence of various factors on the therapeutic outcome of MKTP. METHOD: The single-center retrospective study included stable vitiligo patients who underwent MKTP between June 2009 and June 2018. Univariate and/or multivariable analysis were used to determine the factors affecting the outcome of repigmentation. RESULT: The study comprised 2283 patients who had long-term follow-up data (12-108months). Excellent repigmentation was achieved in 400/606 (66%),788/1341 (58.8%),437/684 (63.9%),18/24 (75%) patients with segmental vitiligo, pre-MKTP phototherapy, younger than 24 years, the lesion on the perineum and scrotum, respectively. However, the patients with a positive family history, Koebner phenomenon responded worse(χ2=29.417, P<0.001; χ2=107.397, P<0.001; respectively). Overall, a significant positive correlation between duration of stability and percentage of repigmentation was found (χ2=42.053, P<0. 001). CONCLUSION: MKTP is efficient and well tolerated for stable vitiligo treatment. Various factors such as duration of disease stability, vitiligo type, family history, site of lesion should be carefully assessed before using MKTP, as it would further improve the post-operative repigmentation.