Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications

Long-term aesthetics, patient-reported outcomes, and auricular sensitivity after microtia reconstruction: A systematic review.

Summary Background Auricular reconstruction for microtia is most frequently performed using autologous costal cartilage (ACC) or porous polyethylene (PPE) implants. Short-term results are generally promising, but long-term results remain unclear. Long-term outcomes were explored in this systematic review, and minimal reporting criteria were suggested for future original data studies. Methods A systematic literature search was conducted in MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials from inception through October 14, 2020. Articles on auricular reconstruction in patients with microtia using ACC or PPE were included if postsurgical follow-up was at least 1 year. Outcome reporting was split into separate publications, and results on complications were reported previously. This publication focused on long-term aesthetic, patient-reported, and sensitivity outcomes. Results Forty-one publications reported on these outcomes. Both materials led to aesthetically pleasing results and high rates of patient satisfaction. ACC frameworks grew similarly to contralateral ears, and the anterior surface of auricles regained sensitivity. Furthermore, postoperative health-related quality of life (HRQoL) outcomes were generally good. Data synthesis was limited due to considerable variability between studies and poor study quality. No conclusions could be drawn on the superiority of either method due to the lack of comparative analyses. Conclusion Future studies should minimally report (1) surgical efficacy measured using the tool provided in the UK Care Standards for the Management of Patients with Microtia and Atresia; (2) complications including framework extrusion or exposure, graft loss, framework resorption, wire exposure and scalp/auricular scar complications and (3) HRQoL before and after treatment using the EAR-Q patient-reported outcome measure (PROM)

Three-dimensionalenvironment and vascularization induce osteogenic maturation of human adipose-derived stem cells comparable to that of bone-derived progenitors

While human adipose-derived stem cells (hADSCs) are known to possess osteogenic differentiation potential, the bone tissues formed are generally considered rudimentary and immature compared with those made by bone-derived precursor cells such as human bone marrow-derived mesenchymal stem cells (hBMSCs) and less commonly studied human calvarium osteoprogenitor cells (hOPs). Traditional differentiation protocols have tended to focus on osteoinduction of hADSCs through the addition of osteogenic differentiation media or use of stimulatory bioactive scaffolds which have not resulted in mature bone formation. Here, we tested the hypothesis that by reproducing the physical as well as biochemical bone microenvironment through the use of three-dimensional (3D) culture and vascularization we could enhance osteogenic maturation in hADSCs. In addition to biomolecular characterization, we performed structural analysis through extracellular collagen alignment and mineral density in our bone tissue engineered samples to evaluate osteogenic maturation. We further compared bone formed by hADSCs, hBMSCs, and hOPs against mature human pediatric calvarial bone, yet not extensively investigated. Although bone generated by all three cell types was still less mature than native pediatric bone, a fibrin-based 3D microenvironment together with vascularization boosted osteogenic maturation of hADSC making it similar to that of bone-derived osteoprogenitors. This demonstrates the important role of vascularization and 3D culture in driving osteogenic maturation of cells easily available but constitutively less committed to this lineage and suggests a crucial avenue for recreating the bone microenvironment for tissue engineering of mature craniofacial bone tissues from pediatric hADSCs, as well as hBMSCs and hOPs

Mosaic BRAF fusions are a recurrent cause of congenital melanocytic naevi targetable by MEK inhibition

Among children with multiple congenital melanocytic naevi (CMN), 25% have no established genetic cause, of which many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of CMN. Here, we study 169 CMN patients, 38 of whom were double wild-type for NRAS/BRAF mutations. Nineteen of these 38 patients had sufficient tissue to undergo RNAseq, which revealed mosaic BRAF fusions in 11/19 patients and mosaic RAF1 fusions in 1/19. Recurrently, fusions involved the loss of the 5’ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate naevi in 5/12 patients, confirming clonality. The absence of the fusion in blood in 8/12 patients indicated mosaicism. Primary culture of BRAF-fusion naevus cells from 3/12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement, and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes

Lop Ear to Conchal Microtia: An Algorithmic Surgical Approach.

BACKGROUND The lop ear deformity is defined by a deficient helix and scapha, underdeveloped anthelix, and downfolding of the helix. The terminology used is still confusing, and the treatment is not entirely structured. The aim of this study was to provide a new systematic surgical approach of this deformity based on our center's experience. MATERIALS AND METHODS All patients undergoing surgical correction of lop ears between 2007 and 2019 at Great Ormond Street Hospital were included. Patients' data, surgical techniques, and postoperative complications were recorded. RESULTS Based on our records, we identified 3 surgical techniques for the correction of lop ears, based on the degree of deformity encountered. In a mild lop ear, correction was achieved with a modified otoplasty technique by improving the definition of the antihelix and superior crus. In a moderate deformity, additional remodeling of the lidded helix was performed (extended otoplasty), whereas for the severe lop ear, the amount of cupping and the deficient cartilage required formal reconstruction using a carved rib cartilage framework. There were a total of 109 patients and 146 lop ears: 58 mild, 27 moderate, and 61 severe lop ears. CONCLUSION We feel that there is a point in the spectrum of congenital ear deformity when a severe lop ear becomes a conchal microtia and recommend this approach to simplify the management of these cases. This is intended to bring greater clarity to how to deal with lop ears, based on the severity of the deformity and the surgical techniques used

Lop Ear to Conchal Microtia — An Algorithmic Surgical Approach

BACKGROUND The lop ear deformity is defined by a deficient helix and scapha, underdeveloped anthelix, and downfolding of the helix. The terminology used is still confusing, and the treatment is not entirely structured. The aim of this study was to provide a new systematic surgical approach of this deformity based on our center's experience. MATERIALS AND METHODS All patients undergoing surgical correction of lop ears between 2007 and 2019 at Great Ormond Street Hospital were included. Patients' data, surgical techniques, and postoperative complications were recorded. RESULTS Based on our records, we identified 3 surgical techniques for the correction of lop ears, based on the degree of deformity encountered. In a mild lop ear, correction was achieved with a modified otoplasty technique by improving the definition of the antihelix and superior crus. In a moderate deformity, additional remodeling of the lidded helix was performed (extended otoplasty), whereas for the severe lop ear, the amount of cupping and the deficient cartilage required formal reconstruction using a carved rib cartilage framework. There were a total of 109 patients and 146 lop ears: 58 mild, 27 moderate, and 61 severe lop ears. CONCLUSION We feel that there is a point in the spectrum of congenital ear deformity when a severe lop ear becomes a conchal microtia and recommend this approach to simplify the management of these cases. This is intended to bring greater clarity to how to deal with lop ears, based on the severity of the deformity and the surgical techniques used

Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications