Priming of adipose-derived stem cells with curcumin prior to cryopreservation preserves their functional potency: Towards an 'Off-the-shelf' therapy for burns.

Stem cells-based treatment for burn wounds require frozen cells as an off-the-shelf therapy; however, cryopreservation-induced oxidative stress resulted in post-thaw cell death or loss of cell functions, thus arrested their clinical practicality. Although antioxidant priming to stem cells increase their resistant to oxidative stress, but this strategy is still unexplored on cryopreserved cells. Herein, we investigated whether curcumin priming before cryopreservation could preserve the therapeutic potency of thawed stem cells. For this, unprimed and curcumin-primed adipose-derived stem cells (ASCs) were cryopreserved for one month. Post-thawing, cells were assessed for viability by trypan blue assay; metabolic activity by MTT assay; senescence by senescence-associated (SA)-ß-galactosidase activity staining assay; migration by scratch healing assay and; mRNA expression by real-time PCR. Subsequently, the healing potential was examined by injecting cells around the wound periphery of acidic burn in rats. Post-healing, skin architecture was histologically examined. Results demonstrated that, curcumin-primed frozen cells (Cryo/Cur-ASCs) showed better post-thaw viability, metabolic activity, migration ability and lower percent of senescence comparative to unprimed frozen cells (Cryo/ASCs). Curcumin priming alleviated the oxidative damage by activating the ROS-reducing cellular antioxidant system as shown by the evident increase in GSH levels and upregulated mRNA expression of glutathione peroxidase (GPx), superoxide dismutases (SOD1, SOD2), and catalase (CAT). Further, invivo findings revealed that Cryo/Cur-ASCs-treated wounds exhibited earlier wound closure with an improved architecture comparative to Cryo/ASCs and depicted healing capacity almost similar to Fresh/ASCs. Our findings suggested that curcumin priming could be effective to alleviate the cryo-induced oxidative stress in post-thawed cells.

Bi-allelic Variants in METTL5 Cause Autosomal-Recessive Intellectual Disability and Microcephaly.

Intellectual disability (ID) is a genetically and clinically heterogeneous disorder, characterized by limited cognitive abilities and impaired adaptive behaviors. In recent years, exome sequencing (ES) has been instrumental in deciphering the genetic etiology of ID. Here, through ES of a large cohort of individuals with ID, we identified two bi-allelic frameshift variants in METTL5, c.344_345delGA (p.Arg115Asnfs∗19) and c.571_572delAA (p.Lys191Valfs∗10), in families of Pakistani and Yemenite origin. Both of these variants were segregating with moderate to severe ID, microcephaly, and various facial dysmorphisms, in an autosomal-recessive fashion. METTL5 is a member of the methyltransferase-like protein family, which encompasses proteins with a seven-beta-strand methyltransferase domain. We found METTL5 expression in various substructures of rodent and human brains and METTL5 protein to be enriched in the nucleus and synapses of the hippocampal neurons. Functional studies of these truncating variants in transiently transfected orthologous cells and cultured hippocampal rat neurons revealed no effect on the localization of METTL5 but alter its level of expression. Our in silico analysis and 3D modeling simulation predict disruption of METTL5 function by both variants. Finally, mettl5 knockdown in zebrafish resulted in microcephaly, recapitulating the human phenotype. This study provides evidence that biallelic variants in METTL5 cause ID and microcephaly in humans and highlights the essential role of METTL5 in brain development and neuronal function.

Salvage Reconstruction of Composite Defects of the Anterior Mandible, Floor of Mouth, and Lip.

Anterior mandible defects result in loss of support for the tongue, floor of the mouth and lower lip, resulting in impairment of airway, feeding, and speech. We treated four patients with these "Andy Gump" deformities. Reconstruction was performed with two free flaps: a fibula osteocutaneous flap for the anterior mandible and floor of the mouth, and a soft tissue free flap for the lip, chin, and anterior neck. The lower lip was suspended cranially with fascia or tendon grafts ± mini-temporalis turndown flaps. All flaps survived completely. All patients were tube feed-dependent before surgery; they all resumed an oral diet. All tracheostomies were decannulated. Lip competence was restored as evidenced by cessation of drooling. Speech improved from unintelligible to intelligible with frequent repetitions. Objective assessment was performed with the functional intraoral Glasgow scale; the mean FIGS score improved from 3.25 (range 3-4) to 11 (range 9-13). We conclude that composite anterior mandible and tongue defects have large tissue requirements that require multiple free flaps. Reconstruction leads to significant improvement in function.

An Algorithm for Reconstruction of Electrical Injuries of the Scalp.

BACKGROUND: Electrical injuries of the scalp are a frequent occurrence in developing countries. Burns can be contact or conductive and result in extensive tissue damage. The authors present their experience with treatment of scalp and calvarial electrical injuries and propose a management algorithm. METHODS: This was a retrospective cohort study comprising all patients with electrical injuries of the scalp treated at the authors' center between January of 2010 and December of 2016. Noncontrast computed tomography scans were obtained to assess viability of the calvarium in patients who presented more than 2 weeks after injury. Single-stage débridement and reconstruction were performed. All nonviable soft tissue and bone was removed. Soft-tissue reconstruction was performed with skin grafts, local scalp flaps, pedicled trapezius flaps, and free flaps (anterolateral thigh, latissimus dorsi, and scapular). Cranioplasty was performed in a delayed manner with autologous bone grafts. RESULTS: Over a 7-year period, a total of 52 patients underwent scalp reconstruction for high-voltage (44 patients) and low-voltage (eight patients) electrical injury. All patients underwent successful soft-tissue reconstruction. Osteomyelitis with draining sinuses developed in three patients; these patients underwent flap re-elevation and bone débridement, which resulted in a healed wound and stable reconstruction. Cranioplasty was performed with split calvarial grafts in two patients and split rib grafts in four patients. One patient underwent scalp tissue expansion for hair restoration. CONCLUSION: The authors propose an algorithm for reconstruction of electrical injuries of the scalp. Thorough débridement of the calvarium is the most important determinant of a successful outcome. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Curcumin preconditioned human adipose derived stem cells co-transplanted with platelet rich plasma improve wound healing in diabetic rats.

AIM: Inflammatory and oxidative microenvironment at diabetic' wound site hinder the therapeutic efficacy of cell-based therapies in diabetic patients. The purpose of this study is to explore the competence of curcumin preconditioned human adipose derived cells (hASCs) in combination with platelet rich plasma (PRP) for the repair of wounds in diabetic rats. MAIN METHODS: The cytoprotective effect of curcumin preconditioning for hASCs against hyperglycemic stress was evaluated through analysis of cell morphology, viability, cytotoxicity, senescence, and scratch wound healing assays. Subsequently, the healing capacity of curcumin preconditioned hASCs (Cur-hASCs) added to PRP was examined in excisional wounded diabetic rat model. Healed skin biopsies were excised to analyze gene and protein expression of wound healing markers by qPCR and western blotting. Histopathological changes were observed through hematoxylin and eosin staining. KEY FINDINGS: We found that Cur-hASCs counteract the glucose stress much better than non-preconditioned hASCs by maintaining their cellular morphology and viability as well as metabolic potential. Further in vivo results revealed that, Cur-hASCs co-injected with PRP resulted in faster wound closure, improved fibroblast proliferation, increased neovascularization, marked reduction in inflammatory cells, and compact extracellular matrix with completely covered thick epithelium. Moreover, Cur-hASCs + PRP treatment significantly improved the expression of key healing markers such as pro-angiogenic (Vegf), dermal matrix deposition (Col1α1), cell migration (bFgf) and cell proliferation (Pcna) at wound site. SIGNIFICANCE: Our findings propose a combinatorial therapy (Cur-hASCs + PRP) as a novel modality to improve the efficacy of hASCs-based therapy for diabetic wounds.

Vitamin E preconditioning alleviates in vitro thermal stress in cultured human epidermal keratinocytes.

AIMS: Thermal burns are the most common type of skin injuries. Clinically, the deteriorating thermal wounds have been successfully treated with skin cell sheets, suspensions or bioengineered skin substitutes. After thermal injury, oxidative microenvironment prevalent in the burnt tissue due to imbalance between production of free radicals and antioxidants defense aiding to destruction of cellular or tissue components. However, depleted antioxidant content particularly vitamin E after heat injury challenges efficient regenerative and healing capacity of transplanted cells. Thus, aim of current study was to pretreat human epidermal keratinocytes with vitamin E in order to enhance their survival rate and therapeutic ability under oxidative microenvironment induced by in vitro heat stress. MAIN METHODS: Keratinocytes were treated with 100 µM vitamin E at 37 °C for 24 h followed by thermal stress at 51 °C for 10 min. Cell viability and cytotoxicity assays, gene expression analysis and paracrine release analysis were performed. KEY FINDINGS: Vitamin E preconditioning resulted in significantly improved cell morphology, enhanced viability and reduced lactate dehydrogenase release. Furthermore, Vitamin E preconditioned cells exposed to thermal stress showed significant down-regulated expression of BAX and up-regulated expression of PCNA, BCL-XL, vascular endothelial growth factor (VEGF), involucrin, transglutaminase 1 (TGM1) and filaggrin (FLG) escorted by increased paracrine release of VEGF, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). SIGNIFICANCE: Results of the current study suggest that clinical transplantation of vitamin E preconditioned keratinocytes alone or in combination with dermal fibroblasts in skin substitutes for the treatment of thermally injured skin.

The efficacy of excision followed by intralesional 5-fluorouracil and triamcinolone acetonide versus excision followed by radiotherapy in the treatment of ear keloids: A randomized control trial.

BACKGROUND: The ear is the common site for keloid formation especially in women after ear piercing. Surgery is the main stay of treatment in these lesions but there are large numbers of treatment failures in surgery alone. OBJECTIVE: The objective of this study was to compare the efficacy of post-excision intralesional 5-fluorouracil/triamcinolone acetonide (5-FU/TAC) and post-excision radiotherapy in the treatment of ear keloids. STUDY DESIGN: A randomized controlled trial. SETTING: The study was conducted from May 2014 to January 2015 at Jinnah Burn and Reconstructive Surgery Centre, Allama Iqbal Medical College, Lahore. SUBJECT & METHODOLOGY: After approval from the hospital ethical committee, 60 patients presented in the outpatient department fulfilling the inclusion criteria were selected and randomly assigned in two groups with the help of the random number table. Patients in group A had excision followed by intralesional 5-FU/TAC injections while patients of group B had excision followed by radiotherapy. Patients were assessed at 6 months after completion of treatment for efficacy (no recurrence within 6 months of treatment). RESULTS: In our study total of 60 patients completed the study, with 30 patients in each group. 7 patients (23.34%) in Group-A and 9 patients (30%) in Group-B were males while 23 patients (76.67%) in Group-A and 21 patients (70%) in Group-B were females i.e. male to female ratio is 1:2.75. Mean age was 31.8+6.48years. The comparison of frequency of efficacy in both groups showed that 73.33% (n=22) in Group-A and 43.33% (n=13) in Group-B had efficacy, p value was calculated as 0.01, showing a significant statistical difference. CONCLUSION: Excision and intralesional 5-FU/TAC is an effective treatment for keloids on the ears.

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

AIMS: Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. MAIN METHODS: Fibroblasts were treated with 100µM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. KEY FINDINGS: Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNß and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. SIGNIFICANCE: The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds.