No / Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase-PCR (qRT-PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)-mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

Yes / Multiple factors and conditions can lead to impaired wound healing. Chronic non-healing wounds are a common problem among the elderly. To identify microRNAs negatively impacting the wound repair, global miRNA profiling of wounds collected from young and old mice was performed. A subset of miRNAs that exhibited an age-dependent expression pattern during wound closure was identified, including miR-31 and miR-200c. The expression of miR-200 family members was markedly downregulated upon wounding in both young and aged mice, with an exception of acute upregulation of miR-200c at the early phase of wound healing in aged skin. In unwounded aged skin (versus unwounded younger skin), the level of miR-200c was also found elevated in both human and mice. Overexpression of miR-200c in human ex vivo wounds delayed re-epithelialisation and inhibited cell proliferation in the wound epithelium. Modulation of miR-200c expression in both human and mouse keratinocytes in vitro revealed inhibitory effects of miR-200c on migration, but not proliferation. Accelerated wound closure in vitro induced by anti-miR-200c was associated with upregulation of genes controlling cell migration. Thus, our study identified miR-200c as a critical determinant that inhibits cell migration during skin repair after injury and may contribute to ageassociated alterations in wound repair. / Supported by a grant from Medical Research Council UK (MR/K011324/1)

Yes / Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re‐epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re‐epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome. / University of Manchester Strategic Fund; Wellcome Trust; BBSRC; Ministry of Higher Education, Malaysia Universiti; Sains Malaysia

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-18T21:33:36Z No. of bitstreams: 2 Tese - Taís Andrade Dias de Souza - 2016.pdf: 2752981 bytes, checksum: caa2c6685930afbfbabce8d4180cf875 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-19T13:00:53Z (GMT) No. of bitstreams: 2 Tese - Taís Andrade Dias de Souza - 2016.pdf: 2752981 bytes, checksum: caa2c6685930afbfbabce8d4180cf875 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-19T13:00:53Z (GMT). No. of bitstreams: 2 Tese - Taís Andrade Dias de Souza - 2016.pdf: 2752981 bytes, checksum: caa2c6685930afbfbabce8d4180cf875 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-29 / Impaired wound healing is one of the main complications of diabetes. It is related to high blood glucose levels that cause metabolic and cellular changes, making the tissue repair process difficult, with long and costly treatment. Chitosan has been investigated for its biological activity when in contact with tissue. However, further elucidation about the mechanisms of chitosan’s action on the wound healing is needed, as well as how its physico-chemical and handling characteristics can offer better performance for the treatment of diabetic wounds. This study aimed to develop and characterize a chitosan hydrogel, and evaluate its angiogenic potential and healing activity in skin wounds of experimentally-induced diabetic rats. To obtain 80, 85 and 90% deacetylation degree, a deacetylation protocol was conducted in basic medium and high temperature. After hydrogels preparation, they were evaluated for pH, viscosity, organoleptic properties, microbiological contamination, and antimicrobial activity for 60 days and stored at room temperature and under refrigeration. The angiogenic potential of each formulation was investigated by the chorioallantoic membrane assay of embryonated chicken eggs, determining the vascular area by fresh analysis and vascular density by histological evaluation. The healing activity of chitosan gels was determined by the treatment of skin wounds promoted at the dorsal region of experimentally-induced diabetic female rats, evaluated at the periods of three, 7 and 14 days postoperatively. The following parameters were investigated: wound contraction rate, microscopic changes in the epidermis and dermis by hematoxylin-eosin, angiogenic activity, and cell proliferation by immunohistochemistry. The methodology used to deacetylation of the chitosan was considered appropriate, and the degrees of deacetylation pretended were obtained. During the 60 days of evaluation, there was a significant change only in the viscosity of samples kept at room temperature and refrigeration was considered the best form of storage. The angiogenic activity of chitosan gels was positive with an increase in the area and vascular density of the chorioallantoic membrane. The higher the degree of deacetylation, the greater the angiogenic response observed, therefore, the 90% deacetylation hydrogel showed the best performance in this analysis. In the treatment of skin wounds in diabetic rats, the chitosan hydrogels increased the rate of contraction of the wounds, had greater angiogenesis indexes, cell proliferation and collagen production resulting in faster healing compared to the control group. Hydrogel higher’s degree of deacetylation seems to optimize the biological activity of the polymer. / O comprometimento da cicatrização de feridas cutâneas é uma das principais complicações decorrentes do diabetes e está relacionada aos elevados níveis de glicose sanguínea que causam alterações metabólicas e celulares tornando o processo cicatricial mais lento e o tratamento longo e dispendioso. A quitosana tem sido investigada quanto à sua atividade biológica quando em contato com tecidos vivos. No entanto, é necessária uma maior elucidação sobre seus mecanismos de ação sobre a cicatrização de feridas e como as suas características físico-químicas podem oferecer um melhor desempenho para o tratamento de feridas diabéticas. O presente estudo teve por objetivo desenvolver e caracterizar um hidrogel de quitosana em diferentes graus de desacetilação e avaliar seu potencial angiogênico e atividade cicatrizante no tratamento de feridas cutâneas de ratas diabéticas induzidas experimentalmente. Para a obtenção dos graus de desacetilação 80, 85 e 90% foi utilizado protocolo de desacetilação em meio básico e temperatura elevada. Após a confecção das formulações, os géis foram avaliados quanto ao pH, viscosidade, propriedades organolépticas, contaminação microbiológica e atividade antimicrobiana durante 60 dias, sendo armazenados à temperatura ambiente e sob refrigeração. O potencial angiogênico de cada formulação foi investigado por meio do ensaio da membrana córioalantoide de ovos embrionados de galinha, determinando-se a área vascular por análise a fresco e a densidade vascular pela avaliação histológica. A atividade cicatrizante dos géis de quitosana foi determinada pelo tratamento de feridas cutâneas promovidas na região dorsal de ratas diabéticas induzidas experimentalmente, sendo avaliados os períodos de três, sete e 14 dias de pós-operatório. Foram investigados os parâmetros: taxa de contração macroscópica das feridas, alterações microscópicas em epiderme e derme pela coloração de hematoxilina-eosina, atividade angiogênica e proliferação celular por imunoistoquímica. A metodologia utilizada para desacetilação da quitosana foi considerada adequada, sendo obtidos os graus de desacetilação desejados. Durante os 60 dias de avaliação, houve alteração significativa apenas na viscosidade das amostras mantidas em temperatura ambiente, sendo a refrigeração considerada melhor forma de armazenamento. A atividade angiogênica dos géis de quitosana foi positiva havendo aumento da área e da densidade vascular da membrana córioalantoide. Quanto maior o grau de desacetilação maior foi a resposta angiogênica do produto, sendo o gel de 90% de desacetilação o que apresentou melhor desempenho nesta análise. No tratamento das feridas cutâneas de ratas diabéticas, os géis de quitosana aumentaram a taxa de contração das feridas, apresentaram maiores índices de angiogênese, proliferação celular e produção de colágeno o que resultou em cicatrização mais rápida quando comparado ao grupo controle. O maior grau de desacetilação do gel parece otimizar a atividade biológica do polímero.