Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds

Skin regeneration remains a challenge, requiring a well-orchestrated interplay of cell–cell and cell–matrix signalling. Cell sheet (CS) engineering, which has the major advantage of allowing the retrieval of the intact cell layers along with their naturally organized extracellular matrix (ECM), has been poorly explored for the purpose of creating skin substitutes and skin regeneration. This work proposes the use of CS technology to engineer cellular constructs based on human keratinocytes (hKC), key players in wound re-epithelialization, dermal fibroblasts (hDFb), responsible for ECM remodelling, and dermal microvascular endothelial cells (hDMEC), part of the dermal vascular network and modulators of angiogenesis. Homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed simultaneously to target wound re-vascularization and re-epithelialization. After implantation of the constructs in murine full-thickness wounds, human cells were engrafted into the host wound bed and were present in the neotissue formed up to 14 days post-implantation. Different outcomes were obtained by varying the composition and organization of the 3-D constructs. Both hKC and hDMEC significantly contributed to re-epithelialization by promoting rapid wound closure and early epithelial coverage. Moreover, a significant increase in the density of vessels at day 7 and the incorporation of hDMEC in the neoformed vasculature confirmed its role over neotissue vacularization. As a whole, the obtained results confirmed that the proposed 3-D CS-based constructs provided the necessary cell machinery, when in a specific microenvironment, guiding both re-vascularization and re-epithelialization. Although dependent on the nature of the constructs, the results obtained sustain the hypothesis that different CS-based constructs lead to improved skin healing.The authors thank Hospital da Prelada (Porto), in particular Dr. Paulo Costa for lipoaspirate collection, and Skingineering (PTDC/SAU-OSM/099422/2008) for to financial support; a Portuguese Foundation for Science and Technology (FCT) funded project. The research leading to these results has also received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS

Human skin cell fractions fail to self-organize within a gellan gum/Hyaluronic acid matrix but positively influence early wound healing

Split-thickness autografts still are the current gold standard to treat skin, upon severe injuries. Nonetheless, autografts are dependent on donor site availability and often associated to poor quality neoskin. The generation of dermal–epidermal substitutes by tissue engineering is seen as a promising strategy to overcome this problematic. However, solutions that can be safely and conveniently transplanted in one single surgical intervention are still very challenging as their production normally requires long culture time, and graft survival is many times compromised by delayed vascularization upon transplantation. This work intended to propose a strategy that circumvents the prolonged and laborious preparation period of skin substitutes and allows skin cells self-organization toward improved healing. Human dermal/epidermal cell fractions were entrapped directly from isolation within a gellan gum/hyaluronic acid (GG-HA) spongy-like hydrogel formed from an off-the-shelf dried polymeric network. Upon transplantation into full-thickness mice wounds, the proposed constructs accelerated the wound closure rate and re-epithelialization, as well as tissue neovascularization. A synergistic effect of the GG-HA matrix and the transplanted cells over those processes was demonstrated at early time points. Despite the human-derived and chimeric blood vessels found, the proposed matrix did not succeed in prolonging cells residence time and in sustaining the self-organization of transplanted human cells possibly due to primitive degradation. Despite this, the herein proposed approach open the opportunity to tackle wound healing at early stages contributing to re-epithelialization and neovascularization.We thank the Hospital da Prelada (Porto), in particular Dr. Paulo Costa for lipoaspirates collection and to financial support by Skingineering (PTDC/SAU-OSM/099422/2008), Portuguese Foundation for Science and Technology (FCT)-funded project. The research leading to these results has also received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS

Interfollicular epidermal stem cells: boosting and rescuing from adult skin

Publicado na série "Methods in Molecular Biology", vol. 989 (2013), ISSN 1064-3745Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the β 1-integrin ligand and Rho- Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f bri /CD71 dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f bri /CD71 dim subpopulations. The stepby- step methodology herein described indulges the boosting and consecutive puri fi cation and separation of interfollicular epidermal stem cells from human keratinocytes cultures.(undefined

In vivo osteogenic differentiation of stem cells inside compartmentalized capsules loaded with co-cultured endothelial cells

Capsules coated with polyelectrolytes and co-encapsulating adipose stem (ASCs) and endothelial (ECs) cells with surface modified microparticles are developed. Microparticles and cells are freely dispersed in a liquified core, responsible to maximize the diffusion of essential molecules and allowing the geometrical freedom for the autonomous three-dimensional (3D) organization of cells. While the membrane wraps all the instructive cargo elements within a single structure, the microparticles provide a solid 3D substrate for the encapsulated cells. Our hypothesis is that inside this isolated biomimetic 3D environment, ECs would lead ASCs to differentiate into the osteogenic lineage to ultimately generate a mineralized tissue in vivo. For that, capsules encapsulating only ASCs (MONO capsules) or co-cultured with ECs (CO capsules) are subcutaneously implanted in nude mice up to 6 weeks. Capsules implanted immediately after production or after 21 days of in vitro osteogenic stimulation are tested. The most valuable outcome of the present study is the mineralized tissue in CO capsules without in vitro pre-differentiation, with similar levels compared to the pre-stimulated capsules in vitro. We believe that the proposed bioencapsulation strategy is a potent self-regulated system, which might find great applicability in bone tissue engineering. Statement of Significance The diffusion efficiency of essential molecules for cell survival is a main issue in cell encapsulation. Former studies reported the superior biological outcome of encapsulated cells within liquified systems. However, most cells used in TE are anchorage-dependent, requiring a solid substrate to perform main cellular processes. We hypothesized that liquified capsules encapsulating microparticles are a promising attempt. Inspired by the multiphenotypic cellular environment of bone, we combine the concept of liquified capsules with co-cultures of stem and endothelial cells. After implantation, results show that co-cultured capsules without in vitro stimulation were able to form a mineralized tissue in vivo. We believe that the present ready-to-use TE strategy requiring minimum in vitro manipulation will find great applicability in bone tissue engineering.The authors acknowledge the financial support by the Portuguese Foundation for Science and Technology (FCT) through the Ph.D. (SFRH/BD/69529/2010-Clara R. Correia) and the Post-doc (SFRH/BPD/96611/2013-Mariana T. Cerqueira) grants, and the funding of RL3-TECT-NORTE-01-0124-FEDER-000020 for Rogério P. Pirraco. The authors are also grateful to Dr. Teresa Oliveira for her valuable help with the tissue processing and histological procedures.info:eu-repo/semantics/publishedVersio

Human embryonic stem cell-derivatives in a hydrogel-based skin model

Cases of massive skin loss upon injury or disease still represent a serious healthcare challenge worldwide, and despite the many alternatives available, results are not yet satisfactory. Skin Tissue Engineering, especially now combined with Stem Cell Research, holds great expectation towards an efficient and high quality skin regeneration. Human embryonic stem cells (hESCs) are quite attractive for this purpose, as they possess both immunoprevileged features and unique self-renewal and differentiation potency, being able to provide unlimited biological material. In this specific work, an innovative hydrogel-based model, together with hESCs-derived epidermal cells is proposed for skin regeneration. hESCs were moved from Mouse embryonic fibroblasts (MEFs), cultured in a feeder-free system comprising Matrigel and mTeSR media, and characterized by immunocytochemistry for pluripotency markers. After expansion, hESCs were differentiated towards the epidermal lineage,by culturing them in Collagen IV coated dishes, and upon supplementation of both Retinoic acid and bone morphogenetic protein 4. Keratin 18/14 positive cells were obtained, confirming the success of the used protocol. Differentiated epidermal cells were purified and cultured onto the hydrogel. Following several culture methodologies optimization, hESCsderived epidermal cells were able to adhere, proliferate and form a stratified epidermis-like structure demonstrating the potential and expectation for Skin Regeneration

Stromal vascular fraction from adipose tissue and cell sheet engineering to build vascularization units for tissue engineering and regenerative medicine

Vascularization holds the gold key for the effective survival and engraftment of complex engineered tissues and organs for Tissue Engineering and Regenerative Medicine. The lack of adequate vascularization post-transplantation often results in cell necrosis and ultimate failure and rejection of the engineered construct. Herein, we propose a strategy capable of surpassing this obstacle. Harnessing easy accessible adipose tissue stromal vascular fraction (SVF) as a source for cells with intrinsic angiogenic potential, and cell sheet technology we were able to engineer cell sheets with high angiogenic potential. SVF was isolated from the adipose tissue of healthy human subjects after enzymatic digestion and 2x105 nucleated cells/well were seeded on 24 well plates for cell sheet formation. To further boost cells’ angiogenic potential, hypoxic conditions of 5% of oxygen were provided to some of the cells while the rest was cultured in typical normoxia, for up to 8 days of culture, in basal medium. Flow cytometry analysis demonstrated the presence of a heterogeneous population of mesenchymal progenitors, endothelial and hematopoietic cells. Furthermore, the proliferation of SVF cells was evaluated through dsDNA quantification, which showed higher numbers for cells in hypoxic conditions, at earlier time points. Immunocytochemistry against CD31 and CD146 revealed the presence of an interconnected and highly branched network of vessel-like structures, more prominent for cells in hypoxia after 5 days of culture and quite similar for both conditions after 8 days, in the absence of any specific media supplementation. In vivo testing using the cell sheets detached from the wells and HIF expression analysis are currently underway. Taken together, the great potentiality of cell sheet technology with SVF cells cultured in hypoxia opens new exciting perspectives and may represent tremendously valuable vascularization units for tissue engineering strategies

Functional cell microcarriers: a new platform for cell separation and expansion

Publicado em "Journal of Tissue Engineering and Regenerative Medicine", vol. 7, supp. 1 (2013)The success of many stem cell applications in the biomedical field is highly dependent on the development of separation techniques for isolation and purification of cells with a very high yield and purity. Despite all the achievements made in the field over the past several years, new systems for effective cell separation are still needed. Previous work from our group demonstrated that functional chitosan films grafted with antibodies promote selective cell adhesion. 1 Herein we developed chitosan microparticles able to capture a specific cell types based in the concept of antibody coating for cell sorting. Our goal was to create new biomaterial surfaces capable of recruit a specific cell population within a mixture, reducing cell manipulation and time-consuming allowing at the same time cell expansion. Such system acts as a microcarrier for cell expansion of a specific cell target. Microcarrier culture system offers the advantage of providing a larger surface area for the growth of anchorage-dependent cells in a suspension culture system. Chitosan was chosen due to the excellent biocompatibility, gel forming properties, chemistry surface and low cell adhesion. This allows the modification with specific biochemical cues, for a controllable cell attachment. Here we develop functional biotinylated microparticles, such system allows tailoring microparticles to a variety of functional biomolecules. Here we tested the immobilization of antibodies to target specific cell types, CD31 for endothelial cells and CD90 for adipose stem cells. Primarily designed for an application in tissue engineering, two main challenges are accomplished with the herein presented microparticles: separation and scale-up expansion of specific cell type. The herein developed polymeric microparticles can also be used for directly deliver cells in vivo to repair and regenerate tissues

Functional chitosan microcarriers for selective cell attachment and expansion

The success of many stem cell applications in the biomedical field is highly dependent on the development of reliable techniques either for isolation or selection of specific cell populations with a very high yield and purity.1 In this work we propose the use of chitosan microparticles (μPs) to capture a specific cell type based in the concept of antibody-antigen binding. Our goal was to create new biomaterials capable of selecting within a heterotypic cell suspension, a specific sub-population, and supporting subsequent cell expansion. Such system simultaneously allows the selection and acts as a microcarrier for a specific target, thus reducing cell manipulation and time-consumption

Detecção molecular do cassava vein mosaic virus (CsVMV) em mandioca (Manihot esculenta CRANTZ).

A mandioca (Manihot esculenta CRANTZ) é uma cultura de grande importância para muitos países subdesenvolvidos e em desenvolvimento, pois constitui uma importante fonte de energia na dieta da população desses países. Entretanto, a produtividade desta cultura vem sendo comprometida pela alta incidência de viroses, já que esta é propagada vegetativamente