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A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. While the main focus in research and clinical application lies in dermal and epidermal substitutes, the development of a subcutaneous replacement, the hypodermis, is often neglected. This chapter describes the use of fibrin sealant as a hydrogel scaffold to generate a three-dimensional skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes are seeded within a fibrin hydrogel. On top, another fibrin clot with incorporated fibroblasts is placed for the construction of the dermal layer. Keratinocytes are added on top of the two-layered construct to form the epidermal layer. The three-layered construct is cultivated for up to 3 weeks with keratinocytes being exposed to air according to the air-liquid interface cultivation model.

Introduction: Generation of adipose tissue for burn patients that suffer from an irreversible loss of the hypodermis is still one of the most complex challenges in tissue engineering. Electrospun materials with their micro- and nanostructures are already well established for their use as extracellular matrix substitutes. Gelatin is widely used in tissue engineering to gain thickness and volume. Under conventional static cultivation methods the supply of nutrients and transport of toxic metabolites is controlled by diffusion and therefore highly dependent on size and porosity of the biomaterial. A widely used method in order to overcome these limitations is the medium perfusion of 3D biomaterial-cell-constructs. In this study we combined perfusion bioreactor cultivation techniques with electrospun poly(l-lactide-co-glycolide) (P(LLG)) and gelatin hydrogels together with adipose-derived stem cells (ASCs) for a new approach in soft tissue engineering., Methods: ASCs were seeded on P(LLG) scaffolds and in gelatin hydrogels and cultivated for 24 hours under static conditions. Thereafter, biomaterials were cultivated under static conditions or in a bioreactor system for three, nine or twelve days with a medium flow of 0.3ml/min. Viability, morphology and differentiation of cells was monitored., Results: ASCs seeded on P(LLG) scaffolds had a physiological morphology and good viability and were able to migrate from one electrospun scaffold to another under flow conditions but not migrate through the mesh. Differentiated ASCs showed lipid droplet formations after 21 days. Cells in hydrogels were viable but showed rounded morphology. Under flow conditions, morphology of cells was more diffuse., Discussion: ASCs could be cultivated on P(LLG) scaffolds and in gelatin hydrogels under flow conditions and showed good cell viability as well as the potential to differentiate. These results should be a next step to a physiological three-dimensional construct for soft tissue engineering and regeneration., (Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.)

An irreversible loss of subcutaneous adipose tissue in patients after tumor removal or deep dermal burns makes soft tissue engineering one of the most important challenges in biomedical research. The ideal scaffold for adipose tissue engineering has yet not been identified though biodegradable polymers gained an increasing interest during the last years. In the present study we synthesized two novel biodegradable polymers, poly(ε-caprolactone-co-urethane-co-urea) (PEUU) and poly[(L-lactide-co-ε-caprolactone)-co-(L-lysine ethyl ester diisocyanate)-block-oligo(ethylene glycol)-urethane] (PEU), containing different types of hydrolytically cleavable bondings. Solutions of the polymers at appropriate concentrations were used to fabricate fleeces by electrospinning. Ultrastructure, tensile properties, and degradation of the produced fleeces were evaluated. Adipose-derived stem cells (ASCs) were seeded on fleeces and morphology, viability, proliferation and differentiation were assessed. The biomaterials show fine micro- and nanostructures composed of fibers with diameters of about 0.5 to 1.3 µm. PEUU fleeces were more elastic, which might be favourable in soft tissue engineering, and degraded significantly slower compared to PEU. ASCs were able to adhere, proliferate and differentiate on both scaffolds. Morphology of the cells was slightly better on PEUU than on PEU showing a more physiological appearance. ASCs differentiated into the adipogenic lineage. Gene analysis of differentiated ASCs showed typical expression of adipogenetic markers such as PPARgamma and FABP4. Based on these results, PEUU and PEU meshes show a promising potential as scaffold materials in adipose tissue engineering.

Background: Adipose-derived stem cells (ASCs) play a key role in tissue engineering approaches and are probably of major importance in the context of autologous fat transfer. A number of different tools for harvesting ASCs-containing fat tissue have been established. Such devices should be easy to handle, time saving, low priced, safe and provide a high amount of viable ASCs in the aspirate. Power-assisted liposuction (PAL) has not yet been described in the literature as a tool for fat harvesting for lipotransfer. Aim of this study was to investigate ASCs' viability in fat tissue harvested using PAL versus manual aspiration (MA)., Methods: Fat tissue was obtained from 9 donors undergoing abdominoplasty. Samples were divided into two sections. Out of each section fat was harvested using either PAL or MA. Number of isolated ASCs was defined, proliferation rate was determined and cell viability was assessed by flow cytometry. The ability of isolated ASCs to differentiate into mature adipocytes was analyzed by gene marker expression., Results: The number of viable ASCs and the proliferation rates did not significantly differ between PAL and MA but cells harvested using PAL showed significantly higher expression levels of differentiation markers adiponectin, GLUT4 and PPARg., Conclusion: Our results show that PAL is a feasible method for harvesting fat tissue containing viable ASCs. Quantity and quality of PAL-harvested ASC is similar or even better, respectively, compared to ASCs harvested by MA., (Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. However, the main focus in research and clinical application lies on dermal and epidermal substitutes whereas the development of a subcutaneous replacement (hypodermis) is often disregarded. In this study we used fibrin sealant as hydrogel scaffold to generate a three-layered skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes were embedded in the fibrin hydrogel and were combined with another fibrin clot with fibroblasts for the construction of the dermal layer. Keratinocytes were added on top of the two-layered construct to form the epidermal layer. The three-layered construct was cultivated for up to 3 weeks. Our results show that ASCs and fibroblasts were viable, proliferated normally, and showed physiological morphology in the skin substitute. ASCs were able to differentiate into mature adipocytes during the course of four weeks and showed morphological resemblance to native adipose tissue. On the surface keratinocytes formed an epithelial-like layer. For the first time we were able to generate a three-layered skin substitute based on a fibrin hydrogel not only serving as a dermal and epidermal substitute but also including the hypodermis. [ABSTRACT FROM AUTHOR]