RESUMEN
BACKGROUND AIMS: Complex injuries of the upper and lower extremities often result in scarring and subsequent adhesion formation, which may cause severe pain and distinctly reduce range of motion. In revision surgery, replacement of the missing subcutaneous tissue is desirable to prevent new adhesions, to cushion scarred tendons and nerves and to regain tissue elasticity. Therefore, the objective of this study was the in vitro evaluation of cell-seeded collagen matrices to serve as the basis for the reconstruction of the subcutaneous adipose tissue layer. METHODS: Five commercially available acellular dermal collagen matrices were seeded with human adipose-derived stromal cells (hASC). Size and shape stability of cell-matrix constructs were assessed and cell adhesion onto the matrix surface was evaluated histologically. Adipogenic differentiation of hASC on matrices was evaluated by means of histological staining, triglyceride quantification, and quantitative real-time polymerase chain reaction gene expression analysis. RESULTS: The collagen matrix Permacol was the only cell-seeded material that exhibited excellent size and shape stability. For Permacol and Strattice, successful seeding with continuous cell layers on top of the matrices was observed. For both matrices, histological staining, triglyceride quantification and messenger RNA expression of adipogenic transcription factors indicated substantial adipogenic differentiation of hASC after long-term induction as well as after short-term induction of only 4 days. CONCLUSIONS: Of all matrices investigated, only Permacol exhibited adequate handling stability and the development of a thin adipose tissue layer on top of the matrix. Thus, this matrix appears promising to be used in the development of a subcutaneous cushioning layer after complex injuries involving large scar formation.
Asunto(s)
Colágeno/química , Matriz Extracelular/química , Células Madre Mesenquimatosas/metabolismo , Grasa Subcutánea/metabolismo , Adulto , Técnicas de Cultivo de Célula , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Grasa Subcutánea/citologíaRESUMEN
Adipose-derived mesenchymal stromal/stem cells (ASCs) represent a commonly used cell source for adipose tissue engineering. In this context, ASCs have routinely been cultured in conventional 2D culture and applied as single cell suspension for seeding onto scaffold materials or direct injection. However, this approach is associated with the loss of their intrinsic 3D microenvironment and leads to impaired regenerative capacity of the cells. Thus, the application of ASCs as self-assembled 3D spheroids with cells residing in their own matrix is an attractive alternative. However, characterization of the structural features and differentiation capacity of the spheroids is necessary to effectively apply them as building blocks in adipose tissue engineering. In this study, we focus on extracellular matrix (ECM) development in ASC spheroids, as well as adipogenic differentiation in comparison to conventional 2D culture using different induction protocols. Reproducible assembly of ASCs into spheroids was achieved within 24 h using the liquid overlay technique. Undifferentiated spheroids displayed a stromal ECM pattern, with fibronectin, collagen V, and VI as the main components. In the course of adipogenesis, a dynamic shift in the ECM composition toward an adipogenic phenotype was observed, associated with enhanced expression of laminin, collagen I, IV, V, and VI, similar to native fat. Furthermore, adipogenic differentiation was enhanced in spheroids as compared with 2D cultured cells, with the spheroids needing a distinctly shorter adipogenic stimulus to sustain adipogenesis, which was demonstrated based on analysis of triglyceride content and adipogenic marker gene expression. In summary, culturing ASCs as spheroids can enhance their adipogenic capacity and generate adipose-like microtissues, which may be a promising cell delivery strategy for adipose tissue engineering approaches. Impact statement Adipose-derived mesenchymal stromal/stem cells (ASCs) as a widely used cell source for adipose tissue engineering have been shown to be limited in their regenerative capacity when applied as single cells. As an alternative approach, the delivery as spheroids, consisting of cells in a 3D context, may be favorable. However, insights into extracellular matrix (ECM) development and efficient adipogenic differentiation are required for their effective application. In this study, we show that differentiated ASC spheroids develop an ECM, resembling native adipose tissue. Furthermore, the ASC spheroids exhibited a superior differentiation capacity as compared with conventional 2D culture, and required only a short adipogenic induction stimulus. Our results identify ASC-derived spheroids as an attractive cell delivery method for adipose tissue engineering approaches.
Asunto(s)
Adipogénesis , Tejido Adiposo , Matriz Extracelular , Células Madre Mesenquimatosas , Diferenciación Celular , Células Cultivadas , Humanos , Ingeniería de TejidosRESUMEN
Adipose tissue engineering aims at the restoration of soft tissue defects and the correction of contour deformities. It is therefore crucial to provide functional adipose tissue implants with appropriate volume stability. Here, we investigate two different fibrin formulations, alone or in combination with biodegradable polyurethane (PU) scaffolds as additional support structures, with regard to their suitability to generate volume-stable adipose tissue constructs. Human adipose-derived stem cells (ASCs) were incorporated in a commercially available fibrin sealant as well as a stable fibrin hydrogel previously developed by our group. The composite constructs made from the commercially available fibrin and porous poly(ε-caprolactone)-based polyurethane scaffolds exhibited increased volume stability as compared to fibrin gels alone; however, only constructs using the stable fibrin gels completely maintained their size and weight for 21 days. Adipogenesis of ASCs was not impaired by the additional PU scaffold. After induction with a common hormonal cocktail, for constructs with either fibrin formulation, strong adipogenic differentiation of ASCs was observed after 21 days in vitro. Furthermore, upregulation of adipogenic marker genes was demonstrated at mRNA (PPARγ, C/EBPα, GLUT4 and aP2; qRT-PCR) and protein (leptin; ELISA) levels. Stable fibrin/PU constructs were further evaluated in a pilot in vivo study, resulting in areas of well-vascularized adipose tissue within the implants after only 5 weeks. Copyright © 2013 John Wiley & Sons, Ltd.