Abstract
Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues.
Highlights
Tissue engineering is ‘an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ’ [1]
Isolated stem cells from human adipose tissue were expanded and their specific surface antigens were characterized by flow cytometry, which was essential for ensuring the purity of the cell population
When the endothelial differentiation was performed in cells cultured without the fiber mesh, no difference on the proliferation comparing with the basal medium was detected (Figure 4A) cell viability assay employing Calcein-AM staining was performed to ensure the viability and assess the morphology and distribution of human adipose tissue-derived stem cells (hASCs) after 21 days (Figure 4B and C). These results showed that the cells cultured on electrospun PHB/PHB-HV fiber mesh with basal medium are well distributed and the cells cultured on electrospun PHB/PHB-HV fiber mesh with endothelial differentiation medium rearrange themselves forming circle-like structures that are characteristic of endothelial cells organization, mimicking the tubular organization of blood vessels
Summary
Tissue engineering is ‘an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ’ [1]. To become widely used in clinical practice, bone tissue-engineering products must overcome a series of challenges, the completely supply of nutrients and metabolites diffusion being one of the most important. The vascularization of cell-seeded implants plays an important role in cell survival, as these cells require access to substrate molecules (oxygen, glucose and amino acids) and clearance of products of metabolism (CO2, lactate and urea) [7,8]. Due to the limitations of oxygen diffusion, most cells cannot survive at distances greater than or equal to 150 mm from a capillary [4] and blood vessels have important metabolic and rheological functions that are organ-specific and important for the regeneration of tissue [9]
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