Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Platelet lysate-based pro-angiogenic nanocoatings

Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenicassociated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. Statement of Significance The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures.

Multiphasic, multistructured and hierarchical strategies for cartilage regeneration

Cartilage tissue is a complex nonlinear, viscoelastic, anisotropic, and multiphasic material with a very low coefficient of friction, which allows to withstand millions of cycles of joint loading over decades of wear. Upon damage, cartilage tissue has a low self-reparative capacity due to the lack of neural connections, vascularization, and a latent pool of stem/chondroprogenitor cells. Therefore, the healing of articular cartilage defects remains a significant clinical challenge, affecting millions of people worldwide. A plethora of biomaterials have been proposed to fabricate devices for cartilage regeneration, assuming a wide range of forms and structures, such as sponges, hydrogels, capsules, fibers, and microparticles. In common, the fabricated devices were designed taking in consideration that to fully achieve the regeneration of functional cartilage it is mandatory a well-orchestrated interplay of biomechanical properties, unique hierarchical structures, extracellular matrix (ECM), and bioactive factors. In fact, the main challenge in cartilage tissue engineering is to design an engineered device able to mimic the highly organized zonal architecture of articular cartilage, specifically its spatiomechanical properties and ECM composition, while inducing chondrogenesis, either by the proliferation of chondrocytes or by stimulating the chondrogenic differentiation  of stem/chondro-progenitor cells. In this chapter we present the recent advances in the development of innovative and complex biomaterials that fulfill the required structural key elements for cartilage regeneration. In particular, multiphasic, multiscale, multilayered, and hierarchical strategies composed by single or multiple biomaterials combined in a welldefined structure will be addressed. Those strategies include biomimetic scaffolds mimicking the structure of articular cartilage or engineered scaffolds as models of research to fully understand the biological mechanisms that influence the regeneration of cartilage tissue.

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Lack of the transcription factor hypoxia-inducible factor (HIF)-1α in macrophages accelerates the necrosis of Mycobacterium avium-induced granulomas

Accepted Manuscript

Gellan gum-hyaluronate spongy-like hydrogels promote angiogenesis in hindlimb ischemia

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Macroscopic and histopathological study of the placenta: an essential resource in litigation processes

The pathological study of the placenta is of upmost importance in cases of unexplained fetal/perinatal loss and often these carry litigation implications. Integrating pathological findings and the underlying pathophysiological processes, leading to placental lesions, is fundamental for the evaluation of poor fetal and perinatal outcomes and to distinguish from cases of true negligence.