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.

Tendon regeneration through a scaffold-free approach: development of tenogenic magnetic hASCs sheets

Tendon's regeneration is limited, demanding for cell-based strategies to fully restore their functionality upon injury. The concept of magnetic force-based TE(1), generally using magnetic nanoparticles may enable, for example, stem cell stimulation and/or remote control over TE constructs. Thus, we originally propose the development of magnetic cell sheets (magCSs) with tenogenic capability, aimed at promoting tendon's regeneration. A Tenomodulin (TNMD+) subpopulation was sorted from human adipose stem cells (hASCs), using TNMD-coated immunomagnetic beads(2) and used as cell source for the development of magCSs. Briefly, cells were labeled with iron oxide composite particles (Micromod) and cultured for 7 days in α-MEM medium with or without magnetic stimulation provided by a magnetic device (nanoTherics). CSs were retrieved from the plates using magnet attraction as contiguous sheets of cells within its own deposited ECM.

Cryopreservation of Cell Sheets of Adipose Stem Cells: Limitations and Successes

Cell Sheets of hASCs (hASCs-CS) have been previously proposed for wound healing applications(1, 2) and despite the concern for production time reduction, the possibility of having these hASCs-CS off-the-shelf is appealing. The goal of this work was to define a cryopreservation methodology allowing to preserve cells viability and the properties CS matrix. hASCs-CS obtained from three different donors were created in UP-cell thermoresponsive dishes(Nunc, Germany) as previously reported(1,2). Different cryopreservation conditions were considered: i)FBS plus DMSO(5% and10%); ii)0.4M of Trehalose plus DMSO (5% and 10%); iii)cryosolution PLL (Akron Biotech, USA); and iv)vitrification. The cryopreservation effect was first assessed for cellular viability by flow cytometry using 7-AAD, and after dissociating the hASCs-CS with collagenase and trypsin-EDTA 0.25%. The expression (RT-PCR) and deposition (western blot and immunocytochemistry) of collagen type I, laminin and fibronectin, and the organization (TEM) of the extracellular matrix was further assessed before and after hASCs-CS cryopreservation to determine a potential effect of the method over matrix composition and integrity. The obtained results confirmed that cell viability is affected by the cryopreservation methodology, as shown before for different CS(3). Interestingly, the matrix properties were not significantly altered and the typical cell sheetâ s easiness of manipulation for transplantation was not lost.

Skin-derived cell-sheets as powerful tools to engineer skin analogues

Cell/cell-extracellular matrix (ECM) dynamic interactions appear to have a major role in regulating communication through soluble signaling, directing cell binding and activating substrates that participate in the highly organized wound healing process. Moreover, these interactions are also crucial for in vitro mimicking cutaneous physiology. Herein we explore cell sheet (CS) engineering to create cellular constructs formed by keratinocytes (hKC), fibroblasts (hDFB) and dermal microvascular endothelial cells (hDMEC), to target skin wound healing but also the in vitro recreation of relevant models. Taking advantage of temperature-responsive culture surfaces, which allow harvesting cultured cells as intact sheets along with the deposited native ECM, varied combinations of homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed. Constructs combining one CS of keratinocytes as an epidermis-like layer plus a vascularized dermis composed by hDFB and hDMECs were assembled as skin analogues for advancing in vitro testing. Simultaneously both hKC and hDMEC were shown to significantly contribute to the re-epithelialization of full-thickness mice skin wounds by promoting an early epithelial coverage, while hDMEC significantly lead to increased vessels density, incorporating the neovasculature. Thus, although determined by the cellular nature of the constructs, these outcomes demonstrated that CS engineering appear as an unique technology that open the possibility to create numerous combinations of 3D constructs to target defective wound healing as well as the construction of in vitro models to further mimic cutaneous functions crucial for drug screening and cosmetic testing assays.

Plastic-damage smeared crack model to simulate the behaviour of structures made by cement based materials

This work proposes a constitutive model to simulate nonlinear behaviour of cement based materials subjected to different loading paths. The model incorporates a multidirectional fixed smeared crack approach to simulate crack initiation and propagation, whereas the inelastic behaviour of material between cracks is treated by a numerical strategy that combines plasticity and damage theories. For capturing more realistically the shear stress transfer between the crack surfaces, a softening diagram is assumed for modelling the crack shear stress versus crack shear strain. The plastic damage model is based on the yield function, flow rule and evolution law for hardening variable, and includes an explicit isotropic damage law to simulate the stiffness degradation and the softening behaviour of cement based materials in compression. This model was implemented into the FEMIX computer program, and experimental tests at material scale were simulated to appraise the predictive performance of this constitutive model. The applicability of the model for simulating the behaviour of reinforced concrete shear wall panels submitted to biaxial loading conditions, and RC beams failing in shear is investigated.

Can oil, plastic and RAP wastes have a new life in novel asphalt mixtures?

The pavement recycling allows to reuse reclaimed asphalt pavement (RAP) or other waste materials in new asphalt mixtures for road construction or rehabilitation, thus re-ducing the use of virgin materials (aggregates and bitumen). Thus, the main aim of this study is to minimize the use of natural resources through the reuse of three waste materials: HDPE, mo-tor oil and RAP. Different amounts of waste motor oil and HDPE were added to an asphalt binder with 50% aged bitumen. The best solutions to produce the modified binders (4.5 to 5.0% HDPE and 10 % waste motor oil) performed as well as a conventional bitumen although they only used 35 % of virgin bitumen. Asphalt mixtures with 50 % RAP were produced with the selected modified binders, improving some characteristics in comparison with conventional asphalt mixtures. In conclusion, these wastes can revive in new asphalt mixtures.

Plastic waste use as aggregate and binder modifier in open-graded asphalts

Road pavements are very important infrastructures for the Society, but they can cause serious environmental impacts during construction, operation and rehabilitation phases. Thus, it is essential to develop surface paving solutions that promote not only the durability but also a comfortable and safe use. In fact, this work aims to study the properties of new opengraded mixtures for surface layers produced with plastic wastes. First, HDPE and EVA wastes were used as bitumen modifiers, and then another plastic waste (PEX) replaced part of the aggregates. After studying the modified binders, the open-graded mixtures were designed, and then they were tested concerning their particle loss, rutting resistance, surface texture and damping effect. It was concluded that both ways of using the plastic wastes can improve the mechanical and functional properties of the open-graded mixtures related to the pavement performance.

Mechanical analysis of asphalt mixtures produced with waste plastic modified binders

This work compares the viscoelastic properties of an asphalt binder (70/100 pen) modified with different waste plastics and the mechanical properties of the resultant asphalt mixtures. Two different plastic wastes were used, namely recycled HDPE and EVA. Three different polymer modified binders were produced with these plastic wastes: i) 5% HDPE modified binder (P5); ii) 5% EVA modified binder (E5) and; iii) a modified binder with 4% of EVA and 2% HDPE (E4P2). Asphalt mixtures were produced with these modified binders, and their mechanical properties were analysed and compared with a conventional mixture produced with a 30/50 pen bitumen. It was possible to conclude that these recycled polymers are able to improve the mechanical performance of the asphalt mixtures used in road paving.

Study of the interaction between asphalt and plastic wastes in new polymer modified binders (PMB)

The aim of this study is evaluating the interaction between several base pen grade asphalt binders (35/50, 50/70, 70/100, 160/220) and two different plastic wastes (EVA and HDPE), for a set of new polymer modified binders produced with different amounts of both plastic wastes. After analysing the results obtained for the several polymer modified binders evaluated in this study, including a commercial modified binder, it can be concluded that the new PMBs produced with the base bitumen 70/100 and 5% of each plastic waste (HDPE or EVA) results in binders with very good performance, similar to that of the commercial modified binder.

Adipogenic cell sheets to recreate in vitro adipose tissue microenvironments

Cell sheet (CS) engineering, taking advantage of cellular self-matrix organized as in native tissue, has been largely explored, including by us, for different purposes [1â 3]. Herein we propose for the ï¬ rst time, the use of human adipose stem cells (hASCs)-derived CS to create adipose tissue analogues with different levels of maturation. hASCs were cultured on UpCellTM thermo-responsive dishes for 1, 3 and 5 days under basal conditions previously established by us [3]. The inï¬ uence of pre-differentiation time and respective cell number, over CS stability and differentiation was assessed. Mechanically robust CS were only obtained with 5 days pre-differentiation period. Adipogenesis was followed along the culture assessing the variation of expression of mesenchymal (CD73, CD105 but not CD90) and adipogenic (PPARg, FABP4 and LPL) markers by ï¬ ow cytometry, immunocytochemistry and RT-PCR. Increased ratio of differentiated cells was achieved for longer pre-differentiation periods, while maturation degree was modulated by the maintenance medium. Independently of the overall CS differentiation/maturation level, 3D constructs were fabricated by stacking and further culturing 3 CS. Thus, by varying the culture conditions, different 3D adipose tissue-like microenvironments were recreated, enabling future development of new tissue engineering strategies, as well as further study of adipose tissue role in the regeneration of different tissues.