Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

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.

Silica nanoparticles as dexamethasone delivery systems able to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Bioactive glasses, especially silica-based materials, are reported to pres- ent osteoconductive and osteoinductive properties, fundamental char- acteristics in bone regeneration [1,2]. Additionally, dexamethasone (Dex) is one of the bioactive agents able to induce the osteogenic differ- entiation of mesenchymal stem cells by increasing the alkaline phos- phatase activity, and the expression levels of Osteocalcin and Bone Sialoprotein [3]. Herein, we synthesised silica (SiO2) nanoparticles (that present inherent bioactivity and ability to act as a sustained drug delivery system), and coated their surface using poly-L-lysine (PLL) and hyaluronic acid (HA) using the layer-by-layer processing technique. Further on, we studied the influence of these new SiO2-polyelectrolyte coated nanoparticles as Dex sustained delivery systems. The SiO2 nanoparticles were loaded with Dex (SiO2-Dex) and coated with PLL and HA (SiO2-Dex-PLL-HA). Their Dex release profile was evaluated and a more sustained release was obtained with the SiO2-Dex-PLL-HA. All the particles were cultured with human bone marrow-derived mes- enchymal stem cells (hBMSCs) under osteogenic differentiation culture conditions. hBMSCs adhered, proliferated and differentiated towards the osteogenic lineage in the presence of SiO2 (DLS 174nm), SiO2-Dex (DLS 175nm) and SiO2-Dex-PLL-HA (DLS 679nm). The presence of these materials induced the overexpression of osteogenic transcripts, namely of Osteocalcin, Bone Sialoprotein and Runx2. Scanning Elec- tron Microscopy/Electron Dispersive Spectroscopy analysis demon- strated that hBMSCs synthesised calcium phosphates when cultured with SiO2-Dex and SiO2-Dex-PLL-HA nanoparticles. These results indi- cate the potential use of these SiO2-polyelectrolytes coated nanoparti- cles as dexamethasone delivery systems capable of promoting osteogenic differentiation of hBMSCs.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

Enhancement of adhesion and promotion of osteogenic differentiation of human adipose stem cells by poled electroactive poly(vinylidene fluoride)

Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Non-electroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and focal adhesion size and number, total adhesion area, cell size, cell aspect ratio and focal adhesion density were estimated using cells expressing EGFP-vinculin. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix (ECM) proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation.

Dynamic piezoelectric stimulation enhances osteogenic differentiation of human adipose stem cells

This work reports on the influence of the substrate polarization of electroactive β-PVDF on human adipose stem cells (hASCs) differentiation under static and dynamic conditions. hASCs were cultured on different β-PVDF surfaces (non-poled and “poled -”) adsorbed with fibronectin and osteogenic differentiation was determined using a quantitative alkaline phosphatase assay. “Poled -” β-PVDF samples promote higher osteogenic differentiation, which is even higher under dynamic conditions. It is thus demonstrated that electroactive membranes can provide the necessary electromechanical stimuli for the differentiation of specific cells and therefore will support the design of suitable tissue engineering strategies, such as bone tissue engineering.

Effector memory CD4+ T cells are associated with cognitive performance in a senior population

Objective: Immunosenescence and cognitive decline are common markers of the aging process. Taking into consideration the heterogeneity observed in aging processes and the recently described link between lymphocytes and cognition, we herein explored the possibility of an association between alterations in lymphocytic populations and cognitive performance. Methods: In a cohort of cognitively healthy adults (n = 114), previously characterized by diverse neurocognitive/psychological performance patterns, detailed peripheral blood immunophenotyping of both the innate and adaptive immune systems was performed by flow cytometry. Results: Better cognitive performance was associated with lower numbers of effector memory CD4(+) T cells and higher numbers of naive CD8(+) T cells and B cells. Furthermore, effector memory CD4(+) T cells were found to be predictors of general and executive function and memory, even when factors known to influence cognitive performance in older individuals (e.g., age, sex, education, and mood) were taken into account. Conclusions: This is the first study in humans associating specific phenotypes of the immune system with distinct cognitive performance in healthy aging.

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Mesenchymal stem cells culture on poly(vinylidene fluoride) piezoelectric microspheres

Dissertação de mestrado em Biofísica e Bionanossistemas

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (a-cyano-4-hydroxycinnamic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.

Osteogenic differentiation of human mesenchymal stem cells in the absence of osteogenic supplements: a surface-roughness gradient study

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range ( 0.5–4.7 lm), and mean distance between peaks (RSm) gradually varying from 214 lm to 33 lm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra 1.53 lm/RSm 79 lm in Dex-deprived OI medium, and Ra 0.93 lm/RSm 135 lm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone.

Highly functional and biodegradable nanofibrous scaffolds combined with stem cells for bone and cartilage tissue engineering

Among the various possible embodiements of Advanced Therapies and in particular of Tissue Engineering the use of temporary scaffolds to regenerate tissue defects is one of the key issues. The scaffolds should be specifically designed to create environments that promote tissue development and not merely to support the maintenance of communities of cells. To achieve that goal, highly functional scaffolds may combine specific morphologies and surface chemistry with the local release of bioactive agents. Many biomaterials have been proposed to produce scaffolds aiming the regeneration of a wealth of human tissues. We have a particular interest in developing systems based in nanofibrous biodegradable polymers1,2. Those demanding applications require a combination of mechanical properties, processability, cell-friendly surfaces and tunable biodegradability that need to be tailored for the specific application envisioned. Those biomaterials are usually processed by different routes into devices with wide range of morphologies such as biodegradable fibers and meshes, films or particles and adaptable to different biomedical applications. In our approach, we combine the temporary scaffolds populated with therapeutically relevant communities of cells to generate a hybrid implant. For that we have explored different sources of adult and also embryonic stem cells. We are exploring the use of adult MSCs3, namely obtained from the bone marrow for the development autologous-based therapies. We also develop strategies based in extra-embryonic tissues, such as amniotic fluid (AF) and the perivascular region of the umbilical cord4 (Whartonâ s Jelly, WJ). Those tissues offer many advantages over both embryonic and other adult stem cell sourcess. These tissues are frequently discarded at parturition and its extracorporeal nature facilitates tissue donation by the patients. The comparatively large volume of tissue and ease of physical manipulation facilitates the isolation of larger numbers of stem cells. The fetal stem cells appear to have more pronounced immunomodulatory properties than adult MSCs. This allogeneic escape mechanism may be of therapeutic value, because the transplantation of readily available allogeneic human MSCs would be preferable as opposed to the required expansion stage (involving both time and logistic effort) of autologous cells. Topics to be covered: This talk will review our latest developments of nanostructured-based biomaterials and scaffolds in combination with stem cells for bone and cartilage tissue engineering.