Immunological Compatibility Status of Placenta-Derived Stem Cells is Mediated by Scaffold 3D Structure

Placenta-derived amniotic epithelial cells (AECs), a great cell source for tissue engineering and stem cell therapy, are immunologically inert in their native state; however, immunological changes in these cells after culture and differentiation have challenged their applications. The aim of this study was to investigate the effect of 2D and 3D scaffolds on human lymphocyte antigens (HLA) expression by AECs. The effect of different preparation parameters including pre-freezing time and temperature was evaluated on 3D chitosan–gelatine scaffolds properties. Evaluation of MHC class I, HLA-DR and HLA-G expression in AECs after 7 d culture on 2D bed and 3D scaffold of chitosan–gelatine showed that culture of AECs on the 2D substrate up-regulated MHC class I and HLA-DR protein markers on AECs surface and down-regulated HLA-G protein. In contrast, 3D scaffold did not increase protein expression of MHC class I and HLA-DR. Moreover, HLA-G protein expression remained unchanged in 3D culture. These results confirm that 3D scaffold can remain AECs in their native immunological state and modification of physical properties of the scaffold is a key regulator of immunological markers at the gene and protein expression levels; a strategy which circumvents rejection challenge of amniotic stem cells to be translated into the clinic

Expression, purification and in vitro biological activity from human recombinant BMP-2 produced by a novel approach

Bone morphogenetic proteins have promoted great biomedical interest due to their ability in inducing new bone formation when used as powerful osteoinductive components of several late-stage bone grafting products. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is obtained from mammalian cell expressing systems in low amounts or from bacteria inclusion bodies after timeconsuming refolding methods. Thus, there is a need to establish novel approaches for producing rhBMP-2 in high yields by simple and cheap procedures.Portuguese Foundation for Science and Technolog y, FCT (PhD Grant to PC Bessa , SFRH/BD/17049/2004). This work was also partially supported by the European STREP HIPPOCRATES (NMP3 - CT - 2003 - 505758) and carried out under the scope of European NoE EXPERTISSUES (NMP3 - CT - 2004 - 500283).info:eu-repo/semantics/publishedVersio

Human amniotic membrane as newly identifed source of amniotic fuid pulmonary surfactant

Pulmonary surfactant (PS) reduces surface tension at the air-liquid interface in the alveolar epithelium of the lung, which is required for breathing and for the pulmonary maturity of the developing foetus. However, the origin of PS had never been thoroughly investigated, although it was assumed to be secreted from the foetal developing lung. Human amniotic membrane (hAM), particularly its epithelial cell layer, composes the amniotic sac enclosing the amniotic fuid. In this study, we therefore aimed to investigate a potential contribution of the cellular components of the hAM to pulmonary surfactant found in amniotic fuid. We identifed that cells within the native membrane contain lamellar bodies and express all four surfactant proteins as well as ABCA3. Lipidomic profling by nanoESI – MS/MS revealed the presence of the essential lipid species as found in PS. Also, the biophysical activity of conditioned cell culture supernatant obtained from hAM was tested with captive bubble surfactometry. hAM supernatant showed the ability to reduce surface tension, similar to human PS obtained from bronchoalveolar lavage. This means that hAM produces the essential PS-associated components and can therefore contribute as second potential source of PS in amniotic fuid aside from the foetal lung

A novel approach for the production of human recombinant BMP-2 for bone tissue engineering applications

Bone tissue engineering has been an increasing field of research during the last years. The ideal approach for a regenerative application would consist in the use of cells from the patient, scaffolding materials and differentiation growth factors. Bone morphogenetic protein-2 (BMP-2) is one such growth factors with a strong ability to induce new bone and cartilage formation and has been used as a powerful osteoinductive component of several late-stage tissue engineering products for bone grafting. In this work, we aimed at obtaining high yields of human recombinant BMP-2 in a stable, pure and biologically active form by use of a new bacteria expression system that circumvents the disadvantages of conventional recombinant protein preparation methods and to perform a study of the stability conditions and the functionality of these peptides in vitro in human mesenchymal stem cells and C2C12 murine cell line.info:eu-repo/semantics/publishedVersio

Engineering Blood and Lymphatic Microvascular Networks in Fibrin Matrices

Vascular network engineering is essential for nutrient delivery to tissue-engineered constructs and, consequently, their survival. In addition, the functionality of tissues also depends on tissue drainage and immune cell accessibility, which are the main functions of the lymphatic system. Engineering both the blood and lymphatic microvasculature would advance the survival and functionality of tissue-engineered constructs. The aim of this study was to isolate pure populations of lymphatic endothelial cells (LEC) and blood vascular endothelial cells (BEC) from human dermal microvascular endothelial cells and to study their network formation in our previously described coculture model with adipose-derived stromal cells (ASC) in fibrin scaffolds. We could follow the network development over a period of 4 weeks by fluorescently labeling the cells. We show that LEC and BEC form separate networks, which are morphologically distinguishable and sustainable over several weeks. In addition, lymphatic network development was dependent on vascular endothelial growth factor (VEGF)-C, resulting in denser networks with increasing VEGF-C concentration. Finally, we confirm the necessity of cell–cell contact between endothelial cells and ASC for the formation of both blood and lymphatic microvascular networks. This model represents a valuable platform for in vitro drug testing and for the future in vivo studies on lymphatic and blood microvascularization

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2 : in vitro and in vivo bioactivity

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.The authors acknowledge Anna Hofmann and Anna Khadem for additional help with some experiments, and Karin Brenner for animal maintenance. This work was supported by Fundacao para a Ciencia e Tecnologia (Ph.D. grant SFRH/BD/17049/2004), project ElastM (POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia), Marie Curie Alea Jacta EST short-term grant (MEST-CT-2004-8104), and European STREP Project HIP-POCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

A luciferase-based quick potency assay to predict chondrogenic differentiation.

Chondrogenic differentiation of adipose derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore quick potency assays are required in order to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation potential are time consuming and thus, a novel enhancer and tissue-specific promoter combination was employed for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were co-transfected with the Metridia luciferase based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three dimensional (3D) culture conditions and in a second approach also under 2D culture conditions. With our newly developed quick potency bioassay we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies, in a very short time and at low costs

A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture