Viability of mesenchymal stem cells during electrospinning

Tissue engineering is a technique by which a live tissue can be re-constructed and one of its main goals is to associate cells with biomaterials. Electrospinning is a technique that facilitates the production of nanofibers and is commonly used to develop fibrous scaffolds to be used in tissue engineering. In the present study, a different approach for cell incorporation into fibrous scaffolds was tested. Mesenchymal stem cells were extracted from the wall of the umbilical cord and mononuclear cells from umbilical cord blood. Cells were re-suspended in a 10% polyvinyl alcohol solution and subjected to electrospinning for 30 min under a voltage of 21 kV. Cell viability was assessed before and after the procedure by exclusion of dead cells using trypan blue staining. Fiber diameter was observed by scanning electron microscopy and the presence of cells within the scaffolds was analyzed by confocal laser scanning microscopy. After electrospinning, the viability of mesenchymal stem cells was reduced from 88 to 19.6% and the viability of mononuclear cells from 99 to 8.38%. The loss of viability was possibly due to the high viscosity of the polymer solution, which reduced the access to nutrients associated with electric and mechanical stress during electrospinning. These results suggest that the incorporation of cells during fiber formation by electrospinning is a viable process that needs more investigation in order to find ways to protect cells from damage.

CTLA4 enhances the osteogenic differentiation of allogeneic human mesenchymal stem cells in a model of immune activation

Allogeneic mesenchymal stem cells (allo-MSCs) have recently garnered increasing interest for their broad clinical therapy applications. Despite this, many studies have shown that allo-MSCs are associated with a high rate of graft rejection unless immunosuppressive therapy is administered to control allo-immune responses. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a co-inhibitory molecule expressed on T cells that mediates the inhibition of T-cell function. Here, we investigated the osteogenic differentiation potency of allo-MSCs in an activated immune system that mimics the in vivo allo-MSC grafting microenvironment and explored the immunomodulatory role of the helper T cell receptorCTLA4 in this process. We found that MSC osteogenic differentiation was inhibited in the presence of the activated immune response and that overexpression of CTLA4 in allo-MSCs suppressed the immune response and promoted osteogenic differentiation. Our results support the application of CTLA4-overexpressing allo-MSCs in bone tissue engineering.

Single-walled carbon nanotubes functionalized with sodium hyaluronate enhance bone mineralization

The aim of this study was to evaluate the effects of sodium hyaluronate (HY), single-walled carbon nanotubes (SWCNTs) and HY-functionalized SWCNTs (HY-SWCNTs) on the behavior of primary osteoblasts, as well as to investigate the deposition of inorganic crystals on titanium surfaces coated with these biocomposites. Primary osteoblasts were obtained from the calvarial bones of male newborn Wistar rats (5 rats for each cell extraction). We assessed cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and by double-staining with propidium iodide and Hoechst. We also assessed the formation of mineralized bone nodules by von Kossa staining, the mRNA expression of bone repair proteins, and the deposition of inorganic crystals on titanium surfaces coated with HY, SWCNTs, or HY-SWCNTs. The results showed that treatment with these biocomposites did not alter the viability of primary osteoblasts. Furthermore, deposition of mineralized bone nodules was significantly increased by cells treated with HY and HY-SWCNTs. This can be partly explained by an increase in the mRNA expression of type I and III collagen, osteocalcin, and bone morphogenetic proteins 2 and 4. Additionally, the titanium surface treated with HY-SWCNTs showed a significant increase in the deposition of inorganic crystals. Thus, our data indicate that HY, SWCNTs, and HY-SWCNTs are potentially useful for the development of new strategies for bone tissue engineering.

Biomechanical and histological evaluation of hydrogel implants in articular cartilage

We evaluated the mechanical behavior of the repaired surfaces of defective articular cartilage in the intercondylar region of the rat femur after a hydrogel graft implant. The results were compared to those for the adjacent normal articular cartilage and for control surfaces where the defects remained empty. Hydrogel synthesized by blending poly(2-hydroxyethyl methacrylate) and poly(methyl methacrylate-co-acrylic acid) was implanted in male Wistar rats. The animals were divided into five groups with postoperative follow-up periods of 3, 5, 8, 12 and 16 weeks. Indentation tests were performed on the neoformed surfaces in the knee joint (with or without a hydrogel implant) and on adjacent articular cartilage in order to assess the mechanical properties of the newly formed surface. Kruskal-Wallis analysis indicated that the mechanical behavior of the neoformed surfaces was significantly different from that of normal cartilage. Histological analysis of the repaired defects showed that the hydrogel implant filled the defect with no signs of inflammation as it was well anchored to the surrounding tissues, resulting in a newly formed articular surface. In the case of empty control defects, osseous tissue grew inside the defects and fibrous tissue formed on the articular surface of the defects. The repaired surface of the hydrogel implant was more compliant than normal articular cartilage throughout the 16 weeks following the operation, whereas the fibrous tissue that formed postoperatively over the empty defect was stiffer than normal articular cartilage after 5 weeks. This stiffness started to decrease 16 weeks after the operation, probably due to tissue degeneration. Thus, from the biomechanical and histological point of view, the hydrogel implant improved the articular surface repair.

Whole transcriptome organisation in the dehydrated supraoptic nucleus

The supraoptic nucleus (SON) is part of the central osmotic circuitry that synthesises the hormone vasopressin (Avp) and transports it to terminals in the posterior lobe of the pituitary. Following osmotic stress such as dehydration, this tissue undergoes morphological, electrical and transcriptional changes to facilitate the appropriate regulation and release of Avp into the circulation where it conserves water at the level of the kidney. Here, the organisation of the whole transcriptome following dehydration is modelled to fit Zipf's law, a natural power law that holds true for all natural languages, that states if the frequency of word usage is plotted against its rank, then the log linear regression of this is -1. We have applied this model to our previously published euhydrated and dehydrated SON data to observe this trend and how it changes following dehydration. In accordance with other studies, our whole transcriptome data fit well with this model in the euhydrated SON microarrays, but interestingly, fit better in the dehydrated arrays. This trend was observed in a subset of differentially regulated genes and also following network reconstruction using a third-party database that mines public data. We make use of language as a metaphor that helps us philosophise about the role of the whole transcriptome in providing a suitable environment for the delivery of Avp following a survival threat like dehydration.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.