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.The study was supported financially by the Academy of Finland (136288, 140978 and 256931), the Sigrid Jusélius Foundation, the Pirkanmaa Hospital District and the Finnish Funding Agency for Technology and Innovation (TEKES). This study was also supported by FEDER through the COMPETE Program, by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Project PEST- C/FIS/UI607/2011 and by projects NANO/NMed-SD/0156/2007 and PTDC/CTM NAN/112574/2009. The autors also thank the project Matepro – Optimizing Materials and Processes”, ref. NORTE-07-0124-FEDER-000037”, co-funded by the “Programa Operacional Regional do Norte” (ON.2 – O Novo Norte), under the “Quadro de Referência Estratégico Nacional” (QREN), through the “Fundo Europeu de Desenvolvimento Regional” (FEDER). V.S. and C.R. thank the FCT for the SFRH/BPD/63148/2009 and SFRH/BPD/90870/2012 grants, respectively

Tissue Engineering in Oral and Maxillofacial Surgery : From Lab to Clinics

Regenerative medicine aims at the functional restoration of tissue malfunction, damage or loss, and can be divided into three main approaches. Firstly, the cell-based therapies, where cells are administered to re-establish a tissue either directly or through paracrine functions. Secondly, the often referred to as classical tissue engineering, consisting of the combined use of cells and a bio-degradable scaffold to form tissue. Thirdly, there are material-based approaches, which have made significant advances which rely on biodegradable materials, often functionalized with cellular functions (De Jong et al. 2014). In 1993, Langer and Vacanti, determined tissue engineering as an “interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function”. They published this definition in Science in 1993. Tissue engineering has been classically thought to consist of three elements: supporting scaffold, cells and regulating factors such as growth factors (Fig. 1). Depending on the tissue to be regenerated, all three vary. Currently, it is known, that many other factors may have an effect on the outcome of the regenerate. These include factors enabling angiogenesis, physical stimulation, culture media, gene delivery and methods to deliver patient specific implants (PSI) (Fig. 2). During the past two decades, major obstacles have been tackled and tissue engineering is currently being used clinically in some applications while in others it is just taking its first baby steps.Peer reviewe

Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

Bioactive glasses (BaGs) are widely utilised in bone tissue engineering (TE) but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs) on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-%)) and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2) in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP) and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene). Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK)-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK) inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments

Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

Bioactive glasses (BaGs) are widely utilised in bone tissue engineering (TE) but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs) on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-%)) and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2) in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP) and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene). Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK)-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK) inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments

Retinoic acid-stimulated sequential phosphorylation, PML recruitment, and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression

We previously reported an intricate mechanism underlying the homeostasis of Oct4 expression in normally proliferating stem cell culture of P19, mediated by SUMOylation of orphan nuclear receptor TR2. In the present study, we identify a signaling pathway initiated from the nongenomic activity of all-trans retinoic acid (atRA) to stimulate complex formation of extracellular signal-regulated kinase 2 (ERK2) with its upstream kinase, mitogen-activated protein kinase kinase (MEK). The activated ERK2 phosphorylates threonine-210 (Thr-210) of TR2, stimulating its subsequent SUMOylation. Dephosphorylated TR2 recruits coactivator PCAF and functions as an activator for its target gene Oct4. Upon phosphorylation at Thr-210, TR2 increasingly associates with promyelocytic leukemia (PML) nuclear bodies, becomes SUMOylated, and recruits corepressor RIP140 to act as a repressor for its target, Oct4. To normally proliferating P19 stem cell culture, exposure to a physiological concentration of atRA triggers a rapid nongenomic signaling cascade to suppress Oct4 gene and regulate cell proliferation

Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9 ▿

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis