Modification of Poly(propylene fumarate)–Bioglass Composites with Peptide Conjugates to Enhance Bioactivity

Poly­(propylene fumarate) (PPF) has been highlighted as one of the most promising materials for bone regeneration. Despite the promising advantages of using polymer scaffolds for biomedical applications, their inherent lack of bioactivity has limited their clinical application. In this study, PPF was successfully functionalized with Bioglass and a novel catechol-bearing peptide bioconjugate containing bioactive short peptide sequences of basic fibroblast growth factor, bone morphogenetic protein 2, and osteogenic growth peptide. The binding affinity was assessed to be around 110 nmol/cm² with the Bioglass content at 10 wt %. Fluorescence imaging studies show that the catechol-bearing modular peptide binds preferentially to the Bioglass. A 4 week in vitro cell study using human mesenchymal stem cells showed that cell adhesion, spreading, proliferation, and osteogenic differentiation at both gene and protein levels were all improved by the introduction of peptides, demonstrating the potential approach of dually functionalized polymers for bone regeneration

Synthesis and Biological Evaluation of Well-Defined Poly(propylene fumarate) Oligomers and Their Use in 3D Printed Scaffolds

A ring opening polymerization method for synthesizing oligomeric poly­(propylene fumarate) (PPF) provides a rapid, and scalable method of synthesizing PPF with well-defined molecular mass, molecular mass distribution (<i>Đ</i>_m), and viscosity properties suitable for 3D printing. These properties will also reduce the amount of solvent necessary to ensure sufficient flow of material during 3D printing. MALDI mass spectrometry precisely shows the end group fidelity, and size exclusion chromatography (SEC) demonstrates narrow mass distributions (<1.6) of a series of low molecular mass oligomers (700–3000 Da). The corresponding intrinsic viscosities range from 0.0288 ± 0.0009 dL/g to 0.0780 ± 0.0022 dL/g. The oligomers were printed into scaffolds via established photochemical methods and standardized ISO 10993-5 testing shows that the 3D printed materials are nontoxic to both L929 mouse fibroblasts and human mesenchymal stem cells

Additional file 2: Figure S2. of Restoring the quantity and quality of elderly human mesenchymal stem cells for autologous cell-based therapies

Showing cytokines assayed in the conditioned media of young, elderly, and small(+) MSCs cultured on TCP or ECM substrates. (TIF 4048 kb

Additional file 3: Figure S3. of Restoring the quantity and quality of elderly human mesenchymal stem cells for autologous cell-based therapies

Showing heat map representation of cytokine release by young, elderly, and small(+) MSCs cultured on TCP or ECM substrates. (TIF 3807 kb

Effect of Chemical and Physical Properties on the In Vitro Degradation of 3D Printed High Resolution Poly(propylene fumarate) Scaffolds

Two distinct molecular masses of poly­(propylene fumarate) (PPF) are combined with an additive manufacturing process to fabricate highly complex scaffolds possessing controlled chemical properties and porous architecture. Scaffolds were manufactured with two polymer molecular masses and two architecture styles. Degradation was assessed in an accelerated in vitro environment. The purpose of the degradation study is not to model or mimic in vivo degradation, but to efficiently compare the effect of modulating scaffold properties. This is the first study addressing degradation of chain-growth synthesized PPF, a process that allows for considerably more control over molecular mass distribution. It demonstrates that, with greater process control, not only is scaffold fabrication reproducible, but the mechanical properties and degradation kinetics can be tailored by altering the physical properties of the scaffold. This is a clear step forward in using PPF to address unmet medical needs while meeting regulatory demands and ultimately obtaining clinical relevancy

IL-13 Induces YY1 through the AKT Pathway in Lung Fibroblasts

<div><p>A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13’s downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 <i>in vitro</i>. <i>In vivo</i> YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.</p></div

IL-13-induced YY1 expression is regulated by AKT pathway.

<p>MRC-5 cells were transfected with AKT and pCDNA1 control plasmids with electroporation. (A). At 24 h after transfection, the cells were starved for 24 h. IL-13 (30 ng/ml) was added to the cells for 12 h, The cells were lyzed, and the levels of YY1, AKT, p-AKT and β-actin were determined by Western blot. (B). YY1 and β-actin expression from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119039#pone.0119039.g004" target="_blank">Fig. 4A</a> were scanned and were conducted a densitometric analysis with Image J. YY1 expression was normalized to β-actin. Scanned data were analyzed by T-test. ** indicate p value <0.01. (C) After MRC-5 cells were starved for 24 h without serum, cells were pre-treated with or without wortmannin (10 μM) for 1 h, and then treated with or without IL-13 (30 ng/ml) for 12 h in serum-free DMEM. Whole cell extracts were subjected to Western blot analysis for determining the levels of YY1, α-SMA, p-AKT, collagen and β-actin. (D). the mRNA expression of YY1 was detected by quantitative PCR. GAPDH mRNA expression was used as an internal control. The data are presented with standard errors derived from at least three independent experiments, each performed in triplicate; n = 3 and <i>**p</i> < 0.01.</p

Mapping the Mechanome of Live Stem Cells Using a Novel Method to Measure Local Strain Fields <em>In Situ</em> at the Fluid-Cell Interface

<div><p>During mesenchymal condensation, the initial step of skeletogenesis, transduction of minute mechanical forces to the nucleus is associated with up or down-regulation of genes, ultimately resulting in formation of the skeletal template and appropriate cell lineage commitment. The summation of these biophysical cues affects the cell's shape and fate. Here, we predict and measure surface strain, in live stem cells, in response to controlled delivery of stresses, providing a platform to direct short-term structure - function relationships and long-term fate decisions. We measure local strains on stem cell surfaces using fluorescent microbeads coated with Concanavalin A. During delivery of controlled mechanical stresses, 4-Dimensional (x,y,z,t) displacements of the bound beads are measured as surface strains using confocal microscopy and image reconstruction. Similarly, micro-particle image velocimetry (μ-piv) is used to track flow fields with fluorescent microspheres. The measured flow velocity gradient is used to calculate stress imparted by fluid drag at the surface of the cell. We compare strain measured on cell surfaces with those predicted computationally using parametric estimates of the cell's elastic and shear modulus. Finally, cross-correlating stress - strain data to measures of gene transcription marking lineage commitment enables us to create stress - strain - fate maps, for live stem cells <em>in situ</em>. The studies show significant correlations between live stem cell stress - strain relationships and lineage commitment. The method presented here provides a novel means to probe the live stem cell's mechanome, enabling mechanistic studies of the role of mechanics in lineage commitment as it unfolds.</p> </div

Fibroblast specific protein 1 (FSP1), α-SMA, YY1, and collagen were increased in CC10-IL-13 transgenic mice.

<p><b>(A)</b> Lung tissues were stained with Hematoxylin and eoxin and Masson trichrome. The histological features and collagen were compared in between wild type (WT) control mice and CC10-IL-13 transgenic mice. All figures are at original magnification with a ruler (upper panel) and with a ruler (lower panel) on the images. <b>(B)</b> Immunohistochemical staining was used to localize the sites of FSP1, α-SMA, and YY1 in lungs of CC10-IL-13 transgenic mice and WT l mice. Collagen stains blue in these panels. All figures are at original magnification: 10 × (left panel) and 63 × (right panel). YY1, FSP1, and α-SMA expression are increased in lung tissue from IL-13 transgenic mice (n = 4). <b>(C)</b> Lung tissues from IL-13 transgenic (IL-13tg) and wild-type mice were stained by immunofluorescence for YY1 (Red), FSP1 (Green) and DAPI (blue). The magnification is 63x. Low magnification 10x showed in supplemental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119039#pone.0119039.s001" target="_blank">S1 Fig.</a></p

<i>In situ</i> mapping of stem cell stresses and strains.

<p>4D (x,y,z,t) image of microsphere displacements (<b>A, C, E</b>) and microbead displacements (<b>B, D, F</b>) shows flow fields and surface strains, respectively, in live stem cells subjected to fluid flow at low (<b>A,B</b>), high (<b>C,D</b>), and very high (<b>E,F</b>) densities. Calcein Green stains live cells. Red and white arrows indicate velocity of flow (microsphere displacement and direction: <b>A, C, E</b>) and strain (microbead displacements: <b>B, D, F</b>), respectively. Red, green, and white dots (<b>B, D, F</b>) show respective microbead positions at 0, 30, and 60 minutes after flow. Stresses imparted by flow at cell surfaces are calculated from the experimentally determined velocity gradient (slope of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043601#pone.0043601.s001" target="_blank">Fig. S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043601#pone.0043601.e003" target="_blank">Equation 3</a>). Cell surface strains (deformations) are calculated using experimentally measured microbead displacements on cell surfaces.</p