Engineering Vascularized Bone Grafts by Integrating a Biomimetic Periosteum and β‑TCP Scaffold

Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (β-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical β-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the β-TCP scaffold. A nonperiosteum structural cell sheets-covered β-TCP and plain β-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/β-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered β-TCP graft is a promising approach for bone regeneration

dsDNA Assay.

<p>Graph shows cell number at each time point. Starred bars designate significant differences between groups within a single time point (n = 3).</p

RT-PCR Assay.

<p>Four genes were analyzed: ngf-β (a), nt–3 (b), vegf-a (c), and pdgf-bb (d) with GAPDH used as a house-keeping gene. Data is shown as expression relative to GAPDH at 7 and 14 days. Starred bars specify significant differences between groups.</p

Immunostaining for p75^LNGFR(green) and S100-β (red) for days 7 (a-c) and 14 (d-e).

<p>Panels are divided horizontally by group and vertically by stain. The final column is a co-stain of DAPI, S100-β, and p75^LNGFR. Majority of cells stain positive for both SC markers suggesting the scaffolds do not significantly influence cell character. Scale bar = 100μm.</p

Live/ Dead cytotoxicity viability assay at days 3 (a, b, c), 7 (d, e, f), and 14 (g, h, i).

<p>Staining also suggested directed growth of the cells on the 3D-printed scaffold in parallel with the struts. Staining highlights pattern of clustered growth on the scaffolds (e, f, h, i). Scale bar = 100μm.</p

SEM and Microscopy Imaging.

<p>Micro and SEM images of the printed and template casted scaffolds at low (a, b) and high magnification (c, d) and at day 5 (e-h). Day 5 images show visual alignment of SCs on the printed scaffold. Arrows indicate SCs on scaffolds. Scale bar on a, b = 2mm; on c, d = 500μm; on e, g = 100μm; on f, h = 25μm.</p

ELISA Assays for β-NGF (a) and VEGF-A (b).

<p>Protein concentration per cell in the growth media at days 3, 7, and 14, was normalized using the cell numbers from the dsDNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139820#pone.0139820.g005" target="_blank">Fig 5</a>) results. Data collected from three different samples at each time point. Starred bars indicate significant difference between groups at each time point. Significant differences between time points not shown.</p

Cell Alignment.

<p>Results show the percentage of cells in a given random viewing field within a certain degree of parallel with the major scaffold struts. Data is from DAPI images taken at 7 (a) and 14 (b) days from 4 different fields per time point. Fields analyzed were different than those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139820#pone.0139820.g003" target="_blank">Fig 3</a>. Schematic (c-d) shows the method of measuring the angle between the cell axis in relation to strut. X marks the location of the cell being measured. A line was draw through the major axis of the cell’s nucleus and the angle this line formed with the indicated strut was recorded. The shaded region denoted by Z indicates the top facing regions of the strut from which nuclei were measured.</p

Regulating Functional Groups Enhances the Performance of Flexible Microporous MXene/Bacterial Cellulose Electrodes in Supercapacitors

Ultrathin MXene-based films exhibit superior conductivity and high capacitance, showing promise as electrodes for flexible supercapacitors. This work describes a simple method to enhance the performance of MXene-based supercapacitors by expanding and stabilizing the interlayer space between MXene flakes while controlling the functional groups to improve the conductivity. Ti3C2Tx MXene flakes are treated with bacterial cellulose (BC) and NaOH to form a composite MXene/BC (A-M/BC) electrode with a microporous interlayer and high surface area (62.47 m2 g–1). Annealing the films at low temperature partially carbonizes BC, increasing the overall electrical conductivity of the films. Improvement in conductivity is also attributed to the reduction of −F, −Cl, and −OH functional groups, leaving −Na and −O functional groups on the surface. As a result, the A-M/BC electrode demonstrates a capacitance of 594 F g–1 at a current density of 1 A g–1 in 3 M H2SO4, which represents a ∼2× increase over similarly processed films without BC (309 F g–1) or pure MXene (298 F g–1). The corresponding device has an energy density of 9.63 Wh kg–1 at a power density of 250 W kg–1. BC is inexpensive and enhances the overall performance of MXene-based film electrodes in electronic devices. This method underscores the importance of functional group regulation in enhancing MXene-based materials for energy storage

Pt Nanoparticle–Mn Single-Atom Pairs for Enhanced Oxygen Reduction

The intrinsic roadblocks for designing promising Pt-based oxygen reduction reaction (ORR) catalysts emanate from the strong scaling relationship and activity–stability–cost trade-offs. Here, a carbon-supported Pt nanoparticle and a Mn single atom (PtNP–MnSA/C) as in situ constructed PtNP–MnSA pairs are demonstrated to be an efficient catalyst to circumvent the above seesaws with only ∼4 wt % Pt loadings. Experimental and theoretical investigations suggest that MnSA functions not only as the “assist” for Pt sites to cooperatively facilitate the dissociation of O2 due to the strong electronic polarization, affording the dissociative pathway with reduced H2O2 production, but also as an electronic structure “modulator” to downshift the d-band center of Pt sites, alleviating the overbinding of oxygen-containing intermediates. More importantly, MnSA also serves as a “stabilizer” to endow PtNP–MnSA/C with excellent structural stability and low Fenton-like reactivity, resisting the fast demetalation of metal sites. As a result, PtNPs–MnSA/C shows promising ORR performance with a half-wave potential of 0.93 V vs reversible hydrogen electrode and a high mass activity of 1.77 A/mgPt at 0.9 V in acid media, which is 19 times higher than that of commercial Pt/C and only declines by 5% after 80,000 potential cycles. Specifically, PtNPs–MnSA/C reaches a power density of 1214 mW/cm2 at 2.87 A/cm2 in an H2–O2 fuel cell