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

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

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

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

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

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

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