Nano-Engineered Scaffold for Osteoarticular Regenerative Medicine

In the last decade, regenerative medicine has benefited from the exponential development of nanomaterial sciences, tissue engineering and cell-based therapies. More and more sophisticated designed structures and surface topologies are being developed to basically mimic the extracellular matrix of native tissues such as cartilage and bone. Here we give an overview of the progress made in osteochondral lesion repair, with nano-engineered scaffolds comprising building blocks such as nanoparticles, nanotubes, layer-by-layer nano-assemblies, molecular self-assembly, nanopatterned surfaces…. This nano-engineering technology is coupled with bio-functionalization, by the use of adhesion peptides, growth factors, or deoxyribonucleic acid, to drive cell adhesion, proliferation and behavior towards tissue regeneration. In osteochondral regeneration, the challenge is the simultaneous development of chondrocytes and cartilage extracellular matrix on the one side and a well vascularized bone tissue with osteoblasts on the other sid

Minerva 1934

Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI

e-CSC and l-CSC are of fetal origin.

<p>(<b>a</b>) Confocal immuno-fluorescence for c-KIT (<b>b</b>) Cell morphology of early and late gestation chorionic stem cells (e-CSC and l-CSC respectively) passage 4–5. (<b>c</b>) PCR for Y chromosome specific SRY. Positive and negative controls shown. (<b>d</b>) FISH analysis for X (FITC) and Y (Texas Red) chromosomes indicated with green and red arrows respectively. 100 cells were counted. All scale bars 100 µm.</p

e-CSC and l-CSC have a similar immunophenotype.

<p>(<b>a</b>) Flow cytometry for percent of e-CSC (black) and l-CSC (red) populations positive for surface adhesion markers; CD62P, Integrin β7, CD11a, CD106, CD51/61, CD49b, CXCR4, Integrin β3, CD49f, CD49d, αV Integrin and CD49e. (<b>b</b>) Percentage of e-CSC (black) and l-CSC (red) populations that adhered to fibronectin a-chemotryptic 40 kDa (Fn 40 kDa), fibronectin a-chemotryptic 120 kDa (Fn 120 kDa), whole fibronectin (Fn), collagen I (col I), collagen IV (col IV), human placenta laminin (laminin) and vascular cell adhesion molecule-1 (VCAM-1). Negative control DMEM alone (media). Data. * P<0.05, Student's <i>t</i> test, <i>n</i> = 3 per cell group. Mean ± s.e.m. (<b>c</b>) Confocal immuno-fluorescence for endothelial marker (CD14), hematopoietic markers (CD34 and CD45), MSC-associated markers (CD105, CD73 and CD44), matrix protein (vimentin) and markers found in pluripotent cells as well as MSC (CD29 and CD90) stained with FITC (green). Nuclei stained with DAPI (blue). Scale bar 100 µm. Positive controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043395#pone.0043395.s003" target="_blank">Fig. S3a</a>.</p

Osteogenic, adipogenic and neurogenic differentiation of e-CSC and l-CSC.

<p>(<b>a</b>) Alizarin red staining (calcium deposits) and von kossa staining (mineralisation) of cells grown in osteogenic permissive media for 2 weeks. (<b>b</b>) Quantitative real time PCR of fold increased expression of osteogenic genes after osteogenic differentiation; osteopontin (OP), procollagen endopeptidase enhancer (PCE), osteocalcin (OC), osterix (OSX), bone sialo-protein-II (BSP) and bone morphogenic protein-2 (BMP2). (<b>c</b>) Western blot of COL1 for e-CSC and l-CSC not differentiated (non-diff) and differentiated to bone for 2 weeks (diff). Positive and negative controls shown. Loading control is β-actin. (<b>d</b>) Oil Red O staining (lipid droplets) of e-CSC and l-CSC grown in adipogenic permissive media for 3 weeks. (<b>e</b>) Quantitative real time PCR of fold increased expression of adipocyte differentiation regulator peroxisome proliferator-activated receptor gamma (PPARγ). (<b>f</b>) Morphology and expression of neuronal markers β-TUBULIN, MAP2 and NMDA receptor NR1 shown with FITC (green) of e-CSC and l-CSC after 5 days neurogenic differentiation. Nuclei stained with DAPI (blue). All quantitative real time PCR data normalised to GAPDH and basal expression levels of differentiation genes (i.e. 2^−ΔΔCt). e-CSC (black) and l-CSC (red). Data. * P<0.05; ** P<0.01, Student's <i>t</i> test, <i>n</i> = 3 per cell group. Mean ± s.e.m. All scale bars 100 µm.</p

e-CSC and l-CSC express markers found in primordial germ cells.

<p>(<b>a</b>) RT-PCR for expression of c-KIT, STELLA, FRAGILIS, NANOS3, SSEA1, VASA and GAPDH. (<b>b</b>) Flow cytometry for percent of e-CSC (black) and l-CSC (red) populations positive for STELLA, FRAGILIS, NANOS3, SSEA1, DAZL, PUM2, VASA, TNAP and BLIMP1. (<b>c</b>) DNA methylation status of imprinted gene <i>H19</i> as percent total input DNA hypermethylated (black), unmethylated (light grey) and intermediately methylated (dark grey). <i>n</i> = 3 per cell group.</p

e-CSC are smaller in size and grow faster than l-CSC.

<p>(<b>a</b>) Image of cells in suspension used for cell size analysis. (<b>b</b>) Average cell size in µm of e-CSC (black) and l-CSC (red) when in suspension. (<b>c</b>) Average growth rate in hours taken for cell population to double during exponential growth phase; i.e. population doubling time (DT). e-CSC (black), l-CSC (red). (<b>d</b>) Cell expansion capacity over 1500 hours measured by average cumulative population doublings (Cumulative PD) of e-CSC (▴) and l-CSC (▾) when passaged at sub-confluence. (<b>e</b>) Cell kinetics measured by average cumulative population doublings of e-CSC (▴) and l-CSC (▾) when seeded at low density and grown beyond confluence for 288 hours. * P<0.05; ** P<0.01, Student's <i>t</i> test, <i>n</i> = 3 different samples per cell group. Mean ± s.e.m. All scale bars 100 µm.</p

e-CSC and l-CSC contain a subpopulation positive for pluripotency markers.

<p>(<b>a</b>) Representative flow cytometry (<i>n</i> = 3) for percent of cells positive for CD24, OCT4, SOX2, CMYC, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 in e-CSC and l-CSC whole populations (isotype control in black). (<b>b</b>) Representative confocal immunofluorescence images for OCT4, SOX2, KLF4, NANOG, REX1, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 stained with FITC (green). Positive cells indicated with green arrow, negative cells with white arrow. Nuclei stained with DAPI (blue). Scale bar 25 µm. Positive controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043395#pone.0043395.s003" target="_blank">Fig. S3b</a>.</p