Journal: Nature Communications
Article Title: Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization
doi: 10.1038/s41467-019-11455-8
Figure Lengend Snippet: Osteogenic lineage commitment, gene, and protein expression in mineralized cell-laden collagen. a Gene expression of osteocalcin ( OCN ) was significantly higher (** p < 0.01) in mineralized collagen than in OIM treated, both of which were much higher than non-mineralized samples (**** p < 0.0001), after 21 days. Expression of osteocyte-related genes ( DMP1 and PDPN ) was also comparable to OIM after 21 days of culture in mineralized collagen and significantly higher at earlier time points ( DMP1 , ** p < 0.01 after 7 days and * p < 0.05 after 14 days). Data are represented as mean ± SD ( N = 3). b A significant increase in the expression of BMP-2 and BMP-6 for cells in both mineralized collagen and in OIM relative to non-mineralized controls is consistent with enhanced bone-specific metabolic activity. A marked increase in the ratio of RANKL/OPG in mineralized samples, however, suggests the stronger potency for cell-mediated bone remodeling via a paracrine signaling in cell-laden mineralized constructs than in the other groups. ( N = 4 for BMPs and N = 5 for others). At a cell-surface level, the expression of OCN and PDPN on day 14 was very low in ( c , f ) non-mineralized controls and significantly higher for both ( d , g ) mineralized and ( e , h ) OIM-treated samples (** p < 0.01, *** p < 0.001) ( N = 3). Similarly, surface expression of DMP1 was comparable for ( k ) OIM-treated cells (**** p < 0.0001) and ( j ) mineralized collagen (* p < 0.05), both of which were significantly higher than ( i ) non-mineralized controls ( N = 4). Scale bar: 200 μm. Alizarin red staining to assay mineralization shows homogeneous and intense red staining even after 7 days in ( m ) mineralized constructs, whereas staining was more diffuse in ( n ) OIM-treated samples and almost non-existent in ( l ) non-mineralized controls. Scale bar: 100 μm. Reflectance confocal microscopy images of F-Actin/DAPI-stained hMSCs in ( o ) non-mineralized, ( p ) mineralized, and ( q ) OIM treated cell-laden collagen, illustrate the dendritic-like extensions of cells after matrix mineralization, reminiscent of osteocyte-like morphology. The above images, with cells digitally removed ( r – t ), show formation of well-defined lacunae-like regions in the locations where cells resided in mineralized matrix ( s ). Scale bar: 30 μm. Quantification of dendrite-like projections is shown in Supplementary Fig. . All comparisons used ANOVA/Tukey. Source data are provided as a Source Data file
Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-OCN (Bioss antibodies, bs4917R) (1:50 dilution), mouse monoclonal anti-PDPN (Origene, DM3500P) (1:100 dilution), Rabbit polyclonal anti-DMP1 (Invitrogen, PA5-57956) (1:200 dilution), mouse monoclonal anti-CD31 (Dako, JC70A) (1:200 dilution), rabbit polyclonal anti-RUNX2/CBFA1 antibody (Novus Biologicals, NBP1-77461) (1:100 dilution), mouse monoclonal anti-αSMA (Invitrogen, MA5-11547) (1:400 dilution), mouse monoclonal anti-NEFL antibody (Thermo Fisher Scientific, MA1-2010) (1:50 dilution), and mouse monoclonal anti-Neuron-specific enolase antibody (Abcam, ab218388) (1:1000 dilution).
Techniques: Expressing, Activity Assay, Construct, Staining, Confocal Microscopy
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