Rapid Rapamycin-Only Induced Osteogenic Differentiation of Blood-Derived Stem Cells and Their Adhesion to Natural and Artificial Scaffolds

Abstract

Stem cells are a centerpiece of regenerative medicine research, and the recent development of adult stem cell-based therapy systems has vigorously expanded the scope and depth of this scientific field. The regeneration of damaged and/or degraded bone tissue in orthopedic, dental, or maxillofacial surgery is one of the main areas where stem cells and their regenerative potential could be used successfully, requiring tissue engineering solutions incorporating an ideal stem cell type paired with the correct mechanical support. Our contribution to this ongoing research provides a new model of in vitro osteogenic differentiation using blood-derived stem cells (BDSCs) and rapamycin, visibly expressing typical osteogenic markers within ten days of treatment. In depth imaging studies allowed us to observe the adhesion, proliferation, and differentiation of BDSCs to both titanium and bone scaffolds. We demonstrate that BDSCs can differentiate towards the osteogenic lineage rapidly, while readily adhering to the scaffolds we exposed them to. Our results show that our model can be a valid tool to study the molecular mechanisms of osteogenesis while tailoring tissue engineering solutions to these new insights.

Figure 1

Blood derived stem cells' (BDSCs) osteogenic differentiation with rapamycin. (a) BDSCs before starting the differentiation protocol. The cells show typical morphological features of stemness such as small size, roundish shape, and, in vitro, a disposition to a “string of pearls” appearance. (b) Alizarin Red S staining on BDSCs after 10 days of osteogenic differentiation, to evaluate inorganic calcium phosphate deposition. (c) Visualization by immunofluorescence analysis through confocal microscopy of a single differentiated cell (60x). (d) Image from three-dimensional stack analysis by confocal microscopy of blood-derived stem cells after ten days of osteogenic differentiation (in green, osteocalcin; in blue, DAPI: merged; (a), (b) 100 μm, (c) bar = 8 μm).

Figure 2

Electron microscopy demonstrating scaffold colonization of BDSCs differentiated with rapamycin, on bone scaffold. (a) Higher magnification of area indicated in the rectangle displays cells forming tissue-like agglomerates. Notably, electron micrograph captures an osteoblast matrix vesicle just as it initiated the exocytosis of hydroxyapatite (EDX spectrum). (b) Rectangles indicate differentiated osteoblast-like cells as shown by transmission electron microscopy analysis. (c) Higher magnification shows electron-dense granules within the mitochondria and the presence of calcium and phosphorus calcium phosphate aggregates typical of an osteoblast (EDX spectrum). (d) TEM image shows a typical osteoblast cell with flat lamellipodia and dendritic filopodia (bar = 20 μm in (a), 100 μm in (b), bar = 5 μm in (c), and 10 μm in (d)).

Figure 3

BDSCs osteogenic differentiation with rapamycin and titanium scaffolds. Nuclear and Alizarin Red S stainings on type 1 titanium after differentiation by confocal microscopy analysis ((a) and (b), resp.). Nuclear staining, osteocalcin expression, and merge analysis on type 2 titanium scaffold ((c), (d), and (e), resp.) (osteocalcin green, DAPI blue; (a), (b) bar = 10 mm; (c), (d) 200 μm 4x).

Figure 4

Titanium scaffolds: Titanium type 1. (a) The original endosseous implants before cutting, (b) titanium after cutting and sterilization, (c) titanium type 1 in a 24-well plate before osteogenic differentiation experiments. Titanium type 2. (d) The original titanium grill before cutting. (e) The dimensions of the different perforations. (f) Titanium type 2 in a 24-well plate before osteogenic differentiation experiments. (g) Titanium type 1 immunofluorescence images by confocal microscopy, the low resolution of the osteocalcin and nuclei signals are evident (green and blue, resp.). (h) Titanium type 2 immunofluorescence images by confocal microscopy, it is evident how the more flat surface of this scaffold is better suited for confocal microscopy analysis.

Figure 5

Time course experiments to detect the BDSCs capacity to adhere to the titanium scaffold. The increasing blue signal (DAPI) along time course analysis shows the cells' adhesion and growth on titanium ((a) = after 1 day, (b) = 2 days, (c) = 3 days, (d) = 5 days, (e) = 7 days). (f) shows a graphical representation of (a)–(e) time course analysis performed by Nikon EZ-C1 viewer software.

Figure 6

Analysis to detect BDSC capacity to differentiate on titanium scaffolds. (A) Merge analysis on type 2 titanium scaffold by osteocalcin expression ((B) osteocalcin green) and nuclear staining ((C) DAPI blue) to demonstrate BDSCs osteogenic differentiation. (D) shows a graphical representation of the two channel light intensity signals (expressed in ADG units ×100). It is evident how the blue peaks (nuclear signal) overlap the green peaks (osteocalcin signal). (E) shows time course experiments for osteocalcin expression (expressed as average channel brightness) ((A): bar = 200 μm, (B), (C): bar = 50 μm).