The Osteogenic Potential of Falciform Ligament-Derived Stromal Cells-A Comparative Analysis between Two Osteogenic Induction Programs

Abstract

Mesenchymal stromal cells (MSCs) have gained special relevance in bone tissue regenerative applications. MSCs have been isolated from different depots, with adipose tissue being acknowledged as one of the most convenient sources, given the wide availability, high cellular yield, and obtainability. Recently, the falciform ligament (FL) has been regarded as a potential depot for adipose tissue-derived stromal cells (FL-ADSCs) isolation. Nonetheless, the osteogenic capability of FL-ADSCs has not been previously characterized. Thus, the present study aimed the detailed characterization of FL-ADSCs' functionality upon osteogenic induction through a classic (dexamethasone-based-DEX) or an innovative strategy with retinoic acid (RA) in a comparative approach with ADSCs from a control visceral region. Cultures were characterized for cell proliferation, metabolic activity, cellular morphology, fluorescent cytoskeletal and mitochondrial organization, and osteogenic activity-gene expression analysis and cytochemical staining. FL-derived populations expressed significantly higher levels of osteogenic genes and cytochemical markers, particularly with DEX induction, as compared to control ADSCs that were more responsive to RA. FL-ADSCs were identified as a potential source for bone regenerative applications, given the heightened osteogenic functionality. Furthermore, data highlighted the importance of the selection of the most adequate osteogenic-inducing program concerning the specificities of the basal cell population.

Keywords: adipose tissue; falciform ligament; mesenchymal stromal cells; osteogenesis; retinoic acid.

Figure 1

Histological analysis of adipose tissue samples harvested from the control and falciform ligament depots. (A). Illustrative images of adipose tissue stained with hematoxylin–eosin (scale bar: 100 µm; magnification 10×). (B). Histogram of the mean adipocyte area in the deposits of the control and the falciform ligament (n = 30).

Figure 2

Cell proliferation (A) and metabolic activity (B) of ADSCs grown from the control and falciform ligament depots. * Significantly different from basal (p < 0.05). # Significantly different between the control and the falciform ligament (p < 0.05). Dexamethasone, β-glycerophosphate, and ascorbic acid-supplemented media (DEX), retinoic acid-supplemented media (RA).

Figure 3

Representative images of ADSCs grown from the control and the falciform ligament (4 days) regarding cellular morphology (A) and mitochondrial morphology and distribution (B). Mitochondria = red; filaments of F-actin = green; and nucleus = blue. The scale bar corresponds to 100 μm. Cell area (C) and cell skewness (D). * Significantly different from basal (p < 0.05). # Significantly different between the control and the falciform ligament (p < 0.05).

Figure 4

Analysis of the gene expression, by RT-qPCR, of osteogenic differentiation markers such as SOX9 (A), RUNX2 (B), COL1A1 (C), SP7 (D) and BGLAP (E), in ADSCs of the control and falciform ligament after 7 days of subculture. * Significantly different from basal (p < 0.05). # Significantly different between the control and the falciform ligament (p < 0.05).

Figure 5

(A). Illustrative images of ADSCs cultures established from the control and falciform ligament, stained for the presence of collagen. Scale bar: 100 μm; magnification 2.5× and 5×. (B). Histogram of the mean staining intensity on the control- and falciform ligament-derived cultures (n = 10). * Significantly different from basal (p < 0.05). # Significantly different between the control and the falciform ligament (p < 0.05).

Figure 6

(A). Illustrative images of ADSCs cultures established from the control and falciform ligament, stained for the activity of ALP. Scale 100 μm. Magnification 2.5× and 5×. (B). Histogram of the mean staining intensity of the control- and falciform ligament-derived cultures (n = 10). * Significantly different from basal (p < 0.05). # Significantly different between the control and the falciform ligament (p < 0.05).