Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery

Abstract

Introduction: Adipose-derived stem cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Despite subcutaneous adipose tissue being more abundant, the buccal fat pad (BFP) is easily accessible for dentists and maxillofacial surgeons. For this reason, considering the need for preclinical study and the swine as an optimal animal model in tissue engineering applications, we compared the features of porcine ASCs (pASCs) from both tissue-harvesting sites.

Methods: ASCs were isolated from interscapular subcutaneous adipose tissue (ScI) and buccal fat pads of six swine. Cells were characterized for their stemness and multipotent features. Moreover, their osteogenic ability when cultured on titanium disks and silicon carbide-plasma-enhanced chemical vapor-deposition fragments, and their growth in the presence of autologous and heterologous serum were also assessed.

Results: Independent of the harvesting site, no differences in proliferation, viability, and clonogenicity were observed among all the pASC populations. Furthermore, when induced toward osteogenic differentiation, both ScI- and BFP-pASCs showed an increase of collagen and calcified extracellular matrix (ECM) production, alkaline phosphatase activity, and osteonectin expression, indicating their ability to differentiate toward osteoblast-like cells. In addition, they differentiated toward adipocyte-like cells, and chondrogenic induced pASCs were able to increase glycosaminoglycans (GAGs) production over time. When cells were osteoinduced on synthetic biomaterials, they significantly increased the amount of calcified ECM compared with control cells; moreover, titanium showed the osteoinductive effect on pASCs, also without chemical stimuli. Finally, these cells grew nicely in 10% FBS, and no benefits were produced by substitution with swine serum.

Conclusions: Swine buccal fat pad contains progenitor cells with mesenchymal features, and they also osteo-differentiate nicely in association with synthetic supports. We suggest that porcine BFP-ASCs may be applied in preclinical studies of periodontal and bone-defect regeneration.

Figure 1

Localization of subcutaneous interscapular and buccal fat pad tissue withdrawal. Anatomic regions of subcutaneous interscapular adipose tissue and buccal fat pad (A). Surgical procedure for tissue collection (B).

Figure 2

Stemness features of ScI- and BFP-pASCs. Cell proliferation expressed as doubling time (DT, hours) of ASCs from II to IV passage (A). Viability assessed by MTT assay at days 1, 3, and 7 (B). Morphology of ScI-pASCs and BFP-pASCs (optical microscopy, 200× magnification; scale bar, 100 μm) (C). Clonogenicity from passage I to IV expressed as colony-forming units (CFU-F) percentage (ratio of number of colonies/number of plated cells × 100) (D, upper panel). Data are expressed as mean ± SEM (n = 6). Representative ScI-pASCs and BFP-pASCs plates stained with crystal violet (D, lower panel).

Figure 3

FACS analysis of ScI- and BFP-pASCs. Expression of specific mesenchymal stem cell markers in ScI- and BFP-pASC populations (n = 2). Size and granularity are shown (upper panels). pASCs stained for CD90 and CD271 are reported (lower panels).

Figure 4

Osteogenic potential of pASCs. Quantification of collagen (A), calcified extracellular matrix (ECM) deposition (C), alkaline phosphatase (ALP) activity (E) in undifferentiated (CTRL, white bars), and osteo-differentiated (OSTEO, dark bars) ScI- and BFP-pASCs; data are expressed as mean ± SEM (n = 12). OSTEO versus CTRL *P < 0.05; **P < 0.01; ***P < 0.001. Images of ScI- and BFP-pASC wells stained with Sirius Red (B, left panel) and Alizarin Red-S (D, left panel) and representative microphotographs of ScI-pASCs (B, D, right panel, optical microscopy, 40× magnification; scale bar, 200 μm). Osteonectin expression of ScI-pASCs and BFP-pASCs analyzed with Western blot; its quantification, normalized to β-actin, is also indicated (F).

Figure 5

Adipogenic and chondrogenic potential of pASCs. Microphotographs of BFP-pASCs and ScI-pASCs maintained for 14 days in control (CTRL) and adipogenic medium (ADIPO; 200× magnification; scale bar, 50 μm), both during culture (A, upper panels) and after lipid vacuoles staining by Oil Red O (B, middle panels). Quantification of lipid vacuoles formation by Oil Red O extraction is shown in A, lower panel. Quantification of glycosaminoglycans (GAGs) production normalized on DNA content in CHONDRO-pASCs after 7, 14, and 21 days of differentiation in pellet culture (B).

Figure 6

Influence of biomaterials on pASC osteogenic differentiation. Calcified ECM deposition in undifferentiated (CTRL, white bars) and osteogenic-differentiated (OSTEO, dark bars) ScI- and BFP-pASCs, cultured for 21 days on monolayer (plastic adherent, PA), or seeded on titanium disks (TIT) or on silicon carbide–plasma-enhanced chemical vapor deposition (SIC) fragments (A). Protein concentration of pASCs cultured either in monolayer or adhering to biomaterial both in CTRL (white row) and OSTEO (dark row) is reported in panel (B). Data are expressed as mean ± SEM (n = 3). OSTEO versus CTRL: **P < 0.01; TIT versus PA; ^§P < 0.05; ^§§P < 0.01; ^§§§P < 0.001.

Figure 7

pASCs cultured in media supplemented with porcine sera. pASCs were grown for 7, 14, and 21 days, in DMEM supplemented with 10% FBS or 5% autologous or heterologous serum. Data are expressed as mean ± SEM (n = 4). Microphotographs of ScI-pASCs in culture for 21 days (lower panel, optical microscopy, 100× magnification, scale bar 50 μm). AS, autologous serum; FBS, fetal bovine serum; HS, heterologous serum.