We investigated the possibility of isolating porcine ASCs from Buccal Fat Pads (BFP-pASCs), which have similar stemness features to the ones isolated from subcutaneous tissue (ScI-pASCs), previously characterized
. Human BFP-ASCs might be quite easily applied in oral tissue engineering, because this tissue is rapidly accessible by dentists and maxillofacial surgeons
. However, before moving to the clinic, it is mandatory to perform approved preclinical studies to validate the safety and efficacy of cellular therapies. The most used large-animal model of human oral bone defects is swine
[31, 42], because these animals present a healing potential comparable to that of the human. Several studies have been conducted by using stem cells in oral diseases and orofacial research: Wilson et al.
 investigated bone regeneration in the pig mandible ramus by either local or systemic ASCs injection, concluding that both treatments accelerate the healing process, without any significant difference between the two routes of administration. In another study, similar results were obtained combining decidua stem cells with a β-TCP scaffold in a minipig model
Here we compared pASCs derived from two different body areas and evaluated their behavior in vitro to identify a convenient source for future preclinical studies. BFP-pASCs were very similar to ScI-pASCs. Although the cellular yield of the porcine ASCs was lower than the human one
, after 30 days in culture, we could have been able to obtain a homogeneous populations of about 108 to 109 cells, with still a pronounced clonogenic ability.
Both cell populations, analyzed at passage 4, were CD90+, CD271-, CD45-, and CD14-. These results are similar to the ones on porcine MSCs from different tissues
, and to our results on human mesenchymal stem cells from the Bichat fat pad that express CD90, CD73, and CD105
, as defined for human mesenchymal stromal cells
In conclusion, both cell populations were highly positive for CD90, one of the main MSC surface antigens, whereas no cross-reactivity has been observed for CD73 and CD105. Although limited, these results are consistent with the ones obtained with porcine MSCs from bone marrow
Furthermore, by a molecular approach of RT-PCR, we have preliminary data on the expression of Kruppel-like factor 4 (Klf-4), a marker of immature stem cells involved in the control of cell multipotency in many development-related processes and in the maintenance of stem cell-associated properties
. The mRNA expression levels in BFP-pASCs are comparable to the ones in human-ASCs. We consider this result interesting, because we recently showed that Klf-4 expression in hASCs seems to be related to the cell proliferation, clonogenic ability, and differentiative potential, and to be downregulated by the pathologic condition (obesity) of patients from which cells were isolated
Besides, all the porcine BFP-ASCs, grown in the presence of inductive stimuli, nicely increased both osteogenic and adipogenic features, as already described for subcutaneous porcine ASCs
[34, 49, 50]. At last, both populations are able to progressively depose GAGs during 3D culture when induced to chondro-differentiate. Altogether, these results suggest our claim that swine buccal fat pad contains progenitor cells of the mesenchymal stromal cell family, similar to the human ones.
Because these cells could be used in preclinical studies of tissue engineering, and their interaction with appropriate supports is essential, we evaluated the ability of both pASCs to grow and differentiate onto two synthetic scaffolds: the former, a widely used biomaterial in dental surgeries (titanium), and the latter, a promising candidate for the coating of some portions of implant (SiC-PECVD). Like human ASCs
, pASCs adhere and differentiate on both scaffolds. Moreover, the osteoinductive properties of titanium on hASCs
, were also observed on both porcine progenitor cells, whereas SIC-PECVD did not modulate their osteogenic differentiation.
Next, testing porcine autologous or heterologous sera, we detected that pASCs proliferated slower than cells cultured in the presence of FBS, and they dramatically stopped growing, changed morphology, and aggregated in clusters. These data are consistent with previous data by Schwarz et al.
, in which equine ASCs cultured with autologous serum proliferate less than with FBS. Differently, our results are in contrast with data obtained with human ASCs, in which it has been shown that the use of autologous serum favors or does not influence ASCs proliferation
[21, 51, 52]. Nevertheless, Kurita et al. showed that among four human ASC populations, only one proliferates faster when cultured with autologous serum. This discrepancy has also been observed for human bone marrow stem cells
[54–57], suggesting that other factors may influence cell growth. This issue requires further investigation to be clarified, although we have shown that both pASCs behaved similarly.