Judging from the positive staining for the endothelial markers, the combination of high concentration of VEGF together with fibronectin substrate produced a reliable EPC phenotype in PBMCs which was maintained longer than under the gelatin culture condition. However, the low expression of CD31 indicates that complete maturation could not be achieved during the 3-week exposure to both differentiating media. Differently from PBMCs, more than 90% of BMMCs could bind BS-I and express VEGFR-2 up to 3 weeks under the gelatin culture condition. Thus, BMMCs are more prone than PBMCs to maintain over time an EPC phenotype when exposed to the gelatin-based differentiating conditions.
Cell proliferation was markedly affected by both the biological sources of mononuclear cells and the culture conditions. PBMCs showed a very low attitude to proliferate, although they reacted better to the gelatin medium. Only under this latter condition was it possible to obtain PBMC confluence in 30% of culture dishes, but only after 20 days since cell seeding. However, confluent cells were not able to proliferate any more. In contrast, more than 60% of BMMCs grown in the fibronectin medium could rapidly expand and reach confluence even at the second passage within 3 weeks. This finding is in agreement with the report of Mertsching et al. [27
] who expanded pig BMMCs under the same culture conditions. We obtained the best proliferation performance with BMMCs exposed to the gelatin-based formulation, since in 100% dishes it was possible to re-split cells and increase cell number over 50% after 1 week from each re-seeding. Although proliferation was better induced by gelatin rather than fibronectin culture condition, the latter composition allowed PBMCs to be more viable up to the first confluence. A possible explanation may be the efficacy by which cells adhere to the culture dish, since a high number of cell contacts stimulate survival pathways in most cells (anoikis) [28
]. Fibronectin is known, indeed, to bind cells through a wide pattern of integrins and other receptor-like structures, hence affecting endothelial cell viability and functions [29
]. Moreover, VEGF was present in the fibronectin medium at a high level, thus improving cell survival [32
In contrast, BMMC viability was positively affected by the gelatin medium based on their more elevated rate of cell proliferation. This was especially evident during the second passage when the exponential growth of BMMCs was more pronounced. To better understand which was the relative involvement of the culture components on the behaviour of BMMCs, we studied a third condition in which cells were grown on fibronectin-coated dishes in the presence of EGM-2 solution. The viability of BMMCs was similar to those obtained with the gelatin-based formulation, suggesting that these two processes were stimulated by the higher amount of IGF-1 and FGF-2, together with EGF, since they synergistically increase cell growth and survival [33
]. The unattached BMMCs which remained suspended in the medium during the first 3 days of culture were also able to subsequently adhere to the coated dishes and expand; thus, we suggest that the amount of BMMC-derived EPCs can be further increased by simultaneously growing both early-suspended and adherent cells under the same culture conditions.
As a consequence of these preliminary results, we decided not to perform further investigations on PBMCs-derived EPCs because they showed a poor proliferative potential. We did not try to mobilize EPCs from bone marrow, i.e. by means of the granulocyte colony-stimulating factor (G-CSF), because the yield of these cells in peripheral blood is, in any case, lower than that registered by using bone marrow itself [13
Among the natural polymers which are commonly used as suitable scaffolds for regenerative medicine, the hyaluronan-based materials show interesting features because they are biocompatible, biodegradable, and can stimulate biological activities through hyaluronan-related cell receptors [36
]. Several approaches of cell therapy need rapid vascularization during the regeneration of the damaged tissue; in this case, hyaluronan plays a beneficial role not only as a cell vehicle but also because it can activate neoangiogenesis through the slow release of its naturally degraded fragments [37
The percentage of the BMMC-derived EPCs which adhered to the scaffold was higher than 90%, irrespective of the differentiating medium, and even using second-passage cells. This latter finding suggests that we can expand post-confluent cells and also obtain an almost complete adhesion of these EPCs to the scaffold. In a previous report, we suggested the transplanting in vivo of HYAFF®
11 engineered with human EPCs after just a few days from cell seeding in order to obtain the best effects on neovascularization [23
]; therefore, we investigated the viability of treated BMMCs within 3 days of culture on the scaffold. As for cultures on coated dishes, the best viability was observed by pre-treating BMMCs under gelatin or intermediate culture condition. Also under these conditions, BMMC growth on a hyaluronan-base polymer was attributable to the positive effects elicited by IGF-1, FGF-2, and EGF.
The ability of BMMCs to generate a tubule-like network was evaluated using Matrigel as a standard substrate. The pre-treatment of BMMCs with the fibronectin medium allowed the formation of three-dimensional rings which were without gaps, like those derived from HUVECs. Under this condition, BMMCs were also able to plug into the HUVEC network, extending thin connections formed by single cell layers such as during the in vivo capillary formation. Therefore, BMMCs treated with the fibronectin medium showed both angiogenic- and vasculogenic-like properties in vitro. In contrast, the gelatin medium was not as efficient as the fibronectin one to prime BMMCs alone for tubule-like formation.
Taken together, these results suggest that a leading protocol to obtain a suitable number of specialized EPCs for neovascularization could be to expand BMMCs with the gelatin-based formulation and to expose them to the fibronectin medium to optimize their ability to produce a reliable capillary network. An alternative strategy was the one adopted by Serrano et al. [38
], which was characterized by exposing pig PMBCs to EGM-2 for 2 weeks and then depriving the medium of FGF-2 with the purpose of enhancing endothelial differentiation in this second phase of culture.
The study of both function and differentiation of MSCs gave further useful information. First of all, MSCs alone and in the absence of any pre-treatment with endothelial differentiating media could form initial and rough tubule-like structures in Matrigel. As a matter of fact, the test we used to evaluate tubule-like formation on Matrigel is not exclusive for capillary structures, but allows the circular joining of other kinds of cells, such as fibroblasts [39
]. Since native MSCs did not show a pattern of endothelial markers, it is likely that their attempt to create a cell network was mainly referred to their mesenchymal phenotype. MSCs, together with EPCs, indeed represent an excellent mixed cell population that has been described as originating capillaries with pericytes [14
] and vessels of middle/large size provided with a complete vascular wall [40
One main limitation of the present work is the lack of investigation about pig EPCs obtained by selecting an antigen expressed by very early immature precursor cells, i.e. CD34 and CD133, or by the expansion of endothelial cell-forming colony units. However, both these protocols yield few EPCs in a short time, thereby reducing their availability for precocious intervention of cardiac regeneration. Nevertheless, these cells can also be greatly expanded, but the time available to obtain a suitable number of functional EPCs for clinical applications is about 2 months. Another limit of this research is the absence of studies related to EPC migration and EPC release of angiogenic factors. The assay of in vitro tubule-like structure formation that was performed in the present work should also include these EPC activities, although caution is needed to directly correlate this morphological assay with endothelial functionality in vivo [41
In conclusion, pig BMMCs are preferentially expanded rather than differentiated to endothelial cells using high concentrations of IGF-1 and FGF-2, in the presence of EGF and a low level of VEGF. In contrast, fibronectin-coated dishes supplemented with an elevated amount of VEGF and lower concentrations of IGF-1 and FGF-2 can be considered a good formulation to produce capillary-like structures by BMMCs. Therefore, these two different protocols could be followed and integrated to prepare a large number of specialized EPCs as a useful strategy to modulate neovascularization.