Histomorphometric analysis of the density of OEC-derived human vessels within the implantation bed was performed from a total scan of a histological slide of the implantation bed that was stained with an antibody to human-specific CD31 (Figure 2). In total, n = 2 animals/donor/time point were used. At day 2 after implantation, there were already 2.7 ( ± 0.6) and 4.0 ( ± 0.7) OEC-derived vessels/mm2 for donors 1 and 2, respectively. This number continued to increase, and by day 14 there were 11.4 ( ± 1.8) and 17.8 ( ± 0.8) OEC-derived vessels/mm2, for OECs from each of the two donors. Regardless of donor, the trend was for an increase in OEC-derived vascular structures over the implantation time. Quantification of vascularization arising from murine endothelial cells demonstrated similar trends (Figure 2). By day 14 of implantation, there were a total of 13.8 ( ± 3.8) and 11.9 ( ± 1.9) murine vessels/mm2 for constructs from each of the two OEC donors, respectively. The rate of vascularization and number of vascular structures was similar for both human- and murine-derived vessels. By the end of the study, vascularization arising from OECs accounted for 44.9% and 60.0% of total scaffold vascularization for donors 1 and 2, respectively. This indicates a substantial functional contribution of the precultured OECs towards overall scaffold vascularization.
The potential of human outgrowth endothelial cells to contribute to vascularization in complex tissue-engineering strategies has been established by our group and others (Fuchs et al., 2006a, 2009a). Additionally, we have demonstrated potential for mature endothelial cells derived from human dermal tissue to act in a similar capacity (Unger et al., 2004, 2005, 2007). Observations with both OECs and other primary endothelial cells, originally demonstrated in vitro, have also been verified in vivo (Fuchs et al., 2009c; Unger et al., 2010). In one such study, we found co-cultures of OEC and pOB on SPCL scaffolds to have a high potency for the formation of human-specific vascular structures when transplanted in vivo (Fuchs et al., 2009c). This study, which focused on a single histological endpoint, indicated both the formation of OEC-derived vessels and a contribution to this phenomenon from the osteoblast component, acting as a natural source for potent angiogenic signalling within the co-culture. However, further mechanisms regarding the time course of the OEC-derived vessel formation in vivo and the further potential roles of osteoblasts were not completely elucidated. In the present study, we have examined the formation of these OEC-derived vessels in a more dynamic approach, with several histological time points and total implant histomorphometric analysis at each time point, in order to determine the time course of OEC-derived vessel formation along with the dynamics of total scaffold vascularization by both OEC- and murine-derived vessels. Additionally, we have used vimentin human-specific staining, which was not performed in our previous study, coupled with CD 31 staining to specifically detect pOBs within the co-culture, in order to determine the specific location and potential contribution of these cells to OEC-derived vessel formation.
In this investigation, along with previous studies by our group (Fuchs et al., 2007), it is apparent that even prior to implantation, the OEC component was primed for the formation of vascular structures. The bud- or sprout-like structures seen in the histology of these scaffolds, following in vitro co-culture but prior to implantation, are reminiscent of the first stages of angiogenesis, where endothelial cells begin to reorganize and form sprouts that eventually lead to new vessels (Carmeliet, 2005; Folkman, 2003). Following 2 days of implantation, there was evidence of OEC-specific vessels with apparent lumena. The presence of erythrocytes within these lumena indicates that some human-derived vascular structures were already anastomosed to the host blood supply and were functionally contributing to vascularization of the implantation bed after only 48 h in vivo. These findings indicate the potential of OECs to form microvessel-like structures during in vitro cultivation that enable rapid anastomoses upon implantation, suggesting this as a potential ‘natural’ anastomosis technique for tissue engineering. Histomorphometric quantification of human-specific vessels demonstrated that the number of perfused human-derived vascular structures increased approximately four-fold from day 2 to day 14 of implantation. Although there appeared to be some differences in cell potency between the two OEC donor sources, both donors demonstrated an increase in human-derived vascularization of the scaffold over the evaluation period. One of the main differences between the two donors (Figure 2) is that in one the principal acceleration of vessel growth took place between days 2 and 5, whereas in the other donor this took place later, that is, between days 5 and 8. Moreover, the OEC-derived vessels made a substantial contribution to overall scaffold vascularization, as the number of vessels arising from OECs was similar to the number arising from the host. This is further evidence of the potency of this cell population to contribute to scaffold vascularization. The present study demonstrates the potential of preseeded OECs in cell-based tissue engineering strategies as a supplemental vascularization source, especially when host-mediated vascularization proves insufficient by itself, to fully integrate a biomaterial within the implantation bed in an appropriate time frame and with a sufficient vascular density.
Another finding in this study could help elucidate the contribution of co-cultured osteoblasts to OEC-derived vessel formation. Osteoblasts are known to produce a number of signals, including paracrine factors such as VEGF and structural components such as collagen I, which exert a pro-angiogenic effect on the co-cultured endothelial cells and enhance the formation of vessel-like structures (Fuchs et al., 2009c; Unger et al., 2007). Our histological evidence from serial sections demonstrates co-localization of osteoblasts and OECs, with the osteoblasts forming a surrounding layer on the OEC-derived microvessels. These findings suggest that the contribution of osteoblasts goes beyond only a paracrine effect, and that the cell body of the osteoblasts may serve a structural role in the formation of these new vessels. Thus, osteoblasts may act as pericyte-like cells within these co-cultures by contributing structural and paracrine support to stabilize the endothelial-derived vascular structures. Such a role may be similar to that of an adventitial layer in normal vessel physiology, where mural cells, including fibroblasts, smooth muscle cells and other pericytes, provide structural support to vessels and enhance angiogenesis (Gerhardt and Betscholtz, 2003; Shepro and Morel, 1993). A connection between osteoblasts and pericytes is not without precedent. However, in this respect most literature findings indicate the potential of pericytes as pluripotent progenitors that can undergo osteogenic differentiation (Brighton et al., 1992; Doherty et al., 1998; Reilly et al., 1998). To our knowledge, there is not a significant body of literature that suggests the potential of osteoblasts to act in a pericyte-like role. However, given the demonstrated similarities in phenotypic expression (Reilly et al., 1998), perhaps osteoblasts do have some capability to serve in this pericyte-like role. This phenomenon may arise specifically in co-cultures, where no other cell population exists to support the endothelial cells in the vessel formation process. Therefore, in co-cultures osteoblasts might be committed to this role as the default cell source for cell-based structural support of vessels. Nevertheless, the findings of this study, along with previous work, point to a possible synergy between the pOBs and OECs, in which pOBs may be able to provide both the chemical cues and structural support needed for OEC to rapidly form vessels.
The finding that OECs rapidly vascularize SPCL scaffolds in co-culture with pOB after only 2 days of implantation in vivo is promising for complex tissue engineering strategies, in which a rapid scaffold vascularization is desired. The rapid formation of anastomoses with the host vasculature after only 48 h is quite exciting and points to the potency of this cell source. The number of OEC-derived vessels increased over the implantation time with a density similar that of host-derived vessels. Furthermore, evidence is also provided for the multifunctional role of pOBs in the co-culture, as a structural pericyte-like support for the OEC-derived vessels was observed histologically. These findings, together with previous in vitro and in vivo studies by our group, demonstrate the potential of cellular crosstalk in co-cultures of endothelial cells and osteoblasts to provide an effective cell-based approach for tissue engineering.
The goal of our study was to establish the time scale over which human-specific vessels arise from co-cultures of osteoblasts and endothelial cells in vivo. Additionally, this study further focused on the potential roles osteoblasts may play in the formation of OEC-derived microvessels. Our findings demonstrated that human-specific vascular structures begin to form during in vitro precultivation and that these structures anastomosed with host vasculature within 48 h of implantation, as there was evidence of human-derived vessels visibly perfused with erythrocytes at this early time point. The number of human cell-derived vessels increased with time, irrespective of donor source, although the source seemed to result in different cell potency. An additional exciting finding was made histologically. While a pro-angiogenic paracrine contribution from osteoblasts has been previously established, we have demonstrated here the potential of these cells to serve additionally as structural vessel wall components, similar to that of pericytes in normal vascular physiology. The findings of this study further demonstrate the synergistic and multifunctional role of osteoblasts in co-culture with endothelial cells as an exciting strategy for cell-based complex tissue engineering.