This study evaluated the potential for ECs in combination with MSCs to create microvascular networks in three-dimensional bone tissue engineered constructs. To assess how coculture of MSCs with ECs influenced the phenotype of cells delivered for tissue regeneration in vivo, we studied angiogenic and osteogenic gene-expression profiles after 1 week of dynamic culture in vitro. The results show that formation of an endothelial microvascular network resulted in upregulated expression of human ki67, a well-described biomarker for cellular proliferation . Various genes involved in cell growth and differentiation, were upregulated in MSC/EC constructs, and the influence of ECs on both angiogenic and osteogenic gene-expression profiles was evident. The results of the in vivo experiment showed that the response to the angiogenic signal on ingrowth of CD31-positive cells was similar for both experimental groups, but that implanted human MSCs could support functional blood vessels as perivascular cells. Histologic evaluation showed that generation of ectopic bone in poly(LLA-co-DXO) scaffolds required a local osteoinductive environment. However, increased osteogenic potential was found for both cellular constructs compared with empty controls, and expression of ALP was significantly upregulated in the presence of endothelial microvascular networks.
The potential use of biodegradable polymers as scaffolds for cell-based tissue regeneration was reviewed by Gunatillake and Adhikari . An important attribute of these materials is their chemical versatility, allowing mechanical properties and material degradation to be tailored to specific clinical conditions. Hence these materials are potentially applicable to regeneration of various types of tissue. The response of both OBs and MSCs to the scaffold used in this report has been investigated in several in vitro studies [13, 15, 19]. The results from these studies suggest that the material enhances osteogenic differentiation of both cell types. Our results show that the osteogenic stimulatory effects of the scaffold material and the cellular interactions are not sufficient to induce ectopic bone formation in NOD/SCID-mice. However, several other authors have used polymer scaffolds to deliver osteogenic growth factors and then induce ectopic bone formation [20, 21]. MSC/polymer constructs, cultured with or without osteogenic stimulatory conditions before implantation, have also been investigated for their osteogenic potential, with successful generation of ectopic bone [8, 22].
Complex tissues depend on functional blood vessels for cell survival as well as for tissue organization after in vivo implantation. Prevascularization, in which endothelial cell microvascular networks are developed in vitro, have been attempted with different cells and materials. Microvascular networks were created by using MSC/EC coculture spheroids, in which limited functionality was found in vivo. Asakawa et al. were able to generate a three-dimensional tissue by using dermal fibroblasts as supporting cells for developing endothelial microvascular networks, whereas Yu et al. seeded ECs with OBs in poly-ϵ-caprolactone and hydroxyapatite scaffolds, and subsequently demonstrated enhanced osteogenesis in rat long-bone defects. Thus, for development of a de novo microvasculature, ECs depend on supporting cells, a role that can be undertaken by several cell types [26, 27]. Our results show that a poly(LLA-co-DXO) scaffold can support the formation of EC/MSC microvascular networks in three dimensions, suggesting that prevascularized tissue regeneration with this material is feasible.
In the present study, we cocultured cells for 1 week before in vivo implantation and found altered gene-expression profiles with reference to both angiogenesis and osteogenesis. Genes related to skeletal development and the ECM were highly expressed in monocultured MSCs. These results are in agreement with an earlier study by Fuchs et al., showing that the expression of osteogenic markers from OBs was downregulated in the presence of ECs in 4-week cultures. Long-term incubation in vitro is beneficial when studying the mineralization process, but might be considered less practical for clinical applications and have less potential for regenerating bone . Although an overview of osteogenic and angiogenic gene-expression profiles were generated in the present work, functional evaluations of candidate targets might have been of interest to assess further the effect from regulation of individual genes. Evaluations on tissue development were made on the gene, protein, and morphologic level, to determine the effect of the paracrine signal from all biologic factors delivered by the tissue-engineered constructs.
Murine models of ectopic bone formation are widely used to evaluate osteogenic potential in bone-tissue engineering. By using a subcutaneous mouse model to compare implantation of MSCs and OBs, Tortellini et al. reported that MSCs enhanced vascularization through increased recruitment of host ECs. MSCs are known to secrete multiple paracrine factors stimulating EC migration and wound healing , and this might be downregulated when ECs are already present in the construct. In the present study, angiogenic gene expression was higher in monocultured MSCs at the moment of implantation, suggesting that increased angiogenic stimulation could be delivered to the host bed from MSCs alone. With ECs already present, paracrine signals to attract and activate ECs might be less relevant in the coculture system.
An initial response from the host circulation was also found as higher expression of mouse VEGF at 1 week, when compared with cocultured constructs. The opposite regulation of human VEGF was observed, with upregulation in the coculture group at the end of the experimental period. These findings might be interpreted as cell/scaffold constructs containing ECs having a stronger ability to maintain a proangiogenic signal after implantation. However, the expression of mouse CD31 was not notably different for the two experimental groups, suggesting a similar vascular host response with regard to the presence of ECs. The total number of blood vessels was not quantified, but no obvious difference could be observed.
Recruitment of perivascular cells with subsequent production of basement membrane proteins are key events in the maturation of developing vasculature . The α-SMA originated from mouse cells was similarly expressed on the mRNA level in MSC and MSC/EC constructs, with an expected increase for both groups between 1 and 3 weeks, as scaffolds were increasingly penetrated with tissue. MSCs in cell/scaffold constructs responded to the presence of ECs by upregulating the expression of α-SMA at all time points, although the differences were not statistically significant. In vitro evaluation showed that α-SMA was expressed also in undifferentiated MSCs, whereas the results from the in vivo experiment showed that adding ECs resulted in further upregulation of α-SMA. However, the ability of MSCs to function as perivascular cells for developing vessels did not depend on the coimplantation of ECs. Gene expression of α-SMA was strongly downregulated for both constructs when compared with empty controls, suggesting less demand for transcription of α-SMA after implantation of MSCs. Furthermore, α-SMA-positive cells surrounding functional blood vessels were found well distributed in the connective tissue for both experimental groups. In addition to α-SMA, positive staining of human vimentin was demonstrated, showing that implanted MSCs were a viable part of the connective tissue.
Kaigler et al. studied dermal microvascular endothelial cells and MSCs seeded on poly(lactic co-glycolic acid) scaffolds implanted subcutaneously in immunodeficient mice. No significant difference was observed in the number of total blood vessels at 2 and 4 weeks, but in line with our findings, a higher percentage of human-derived vessels were found in MSC/EC constructs. At 4 weeks, the area of bone as a percentage of the total tissue area was as high as 35% in the MSC/EC group. In the present study, we cultured cells for 1 week before implantation in vivo for 3 weeks, but no osteoid formation was detectable within the constructs. We used MSCs at a lower passage, and this might have influenced the differentiation stage of cells at implantation and, subsequently, the osteogenic potential. In addition, the difference in density of cells with regenerative potential should be considered in the interpretation of the results. When evaluating the expression of osteogenic biomarkers, we found an upregulation of ALP for MSC/EC-constructs compared with both monocultured MSCs and empty control scaffolds. These results are in accordance with those of Xue et al. with a two-dimensional coculture MSC/EC model. Compared with monocultured MSCs, at 5 days, multiple osteogenic genes from the cocultured MSC/EC exhibited downregulation, with the exception of ALP . The proposed mechanism for endothelial influence on osteogenic differentiation was therefore that ECs promoted maintenance of MSCs at a proliferative stage rather than inducing terminal differentiation. Although full penetration of tissue within scaffolds was confirmed at the end of the experiment in the present work, longer observation periods might have been of interest to follow further the remodeling and maturation of the tissue.
Positive Alizarin Red staining was found in both cell/scaffold constructs, but not in scaffolds implanted without cells. The majority of the area within the material was filled with connective tissue, with interspersed calcified nodules. The total area of positive staining was not significantly different for the two experimental groups. Both MSCs and MSC/EC thus enhanced the osteogenic potential of poly(LLA-co-DXO) scaffolds after ectopic implantation.