Since the discovery of stem cells, scientists have been devoted their efforts to the replacement of damaged breasts using tissue engineering and regenerative medicine. The adoption of stem cells to replenish the desired breast tissue could eliminate the long-term complications of breast reconstruction, including capsular contracture or donor site morbidity. The benefits of hUCMSCs such as low immunogenicity, non-invasive access, large-scale production, and multilineage differentiation, implicate them as a promising better choice for breast reconstruction. In this study, we found that hUCMSCs could promote vascularization via the integrin β1/ERK1/2/HIF-1α/VEGF-A signal pathway in endothelial cells. hUCMSCs could also directly differentiate into adipocytes and endothelial cells in fat grafts. We adopted the hUCMSCs as seed cells to reconstruct breast with tissue engineering strategy successfully.
In the preclinical studies of various ischemic diseases, hUCMSCs have promoted blood perfusion in ischemic tissues[27; 28]. However, the effects of hUCMSCs on the characteristics of endothelial cells in vitro are unknown. Our results demonstrated that hUCMSC-CM potentially promoted the migration, proliferation, and tubular formation of HUVECs. There was no direct contact between the two cell types in the coculture system, therefore, hUCMSCs may promote vascularization via the paracrine effect.
Furthermore, we attempted to elucidate one of the essential signaling pathways in HUVECs activated by the paracrine secretion of hUCMSCs. Paracrine factors of hUCMSCs have been revealed to promote angiogenesis, such as VEGF-A, bFGF, Ang-1, aFGF, PDGF, etc[29]. In particular, VEGF-A is the most important of these members as it plays a vital function in both vasculogenesis (de novo vasculature formation) and angiogenesis (vessel formation sprouting from existing vasculature)[30]. VEGF-A ensures the migration of endothelial cells, promotes vessel branching and is indispensable for recruitment, retention, proliferation, migration, and differentiation of endothelial progenitor cells[31]. Besides, VEGF-A expression is intensively influenced by HIF-1α, a transcriptional activator which is unstable under normal condition due to the degradation pathway of the ubiquitin-proteasome pathway[32]. Our results demonstrated that hUCMSC-CM could promote migration, tubule formation and VEGF-A secretion in HUVECs, and which could be suppressed by HIF-1α inhibition.
Integrins, a class of transmembrane proteins consisting of α and β subunits, are greatly involved in cell adhesion, differentiation, growth, migration, and angiogenesis via transmitting extracellular signals across the membrane[33]. Our results showed that the expression of HIF-1α in HUVECs was suppressed followed inhibition of integrin β1. Thus, hUCMSC-CM were suggested to stimulate integrin β1 and then stabilize HIF-1α mediated VEGF-A expression in HUVECs. ERK1/2 and AKT are common downstream effectors of integrin, and they also participate HIF-1α regulation. Our results demonstrated hUCMSC-CM induced phosphorylation of ERK1/2 and this could be suppressed by inhibition of integrin β1. Meanwhile, hUCMSC-CM have no affection on AKT activation. Therefore, hUCMSCs potentially promote angiogenesis via paracrine-induced activation of the integrin β1/ ERK1/2/HIF-1α/ VEGF-A signaling pathway in endothelial cells.
Moreover, we evaluated the roles of hUCMSCs injection in fat graft without breast scaffold. The volume retention rates of fat grafts in the Low hUCMSC group and the Medium hUCMSC group significantly improved after 12 weeks of implantation. Of note, the number of hUCMSCs exhibited no potential positive correlation with the volume retention rate of lipotransfer, whereas fat grafts in the Low hUCMSC group owned the highest volume retention rate. Through HE staining and quantitative analysis, we demonstrated that the Low hUCMSC group holds the lowest cavitary necrosis area and fibrosis degree, but the highest fat tissue occupation and the best adipocytes integrity. The proportion of adipocytes within the diameter of mature adipocytes was 86%, and this was the highest among the four groups. Collectively, the adipose tissue in the Low hUCMSC group remained more intact than other groups.
We employed immunofluorescence staining and cell tracing methods to focus on the histological effects and mechanisms of MSC implantation. The immunofluorescence staining of Perilipin confirmed that large areas of complete active adipose tissue of fat grafts in the Low hUCMSC group were preserved, which was consistent with HE staining results mentioned above. Blood vessels were observed through immunofluorescence staining of CD31. Quantitative analysis revealed significantly higher proportions of the red fluorescence (CD31+) areas in all three hUCMSC groups compared to that of the Control group. hUCMSCs could, therefore, effectively promote the vascularization and the retention of fat grafts. However, no significant difference in blood vessel areas was found between the three hUCMSC groups. The possible reason might be related to the local inflammation environment following transplantation of different amounts of hUCMSCs. We proceeded with the immunohistochemical staining of macrophages. and revealed that the numbers of M1 macrophages in the Low hUCMSC group and High hUCMSC group were significantly lower than the Control group. Besides, the number of M1 macrophages in the Low hUCMSC group was the least among four groups. The number of M1 macrophages in the High hUCMSC group and the Control group showed no statistical difference but were significantly higher than that in the Low hUCMSC group. For M2 macrophages, we reported no significant statistical difference between all four groups. M1 macrophages express proinଂammatory cytokines and are characterized by long-term inflammation, therefore, they could cause tissue damage. Contrarily, M2 macrophages potentially promote tissue remodeling and exert anti-inflammatory and immunoregulatory effects[34]. The degree of fibrosis and calcification of adipose tissue is also related to M2 macrophages[35]. Therefore, a lower amount of hUCMSCs can reduce the number of M1 macrophages to inhibit the inflammatory response, this consequently would reduce the loss of blood vessels and adipocytes.
In addition, we examined the differentiation of hUCMSCs in vivo[36]. Our results showed that numerous hUCMSCs survived in the grafts after 12 weeks of implantation and some positively expressed Perilipin or CD31. The findings confirmed that hUCMSCs could differentiate into adipocytes and vascular endothelial cells in vivo. However, no typical adipocyte morphology or complete vascular ring structure of these hUCMSCs were observed. Therefore, it was concluded that after 12 weeks of implantation, these hUCMSCs might be in the differentiation stage. Whether the differentiated cells exerted the corresponding functions and the stage at which they could change their morphologies into mature cells warrants further exploration.
The present study also explored the effects of hUCMSCs transplantation in breast reconstruction using the combination of lipotransfer and 3D-printed breast scaffolds. TPU materials and 3D printing technology were selected to construct diamond-shaped (N5S4) breast scaffolds. Our previous study demonstrated that the scaffolds with N5S4 architecture that mimic the lattice microstructure of diamond are soft and resilient enough for breast reconstruction[24]. Since the ratio of 0.5×106 hUCMSCs to 0.5ml fat achieves the highest volume retention rate of the graft among all three concentrations and the diameter of the breast scaffold is 1cm depending on the size of the nude mice, we injected 0.2ml of fat. The number of hUCMSCs injected was 0.2×106 per scaffold. After 4 weeks of implantation, normal breast shape was maintained in the hUCMSC group, while peripheral scaffolds in the Control group were exposed, as such, they lost the normal breast shape. The measure of the weights and volumes of these implants also demonstrated more integrity with the injection of hUCMSCs. Furthermore, HE staining revealed that the proportion of adipose tissue in the scaffolds of the hUCMSC group was significantly higher than that of the Control group, and the degree of fibrosis was significantly reduced. hUCMSCs were suggested to potentially improve the volume retention rate of adipose tissue in the scaffold and inhibit fibrosis. We also noted that adipose tissue in the scaffold reduced from 4 weeks to 12 weeks of transplantation. The immunofluorescence staining of CD31+ revealed that the proportion of green fluorescence (CD31+) was reduced to 22.12% of the 4-week-value in the hUCMSC group after 12 weeks of implantation. Most of the neovascularization disappeared and at this time point, no significant difference was reported between the hUCMSC group and the Control group. We concluded that hUCMSCs could only promote the rapid neogenesis of blood vessels in the early period of implantation, but stable and mature blood vessels could not be maintained in the long term, thereby reducing the adipose tissue. In addition, surviving fat grafts were associated with exclusively donor-derived vasculature. As the donor-derived microvasculature decreased the graft perfusion at an early stage, large proportions of adipose tissue were lost[37]. Hence, the function of hUCMSCs in promoting rapid vascularization at an early stage is vital to the retention of fat tissue. Also, the number of CD68+ macrophages associated with the severe degree of inflammatory response[38] in the tissue engineered breast was significantly reduced after hUCMSCs transplantation. This demonstrated that hUCMSCs could significantly reduce the number of CD68+ macrophages in the compound and impeded inflammatory responses, which consequently reduced the damage of adipose tissue. Therefore, to improve the effect of breast reconstruction, hUCMSCs potentially accelerates early vascularization and reduces the inflammatory response.
The clinical therapeutic advantages of stem cells have become increasingly apparent and significant breakthroughs have been made in the management of refractory diseases and tissue engineering[39]. Nevertheless, many stem cells have some limiting factors for their clinical applications, including ethical issues and invasive procedures. HUCMSCs have abundant resources and hold non-invasive extraction procedure[40]. Currently, large-scale production and storage of hUCMSCs are achievable, and hUCMSCs suspension products have been adopted in clinical trials[41; 42]. Importantly, hUCMSCs maintain an earlier embryologic phase, which is much younger and can secrete a wide range of multifunctional factors[29]. Studies have also demonstrated that hUCMSCs are promising in tissue engineering and cell-based therapy. It was once thought that the function of MSCs in the damaged tissue was cell replacement for resident cells, it is now widely confirmed that the more immediate principal mechanism of action of MSCs in vivo is via paracrine[40]. The critical parameters in their ability to modify the function of host cells and tissues are the secretion of multifunctional factors and extracellular vesicles[43]. We have confirmed in the present study that, hUCMSCs can promote vascularization in vitro via paracrine, which potentially activates the vital signal pathway of integrin β1/ERK1/2/HIF-1α/VEGF-A in endothelial cells. HUCMSCs could also differentiate into adipocytes and vascular endothelial cells in fat tissue, and exert immunomodulatory effects by inhibiting the infiltration of M1 macrophages.