Bone is a dynamic, highly vascularized tissue with the unique capacity to heal and to remodel without leaving a scar . Large bone defects, caused by trauma, tumor resection, or infections, require implants to restore the loss of function. Current treatments of large bone defects are based on autogene or allogene bone grafts and metal or ceramic implants that still have several limitations. Bone tissue engineering could provide an alternative to conventional treatments of fracture healing. However, this approach requires a matrix to allow progenitor cell delivery and support tissue invasion.
Bone defect repair is one of the major targets of tissue engineering research . Pre-seeding a biomaterial with tissue-specific cells prior to implant is one method that is being examined to speed up tissue regeneration . However, one of the major problems after the implantation of large biomaterials is the slow vascularization of the material .
Vascularization is a key process in tissue engineering and regeneration and represents one of the most important issues in the field of regenerative medicine. Thus, several strategies to improve vascularization are currently under clinical evaluation.
In fact, approximately 10% of fractures are slow or nonhealing  and may be characterized by poor vascularization and a resulting hypoxic local microenvironment . The absence of a sufficient vasculature impairs native healing processes by limiting the availability of nutrients to resident progenitor cells and infusion of inductive factors from the systemic circulation that promote healing. The consequences of a low-oxygen environment on cells are unclear but may include enhanced apoptosis, reduced proliferation, and inhibited differentiation; all of which ultimately impact tissue repair.
Cell transplantation is a promising alternative to the gold standard of autologous bone grafting to stimulate bone repair. Human DPSCs or AFSCs are of great interest in bone reconstruction, having demonstrated efficacy in both preclinical and clinical models [18, 19, 37, 38]. These stem cells can be easily isolated and expanded in culture without ethical problems. Autologous transplantation can be performed with both cell types. AFSCs can be used in neonatal surgery to treat congenital malformation. DPSCs, available from puberty onwards, can be used to heal pathologies occurring in adult life.
The objective of this study was to investigate whether the application of collagen sponge scaffolds, combined with expanded and osteogenic precultured human stem cells, had an effect on vascularization and the osteogenic potential of the scaffolds in vivo after implantation into parietal bone defect of immunodeficient rats. We discuss which combination of scaffold/cells shows the highest potential for further investigations. Adequate porosity and surface properties are recognized as important parameters in identifying scaffolds for tissue engineering. Not fully interconnected and irregularly shaped pores often lead to insufficient vascularization . Depending on the site of implantation, vascularization might be the critical step for successful tissue engineering.
The collagen sponge scaffold, currently used in several therapeutic practice, has been largely investigated in the literature – some authors have reported that it is not appropriate for successful new bone formation, because a mineral matrix, or a bone morphogenetic protein-expressing nonmineral matrix, or production of bone morphogenetic protein-2 by genetically engineered cells is also required . However, our results contradict these previous data, because the cell-free collagen sponge showed an excellent healing bone process, especially at the longer time point. Our data therefore support the hypothesis that collagen membranes are a good scaffold, facilitating cell adhesion and bone-forming cells in the defect site.
However, our data show that cell-seeded scaffolds perform a better and faster bone reconstruction capability than collagen alone. This improvement can be due to both the osteogenic and the vascular differentiation potential of DPSCs and AFSCs, even if AFSCs seems to show a better result. Histological staining of human mitochondria confirmed the presence of human stem cells after 8 weeks of implantation in the new bone tissue and also in vessels, creating the endothelium of the new vessel network in the scaffold. A recent study showed potential for AFSCs in bone regeneration and angiogenesis in bone defects , while a demonstration of the role of AFSCs in both the processes was lacking. Our data demonstrate that human cells, present in the graft after 2 months, are also expressing osteocalcin inside the new bone and von Willebrand factor around vessels, being responsible for osteo and vascular formations. This may be due to the choice of implanting AFSCs after 1 week of osteogenic differentiation in vitro. In this condition osteogenic markers are not yet increased, and therefore a subpopulation of undifferentiated AFSCs could generate vessels, responding to local environmental signals. Vascularization of cell-seeded implants can play an important role for cell survival and differentiation in the scaffold, because these cells need oxygen to form osteoblasts.
The AFSC and DPSC populations employed here are c-kit+ and ckit+-CD34+, respectively, being superficial markers expressed in hematopoietic and vascular-associated tissue .