In Vitro Prevascularization of Self-Assembled Human Bone-Like Tissues and Preclinical Assessment Using a Rat Calvarial Bone Defect Model

Abstract

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Recent Advances on Cell-Based Co-Culture Strategies for Prevascularization in Tissue Engineering

“…Formation of new vessels can be increased by direct or indirect role of growth factors. VEGF and bFGF show direct effect on angiogenesis ( Choi et al, 2018 ; Kawecki et al, 2021 ). They stimulate mobilization of EPCs that speed up the initiation of angiogenesis.…”

Preclinical Evaluation of BMP-9-Treated Human Bone-like Substitutes for Alveolar Ridge Preservation following Tooth Extraction

“…These pro-angiogenic properties are expected to enhance therapeutic outcomes when substitutes are implanted in vivo, in particular by promoting graft neovascularization that would favor bone healing [ 57 , 58 , 59 ]. Since we previously showed in a rodent calvarial bone defect model that prevascularized bone-like substitutes improved the graft survival after 12 weeks of implantation [ 25 ], we hypothesize that adding a rhBMP-9 treatment to our protocols would allow the production of a prevascularized bone-like substitute with superior in vivo bone healing level and graft survival.…”

“…For in vitro and in vivo studies, substitutes were assigned to four experimental groups: stromal substitutes, bone-like substitutes, BMP-9-treated stromal substitutes, and BMP-9-treated bone-like substitutes ( Table 2 ). Tissues were obtained by seeding hASCs at a density of 4000 cells/cm 2 (Passage 7) in 6-well plates containing a peripheric anchorage device (Whatman paper, Fisher Scientific, Quebec City, QC, Canada), as previously described [ 23 , 24 , 25 , 61 ]. Cells were first grown in DMEM supplemented with 10% FBS, antibiotics, and 1.8 mM calcium chloride (CaCl 2 ) (basal stromal control medium).…”

“…Over the years, our research team has developed all-natural bone-like substitutes based on cell-assembled extracellular matrix components surrounding live osteogenically induced human adipose-derived stromal/stem cells (hASCs) [ 23 , 24 , 25 ]. The capacity to differentiate towards the osteogenic lineage had already been established for this mesenchymal cell population [ 26 ].…”

Biofabricating the Vascular Tree in Engineered Bone Tissue

“…Firstly, microvascular structures need perfusion to maintain viability of cells and drive maturation in vitro within a large fabricated 3D tissue engineered construct. Thus far, a majority of studies have focused on generating microvascular networks without any attention to the spatial organization of formed microvessels, thereby failing to provide a natural inlet and outlet for perfusion during preculture in the lab or for in vivo anastomosis [28][29][30][31]. Secondly, inosculation with host capillary bed takes at least 48 hours resulting in reliance on diffusion for nutrient transport during this period, which is inadequate when upscaling to larger constructs [32,33].…”