Hypoxia Impedes Vasculogenesis of In Vitro Engineered Bone

Abstract

To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, a-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1a was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in cocultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1a expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.

Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures

“…This finding is in line with other reports of successful vasculogenesis in cocultures in osteogenic medium (Gawlitta et al, , reviewed by Liu et al, , Liu et al, ). Our results indicate an extensive context‐dependent communication between MSCs and endothelial cells, creating a mutually stimulating environment likely involving (growth) factors and/or cell–cell contacts, influencing both mural cell differentiation, and vasculo‐angiogenic and osteogenic processes (Bidarra et al, , reviewed by Hirschi, Ingram, & Yoder, , Loibl et al, , Nassiri & Rahbarghazi, ).…”

“…Multipotency of MSCs was examined via differentiation towards adipogenic, osteogenic, and chondrogenic lineages as described previously (Gawlitta et al, ). Briefly, osteogenesis was examined by staining for ALP activity (Vector SK5100 kit, Vector Laboratories) after culturing for 14 days under osteogenic differentiation medium (ODM), which consisted of α‐MEM (Gibco Paisley, 22561), 10% heat‐inactivated FBS, 0.2 mM ASAP, 100 U/ml‐100 μg/ml PenStrep,10 mM β‐glycerophosphate (Sigma), and 10 nM dexamethasone (Sigma).…”

“…A specific type of EPC named “endothelial colony forming cells” (ECFCs), especially those derived from cord blood, showed robust proliferative potential and inherent vasculogenic and angiogenic capacity and can contribute to de novo blood vessel formation in vitro and in vivo, in contrast to mature ECs (reviewed by Banno & Yoder, ). However, only few studies have addressed the simultaneous formation of in vitro prevascular networks and osteogenesis with these type of cells (Gawlitta et al, , reviewed by Liu et al, , Liu et al, ).…”

Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems

“…A recent study investigated the effect of hypoxic cultures on MSC/EPCs based on the hypothesis that a hypoxic environment contributed to prevascularization. Results, however, showed an impediment of vasculogenesis following hypoxia stimulation compared to normoxic cultures, which supported the formation of prevascularized structures (Gawlitta et al ., ). Additional studies are required to confirm the importance of a hypoxic stimulus for prevascularizing tissue‐engineered co‐culture constructs towards facilitating bone regeneration and fracture repair response in vivo .…”

Characterization of Adipose-Derived Mesenchymal Stem Cell Combinations for Vascularized Bone Engineering

“…[3][4][5][6][7] In vivo research has been limited by the fact that neovascularization is a complex process dependent on a conducive microenvironment consisting of optimal oxygenation, growth factors, and cell-cell interactions. [8][9][10][11] Additionally, studying bone formation in two-dimensional (2D) mono-culture is hindered by the mineralized nature of the bone matrix, and recapitulating this process in vitro has proven difficult. 8 Since engineered three-dimensional (3D) bone tissue constructs require adequate chemotransportation, development of an early vascular network within the tissue construct will be necessary.…”