VEGF Over-Expression by Engineered BMSC Accelerates Functional Perfusion, Improving Tissue Density and In-Growth in Clinical-Size Osteogenic Grafts

Abstract

The first choice for reconstruction of clinical-size bone defects consists of autologous bone flaps, which often lack the required mechanical strength and cause significant donor-site morbidity. We have previously developed biological substitutes in a rabbit model by combining bone tissue engineering and flap pre-fabrication. However, spontaneous vascularization was insufficient to ensure progenitor survival in the core of the constructs. Here, we hypothesized that increased angiogenic stimulation within constructs by exogenous VEGF can significantly accelerate early vascularization and tissue in-growth. Bone marrow stromal cells from NZW rabbits (rBMSC) were transduced with a retroviral vector to express rabbit VEGF linked to a truncated version of rabbit CD4 as a cell-surface marker. Autologous cells were seeded in clinical-size 5.5 cm 3 HA scaffolds wrapped in a panniculus carnosus flap to provide an ample vascular supply, and implanted ectopically. Constructs seeded with VEGFexpressing rBMSC showed significantly increased progenitor survivival, depth of tissue ingrowth and amount of mineralized tissue. Contrast-enhanced MRI after 1 week in vivo showed significantly improved tissue perfusion in the inner layer of the grafts compared to controls. Interestingly, grafts containing VEGF-expressing rBMSC displayed a hierarchically organized functional vascular tree, composed of dense capillary networks in the inner layers connected to large-caliber feeding vessels entering the constructs at the periphery. These data constitute proof of principle that providing sustained VEGF signaling, independently of cells experiencing hypoxia, is effective to drive rapid vascularization and increase early perfusion in clinical-size osteogenic grafts, leading to improved tissue formation deeper in the constructs.

Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

“…A clear interdependency of angiogenesis and osteogenesis occurs upon bone formation 35,36 . For this reason, VEGF-enrichment has been naturally proposed as a strategy to accelerate or increase bone graft vascularization 37,38 . Our study demonstrates that VEGF knocked-out cartilage templates retained full osteoinductive potential in a challenging ectopic environment.…”

A chimeric feeder comprising transforming growth factor beta 1‐ and basic fibroblast growth factor‐primed bone marrow‐derived mesenchymal stromal cells suppresses the expansion of hematopoietic stem and progenitor cells

“…Several approaches are taken to overcome these problems: viz. optimization of the culture media to get higher yields at earlier passages (Basoli et al, 2021;Hoch & Leach, 2014); use of priming agents to improve their functionality (Brunner et al, 1993;Miceli et al, 2021;Noronha et al, 2019), use of specific scaffolds to grow them in three-dimensional cultures (Kouroupis & Correa, 2021;Sharma et al, 2012), and genetic manipulations (Damasceno et al, 2020;Huang et al, 2021;Largo et al, 2020).…”

Frontiers of Hydroxyapatite Composites in Bionic Bone Tissue Engineering

“…Due to the presence of the blood supply outside the tissue-engineered bone, the regeneration of the internal blood vessels and the formation of the bones are promoted. Largo et al [ 167 ] co-cultured vegf-transfecting rBMSCs with HA and placed them in the muscle of rabbits. After one week, MRI examination revealed increased tissue perfusion and generation of a dense capillary network in the graft.…”

“…It also suggests that nerves are important in bone homeostasis [ 182 , 183 ]. In recent years, studies in the field of tissue engineering have found that in addition to vascularization, neurogenesis may also be important in promoting the formation of bone [ 167 , 184 , 185 ].…”