Bone Tissue Engineering with Multilayered Scaffolds—Part I: An Approach for Vascularizing Engineered Constructs In Vivo

Abstract

Obtaining functional capillaries through the bulk has been identified as a major challenge in tissue engineering, particularly for critical-sized defects. In the present study, a multilayered scaffold system was developed for bone tissue regeneration, designed for through-the-thickness vascularization of the construct. The basic principle of this approach was to alternately layer mesenchymal stem cell-seeded nanofibers (osteogenic layer) with microfibers or porous ceramics (osteoconductive layer), with an intercalating angiogenic zone between the two and with each individual layer in the microscale dimension (100-400 mm). Such a design can create a scaffold system potentially capable of spatially distributed vascularization in the overall bulk tissue. In the cellular approach, the angiogenic zone consisted of collagen/fibronectin gel with endothelial cells and pericytes, while in the acellular approach, cells were omitted from the zone without altering the gel composition. The cells incorporated into the construct were analyzed for viability, distribution, and organization of cells on the layers and vessel development in vitro. Furthermore, the layered constructs were implanted in the subcutaneous space of nude mice and the processes of vascularization and bone tissue regeneration were followed by histological and energy-dispersive X-ray spectroscopy (EDS) analysis. The results indicated that the microenvironment in the angiogenic zone, microscale size of the layers, and the continuously channeled architecture at the interface were adequate for infiltrating host vessels through the bulk and vascularizing the construct. Through-thethickness vascularization and mineralization were accomplished in the construct, suggesting that a suitably bioengineered layered construct may be a useful design for regeneration of large bone defects.

Modulating microfibrillar alignment and growth factor stimulation to regulate mesenchymal stem cell differentiation

“…Sterile scaffolds were hydrated and conditioned by incubating in expansion media overnight before cell 6 seeding. At passage 2, MSCs were trypsinized and seeded onto one side of the electrospun scaffolds at a density of 500,000 cells per scaffold or 125,000 cells per scaffold using custom designed scaffold holders as previously reported [36]. Scaffolds retained 82 -87% of the seeded cells (data not shown).…”

Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering

“…Early osteoblastic and 24 endothelial differentiations were observed and cell pro- 25 liferation was increased after 7 days of culture. In 3D, cell 26 migration was observed between layers of LBL constructs, 27 as well as an osteoblastic differentiation.…”

“…"Bioassembly" is based on self-induced assembly of cellu- plays an important role in order to obtain the best final 73 implantable construct [25]. in all four layers (Fig.…”

Angiogenesis in Tissue Engineering: As Nature Intended?

“…For example, Wang et al (2017a) demonstrated that the addition of pericytes to a co-culture system seeded into a calcium phosphate cement scaffold increased vessel density by over 65% after 12 weeks. Sathy et al (2015) also reported promising results following incorporation of pericytes into multi-layered scaffolds designed for tissue engineered bone. The authors were able to produce vascularized bone constructs of up to 400 µm in thickness.…”