Vascularization of Natural and Synthetic Bone Scaffolds

Abstract

Vascularization of engineered bone tissue is critical for ensuring its survival after implantation. In vitro pre-vascularization of bone grafts with endothelial cells is a promising strategy to improve implant survival. In this study, we pre-cultured human smooth muscle cells (hSMCs) on bone scaffolds for 3 weeks followed by seeding of human umbilical vein endothelial cells (HUVECs), which produced a desirable environment for microvasculature formation. The sequential cell-seeding protocol was successfully applied to both natural (decellularized native bone, or DB) and synthetic (3D-printed Hyperelastic "Bone" scaffolds, or HB) scaffolds, demonstrating a comprehensive platform for developing natural and synthetic-based in vitro vascularized bone grafts. Using this sequential cell-seeding process, the HUVECs formed lumen structures throughout the DB scaffolds as well as vascular tissue bridging 3D-printed fibers within the HB. The pre-cultured hSMCs were essential for endothelial cell (EC) lumen formation within DB scaffolds, as well as for upregulating EC-specific gene expression of HUVECs grown on HB scaffolds. We further applied this co-culture protocol to DB scaffolds using a perfusion bioreactor, to overcome the limitations of diffusive mass transport into the interiors of the scaffolds. Compared with static culture, panoramic histological sections of DB scaffolds cultured in bioreactors showed improved cellular density, as well as a nominal increase in the number of lumen structures formed by ECs in the interior regions of the scaffolds. In conclusion, we have demonstrated that the sequential seeding of hSMCs and HUVECs can serve to generate early microvascular networks that could further support the in vitro tissue engineering of naturally or synthetically derived bone grafts and in both random (DB) and ordered (HB) pore networks. Combined with the preliminary bioreactor study, this process also shows potential to generate clinically sized, vascularized bone scaffolds for tissue and regenerative engineering.

Physical and Mechanical Properties of Composite Scaffolds with or without Collagen Impregnation

“…Moreover, osteoblasts and fibrovascular tissue seem to exhibit a preference for binding to biomaterials with pore sizes significantly larger than the own cell size [39]. An ideal bone substitute material for oral and maxillofacial use should mimic the structure of human trabecular bone, acting as a proper support for vascularization and bone ingrowth [36]. The newly developed composites evaluated in the present study showed high interconnectivity that lead to a larger surface area and greater fluid retention capacity than control group.…”

“…For a successful osteoconduction, the 3D structure of a scaffold should support cell penetration and proliferation as well as neovascularization, enabling an adequate diffusion of nutrients to the new cells and preventing the failure of bone repair [4,[34][35][36][37]. Moreover, scaffolds should present interconnected pores of sizes and morphologies adequate to ensure the diffusion of waste products out of the scaffold without interfering with other surrounding tissues [34].…”

Strategy for the Generation of Engineered Bone Constructs Based on Umbilical Cord Mesenchymal Stromal Cells Expanded with Human Platelet Lysate

“…Furthermore, we were also able to show improved proangiogenic activity of the construct together with the absence of inflammatory response from the host. Vascularization of the bone scaffold is critical for the generation of viable and functional constructs [41–43]. Here, not only the differentiation properties of the implanted cell but also the release of key angiogenic and growth factors are relevant to preserve the minimal conditions for bone induction of the engineered construct in vivo [44–46].…”

Functional Biomaterials for Bone Regeneration: A Lesson in Complex Biology

“…[375] Vascularization responses in such constructs has also been reported to be improved through the addition of smooth muscle cells prior to HUVEC seeding. [376] Of further interest, through the use of a perfusable bioreactor to introduce hydrodynamic shear into the construct, cell survival and lumen formation could be enhanced in the central portions of the scaffold. Thus, the authors demonstrated that mechanical stimulation plays a vital role in the angiogenic capacity of such tissue engineered constructs and should be further investigated, particularly in the context of bone tissue engineering.…”

“…Only in vitro and not tested in combination with other materials Zhang et al 2017 [375] Creation of prefabricated vascularized tissue engineered graft for large bone defect [452] Testing of co-culture systems to create a prevascularized bone implant [376] Understanding the role of hydrodynamic shear on natural and synthetic prevascularized bone grafts musculoskeletal regeneration strategies. [382] A summary of the aforementioned papers is reported in Table 2.…”