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.

Keywords: 3D printing bone scaffold; bone regeneration; decellularized bone scaffold; endothelial cell; vascularized bone graft.

Figure 1.

Imaging of decelluarized bone scaffolds (A: SEM and B: photograph) and HB scaffolds (C: SEM and D: photograph). Scale bars in panels A and C are 500 μm.

Figure 2.

Cell culture protocol timelines. (A) Human SMCs were seeded onto the bone scaffold—either DB or HB—and maintained in SMC medium for 3 weeks. Afterwards, HUVECs were seeded, and the constructs cultured in EC medium for 6 days. Fibrin hydrogel was finally added, with ongoing culture in EC medium, for 1–5 days. (B) HUVEC-only culture timeline. DB scaffold was pre-treated with fibronectin for 1 h, and then only HUVECs were seeded and cultured for 6 days, followed by application of fibrin sealant for 1–5 days. Red arrows indicate the work flow direction.

Figure 3.

A. Schematic showing the assembled perfusion bioreactor with syringe chamber inside, connected to flow pump and medium flow through tubing in direct of yellow arrows. B. Assembled glass bioreactor, black arrow indicates the syringe chamber.

Figure 4.

Vascular cell culture on DB scaffolds. (A) Human SMCs cultured for 3 weeks on DB scaffold show extensive coverage of the matrix by H&E, SEM, and phalloidin staining. (B) Human EC (HUVECs) cultured on DB scaffolds for 6 days, with or without fibrin, showed overall little cell growth and a slight tendency for lumen formation (arrows).

Figure 5.

Vascular cell co-culture on DB scaffolds. A: SMC and EC show few lumen-like structures (asterisks); B: One day after fibrin addition shows multiple small lumens appearing (arrows); C, D: Three and 5 days after fibrin addition shows increasing numbers and sizes of lumen-like structures (asterisks).

Figure 6.

Immunostaining for EC markers in SMC-EC-fibrin cultures. A: vWF (green) and DAPI for nuclei (blue) shows green-lined lumens (asterisks) in scaffold interstices after 3 days of fibrin culture. Slight non-specific staining of DB matrix by DAPI is visible. B, C: CD31 (red), vWF (green) and DAPI staining show EC-lined luminal structures.

Figure 7.

Histological staining of hSMCs/ECs/fibrin/DB constructs cultured under static (A) and bioreactor flow conditions (B). Upper right insets in both panels show enlarged image of central DB scaffold area. Yellow arrows show lumen formation, which is absent in the center of the static culture (A). Lower left insets show immunostaining of representative luminal structures, with vWF (green) and CD31 (red).

Figure 8.

hSMCs cultured on HB scaffolds of varying pore sizes for 1–3 weeks, evaluated by confocal imaging. A: hSMC cultured on HB scaffold with 300 µm pore size for 1 week. B: hSMC cultured on 700 µm pore size scaffold for 1 week; and C: hSMC cultured on 1000 µm scaffold for 1 week. D: Cells on HB with 1,000 µm pore size at 3 weeks, 3D reconstruction of several fibers using confocal microscopy. Blue is DAPI stain (nuclei); red is phalloidin (cytoskeleton).

Figure 9.

In vitro EC cellular network formation on HB scaffolds. A1, B1, C1, D1: SEM images of HB scaffolds, yellow arrows indicating the corresponding confocal scanning area in subsequent panels. Pore sizes for A1–D1 are 700 μm, 300 μm, 700 μm, and 1,000 μm, respectively. A2–A5: Confocal scanning of an individual HB scaffold fiber with hSMC and HUVEC co-cultured. B2–B5: Confocal scanning of HB scaffold 300 µm pore spacing with hSMC and HUVEC co-culture. C2–C5: Confocal scanning of HB scaffold with 700 µm pore spacing. D2–D5: Confocal scanning of HB scaffold 1000 µm pore spacing. Blue is DAPI staining for nuclei, green is VE-cadherin (EC marker), red is phalloidin (cytoskeletal marker) and yellow is vWF (EC marker).

Figure 10.

A: EC marker expression level differences between co-culture and mono-culture in HB scaffolds with various pore sizes, exhibiting gene up-regulation during co-culture, the degree of which depends on the pore size. Values of three independent experiments from the triplicate PCR reactions for genes of interest were normalized against average GAPDH Ct values from the same cDNA sample. Fold change of GOI transcript levels between co-culture and monoculture equals to 2^-ΔΔCt, where ΔCt = Ct(GOI) – Ct(GAPDH) and ΔΔCt = ΔCt(co-culture) – ΔCt(monoculture). (n = 3, error bars refer to SD, *p < 0.05). B: EC marker expression level of HUVECs monoculture on HB scaffolds with various pore sizes, showing pore size independent expression levels.