A dual-ink 3D printing strategy to engineer pre-vascularized bone scaffolds in-vitro

Abstract

A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.

Keywords: 3d printing; Bone; GelMA; Hydrogel; Scaffold; Vascularization.

Figure 1.

Steps for dual-ink 3D printing of pre-vascularized CaP scaffolds. (A) Strands of CaP ink (Osteoink) and a semi-sacrificial alginate hydrogel material are extruded using a dual-head 3D printer. (B) The 3D printed fibers are fully covered with a GelMA hydrogel precursor, which is photopolymerized using UV light. (C) The sacrificial alginate ink is physically removed. (D) The hollow microchannels are seeded with a co-culture of hMSCs and ECs.

Figure 2.

Photographs of dual-ink versus single ink 3D printed scaffolds. (A) Dual head 3D printing of CaP ink, followed by (B) printing of semi-sacrificial alginate ink loaded with green fluorescent microparticles, and coverage with a GelMA hydrogel prepolymer (transparent). Upon photopolymerization (C) the alginate fiber is removed, forming interconnected hollow channels perfused with a pink fluorescent microparticle solution between the strands of CaP. In the single ink printed scaffolds (D) the green fluorescent microparticle-laden alginate is extruded, (E) covered with a GelMA hydrogel prepolymer, and (F) pink fluorescent microparticle loaded microchannels are formed, without neighboring CaP strands.

Figure 3.

Cayman’s malachite green phosphate assay results show an increase in phosphate ion concentration in the media that reached to a plateau after 3 days

Figure 4.

Endothelial monolayer formation in microchannels 3D printed either in the presence or absence of the CaP ink. (A-D) ECs without adjacent CaP ink fibers formed a complete monolayer after approximately 3 days. (A) Cells appeared to accumulate at the bottom of the channels, (B) form patches of denser cell numbers with regions completely void of cells on day 1, (C) followed by complete coverage of the empty areas by days 3 and (D) 5. (E-H) A similar pattern of EC morphogenesis was seen in samples fabricated with the 3D printed CaP ink. (I-J) No significant differences were detected for either average sprout length or the average number of sprouts for samples 3D printed with or without the CaP ink.

Figure 5.

Confocal images of 3D printed endothelialized microchannels supported by α-SMA positive hMSCs in control samples (A-C), and adjacent to CaP fibers (D-F). These images suggest that cells have the archetypal morphology and protein expression of perivascular pericytes in both cases.

Figure 6.

Gene expression analyses for pericyte-related markers comparing hMSCs and ECs co-cultured on GelMA hydrogels either with or without a 3D printed CaP ink. (A) ACTA2 and (B) NG2 were visibly lower when in the presence of CaP scaffolds, but not significantly reduced. However, (C) CD13 was significantly higher in control samples than in samples cultured with CaP scaffolds (**p<0.01).