Trabecular-bone mimicking osteoconductive collagen scaffolds: an optimized 3D printing approach using freeform reversible embedding of suspended hydrogels

Abstract

Background: Technological constraints limit 3D printing of collagen structures with complex trabecular shapes. However, the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method may allow for precise 3D printing of porous collagen scaffolds that carry the potential for repairing critical size bone defects.

Methods: Collagen type I scaffolds mimicking trabecular bone were fabricated through FRESH 3D printing and compared either with 2D collagen coatings or with 3D-printed polyethylene glycol diacrylate (PEGDA) scaffolds. The porosity of the printed scaffolds was visualized by confocal microscopy, the surface geometry of the scaffolds was investigated by scanning electron microscopy (SEM), and their mechanical properties were assessed with a rheometer. The osteoconductive properties of the different scaffolds were evaluated for up to four weeks by seeding and propagation of primary human osteoblasts (hOBs) or SaOS-2 cells. Intracellular alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were measured, and cells colonizing scaffolds were stained for osteocalcin (OCN).

Results: The FRESH technique enables printing of constructs at the millimetre scale using highly concentrated collagen, and the creation of stable trabecular structures that can support the growth osteogenic cells. FRESH-printed collagen scaffolds displayed an intricate and fibrous 3D network, as visualized by SEM, whereas the PEGDA scaffolds had a smooth surface. Amplitude sweep analyses revealed that the collagen scaffolds exhibited predominantly elastic behaviour, as indicated by higher storage modulus values relative to loss modulus values, while the degradation rate of collagen scaffolds was greater than PEGDA. The osteoconductive properties of collagen scaffolds were similar to those of PEGDA scaffolds but superior to 2D collagen, as verified by cell culture followed by analysis of ALP/LDH activity and OCN immunostaining.

Conclusions: Our findings suggest that FRESH-printed collagen scaffolds exhibit favourable mechanical, degradation and osteoconductive properties, potentially outperforming synthetic polymers such as PEGDA in bone tissue engineering applications.

Keywords: Additive manufacturing; Bioprinting; Collagen; FRESH; Tissue engineering.

Fig. 1

Trabecular bone-mimicking scaffolds fabricated with 3D printing techniques using PEGDA or type I collagen. Top view (A) and side view (B) of the CAD model used for 3D printing of PEGDA and collagen scaffolds. (C, E, G) PEGDA scaffolds. (D, F, H) Collagen scaffolds. The trabecular model was sliced using either the PreForm software for SLA printing (C) or the Simplify3D software for FRESH printing (D). A cross-sectional view of the middle of the model is shown. Top view of the printed PEGDA scaffold (E) and printed collagen scaffold (F). Porosity of the PEGDA (G) and collagen (H) scaffolds as captured by confocal microscopy. Z projection images corresponding to one printed layer of the PEGDA structure stained with TRITC (Tetramethylrhodamine isothiocyanate) and one printed layer of the collagen scaffold stained with Picrosirius red. Scale bars: 500 μm (A, B, G and H), 2 mm (E and F)

Fig. 2

Scanning electron microscopy imaging of 3D-printed PEGDA and collagen scaffolds. Samples of PEDGA scaffolds (A–C) were freeze-dried, and samples of FRESH-printed collagen scaffolds (D–F) were prepared using ethanol and acetone dehydration followed by critical point drying. Scale bars: 100 μm (A and D), 2 μm (B, C, E, and F)

Fig. 3

Rheological, mechanical and degradation characterization of 3D-printed collagen scaffolds. Storage (G′) and loss (Gʺ) modulus of collagen scaffolds were determined from an amplitude sweep at 0.1 Hz, at a strain of 1% (A). The compressive Young’s modulus of collagen scaffolds was determined using an unconfined static compression at a strain rate of 5000 μm min^− 1 (B). Tests were performed on four different scaffolds. The mass changes of the collagen scaffolds and PEGDA scaffolds (C) were determined by weighting the dry mass of the scaffolds after printing and after 4 weeks in PBS; *p < 0.05

Fig. 4

Osteoblastic activity of hOBs and SaOS-2 cells grown on 2D or on 3D-printed collagen, or on PEGDA scaffolds, assessed by ALP and LDH activity measurement. ALP activity normalized by LDH activity in hOBs (A) and SaOS-2 cells (B) grown in 2D in non-coated wells or in collagen-coated wells, and in 3D on PEGDA and collagen scaffolds. hOBs, human osteoblasts; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; *p < 0.05, **p < 0.005

Fig. 5

Immunocytochemistry of hOBs and SaOS-2 cells grown on FRESH-printed collagen scaffolds. Maximum intensity z projection images captured by confocal microscopy of hOBs and SaOS-2 cells grown on collagen scaffolds for 1 week (A) or 4 weeks (B), and stained for cell nuclei (DAPI), cytosol (carboxyfluorescein diacetate [CFDA]), and the osteoblast marker osteocalcin (OCN). hOBs and SaOS-distribution along the z axis after 1 week (C) or 4 weeks (D). (C-D) Images obtained from maximal intensity x projections using the CFDA channel. Scale bars: 200 μm (A-B), 100 μm (C-D). Both hOBs and SaOS-2 cells colonized the scaffolds within a 4-week timeframe. hOB, human osteoblast