The Use of High-Concentration Collagen-Based Composition and Gelatin Granules as Bioinks for Extrusion 3D Bioprinting...

The aim of the study was to analyze the composition feasibility of the bioinks based on high-concentration collagen (40 mg/ml) and gelatin granules (6.25 mg/ml) for bioprinting of porous-structured hydrogel constructs using an extrusion 3D bioprinter.

Bioprinting was performed on a 3D Invivo bioprinter (Rokit, South Korea). We assessed the filament continuity during extrusion, the changes in its thickness after test printing and incubation, as well as the biodegradation of prepared scaffolds. The hydrogel cytocompatibility was studied by the proliferation of adipose-derived stem cells (ADSCs) incorporated into the scaffolds. Flow cytometry was performed to determine the immunophenotype of ADSCs. Cell proliferation in the scaffold structure was studied in vitro during 28 days spectrophotometrically after adding PrestoBlue reagent. The expression of target genes was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) on day 21 of cultivation. We used the primers for mRNA encoding the synthesis of chondrogenic factors and metabolites (ACAN, SOX9, COL1A1, COL2A1), surface markers (CD29, CD44, CD73, CD90, CD105), as well as hypoxia (HIF1A), proliferation (PCNA), and apoptosis (BCL2, BAX) factors. The morphology of the scaffolds was studied on day 28 of culturing by light microscopy after fixing and staining the histological sections.

The extrusion of the high concentration collagen-based hydrogel composition (40 mg/ml) and gelatin granules (6.25 mg/ml) during printing was stable, there was no filament breakage. When incubated in phosphate-buffered saline, the filament thickness of the hydrogel was statistically significantly higher than the scaffold thickness after printing. The degradation of the scaffolds from the hydrogel and gelatin in the solution of type I collagenase started earlier than the collagen scaffolds. The incubation in phosphate-buffered saline for 14 days resulted in less mass loss when drying the collagen scaffolds with gelatin granules. The cells isolated from human adipose tissue expressed surface markers characteristic of ADSCs. ADSCs proliferation and differentiation in chondrogenic direction were observed in both groups compared. The differences were in the spatial arrangement of the cells. In the collagen scaffolds the most cells were on the surface, while in the scaffolds from collagen and gelatin the cells were distributed throughout the whole volume. The 2^-ΔΔCt quantitative reverse transcription polymerase chain reaction results showed the increased expression of the transcription factor SOX9 by the cells in the collagen and gelatin scaffolds, as well as the decreased expression of the anti-apoptotic gene BCL2 relative to the collagen scaffolds.

The present study suggested the bioink composition based on high concentration collagen (40 mg/ml) and gelatin granules (6.25 mg/ml) for bioprinting of porous-structured hydrogel constructs. The study showed hydrogel to be appropriate for printing and exhibited the properties of a porous material. The hydrogel provided the uniform distribution of ADSCs in the scaffold volume, contributing to their differentiation in the chondrogenic direction. Thus, the suggested bioink composition appears to be a promising material to be used in tissue engineering.