3D Bioprinted GelMA-Gelatin-Hydroxyapatite Osteoblast Laden Composite Hydrogels for Bone Tissue Engineering.

Abstract

Introduction 3D bioprinting offers a novel solution to critical sized bone defects by allowing the placement of osteogenic cells, additive biomaterials and bioactive signaling that mimic native tissue. We describe the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), and osteoblasts for bone tissue engineering. Methods A mouse calvarial osteoblast-laden GelMA-gelatin-LAP bioink consisting of various concentrations of HA was 3D-bioprinted into a porous hydrogel construct. After days 1, 14, and 28, five constructs from each HA concentration were analyzed. The water weight percent differences of the hydrogels and the degradation behavior in enzyme solution were characterized. An ALP assay and histological analysis were performed. Cell survivability was determined using a LIVE/DEAD Viability/Cytoxicity Kit and AlamarBlue Cell Viability reagent. Real-time polymerase chain reaction (RT-qPCR) was performed to measure expression levels of bone morphogenetic protein-7 (BMP-7) and Osteocalcin (BGLAP). Results The addition of 5, 10, and 20 mg/ml of HA significantly reduced hydrogel swelling (p ≤ 0.01) from baseline GelMA-Gelatin hydrogels (Figure 3A). HA significantly decreased hydrogel breakdown in a concentration dependent manner (Figure 3B, p ≤ 0.001). A significant increase in cell proliferation at day 28 was noted in all groups (Figure 3D). ALP activity (Figure 3E) significantly increased with the addition of 5mg/ml and 20mg/ml of HA at days 7 and 28 (p ≤ 0.05). Live/dead staining at 1, 14, and 28 days showed high chondrocyte viability. The addition of 20mg/ml of HA demonstrated significantly greater BMP7 and BGLAP gene expression at both 14 and 28 days over the hydrogels without HA (Figure 5, p ≤ 0.05). Conclusion The addition of HA to GelMA-gelatin hydrogels significantly decreased hydrogel swelling, improved the ability of the hydrogel to resist enzymatic degradation, increased osteoblastic differentiation and mineralization, and increased osteogenic gene expression while maintaining equal cell viability and proliferation to non-HA hydrogels.