IntroductionThe treatment of large bone defects remains an area of significant challenge in orthopaedic surgery. In cases where critical-sized defects (CSDs) remain, current treatment options are underwhelming and are often fraught with complications. 3D bioprinting offers a novel solution to CSDs 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. MethodsA MC3T3-E1 Subclone 4 mouse calvarial osteoblast-laden GelMA-gelatin bioink consisting of various concentrations of HA was 3D-bioprinted into porous hydrogel constructs using an in-lab modified BIO-X 3D Bioprinter. The 3D-fabricated constructs were cross-linked via photopolymerization in the presence of Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) under UV light (1000 mW/cm2) cultured in complete osteogenic medium. After 1, 14, and 28 days, a cohort of constructs were harvested for analysis. An ALP assay and histological analysis were performed. Cell survivability and proliferation in the composite hydrogels was determined. Real-time polymerase chain reaction (RT-qPCR) was performed to measure expression levels of osteogenic genes bone morphogenetic protein-7 (BMP-7) and Osteocalcin (BGLAP) relative to a housekeeping gene (GAPDH). ResultsThe addition of HA to the bioink significantly decreased swelling from baseline GelMA-Gelatin hydrogels with a slight trend of decreased swelling with increasing HA content. The addition of 5, 10, and 20 mg/mL of HA significantly reduced hydrogel swelling (p ≤ 0.01). There was no difference among the HA groups. Degradation analysis with Type IV collagenase demonstrated that HA significantly decreased hydrogel breakdown in a concentration dependent manner (p ≤ 0.001). Results of the Alamar Blue assay demonstrated significantly increased cell proliferation in all groups at day 28, including the hydrogels without any HA. There was no difference in metabolic activity among the groups (p ≤ 0.01). ALP activity for days 7, 14, and 28 was normalized to Day 1 and found that the addition of 5 mg/mL and 20 mg/mL of HA significantly increased ALP expression at days 7 and 28 (p ≤ 0.05). Live/dead staining at 1, 14, and 28 days after printing showed, in all groups, the majority of the osteoblasts were alive at each time-point. The addition of 20 mg/mL of HA (GG20HA) demonstrated significantly greater BMP7 and BGLAP gene expression at both 14 and 28 days over the hydrogels without HA (p ≤ 0.05). ConclusionIn order for tissue-engineered bone defect repair materials to be successful they need to be biocompatible, biodegradable, similar in strength to bone while allowing for good bone induction, and a three-dimensional porous network structure for host tissue ingrowth. Therefore, 3D bioprinting for bone tissue engineering is an exciting possibility in the treatment of critical-sized bone defects as it presents an opportunity to fulfill these qualities. Although there are many reports on 3D bioprinting of bone-like structures, the ideal bioink(s) for bone tissue regeneration have yet to be discovered. Here we report on the use of a cellular GelMA-gelatin bioink with the addition of HA. In this report we demonstrated the suitability of a GelMA-gelatin-HA bioink for 3D bioprinting of bone tissue engineering applications. The addition of HA to GelMA-gelatin hydrogels significantly (1) decreased hydrogel swelling, (2) improved upon the ability of the hydrogel to resist enzymatic degradation, (3) increased osteoblastic differentiation and mineralization, and (4) increased osteogenic gene expression while maintaining equal cell viability and proliferation to non HA hydrogels. GelMA-gelatin-HA is a promising hydrogel for bone tissue repair.Significance/Clinical Relevance: We demonstrated the suitability of a GelMA-gelatin-HA bioink for 3D bioprinting of bone tissue engineering applications. 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. GelMA-gelatin-HA is a promising hydrogel for bone tissue repair.
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