Bone tissue is a vascularized, highly specialized, and hierarchically organized tissue. Nowadays, Tissue Engineering is challenging to recapitulate the structural complexity of physiological bone extracellular matrix (ECM), and biomaterials have been widely investigated . Herein, methacrylated gelatin (GelMA) was largely involved since physico-chemical and mechanical properties of GelMA-based hydrogels can be finely tuned. For this purpose, a biomaterial ink for bone 3d in vitro models was here designed and developed, by adding hydroxyapatite nanoparticles (nHA) in GelMA solution, photocuring with visible light exposure .
Materials and Methods
GelMA was properly methacrylated and then dissolved (7.5% w/V) in DPBS with Ruthenium/Sodium Persulfate as photoinitiator. nHA, added to mimic bone inorganic composition, was added in different concentrations. Composite solutions were tested by shear rate ramps (0.01-1000 s-1) to assess biomaterial ink viscosity with a rheometer. Hydrogels were consequently obtained by exposing the solution to light, mechanical compressive properties were tested, and weight variations in distilled water were evaluated for up to 6 weeks. The optimized biomaterial ink was investigated in terms of printability with a pneumatic extrusion-based BIO X 3D printer, to optimize printing parameters (i.e., speed, pressure, nozzle gauge). Serpentine models and grids were printed to assess printing accuracy (PA%) . In vitro biological tests were performed by encapsulating SAOS-2 preosteoblastic cells in the nHA/GelMA, and cell viability (%) was qualitatively evaluated by LIVE/DEAD staining.
GelMA methacrylation was successfully checked by H NMR spectroscopy (DoF = 55.32%). Rheological analysis showed that the investigated formulations exhibited a typical shear-thinning behavior, adequate for biomaterial ink printability. Viscosity increased by enhancing nHA concentration since nanoparticles acted as a filler. Contrarily, Ru/SPS addition led to a viscosity decrease in solutions with higher nHA concentration, possibly due to unfavorable electrostatic interactions between Ru/SPS and nHA. Stability tests exhibited a weight decrease in the first hours of water immersion, due to the release of uncrosslinked polymeric chains. By adding nHA, higher Ru/SPS concentration led to a higher swelling since a denser polymeric network allowed for the nHA exclusion, and samples absorbed higher water quantity. Contrarily, lower Ru/SPS concentration formed hydrogels whose meshes retained nHA and determined a lower water uptake. Mechanical tests showed higher Ru/SPS concentration led to a decrease (p < 0.05) in mechanical properties compare to GelMA, validating the weight variations results. Biomaterial ink printability was assessed by printing serpentine, to select optimal parameters set, and grid models as well, showing a PA% equal to 65%. High viability (> 80%) was qualitatively checked by LIVE/DEAD staining up to 7 days of culture when SAOS-2 cells were embedded in the hydrogel.
The developed biomaterial ink resulted in a promising biomaterial to better recapitulate bone tissue ECM. Printability of the materials here developed was successfully evaluated by rheological analysis and printability tests. Moreover, cells showed good viability up to 7 days of culture.
Zhu, L. et al., International Journal of Oral Science, 2019
Lim, K. S. et al., Macromolecular Bioscience, 2019
Negrini, N. C. et al., Elsevier, 2018