Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell morphology and alignment.
The THA-col composite was prepared by mixing THA (2.5% w/v) and col 1 (0.5% w/v) with simultaneous gelation induced by hydrogen peroxide and horseradish peroxidase for THA and neutralization for col. The composite biomaterial ink was characterized for fibrillation by turbidity measurement, fluorescence microscopy and rheological properties (shear thinning behaviour and amplitude sweep) were assessed. Anisotropic properties were introduced upon 3D bioprinting (3D DiscoveryTM, RegenHU) the biomaterial ink. Cellular behaviour was investigated by embedding human mesenchymal stromal cell (MSC) spheroids into the hydrogel. Cell migration as well as chondrogenic differentiations was analysed over time by microscopy with subsequent image analysis (Oval plugin, ImageJ, NIH), gene expression analysis, histological stainings and glycosaminoglycan and DNA quantification. The fibrillation of col 1 and the homogenous distribution of the fibers within THA was shown by second harmonic generation imaging and fluorescent imaging and confirmed by turbidity measurement. After 3D bioprinting an anisotropic alignment of col fibrils was achieved that guided cell migration along the fiber orientation. Cell migration of hMSC spheroids showed similar behavior comparing THA-col and col whereas no migration was present for THA only. Chondrogenic differentiation resulted in an increase in cartilage related genes (col 2, aggrecan) with low tendency of hypertrophy (col X, col I, RunX2). Cartilage like matrix deposition was further corroborated by quantification of GAGs within samples that showed an overall increase within 21 days of culture similar to hMSC pellet control group. Safranin O staining resulted confirmed production of proteoglycans . Extrusion based printing has been investigated to produce scaffolds with anisotropic properties on microscale exploiting the shear forces inducing alignment of col fibres within a shear thinning HA matrix. The combination of the two matrix components brings unique features and advantages addressing cell migration, differentiation and material tissue integration compared to the single polymers. THA-col biomaterial has shown its potential for cartilage tissue engineering and represents a potential injectable material for cell free cartilage treatment.