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Biomimetic scaffolds provide essential biophysical and biochemical cues to guide cell behavior. Although the effects of biomaterial-directed cues on cell response have been widely reported. few studies have sought to decouple these effects to better understand the interplay between the different physicochemical factors on tissue-specific cell function. In this study, beta-tricalcium phosphate (B-TCP) was incorporated into electrochemically aligned collagen (ELAC) and random collagen threads, and the individual and interactive effects of biophysical and biochemical cues on osteoblast proliferation, differentiation, and mineralization were investigated. ELAC threads were prepared from collagen solution with 13-TCP using isoelectric focusing. Pure ELAC threads were prepared without 0-TCP. Random collagen threads with and without 0-TCP were prepared by casting neutralized collagen solution into a PLA mold and incubating at 37 °C for 60 inin to induce fibrillogenesis. Confirmation of B-TCP incorporation and validation of collagen fiber alignment was carried out using scanning electron microscopy (SEM) and polarized light microscopy (PLM). Tensile tests were perforined to assess the mechanical properties of collagen threads. Saos-2 cells were seeded on threads to assess cell proliferation, differentiation. and mineralization. Results from SEM and PLM confirmed that collagen fiber alignment in ELAC was retained upon 13-TCP incorporation. ELAC threads were significantly stronger and stiffer (p < 0,05) than random collagen threads, Alkaline phosphatase (ALP) activity was significantly higher (p < 0,05) in ELAC threads compared to random threads. In addition, M-TCP incorporation into ELAC significantly augmented (p < 0.05) cell metabolic activity, ALP activity, and cell-mediated calcium deposition, In conclusion, results from this study indicate that topographical cues from aligned collagen significantly enhance osteoblast function which was further augmented by D-TCP incorporation. Overall, 13-TCP incorporation into aligned collagen framework can yield biomimetic functional scaffolds for bone regeneration applications. [ABSTRACT FROM AUTHOR]

Anisotropic orientation of collagen fibers is a promising strategy to generate biomimetic constructs with improved mechanical and topographical properties to control cell function. Biofabrication using 3D printing allows for the generation of application-specific tailormade collagenous scaffolds with high geometric precision. However, these scaffolds lack collagen anisotropy. In this work, 3D printing was combined with the magnetic alignment approach in an innovative 4D priiiting scheme to introduce high degree of collagen fiber alignment over a larger-scale custom scaffold by optimizing streptavidin-coated magnetic particle (SMP) concentration and ink composition. Collagen and xanthum gum were combined in varying ratios (Col:XG - 1:1.4:1, 9:1) to which a photoinitiator and SMP (0, 0.2,0.4 Ing/ml) were added. An extrusion-based printer was used to 4D print constructs in the presence of a magnetic field CO.2 T). The prints were incubated at 37 °C to induce gelation followed by UV crosslinking. Rheological analyses were performed to compare the viscosities of inks. Print fidelity was quantified by measuring the areas of the printed constructs. Qualitative analysis of collagen fiber alignment in 4D printed constructs was performed using polarized light microscopy (PLM) and scanning electron microscopy (SEM). Rheological studies revealed that all ink compositions demonstrated shear thinning properties. Use of higher XG composition yielded inks with higher viscosity, printed constructs with greater fidelity, but a lower degree of collagen alignment. Increase in SMP concentration to 0.4 mg/m! resulted in a significant decrease (p < 0.05) in ink viscosity compared to 0 and 0.2 ing/ml SMP concentrations resulting in lower print fidelity. Degree of collagen fiber alignment assessed using SEM and PLM was higher in constructs made using highest SMP concentration. In conclusion, fine-tuning ink composition and SMP concentration can produce 4D printed scaffolds with high degree of collagen fiber alignment. This study presents an innovative strategy for producing biomimetic scaffolds towards tendon tissue engineering applications. [ABSTRACT FROM AUTHOR]

Collagen anisotropy is known to provide the essential topographical cues to guide tissue-specific cell function. Recent work has shown that extrusion-based printing using collagenous inks yield 3D scaffolds with high geometric precision and print fidelity. However, these scaffolds lack collagen anisotropy. In this study, extrusion-based 3D printing was combined with a magnetic alignment approach in an innovative 4D printing scheme to generate 3D collagen scaffolds with high degree of collagen anisotropy. Specifically, the 4D printing process parameters-collagen (Col):xanthan gum (XG) ratio (Col:XG; 1:1, 4:1, 9:1 v/v), streptavidin-coated magnetic particle concentration (SMP; 0, 0.2, 0.4 mg ml -1 ), and print flow speed (2, 3 mm s -1 )-were modulated and the effects of these parameters on rheological properties, print fidelity, and collagen alignment were assessed. Further, the effects of collagen anisotropy on human mesenchymal stem cell (hMSC) morphology, orientation, metabolic activity, and ligamentous differentiation were investigated. Results showed that increasing the XG composition (Col:XG 1:1) enhanced ink viscosity and yielded scaffolds with good print fidelity but poor collagen alignment. On the other hand, use of inks with lower XG composition (Col:XG 4:1 and 9:1) together with 0.4 mg ml -1 SMP concentration yielded scaffolds with high degree of collagen alignment albeit with suboptimal print fidelity. Modulating the print flow speed conditions (2 mm s -1 ) with 4:1 Col:XG inks and 0.4 mg ml -1 SMP resulted in improved print fidelity of the collagen scaffolds while retaining high level of collagen anisotropy. Cell studies revealed hMSCs orient uniformly on aligned collagen scaffolds. More importantly, collagen anisotropy was found to trigger tendon or ligament-like differentiation of hMSCs. Together, these results suggest that 4D printing is a viable strategy to generate anisotropic collagen scaffolds with significant potential for use in tendon and ligament tissue engineering applications., (Creative Commons Attribution license.)