Associate Professor Stony Brook University, United States
Introduction: : Engineering collagen constructs that replicate the mechanical strength and biological functionality of load-bearing tissues like tendon remains a significant challenge. This study establishes a scalable and accessible biofabrication method that combines the precision of a commercial 3D bioprinter with the physicochemical principles of molecular crowding. Using an acidic collagen solution in combination with a hypertonic polyethylene glycol (PEG) bath, we systematically investigate how modulating pulling speed and nozzle diameter dictates the final filament properties. Our results demonstrate that faster pulling speeds and smaller nozzle diameters produce thinner, more densely packed filaments. High-resolution scanning electron microscopy (SEM) and polarised light microscopy reveal a tuneable transition in surface morphology, from a disordered isotropic state to a highly ordered phase, quantitatively confirmed by a sharp increase in birefringence. Critically, we demonstrate the preservation of collagen's native, periodic 66 nm d-banding, an essential motif for cell interaction. This structural refinement translates to a dramatic enhancement of mechanical performance, with the strongest filaments achieving an ultimate tensile strength of 0.64 GPa and a Young's modulus of 8.9 GPa, values that surpass native tendon. Furthermore, the fabricated filaments are highly biocompatible and bio-instructive, promoting robust alignment and elongation of mesenchymal stem cells. This work provides a direct link between bioprocess parameters and the resulting hierarchical structure, offering a versatile platform for fabricating high-performance, biomimetic materials for regenerative medicine.
Materials and
Methods: : Collagen type I filaments were fabricated using a custom pulling technique implemented on a commercial 3D bioprinter. A commercially available Type I collagen solution was extruded through nozzles of varying diameters into a concentrated polyethylene glycol (PEG) coagulation bath, which induces rapid fibrillar self-assembly. Key fabrication parameters, including pulling speed and nozzle diameter, were systematically varied to produce a library of filaments with tuneable properties. The resulting filaments were comprehensively characterised. Mechanical properties were quantified via uniaxial tensile testing to determine ultimate tensile strength, Young’s modulus, and toughness. Structural and morphological features were assessed using scanning electron microscopy (SEM) to analyse fibril organisation and confirm the presence of s-banding. Molecular alignment and compaction were further evaluated using polarised light microscopy. For biological evaluation, murine mesenchymal stem cells (mMSCs) were seeded onto the filaments. Cell viability, morphology, and alignment were then quantified using immunofluorescence staining and confocal microscopy.
Results, Conclusions, and Discussions:: Our fabrication platform demonstrated remarkable tunability, with results showing a direct correlation between process parameters and filament properties. Faster pulling speeds and smaller nozzle diameters consistently produced thinner, more compact filaments. The dimensional change was coupled with a profound structural refinement at the micro- and nano-scale. Scanning electron microscopy and polarised light microscopy revealed a transition from a disordered, isotropic fibril arrangement at low pulling speeds to a highly aligned, nematic-like appearance in filaments produced at high speeds. This structural hierarchy directly translated to a dramatic enhancement in mechanical performance. Our most refined filaments achieved a Young's modulus of up to 8.9 GPa and an ultimate tensile strength of 0.64 GPa, values that significantly exceed those of native tendon tissue. Crucially, our gentle aqueous-based method preserved the collagen's native, periodic 66 nm d-banding—a critical bio-instructive motif often lost in other fabrication techniques. The biological significance of this structural fidelity was confirmed in cell culture experiments. The highly aligned filaments were shown to be powerfully bio-instructive, guiding mesenchymal stem cells to adopt a highly elongated, aligned morphology parallel to the filament axis. In conclusion, this work establishes an accessible bioprinting strategy to produce collagenous filaments that uniquely combine mechanical properties superior to native tissue with preserved, essential biological motifs. This platform represents a significant advance for engineering mechanically robust and functional scaffolds for load-bearing tissue applications.
