Topotactic fibrillogenesis of freeze-casted microridged collagen scaffolds for 3D cell culture

Type I collagen is the main component of the extra-cellular matrix (ECM). In vitro, under a narrow window of physico-chemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state. With the help of the ice-patterned micro-ridge features, Normal Human Dermal Fibroblasts and C2C12 murine myoblasts successfully migrate and form highly-aligned populations within the resulting 3D biomimetic collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore, and are of particular interest for biomedical applications where large scale 3D cell alignment is needed such as for muscular or nerve reconstruction.

💡 Research Summary

This paper addresses a central challenge in collagen‑based biomaterials: achieving native‑like fibrillogenesis while simultaneously shaping the material into a macroporous scaffold without denaturing the protein or relying on chemical cross‑linkers. The authors combine directional freeze‑casting with a novel topotactic conversion step that uses ammonia vapour to trigger fibril formation within the ice‑templated architecture.

First, a 40 mg mL⁻¹ solution of acid‑soluble type‑I collagen (extracted from rat tail tendons) is poured into cylindrical molds and frozen at a controlled rate of –5 °C min⁻¹ from 20 °C down to –60 °C. The directional solidification creates lamellar pores aligned with the temperature gradient and, importantly, a periodic micro‑ridge (micro‑ridge) pattern on the pore walls with spacings ranging from ~7 µm at the bottom to ~18 µm at the top of the scaffold.

The frozen constructs are then placed at 0 °C for 48 h in an atmosphere saturated with 30 % aqueous ammonia. Ammonia vapour raises the local pH at the ice–collagen interface while simultaneously depressing the freezing point of the water‑ammonia mixture, causing the ice front to recede slowly. This controlled melting provides the necessary neutral pH for collagen self‑assembly and, because the collagen is confined between receding ice crystals, the newly formed fibrils inherit the pre‑existing ice‑templated geometry—a process the authors term “topotactic fibrillogenesis.” After the ammonia step, the samples are transferred to a humid 37 °C environment for 24 h, then immersed in 5× PBS for at least two weeks to complete fibril maturation at physiological pH.

Structural characterization shows that the resulting scaffolds retain the original lamellar pores and micro‑ridges, now filled with densely packed collagen fibrils. Second‑harmonic generation (SHG) imaging reveals strong signals characteristic of aligned fibrillar collagen, confirming the preservation of orientation across scales. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrate two distinct fibrillar domains: tightly packed nanofibrils (≈10–30 nm) and larger fibrils displaying the classic 67 nm D‑band periodicity. Birefringence under crossed polarizers further validates the ordered nature of the ridge structures.

Mechanical testing in PBS (wet state) on longitudinal slices shows a markedly improved performance compared with previously reported non‑cross‑linked collagen foams. The scaffolds exhibit an average Young’s modulus of 33 ± 12 kPa, ultimate tensile strength of 33 ± 6 kPa, and can sustain strains up to 105 ± 28 % before failure, with a characteristic multi‑step fracture pattern indicating delamination between lamellae. These values place the material in the same stiffness range as native soft tissues such as muscle or dermis, a significant advance over the sub‑kilopascal stiffness of water‑soluble collagen gels.

Biological evaluation focuses on two cell types. Normal Human Dermal Fibroblasts (NHDF) seeded on whole scaffolds proliferate over three weeks, forming a dense surface layer that aligns along the freezing direction, mirroring the underlying ridge orientation. C2C12 murine myoblasts are cultured for 11 days in proliferation medium followed by 4 days in differentiation medium. After fixation, fluorescence staining (DAPI, phalloidin, Myosin‑specific antibodies) combined with SHG imaging shows that myotubes extend longitudinally along the micro‑ridges, indicating that the physical cues provided by the scaffold guide both migration and differentiation.

The study’s novelty lies in (i) the use of ammonia‑induced topotactic fibrillogenesis to convert a freeze‑cast, non‑cross‑linked collagen foam into a water‑stable, fibrillar scaffold; (ii) the preservation of a hierarchical architecture—from nanometer‑scale fibrils to micrometer‑scale ridges to millimeter‑scale lamellar pores—without additional processing steps; and (iii) the demonstration that this architecture can direct cell alignment and support myogenic differentiation without any chemical cross‑linkers or surface functionalization.

Overall, the work provides a scalable, chemically simple route to fabricate collagen scaffolds that combine high mechanical integrity, macroporosity, and intrinsic cell‑guiding topography. Such scaffolds are especially promising for tissue engineering applications requiring large‑scale 3D cell alignment, such as skeletal muscle repair, peripheral nerve regeneration, or tendon/ligament reconstruction. Future directions include incorporating bioactive cues (growth factors, peptides), testing in vivo integration and vascularization, and extending the method to other extracellular matrix proteins or composite systems.