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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.


Introduction
The fibrillar state of collagens is a key parameter for the mechanical properties of extracellular matrices and their interaction with cells. Being able to achieve fibrillogenesis in tissue-mimicking scaffolds is therefore of paramount importance to develop new materials for tissue engineering applications. In vivo, the synthesis and assembly of type I collagen into organized fibrillar structures is mediated by cells. In accordance with the local requirements of the tissue, collagen biosynthesis results in remarkably different supra-fibrillar architectures, ranging from highly-aligned domains in tendon to cholesteric arrangement of fibers in the bone. Reproducing these architectures in vitro requires a thorough control over the physicochemical conditions before, during and after fibrillogenesis. 1 However, the assembly of collagen fibers into ordered domains in the micron range is in many cases insufficient as scaffolds also need a precise structuration at the macroscopic scale. To date, a vast range of techniques have been proposed to shape fibrillar collagen into dense materials, such as extrusion, 2 electrochemical alignment, 3 cell-mediated gel compaction 4 and plastic compression, 5 among others, with encouraging results already obtained in vivo.
Still, these techniques often prove to be unsuitable for additional internal structuration of the materials. In particular porosity, a key morphological feature in biomaterials as it determines diffusion of nutrients and waste and can promote cell guidance, remains difficult to control, except by top-down 3D methods. 6 In this context, freeze-casting, a technique initially developed for the structuration of colloidal suspensions for the elaboration of ceramic materials, 7 has been adapted to produce scaffolds based on collagen and other biopolymers for tissue engineering purpose. [8][9][10] This technique enables to create oriented macroporous scaffolds from colloidal suspensions or polymer solutions by means of directional freezing followed by ice sublimation. 11 Upon anisotropic cooling, an oriented segregation occurs between the newly-formed ice and the solutes and/or particles initially present in the solution/suspension. Tuning of pore sizes and choice between isotropic or anisotropic global structures with anisotropic substructures can be achieved by playing with different parameters such as solution composition, freezing kinetics and temperature gradient. 12 For collagen-based scaffolds, static freeze-casting of type I collagen solutions with concentrations between 5 and 20 mg/mL [13][14][15][16] led to porosities ranging from 20 µm 16 to 200 µm at the upper extremity of the scaffold 15 . While these works have been successful in building porous materials out of acid soluble type I collagen, they have failed in inducing fibrillogenesis of the obtained porous collagen materials. As a result, the obtained scaffolds were water-soluble and required subsequent cross-linking, either by physical 13,17 or chemical 18,19 methods. The stiffness of the resulting materials usually ranges between a few kPa to a few hundreds of kPa in dry state but only between a few hundreds of Pa to a few kPa following rehydration. 20,21 As an alternative, fibrillogenesis of collagen can be induced before freezing. Using un-controlled freezing process in cylinders with a high aspect ratio, Lowe et al.
demonstrated that comparable mechanical properties could be achieved in non-crosslinked fibrillar collagen aligned materials compared to crosslinked non-fibrillar scaffolds. 22 Fibrillogenesis prior to freezing was also used by Murray et al. in their successful clinical test to avoid risks associated with the use of chemical cross-linkers. [23][24][25] However, so far, no fibrillar macroporous collagen material presenting a controlled architecture at multiple length scales and of oriented porosity has been achieved.
In this work we have developed a new method to trigger type I collagen fibrillogenesis after freezecasting, coupling the macroporous structure induced by the ice crystals to the self-assembly of the collagen molecules into fibers. In our topotactic process, ammonia vapors play a double role: while slowly melting ice crystals it induces fibril formation in the collagen domains condensed in between ice crystals.
The obtained scaffolds present highly-organized suprafibrillar architecture and improved mechanical properties compared to non-fibrillar crosslinked collagen scaffolds reported in the literature. 20,21,26 These 3D scaffolds promote the proper alignment of C2C12 myoblasts and of Normal Human Dermal Fibroblasts in vitro and thus act as new 3D culture biomimetic environments with cell guiding properties.

Materials and Methods
Collagen solution. Collagen I was extracted from young rat's tail tendons. After thorough cleaning of tendons with PBS 1X and 4M NaCl, tendons were dissolved in 3mM HCl. Differential precipitation with 300mM NaCl and 600mM NaCl, followed by redissolution and dialysis in 3mM HCl achieved high collagen purity as confirmed by SDS PAGE. Final collagen concentration of 3.7±0.3 mg/mL was determined using hydroxyproline titration. Acid-soluble type I collagen stock solution was aliquoted and stored at 4°C. Prior to use, stock solution was centrifuged at 3000xg at 10°C in Vivaspin® tubes with a 300 kDa filter cut-off to reach a final concentration of 40 mg/mL. Freeze-casting. Concentrated collagen solutions (0.2 mL, 40 mg/mL) were poured into cylindrical molds.
After centrifugation each mold was individually freeze-cast on a home-made freeze-casting setup made of a copper rod partially immersed in liquid nitrogen, with a heating coil and a thermocouple to monitor the temperature of the cooled plate as previously described ( Figure 1A). 27 The collagen solution was cooled at a -5°C.min -1 rate from 20 to -60°C. The final temperature was kept at -60°C until the sample was entirely frozen. Samples were then kept in freezer at -80°C until further use.
Collagen fibrillogenesis and sample preparation. Fibrillogenesis of collagen was performed in different steps. First, the frozen samples were kept at 0°C under ammonia vapors for 48 h to ensure prefibrillogenesis induced by the pH increase. As ammonia vapors reach the ice/collagen surface, ice starts to melt thanks to the freeze point depression of the water/ammonia liquid mixture that is formed at the interface. Throughout the 48h contact time, where the sample is kept in a closed vessel of around 350mL with approx. 10 mL of 30 % ammonia solution, the ice front progressively recedes and the whole sample is exposed to ammonia. Samples (equivalent to approximately 0.2 ml initial volume) were then transferred to tubes floating on 2L water at 37°C and kept for 24h to remove ammonia. Samples were subsequently dipped into 40mL PBS 5X to complete fibrillogenesis at neutral pH for at least 2 weeks. The resulting scaffolds were 12 to 15 mm long with a diameter between 5 to 6 mm.

Ice-templating of macroporous collagen scaffolds
The home-built freeze casting set-up enabled to control the growth of ice crystals in 40 mg.mL-1 type I collagen solutions and thus to successfully template the resulting scaffold. After lyophilization, a porous scaffold with lamellar pores oriented along the freezing direction and presenting a gradient of porosity was obtained ( Figure 1B). At the bottom of the scaffold, pores were arranged in small domains of parallel sheets 25 to 95 µm wide (10 th -90 th percentile) ( Figure 1B bottom right, Figure 1C orange), randomly distributed in 2D (Supporting Information Figure S1A,C). Following ice growth direction, the domains of parallel sheets broadened (Supporting Information Figure S1B,E) and pore major axis increased between 50 and 270 µm (10 th -90 th percentile), with some pores reaching up to 1.3 mm ( Figure 1B top right, Figure   1C violet). A substructure made of parallel ridges also ran along the same direction of the scaffold ( Figure   1D,E). The ridges only appeared on one side of a wall and were periodically spaced, with the period ranging from 6.9 to 10.0 µm (10 th -90 th percentile), at the bottom, to 14.5 to 18.9 µm (10 th -90 th percentile) at the top of the scaffold ( Figure 1F).  Figure S3A) as opposed to dipping freeze-casted scaffold directly after step 1 into a neutral buffer such as PBS 10X that resulted in occluded pores (Supporting Information Figure   S3B).

Scaffold morphology and mechanics
The opaque water-stable self-standing scaffolds obtained at the end of the above-described process were Mechanical properties were measured performing tensile tests in PBS 1X on longitudinal slices from different samples. Figure 5A displays a typical stress vs. strain curve with corresponding photos of the sample in Figure 5B. The sample undergoes a linear deformation ( Figure 5B1,2) to approximately 105±28 % strain and 33±6 kPa stress in average (Table 1) with a Young modulus reaching 33±12 kPa. After this point, it finally cracks at several levels provoking the delamination of the sample (Figure 5B3,4). Strain at failure (%) N=4 105 28

Normal Human Dermal Fibroblasts
Normal Human Dermal Fibroblasts were seeded and cultured on entire scaffolds to observe cell colonization ( Figure 6A). After three weeks in culture, they showed high proliferation on the scaffold surface, forming a dense layer of cells. Most cells seemed aligned along the principal direction (direction of freezing) as shown on Figure 6A1. When observing the outside of a thin longitudinal upper slice, cells are aligned in the same direction as the ridges ( Figure 6A2). This is confirmed by confocal microscopy coupled with SHG, as seen on Figure 6A3 and on Supporting Information Movie S1A, where cells, which actin filaments are labeled, all adopt a fusiform shape aligned along the main direction of the scaffold.
Recovering internal longitudinal slices of the cellularized scaffolds allowed for the study of deeper colonization. A first and most obvious cell population originated from cell migration through the open pores at each extremity of the scaffold (Figure 6A4 top left). Cells migrated inside the scaffold over a distance up to 1.5 mm, they aligned along the main direction ( Figure 6A5) and adopted fusiform shapes with long cytoplasmic protrusions ( Figure 6A6). It was observed that a deeper colonization involving a higher number of cells occurred from the extremity with the larger pores (top of the scaffold, as displayed on Figure 1B) compared to the one with the smaller pores (bottom of the scaffold). A second type of colonization also occurred through the sides of the scaffold, resulting in a density gradient of cells toward the inner part of the scaffold (Figure 6A4 bottom) over a distance up to 500 µm. In this region, fibroblasts aligned along the main direction as shown on Figure 6A7 and on Supporting Information Movie S1B.
Confocal microscopy enabled to observe cells in the plane of a wall and to show their alignment along the ridges ( Figure 6A8, Supporting Information Movie S1C). Noticeably, when similar NHDF cultures were performed on pre-cut longitudinal slices, high proliferation and significant cell alignment were also observed ( Figure 6B1,2).

C2C12 myoblasts
Similar colonization experiments were carried out using C2C12 murine myoblast cell line. Cells were seeded on whole scaffolds for 11 days. Half of the samples were left in proliferation medium whereas the other half was supplemented with differentiation medium for the last 4 days instead of high serum concentration proliferation medium. No significant differences could be observed between the two conditions. In each case, high proliferation and alignment of myoblasts was observed outside the scaffold ( Figure 7A1,2), in the same way as with NHDF. Some parallel myotubes expressing heavy myosin chains (MF20) could sometimes be observed as indicated by immuno-staining ( Figure 7A). Same internal colonization patterns as for NHDF could also be observed from both scaffold extremities and sides but over a longer distance, reaching 2.5 mm from the extremities and 1.5 mm from sides compared to 1.5 mm and 500 µm for NHDF, respectively ( Figure 7B1,2). Some myotubes positive for MF20 were observed aligned to the main direction of the scaffold ( Figure 7C1,2). Confocal microscopy reconstitutions as shown on Figure 7D1,2 and Supporting Information Movie S2A and Movie S2B illustrate both types of colonization: from an extremity ( Figure 7D1 and Supporting Information Movie S2A), and from a side ( Figure 7D2 and Supporting Information Movie S2B). They also enable to observe the anisotropic shape of the myoblasts.

Scaffold structuration and properties
To address the current challenge of preparing scaffolds combining fibrillar collagen and oriented porosity, we have developed a new topotactic procedure to induce collagen fibrillogenesis after freeze casting and obtain scaffolds presenting anisotropic features at multiple length scales. The protocol enables to produce stable scaffolds in physiological conditions without crosslinking or dehydration process and therefore appears relevant to mimic collagen structures observed in native tissues while preserving collagen bioactivity.
A first challenge addressed was to induce the self-organization of type I collagen molecules into liquid crystal phases within the frozen materials. In collagen solutions at 3 mM HCl, isotropic/anisotropic phase transition was reported to occur at ca. 50 mg/mL. 28 Here, while the initial collagen concentration is smaller (40 mg/mL), phase segregation due to ice crystallization locally concentrates collagen above the critical value, as confirmed by the observation of liquid crystal-like organizations after ice templating. In parallel, the ice crystal growth generated ridges parallel to the main axis. These are the first reported ridges in collagen-based scaffolds obtained by directional freezing. Such features have been previously observed in porous ceramics, where they might even form bridges between lamellae. 7 Micro-ridged pores were also observed after freeze-casting chitosan solutions, with a regular spacing of approximately 10 µm. 9 These overgrown dendrites supposedly arise from local ice crystal tip splitting, due to particleinterface interactions occurring in dense solutions. In fact, this phenomenon is highly dependent on the solvent nature and was not observed when using collagen solutions prepared in acetic acid (data not shown).
Our goal was to stabilize the sample with its hierarchical organization via collagen fibrillogenesis using a topotactic process, i.e. without changing the structure and sub-structure of the walls and pores. Here, the term topotaxis strictly refers to the above definition used in the field of chemistry and not to the single-cell sensitivity to the topography gradient phenomenon introduced by Park et al., 31 which was not addressed in the present work. Dipping the freeze-casted scaffold directly into a neutral buffer such as PBS 10X resulted into a loose material with occluded pores, indicating an unfavorable competition between collagen fibrillogenesis and dissolution (Supporting Information Figure S3B). Similar results were obtained when scaffolds were lyophilized before being placed in the buffer medium. To circumvent this issue we turned our attention to a gas phase process using ammonia vapor. This strategy has been widely used to induce fibrillogenesis in concentrated type I collagen solutions at room temperature. 32,33 In our case, using ammonia vapors presents another key advantage: it lowers the freezing temperature of ice.
This freezing point depression allows to progressively achieve fibrillogenesis of the collagen within the walls of the sample kept at 0 °C (Figure 2 step 2). After 48 h under ammonia vapor, the recovered hydrogel was self-standing and translucent, suggesting that small collagen fibrils had been formed. TEM images confirmed that liquid-crystal organization was preserved during fibrillogenesis. This assumption was supported by observation of semi-thin sections under polarized microscopes showing birefringence.
Two specific patterns stand out from the observations made: the alignment of fibrils on large domains which can reach dimension above the tens of micrometers ( Figure 4C and Supporting Information Figure   S2A), and the presence of wave or arch pattern (Figure 2, Figure 4D and Supporting Information Figure   S2C). The first one would correspond to the stabilization of nematic phases while the second pattern would correspond to cholesteric or precholesteric phases. 29 Both phases have been previously observed in 13 highly concentrated collagen solutions as well as in the stabilized fibrillary state. 28,29 Comparison with the literature suggests that the high compaction of the network together with the small size of fibrils would correspond to collagen concentration higher than 300 mg/mL. 30 The following water vapor step used to rinse off NH3 and decrease the pH of the aqueous phase turned out to be paramount to preserve an open porosity. Dipping the scaffold into PBS 5X directly after exposure to ammonia vapors (step 2) resulted in a swollen translucent scaffold with occluded pores. This may be due to the fact that, since the pH of the solution remains high, collagen molecules and fibrils still bear a significant negative charge and can be destabilized by a rapid increase in ionic strength. In contrast, after the water vapor step has allowed decrease ammonia content, the scaffold can be dipped into PBS 5x without significant change in pH and thus without destabilization. This strategy further favors the growth of fibrils, as evidenced on the TEM images. Noticeably, the macroscopic aspect change of the scaffold from translucent to opaque started instantaneously and could be followed by naked eye observation for a few hours. Nevertheless, the scaffold was left in the buffer medium for 2 weeks before use to ensure complete stabilized fibrillogenesis of the scaffolds as collagen matrix can remodel for days after neutralization. 34,35 From a morphological standpoint, the macroporous materials presented here could be compared to collagen sponges. However, collagen sponge lack the fibrillary structure attained here. Nevertheless collagen sponges are still the most common form for collagen biomaterials used in clinical applications, such as the artificial skin graft Integra®. 36 These materials are usually prepared by freezing a solution of collagen and removing water by lyophilisation or by using an organic solvent. 37 In the absence of collagen fibrillogenesis, the resulting sponges are mechanically weak and soluble in water, requiring additional cross-linking steps 38 . Resulting materials have typical Young moduli of 10 kPa in the dry state that systematically decrease in the wet state. 21,39 While mechanical properties can be improved by addition of glycosaminoglycan (GAG) and additional processing steps, 20,26 the Young modulus value of 33 kPa obtained here is unprecedented for pure collagen hydrated macroporous scaffolds. This emphasizes the benefits of our described protocol that allows for the preparation of chemically-stable and mechanicallystrong collagen materials without any required additional cross-linking step. Moreover, these scaffolds can be easily optimized in terms of pore size and collagen concentration to tune their mechanical properties according to the targeted colonizing cells.

Guiding properties for the cells
The high anisotropy of the pores and the ridges substructure, parallel to the direction of freezing, makes the obtained scaffolds appealing to promote cell alignment, a feature highly sought after in tissue engineering, in particular for cells such as myoblasts, tenocytes or neurons.
In our experiments we observed an alignment of both C2C12 and NHDF cells along the main direction of the scaffold inside and outside the scaffolds. Cell alignment outside the scaffold may come from the outer grooves observed under SEM (Supporting Information Figure S4). Inside, cells colonize from both the sample extremities and the sample sides. In both cases cells align along the freezing direction. While the main migration route for cell colonization is considered to be the materials' macroporosity, it should also be possible for NHDF to migrate through the scaffold walls via collagen degradation/remodeling. 40 The partial colonization of scaffolds internal space without any physical constraints nor biochemical cues except the scaffold's oriented porosity itself to guide the cells in is highly encouraging. While migration from both ends of the material was evidenced, it was not possible to observe fully colonized channels.
This may be explained by the kinetics of colonization: here, a migration of NHDFs over ca. 500 µm was obtained after 11 days of culture, and C2C12 myoblasts could colonize the scaffolds over a three times larger distance. Comparable data for fibroblast or myoblast migration in 3D environments are scarce in the literature because colonization rate is expected to depend on many parameters including exact matrix composition and stiffness. 41 However, NHDFs seeded in diluted collagen hydrogels (2 mg.mL -1 ) within microfluidic devices were reported to migrate at rates between 20 and 200 µm per day. 42 larger pore size constructs. 46 However, tenocyte metabolic activity was reported as being more efficient with smaller pore size. 47 Thus the optimal pore size depends on cell type and freeze casting should offer the possibility to fine-tune it to the desired dimensions.
The generation of micro-ridges regularly spaced on the scaffold walls and oriented parallel to the pore main axis appears to confer an additional advantage to our process as it favors cell alignment by topographical contact guidance. A similar observation was reported for freeze-casted chitosan scaffolds which strongly favored the alignment of chick root dorsal neurites. 9 A particularly striking result we obtained is the clear alignment of NHDFs cells seeded on scaffold slices, i.e. in pseudo 2D conditions.
Indeed, surface patterning either on coated PDMS 48,49 or pHEMA 50 substrates, or from microgrooved collagen scaffolds 51 has been shown to increase muscular cell alignment and the formation of myotubes, with the possibility to optimize the spatial periodicity of the features. 48 Here the secondary guidance provided by the ridges is intrinsic to our materials and can also be optimized depending on the targeted cells.
The oriented porous collagen scaffolds presented in our study could also open the road to new strategies for the in vitro development of larger skeletal muscle 3D tissues with aligned myofibers. The production of such artificial tissues relies on cell mediated and guided gel compaction, [52][53][54] and on the transfer of aligned cell sheets into hydrogels, 55-57 among other techniques. In our study the ability of C2C12 cells to differentiate and merge into myotubes was evidenced with limited success. However, it is likely that a longer differentiation time would allow the formation of myotubes, particularly inside the scaffold where diffusion may delay the change of environment and thus differentiation. Still, this may not fully account for the low differentiation and the main hypothesis is the lack of other ECM components, such as laminin, that were shown to play a paramount role in muscle development. 58 The here-presented scaffolds could then be improved with the addition of these other extracellular matrix proteins to the initial collagen solution or by coating them on the preformed scaffold. 9,59 Pore size and collagen concentration might also direct the mechanical properties of myoblasts' microenvironment and hence their differentiation ability, which was shown to be optimal on gels with stiffness around 12 kPa. 60

Conclusion
We have described here a processing strategy that enabled to obtain highly porous and anisotropic fibrillar collagen biomaterials, with pores patterned with regularly-spaced ridges running all along the scaffold, via a topotactic transformation. The self-standing scaffolds exhibit unprecedented stiffness for macroporous pure-collagen scaffolds in the wet state, sparing the use of any cross-linking technique. They showed high bioactivity towards NHDF and C2C12 cells that can colonize these scaffolds without additional physical, chemical or biological cues. The demonstrated ability to align cells could be of significant interest for muscle, tendon or nerve regeneration. In particular, the pore size gradients generated by the freeze-casting process could be used for the colonization and controlled differentiation of different types of cells and the generation of junctions between different tissues.       Inset: close up where the walls can be observed but the pores are occluded.