(367c) Fabrication of Highly Aligned Collagen Sponges from Self-Assembled, Fibrillar Collagen Gels

Collagen is an attractive biomaterial for many tissue engineering applications because it is biocompatible, supports cellular infiltration and growth, and can be remodeled through natural enzymatic pathways. In addition, collagen has the potential to direct regeneration when aligned fibers provide contact guidance fields. The ability to control cellular alignment and orientation is a critical design factor for scaffolds of many tissue types including nerves and smooth muscle. Alignment of fibrillar collagen has most often been generated during self-assembly with external potential fields, or post self-assembly by external mechanical forces or by cellular traction. Fiber alignment can also be generated via electrospinning, although native collagen fiber architecture is not preserved. We have developed a simple method for fabrication of highly aligned collagen sponge-like scaffolds using freezing sublimation. Unlike other approaches where collagen is not driven to self-assemble but rather is frozen as a suspension, and aligned pores are generated via a temperature gradient, our approach generates alignment from self-assembled fibrillar gels. In this study, we have characterized the architecture and fabrication parameters of our novel aligned collagen fiber-like scaffolds and characterized their ability to direct and enhance the outgrowth of neural cell explants.

 Methods

 Scaffold Fabrication: Highly aligned collagen sponge-like scaffolds were fabricated from self-assembled, fibrillar collagen hydrogels. Briefly, type-I bovine collagen (Elastin Products Company, Owensville, MO) was reconstituted in 0.02N acetic acid at 3.0 mg/mL. Buffered hydrogel solutions were prepared using the following ratios: 20 µL HEPES:130 µL 0.15 N NaOH:100 µL 10x MEM: 56 µL M199: 10 µL L-glutamine: 10 µL penicillin/streptomycin: 677 µL 3.0 mg/mL collagen solution. Cylindrical silicone conduits of various diameter (2380 μm, 3175 μm, 4762 μm, and 6350 μm) and aspect ratio were filled with hydrogel solution and  incubated at 37ºC for 1 hour to allow self-assembly. Conduits were then moved into a -80°C freezer for 3 hours followed by lyophilization overnight.

Parameter Variation in Scaffold Fabrication: Variations to this scaffold fabrication process were made to evaluate the effect of freezing temperature and freezing rate. In the freezing temperature study, silicone conduits were filled and self-assembled as described previously, and placed in a -80°C or -20°C for three hours or snap frozen in liquid nitrogen. To examine the effect of a reduced freezing rate, scaffolds were frozen in a Cryo 1°C Freezing Container (Nalgene) at either -20°C or -80°C. To fabricate randomly aligned collagen sponge control scaffolds, hydrogels were prepared as described as above and placed in 15.6mm diameter wells in a 24 well plate, self-assembled at 37ºC for 1 hour, frozen at -80ºC, and lyophilized overnight.

Scanning Electron Microscopy: Non-hydrated scaffolds were prepared as described above and then sputter coated (SCD 004, Balzers Union Limited, Balzers, Liechtenstein) with gold/palladium and their surface topography imaged via SEM (Amray 1830I, Amray Inc. Bedford, MA). Hydrated scaffolds were prepared as described above and then incubated in phosphate buffered saline at 37°C for 1 hour. Scaffolds were then dehydrated in a series of aqueous acetone solutions (25, 50, 75, 95, and 100%) for 15 minutes each and left in 100% acetone overnight. Samples were critical point dried (CPD 020, Balzers Union Limited, Balzers, Liechtenstein), and sputter coated with gold/palladium and imaged via SEM.

Neural Explant Culture: Dorsal root ganglia (DRG) were isolated from pathogen-free chick eggs at embryonic day 8 (Charles River Laboratories, Cambridge, MA) and cultured in DMEM containing 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin supplemented with 100ng/mL nerve growth factor. DRGs were plated on aligned sponge-like collagen scaffolds as well as randomly aligned collagen sponge scaffolds and maintained in culture for 3 days.  

ImmunocytochemistryCultures were fixed in 4% paraformaldehyde solution and then stained immunohistochemically for neurofilament heavy chain (NFM200) and observed with confocal fluorescent microscopy.

Results and Discussion

SEM imaging revealed that  fabricated collagen sponges demonstrated highly aligned collagen fiber-like architecture with long range uniformity. Scaffolds appear as a cable-like bundle of aligned fiber-like structures. Further, cross-sections revealed a highly porous interior within each fiber-like structure.  Fabricated scaffold diameter demonstrated a strong dependence on the diameter of the silicone conduits used for fabrication. Scaffolds fabricated in 2380 μm, 3175 μm, 4762 μm, and 6350 μm silicone conduits had average diameters of 515 μm, 680 μm, 1560 μm, and 2200 μm respectively, shrinking 20-35% the size of the initial cylindrical hydrogel.  Upon hydrating the scaffolds, the diameter of scaffolds fabricated in 2380 μm, 3175 μm, 4762 μm, and 6350 μm silicone conduits swelled to 767 μm, 1094 μm, 2107 μm, and 2756 μm respectively. Additionally, the diameter of the individual fiber-like structures that comprise the scaffold also varied with silicone conduit diameter. Average fiber size for scaffolds fabricated in 2380μm, 3175μm, 4762μm, and 6350μm silicone conduits were 62 μm, 71 μm, 103 μm and 192 μm respectively. Scaffolds with similar fiber-like morphology were obtained at each of the three freezing temperatures examined (LN2, -20°C freezer, and -80°C freezer). Reducing the freezing rate to 1°C/min had no observable effect on fiber or scaffold diameter; however it did seem to smooth the edges of the fiber-like features within the scaffolds as compared to scaffolds which were placed directly in the freezer. SEM imaging of the hydrated scaffolds revealed that the fiber-like topography and the porous interior of the scaffolds was maintained following hydration. Aligned collagen sponge-like scaffolds scaffolds supported the growth of axons from chick embryo dorsal root ganglia, and the directional growth was enhanced over that within random collagen sponge scaffolds.  This fabrication method to generate highly aligned collagen sponge-like scaffolds can be used to guide axon outgrowth or Schwann cell migration. Overall, this methodology offers rapid production of aligned collagen fibers that retain the strength and biocompatibility compared to current collagen-based scaffold techniques without the need for specialized equipment. Through specific variation of fabrication parameters, the size and topography of resulting scaffolds can be tuned to suit a variety of needs and applications.

 Conclusion

 Herein we present a novel method for the generation of highly aligned collagen fiber structures. Aligned collagen fiber scaffolds fabricated through this simple, rapid technique have the potential to serve as building blocks for regenerative medicine strategies across a variety of tissue types where tissue anisotropy is critical to functional outcome, such as engineering tendons and ligaments or nerve guidance conduits.

Acknowledgements

This work is supported by National Institutes of Health grant NIH-NINDS 1R01NS078385