Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Summary

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Abstract

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

Introduction

The goal of regenerative medicine is to enhance tissue regeneration and healing through the application of engineered therapeutics. One approach involves the fabrication of biomaterial constructs that closely emulate the composition and structure of native tissues. When designing these constructs, a critical consideration is the native underlying extracellular matrix (ECM), which provides instructional cues for cell proliferation, migration, and differentiation, ultimately driving tissue regeneration^1,2. The native ECM is predominantly comprised of fibrillar collagen^3,

Protocol

1. Preparation of dialyzed collagen ( Figure 1)

1. Cut dialysis tubing to a length of approximately 3 inches. Use tubing with the following specifications: 32 mm flat width, 20 mm inflated diameter, and 4.8 nm pore size.
2. Rehydrate dialysis tubing in ultra-pure water.
3. Clip one end of the tubing with a dialysis tubing clip.
4. Transfer 5-6 mL of rat tail collagen-type I (concentration: 9-10 mg/mL in 0.02 N acetic acid) into .......

Discussion

The critical steps of the protocol can be distilled into three main parts: 1) collagen fibrillogenesis, 2) hydrogel mounting, and 3) washing. The process of collagen fibrillogenesis occurs spontaneously under neutralizing conditions and relies on the self-assembly of individual collagen molecules into larger stabilized fibrillar structures^10,11. This is achieved through the application of a neutralizing buffered medium like 10x PBS that is warmed to 37 °C an.......

Materials

Name	Company	Catalog Number	Comments
Collagen I, High Concentration, Rat Tail	Corning	CB354249
Eclipse TE-2000-U microscope	Nikon	TE-2000-U	Inverted microscope
Extra Thick Microscope Slides	Fisher Scientific	22-267-005	Works well for collagen extrusion surface
FEI Helios G3 NanoLab DualBeam	Thermo Fisher Scientific	N/A	Scanning electron microscope
Glass Cutter Tool Set 2mm-20mm Pencil Style Oil Feed Carbide Tip	Amazon/MOARMOR	B07Y1D243H	Option for a glass cutter
Hamilton Kel-F Hub Blunt Point Needles (Luer Lock, 22 G)	Fisher Scientific	14-815-574	Needles for collagen extrusion
Nexterion Slide H 3-D	Schott	NC0782819	Hydrophobic slides
PBS Tablets	Thermo Fisher Scientific	18912014
Polyethylene Glycol 8000	Fisher BioReagents	BP233-1
Seamless Cellulose Dialysis Tubing	Fisher Scientific	S25645G
SnakeSkin Dialysis Clips	Thermo Fisher Scientific	PI68011
Synthware Glass Cutter	Synthware	31-501-927	Option for a glass cutter