Available on-demand - F.SM06.03.04
Impact of Drying Time and Temperature on Lyophilized Silk Fibroin Scaffold Structural Properties
Alycia Abbott1,Mattea Gravina1,Jeannine Coburn1
Worcester Polytechnic Institute1
Lyophilized silk fibroin (silk) scaffolds have been used to develop 3D models for multiple disease states.1 Silk is a natural, biocompatible, FDA approved biomaterial that allows a high degree of control over physical properties.2 Lyophilization of different silk solutions produces porous scaffolds with varying pore sizes and Young’s moduli.3 However, the effect of varying the drying time and temperature during lyophilization on pore size, pore shape, and bulk modulus is not well described. Understanding the impact of drying time and temperature conditioning on silk scaffold structural properties will allow better definition of lyophilization protocols and tailoring of silk scaffolds to specific disease states.
Silk fibroin was extracted from B. Mori silkworm cocoons as previously described.1 Solutions of 3, 6, 9, and 12% silk (w/v) were created by concentrating silk solution or diluting with water. Rate-controlled lyophilization cycles were used to create porous scaffolds (15.6 mm diameter). Primary drying times (18 h – 1 week) and drying temperatures (-45°C to -15°C) were explored to determine the impact on pore size, pore shape, and Young’s modulus. Shelf and sample temperatures were recorded during lyophilization. Pores were evaluated through fluorescent and scanning electron microscopy of scaffolds cut into 200 µm thick sections. An ImageJ protocol was developed to evaluate pore size and shape. Scaffolds were hydrated (impact on secondary structures was examined through FTIR), biopsied (6 mm) and compressed between two parallel plates. The Young’s modulus of the scaffolds was determined through uniaxial compression testing on an Instron (2mm/min to 50% strain). Young’s modulus was calculated from the stress-strain curve.
Not all lyophilization runs produced viable scaffolds from all silk concentrations. Higher silk percentages (9 and 12%) were shown to require longer drying times than lower silk percentages (3 and 6%). A gradient of pore sizes was seen throughout scaffolds with smaller pores being present in the outermost regions. Pore size was non-normally distributed when imaged at low magnifications (4x) but was normally distributed at high magnifications (500x) suggesting multiple scales may be important for pore evaluation. Due to low average circularity values, Feret diameter, area, and perimeter were used to compare scaffolds instead of treating pores as spheres. Preliminary data suggests silk concentration had a greater impact on pore size and shape than drying time and temperature. Young’s moduli values increased as silk concentration increased in agreement with previous literature.3 Young’s moduli values for each silk concentration were significantly different from each other silk concentration (p < 0.01). Scaffolds fabricated from the same silk concentration but with stepwise temperature drying had significantly lower Young’s moduli (6%, p<0.001; 12%, p < 0.0001) than scaffolds dried at a constant temperature.
The impact of lyophilization parameters on Young’s modulus, and pore size and shape for four different silk formulations were evaluated. The Young’s moduli of scaffolds increased as silk concentration increased. Drying temperature significantly affected the Young’s moduli of scaffolds with the same silk concentration. Changes in silk concentration had a larger impact on pore size and shape than lyophilization parameters, although analysis is still ongoing. These results begin to demonstrate the impact of drying time and temperature on silk scaffold properties. This knowledge can be used to tailor lyophilization protocols to produce silk scaffolds with specific properties, such as a soft matrix with a desired pore range for tissue engineering applications.
1.D.N. Rockwood, Nat Protoc.6,1612-31 (2011). 2. J.E. Brown, Adv Healthc Mater.6, 1600762(2017) 3. M. Amirikia, Biologicals.57,1-8(2019)