Rheological characterization, compression, and injection molding of hydroxyapatite-silk fibroin composites

Abstract

Traditional bone fixation devices made from inert metal alloys provide structural strength for bone repair but are limited in their ability to actively promote bone healing. Although several naturally derived bioactive materials have been developed to promote ossification in bone defects, it is difficult to translate small-scale benchtop fabrication of these materials to high-output manufacturing. Standard industrial molding processes, such as injection and compression molding, have typically been limited to use with synthetic polymers since most biopolymers cannot withstand the harsh processing conditions involved in these techniques. Here we demonstrate injection and compression molding of a bioceramic composite comprised of hydroxyapatite (HA) and silk fibroin (SF) from Bombyx mori silkworm cocoons. Both the molding behavior of the HA-SF slurry and final scaffold mechanics can be controlled by modulating SF protein molecular weight, SF content, and powder-to-liquid ratio. HA-SF composites with up to 20 weight percent SF were successfully molded into stable three-dimensional structures using high pressure molding techniques. The unique durability of silk fibroin enables application of common molding techniques to fabricate composite silk-ceramic scaffolds. This work demonstrates the potential to move bone tissue engineering one step closer to large-scale manufacturing of natural protein-based resorbable bone grafts and fixation devices.

Keywords: Bone grafts; Compression molding; Injection molding; Orthopedic implants; Silk-ceramic composites.

Figure 1.. HA-SF processing and characterization.

A. Processing of raw silk fibers from B. mori cocoons via degumming and fiber hydrolysis to produce aqueous regenerated silk fibroin (SF) solution which is later lyophilized and ground into powder. B. Scanning electron microscopy images of hydroxyapatite (HA) and SF powders (processed with either 30 minutes or 60 minutes of boiling during degumming). Scale bars are 10 μm in the HA image and 200 μm in the SF images. C. Particle size distribution of HA and SF powders.

Figure 2.. Shear behavior of HA-SF slurries.

A. Representative curve showing shear thinning behavior at all three P/L ratios of HA-SF slurries made with 10 wt.% 60-mb SF. B. Magnification of the low shear rate regime (boxed region: 0 to 40 s⁻¹) of shear thinning curves for 30- and 60-minute boil SF with varying P/L ratios (0.5, 0.6, and 0.7 ratio of HA:SF) and 1, 10, and 20 wt.% SF content (n = 3, error bars are SD).

Figure 3.. Effects of SF content, SF molecular weight, and P/L ratio on HA-SF slurry viscosity.

A. Influence of SF molecular weight (as a result of silk fibroin degumming time) on the viscosity of HA-SF slurries over a range of three different powder-to-liquid (P/L) ratios. B. Influence of P/L ratio on HA-SF slurry viscosity. Statistical analysis performed with two-way ANOVA with Bonferroni posttest (*** p < 0.001, n = 3, error bars are SD).

Figure 4.. Dynamic rheological behavior of HA-SF slurries.

A. Representative curves from frequency sweep testing (0.1 to 100 rad/s) on HA-SF slurries performed at 0.1% strain showing differences in G’ and G” as a result of SF content. B. Differences in dynamic rheological behavior of HA-SF slurries as a result of SF content and molecular weight. C. Effects of SF content on storage modulus of HA-SF slurries. D. Effects of P/L ratio on the storage modulus of HA-SF slurries. Statistical analysis performed with two-way ANOVA with Bonferroni posttest (** p < 0.01, *** p < 0.001, n = 3, error bars are SD).

Figure 5.. HA-SF composite scaffold characterization.

A. Images of compression molded HA-SF scaffolds with 20, 10, and 1 wt.% SF content made with 60-mb SF at a P/L ratio of 0.7 along with corresponding scanning electron microscopy images of fractured surfaces (arrows indicate SF sheets interwoven within the ceramic matrix). Mechanical testing was performed to evaluate the compressive modulus (B), yield strain (C), and yield stress (D) of the HA-SF scaffolds. Statistical analysis performed with two-way ANOVA with Bonferroni posttest (* p < 0.05, ** p < 0.01, n = 6, error bars are SD).