Increasing the strength and bioactivity of collagen scaffolds using customizable arrays of 3D-printed polymer fibers

Abstract

Tendon is a highly aligned connective tissue which transmits force from muscle to bone. Each year, people in the US sustain more than 32 million tendon injuries. To mitigate poor functional outcomes due to scar formation, current surgical techniques rely heavily on autografts. Biomaterial platforms and tissue engineering methods offer an alternative approach to address these injuries. Scaffolds incorporating aligned structural features can promote expansion of adult tenocytes and mesenchymal stem cells capable of tenogenic differentiation. However, appropriate balance between scaffold bioactivity and mechanical strength of these constructs remains challenging. The high porosity required to facilitate cell infiltration, nutrient and oxygen biotransport within three-dimensional constructs typically results in insufficient biomechanical strength. Here we describe the use of three-dimensional printing techniques to create customizable arrays of acrylonitrile butadiene styrene (ABS) fibers that can be incorporated into a collagen scaffold under development for tendon repair. Notably, mechanical performance of scaffold-fiber composites (elastic modulus, peak stress, strain at peak stress, and toughness) can be selectively manipulated by varying fiber-reinforcement geometry without affecting the native bioactivity of the collagen scaffold. Further, we report an approach to functionalize ABS fibers with activity-inducing growth factors via sequential oxygen plasma and carbodiimide crosslinking treatments. Together, we report an adaptable approach to control both mechanical strength and presence of biomolecular cues in a manner orthogonal to the architecture of the collagen scaffold itself.

Statement of significance: Tendon injuries account for more than 32 million injuries each year in the US alone. Current techniques use allografts to mitigate poor functional outcomes, but are not ideal platforms to induce functional regeneration following injury. Tissue engineering approaches using biomaterial substrates have significant potential for addressing these defects. However, the high porosity required to facilitate cell infiltration and nutrient transport often dictates that the resultant biomaterials has insufficient biomechanical strength. Here we describe the use of three-dimensional printing techniques to generate customizable fiber arrays from ABS polymer that can be incorporated into a collagen scaffold under development for tendon repair applications. Notably, the mechanical performance of the fiber-scaffold composite can be defined by the fiber array independent of the bioactivity of the collagen scaffold design. Further, the fiber array provides a substrate for growth factor delivery to aid healing.

Keywords: Composite CG scaffolds; Tendon tissue engineering; Three-dimensional printing; Tunable mechanics.

Fig. 1

Structurally reinforced scaffold-fiber composite. (A) Schematic for creating CG scaffolds without (left) and with (right) ABS polymeric fibers embedded within. Both contain ABS polymer end blocks to facilitate mechanical testing. (B) ABS-fiber reinforced scaffold; fibers are completely incorporated within the scaffold. (C) Representative scanning electron microscopy (SEM) image of transverse section thought fiber-reinforced scaffold showing cross-section of ABS fibers and CG scaffold. Scale bar: 200 µm. (D) ABS-fiber reinforced scaffold strained to 20% strain in tension; although the scaffold would have failed, fiber reinforcement holds the composite together well past physiological strains.

Fig. 2

Mechanical behavior of fiber-reinforced scaffolds. (A) The tensile behavior of ABS–CG composites was assessed for four discrete: scaffold alone; 2 mm sinusoidal fiber array; 1 mm sinusoidal fiber array; straight fiber array. All fiber-reinforced variants were contained 9 parallel fibers (each 1 mm diameter) connecting ABS end blocks to facilitate clamping during tensile tests. The scaffold alone variants also contained ABS end blocks to facilitate clamping. (B) Representative stress–strain curves of all four variants. (C) Diagram depicting how the mechanical properties for each sample were measured. Elastic modulus was taken as the slope of the linear elastic region of the curve. Peak stress was taken as the maximum stress the sample reached, and strain at peak stress was taken as the strain (extension divided by cross-sectional area) at this same point. Finally, toughness was taken as the area under the stress–strain curve before failure.

Fig. 3

Mechanical properties of ABS-fiber reinforced CG scaffold. Elastic modulus (A) and peak stress (B) increased significantly with increasing degree of fiber reinforcement. (C) Strain at peak stress remained unchanged with the exception of the most compliant fiber-reinforcement variant. (D) The toughness of the fiber-reinforced composites was significantly greater than the scaffold alone and increased with degree of fiber reinforcement. ^: p < 0.05; *: p < 0.01; **: p < 0.001.

Fig. 4

Covalent attachment of biomolecular cues via ABS-fibers. (A) Attachment of a model protein (BSA) to ABS substrates via sequential oxygen plasma treatment followed by carbodiimide crosslinking in the presence of fluorescently labeled BSA. Both plasma treatment then carbodiimide crosslinking are required to facilitate covalent biomolecule incorporation. (B) Representative fluorescent images of control ABS fibers (left) and BSA-functionalized ABS fibers (right). *: p < 0.001.

Fig. 5

Covalent presentation of PDGF promotes changes in cell number and metabolic activity. (A) pASCs were cultured on ABS or PGDF-functionalized ABS substrates (2D); pASCs were subsequently cultured in CG scaffolds containing ABS fiber reinforcement or PDGF-functionalized ABS fiber reinforcement. (B) The metabolic activity of pASCs was higher when cultured on PDGF-decorated ABS substrates than ABS substrate controls at each time point. In both cases, the highest metabolic activity was observed at day 4. (C) The metabolic activity of pASCs significantly increased over 7 day culture in CG scaffolds containing either ABS fibers (composite) or PDGF-decorated ABS fibers (composite + PDGF). (D) Additionally, the overall cell number was significantly higher after 7 days in CG scaffolds containing PDGF-decorated fibers. *: p < 0.05; ^: p < 0.01 compared to other timepoints for the same ABS group; ^^: p < 0.001 compared to other timepoints for the same ABS group; ** p < 0.05 compared to days 1 and 4.