Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue

Abstract

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

Keywords: bioprinting; chondroitinase ABC; collagen; melt electrowriting (MEW); meniscus.

Figure 1

Development of the guiding microchamber system and inkjet bioprinting. (a, b) Stereomicroscope and SEM images of the MEW scaffold. Scale bars are equal to 800 μm. (c) Live/dead images (z-stacks) at day 7 after the inkjet step. Scale bar is equal to 800 μm. (d) SEM images of the cells interacting with the MEW scaffold. Scale bar is equal to 20 μm.

Figure 2

Biochemical properties of the engineered cartilage tissue following 5 weeks of in vitro culture. Quantification of (a) DNA, (b) sGAG, and (c) collagen. (d) Collagen/sGAG ratio. (e) sGAG and (f) collagen content normalized to the amount of DNA per construct. All error bars denote standard deviation, and significance was considered p < 0.05, n = 4.

Figure 3

Histological analysis of the engineered cartilage tissue following 5 weeks of in vitro culture. Stained for alcian blue (sGAG), alizarin red (Calcium), collagen type I, II, and X. Scale bar is equal to 800 μm.

Figure 4

Collagen organization within the engineered tissues following 5 weeks of in vitro culture. (A) Picrosirius red staining, polarized light, and color map imaging of collagen fiber distributions. Scale bar is equal to 800 μm. (B) Collagen fiber directionality. (C) Fiber coherency quantification, where a value of 1 indicates fibers are aligned in the same direction, while a value of 0 indicates dispersion of fibers in all directions. All error bars denote standard deviation.

Figure 5

SEM analysis of the collagen organization within the engineered tissues. (A) Representative SEM images. Scale bar is equal to 5 μm on the left, and 1 μm on the images on the right. (B) Quantification of the collagen fiber diameter. All error bars denote standard deviation, significance was considered p < 0.05.

Figure 6

Biomechanical properties of the engineered tissues following 5 weeks of in vitro culture. (A) Ramp modulus. (B) Equilibrium modulus. (C) Dynamic modulus. (D) Tensile modulus. All error bars denote standard deviation, significance was considered p < 0.05, n = 4 for the compression tests and n = 3 for the tensile samples.