Effects of agarose mould compliance and surface roughness on self-assembled meniscus-shaped constructs

Abstract

The meniscus is a fibrocartilaginous tissue that is critically important to the loading patterns within the knee joint. If the meniscus structure is compromised, there is little chance of healing, due to limited vascularity in the inner portions of the tissue. Several tissue-engineering techniques to mimic the complex geometry of the meniscus have been employed. Of these, a self-assembly, scaffoldless approach employing agarose moulds avoids drawbacks associated with scaffold use, while still allowing the formation of robust tissue. In this experiment two factors were examined, agarose percentage and mould surface roughness, in an effort to consistently obtain constructs with adequate geometric properties. Co-cultures of ACs and MCs (50:50 ratio) were cultured in smooth or rough moulds composed of 1% or 2% agarose for 4 weeks. Morphological results showed that constructs formed in 1% agarose moulds, particularly smooth moulds, were able to maintain their shape over the 4 week culture period. Significant increases were observed for the collagen II:collagen I ratio, total collagen, GAG and tensile and compressive properties in smooth wells. Cell number per construct was higher in the rough wells. Overall, it was observed that the topology of an agarose surface may be able to affect the phenotypic properties of cells that are on that surface, with smooth surfaces supporting a more chondrocytic phenotype. In addition, wells made from 1% agarose were able to prevent construct buckling potentially, due to their higher compliance.

Figure 1

Meniscus mold-maker. (A) AutoCAD drawing of the mold-maker (negative of the idealized meniscus shape) (B) Mold-maker fabricated with smooth topology using a ZPrinter 310 rapid prototyping machine (scale bar = 2 mm). (C) Mold-maker fabricated with rough topology using a Dimension 768 SST rapid-prototyping machine (scale bar = 2 mm).

Figure 2

Gross morphology and histology of constructs cultured for 4 wks. Picrosirius red, polarized light and safranin O / fast-green staining of constructs at 10X magnification.

Figure 3

Biochemical characterization of constructs at t = 4 wks. (A) Cell number per construct. Dashed line indicates original cell seeding density of ~20 million cells per construct. (B) Collagen content normalized to construct wet weight. (C) GAG content normalized to construct wet weight. (D) Collagen II per construct normalized to collagen I per construct. Data presented as mean ± SD with significance among groups labeled with different letters (p < 0.05).

Figure 4

Biomechanical characterization of constructs at t = 4 wks. (A) Tensile modulus and ultimate strength of constructs. (B) Aggregate modulus determined from creep indentation testing. Data presented as mean ± SD with significance between groups labeled with different letters (p < 0.05).