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 molds avoids drawbacks associated with scaffold use while still allowing formation of robust tissue. In this experiment two factors were examined, agarose percentage and mold 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 molds composed of 1% or 2% agarose for 4 wks. Morphological results showed that constructs formed in 1% agarose molds, particularly smooth molds, were able to maintain their shape over the 4 wk culture period. Significant increases were observed for the collagen II to 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.

Regional Effects of Enzymatic Digestion on Knee Meniscus Cell Yield and Phenotype for Tissue Engineering

“…Primary meniscus cells are often used for this purpose, and have shown great promise in creating tissue engineered constructs with properties similar to native cartilage. [1][2][3][4][5][6][7][8] To obtain meniscus cells for tissue engineering efforts, enzymatic digestion is needed, but little is known about the effects of isolation on the phenotype of meniscus cells, or which digestion technique is most effective for extracting cells from the meniscus. Isolating cells from fibrocartilaginous tissues such as the meniscus is difficult due to the abundance of fibrous extracellular matrix (ECM).…”

Biological Augmentation and Tissue Engineering Approaches in Meniscus Surgery

“…[40][41][42] At the same time, 3D woven PCL scaffolds have conferred increased biomechanical properties in a composite preparation with cartilage-derived matrix, obviating the need for growth factor augmentation, and may certainly be applied to meniscus restoration. 43,44 Gunja et al [45][46][47][48] have conducted work using nonwoven poly-L-lactic acid scaffolds exploring the positive synergistic effects of bFGF and hypoxia, hydrostatic pressure, and TGF-ß and coculture (chondrocytes and meniscus cells) using in vitro models. Ultimately, it is hoped that cellular infiltration and matrix deposition may restore some of the viscoelastic properties of meniscus tissue in which the fluid phase carries a significant amount of the load.…”

Building an Anisotropic Meniscus with Zonal Variations

“…24,25 An additional positive die representing the inner one-third of the rabbit meniscus was similarly created. The positive die was plunged into molten 2% agarose (Fisher Scientific), and the agarose was allowed to set.…”

“…Previous work has shown that this ratio of cells yields constructs with both collagen types I and II, consistent with the fibrocartilage matrix content. 25,27,28 These cells were seeded into the meniscus-shaped agarose wells. After 7d, the intact control meniscus constructs were removed from the wells and placed into plastic six-well plates (BD Biosciences) coated with 2% agarose to prevent construct adhesion, cell migration, and construct deformation through outgrowth of their meniscus-shaped wells.…”