siRNA Treatment Enhances Collagen Fiber Formation in Tissue-Engineered Meniscus via Transient Inhibition of Aggrecan Production.

Abstract

The complex collagen network of the native meniscus and the gradient of the density and alignment of this network through the meniscal enthesis is essential for the proper mechanical function of these tissues. This architecture is difficult to recapitulate in tissue-engineered replacement strategies. Prenatally, the organization of the collagen fiber network is established and aggrecan content is minimal. In vitro, fibrochondrocytes (FCCs) produce proteoglycans and associated glycosaminoglycan (GAG) chains early in culture, which can inhibit collagen fiber formation during the maturation of tissue-engineered menisci. Thus, it would be beneficial to both specifically and temporarily block deposition of proteoglycans early in culture. In this study, we transiently inhibited aggrecan production by meniscal fibrochondrocytes using siRNA in collagen gel-based tissue-engineered constructs. We evaluated the effect of siRNA treatment on the formation of collagen fibrils and bulk and microscale tensile properties. Specific inhibition of aggrecan production by fibrochondrocytes via siRNA was successful both in 2D monolayer cell culture and 3D tissue culture. This inhibition during early maturation of these in vitro constructs increased collagen fibril diameter by more than 2-fold. This increase in fibril diameter allowed these tissues to distribute strains more effectively at the local level, particularly at the interface of the bone and soft tissue. These data show that siRNA can be used to modulate the ECM to improve collagen fiber formation and mechanical properties in tissue-engineered constructs, and that a transient decrease in aggrecan promotes the formation of a more robust fiber network.