Collagen Deposition, Cellular and Molecular characterisation of self-assembled tendon constructs grown in vitro from embryonic chick tenocyte cells

Abstract

Tendons are fibrous connective tissues that connect and transmit forces between muscle and bone. Following injury, they are very difficult to repair surgically, often resulting in tissue that is not comparable to native tendons in structural integrity and mechanical strength and are therefore prone to re-injury. A tissue engineered approach that would provide tendon tissue regeneration would be enormously valuable. A step toward this goal would be establishment of an in vitro system that could replicate tendon development and maturation from progenitor cells, generating tissue for transplantation capable of integrating and repairing damaged tendons. Limited progress in this area has been made to date and no system can as yet support tendon tissue maturation. In this study, I have focused on the characterisation and optimisation of a protocol using tissue engineered constructs designed to mimic tendon development, monitoring cell self-assembly and collagen deposition. Primary embryonic chick tendon cells (tenocytes) isolated at embryonic day 14 (E14), from the metatarsal tendons plated within a fibrin gel, that provides a natural substrate for the cells to self-assemble into tendon-like constructs. The cell-gel mixture was plated in between two sets of pins and suture and left in culture for up to 28- days. I show that regions of high collagen deposition are present in distinct regions within tendon constructs, centrally and peripherally. Further analysis of these regions of high collagen deposition was a focus with the goal of understanding and replicating these conditions throughout constructs. Alignment of pins in the experimental set up was important for consistent central deposition of collagen with fibres that were aligned along the long axis of the construct. While improvement of the culture system is required to produce more homogeneous tendon-like tissue, it provides an opportunity to examine the patterns of collagen deposition and uncover the conditions that best support collagen. The establishment and characterisation of these mini-tendon constructs provides an in vitro system to further our understanding of the self-assembly process, to work toward improving tendon-like tissue generation, as well as providing a tool for in vitro testing of molecular pathways and processes required.