Design and characterization of an aligned collagen-GAG scaffold-membrane composite with soluble factor presentation for tendon tissue engineering

摘要

With over 32 million tendon and ligament injuries in the US each year with associated costs of $30 billion, the need for viable tendon repair strategies is becoming increasingly important. Current surgical and tissue engineering approaches have had limited success; repeat injuries and generally poor clinical outcomes are common with failure rates as high as 94%. This thesis discusses the development of collagen-glycosaminoglycan (CG) biomaterial scaffolds for tendon tissue engineering. Key elements incorporated into the design of these biomaterials include an aligned microstructure to mimic healthy tendon, integration of therapeutic biomolecules to increase tendon cell migration and metabolic activity, and enhancement of construct mechanical competence through the addition of a novel CG membrane shell. CG scaffolds have been utilized to regenerate a variety of tissues, including dermis, peripheral nerves, conjunctiva, and cartilage. These materials have numerous advantages as tissue engineering scaffolds, including high porosity (> 95%) and an abundance of native ligands for cells to attach onto. CG scaffolds are manufactured by freeze-drying a suspension of collagen and glycosaminoglycan co-precipitate. The freezing step results in an interpenetrating network of ice crystals surrounded by CG content; sublimation leaves behind a scaffold with interconnected pores. This thesis describes the development of specialized freeze-drying techniques that utilize unidirectional heat transfer to produce scaffolds with aligned pores that mimic the native microstructure of tendon. Modulation of the final freezing temperature enables fabrication of a series of aligned CG scaffolds with constant relative density but a wide range of pore sizes (55-243 μm). The addition of chemotactic growth factors to aligned CG scaffolds is also shown to enhance tendon cell migration, viability, and metabolic activity. This thesis also details the creation of CG scaffold-membrane core-shell composites with improved mechanical integrity for tendon tissue engineering. While CG scaffolds possess many advantageous qualities for tissue engineering applications, their mechanical properties are typically orders of magnitude lower than native tendon. Taking inspiration from core-shell composites like plant stems in nature, scaffold-membrane composites composed of a low density aligned CG scaffold core surrounded by a high density CG membrane shell were synthesized. Fabrication and characterization of the novel CG membrane is discussed in detail. The addition of an optimized membrane shell is shown to increase scaffold tensile elastic modulus by a factor of 36 while also maintaining adequate permeability to support tendon cell viability after 14 days of in vitro culture.