A Gene Therapy Application for Ligament Wound Healing: In Vitro scAAV-IGF-1 delivery to Rodent Medial Collateral Ligament Fibroblasts

INTRODUCTION: Mid-substance and avulsion ruptures of cruciate and collateral ligaments are common knee injuries that require surgical repair and can often lead to a series of complex biomechanical, microarchitectural and extracellular matrix compositional changes that can induce osteoarthritis in 50% or more of affected patients. Rehabilitation time, compromised quality of the dense fibrous connective tissue repair, and reduced ligament strength are common side effects of tendon grafts and suture repairs. In the fields of tissue engineering and orthopaedics, there is a great interest in viable growth and healing factors to aid in connective tissue repair process. IGF-1 has been shown to increase cell proliferation and extracellular matrix synthesis in ligament and tendon fibroblasts. Work from our laboratory has shown that the ex vivo systemic administration of IGF-1 can enhance the strength of ligament, upregulate collagen gene expression (Col1a2, Col3a1) and improve mature collagen cross-link formation in healing rodent medial collateral ligament (MCL) tissues. Recent evidence suggests that splice variants of the igf1 gene may be influential in specific aspects of the growth and repair processes in skeletal muscle. Also, advances in gene therapy have improved such that the use of adeno-associated viruses (AAVs) to aid in growth factor delivery has proven to be none pathogenic in humans, while delivering molecular cargos of up to 5 kb. Therefore, the purpose of this study is two-fold: 1) To create a successful IGF-1 ex vivo delivery method for injured ligaments using an adeno-associated virus-2 (AAV2IGF-1) gene therapy and, 2) To compare the in vitro effects of two splice variants of IGF-1: the IGF-1A isoform and the IGF-1B isoform (also known as mechano-growth factor [MGF]). We hypothesize that IGF-1 gene therapy will significantly improve in vitro MCL fibroblast IGF-1 secretion using scAAV transfection methodology.