Hyaluronic acid-binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein
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ORIGINAL ARTICLE
Hyaluronic acid‑binding insulin‑like growth factor‑1: Creation of a gene encoding a bifunctional fusion protein Shuiliang Shi1,2 · Congrong Wang2 · Stephen B. Trippel2,3 Received: 10 August 2020 / Accepted: 24 November 2020 / Published online: 2 December 2020 © Springer Nature B.V. 2020
Abstract Chondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair. Keywords Genetic engineering · Articular chondrocyte · Insulin like growth factor-1 · Versican · Hyaluronic acid Abbreviations AAV Adeno-associated virus AG1 Aggrecan G1 domain AU Arbitrary unit CM Conditioned medium DMB Dimethylmethlyene blue DMEM Dulbecco’s minimum essential medium Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-06034-w) contains supplementary material, which is available to authorized users. * Shuiliang Shi [email protected] Congrong Wang [email protected] Stephen B. Trippel [email protected] 1
Present Address: MaAnShan, BeiCheng, HuangYan, TaiZhou 318020, ZheJiang, China
2
Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA
3
Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA
EDTA Ethylenediaminetetraacetic acid F Forward primer FBS Fetal bovine serum G1 Globular domain 1 GAG Glycosaminoglycan HA Hyaluronic acid HB-EGF Heparin-binding epidermal-like growth factor HEK-293 Human embryonic kidney 293 cells HRP Horseradish peroxidase IGF-1 Insulin-like growth factor-1 Ka Equilibrium association constant PBS Phosphate buffered saline R Reverse primer SDS Sodium dodecyl sulfate/sulphate TBST Tris-buffered saline/Tween VG1 Versican G1 domain
Introduction Articular cartilage provides a gliding surface that enables pain-free motion of diarthrodial j
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