Adenovirus-mediated tissue inhibitor of metalloproteinase-3 gene transfection inhibits rabbit intervertebral disc degeneration in vivo

Abstract

The aim of this study was to investigate the inhibitory effects of recombinant adenovirus vector carrying tissue inhibitor of metalloproteinase-3 (RAd-TIMP-3) against degeneration of rabbit intervertebral disc. Thirty Japanese white rabbits of 4 months old were randomly divided into 5 groups. Mild or moderate rabbit lumbar disc degeneration model was constructed with the controllable axial loading device by imposing 98 N pressure at the discs for 2 weeks. Various doses of virus were injected into the degenerated discs as follows: 20 μL of normal saline in group 1; 20 μL of RAd66 (an empty adenovirus vector, 1.0Â10 10 OPU/mL) in group 2; and 20, 10, and 5 μL of RAdTIMP-3 (1.0Â10 10 OPU/mL) in groups 3, 4, and 5, respectively. Two weeks after the injection, the discs were collected for investigations, including assessment of degeneration degrees according to the Thompson's grading system, reverse-transcription polymerase chain reaction (RT-PCR) assay for TIMP-3 gene, Safranin O-Fast green staining, and immunohistochemical staining for TIMP-3 and type II collagen. According to Thompson's criteria, the degeneration of groups 3, 4, and 5, especially group 3, was alleviated as compared with groups 1 and 2. RT-PCR revealed that the expression of TIMP-3 in groups 3, 4, and 5, especially in group 3, was significantly enhanced as compared with group 1 (P < 0.01). Both Safranin O-Fast green staining and type II collagen staining demonstrated better reserved integrity of disc matrix in groups 3, 4, and 5 than in groups 1 and 2. TIMP-3 staining exhibited an obvious increase of positive-staining rate in groups 3, 4, and 5 as compared with group 1. The positive-staining rate in group 3 (79.42%AE1.35%) was about 3 times that of group 1 (25.47%AE5.46%, P < 0.01). RAdTIMP-3 can effectively protect the matrix of rabbit intervertebral disc against overloading-induced degeneration in a dose-dependent manner, resulting in the alleviation of disc degeneration.

Biomimetic Proteoglycans Mimic Macromolecular Architecture and Water Uptake of Natural Proteoglycans

“…In natural tissue remodeling, proteoglycans are enzymatically digested along the protein core. ,− However, during degeneration, the enzymatic digestion of proteoglycans outpaces the cellular synthesis, leading to loss of these important matrix macromolecules, which can result in a host of mechanical, hydration and nutritional deficits to tissue function that, in turn, are manifested clinically as degenerative conditions, including arthritis, intervertebral disc degeneration, skin aging, and urinary incontinence, among others. ,− In an attempt to restore proteoglycan content to the matrix, researchers have used tissue engineering, − genetic engineering, − and growth factors − to stimulate intracellular proteoglycan synthesis, in addition to enzymatic inhibitors − to suppress further degradation of the macromolecules in the matrix. While intriguing, these technologies are still not widely used clinically.…”