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Osteoarthritis (OA) is a degenerative as well as inflammatory disease caused by an imbalance between both catabolic and anabolic factors, affecting both humans and canines alike. The main catabolic mediators, interleukin (IL) -1β and tumor necrosis factor α (TNFα), propagate the expression of other inflammatory mediators that are involved in the degradation of cartilage matrix proteins and ultimately the loss of cartilage. To combat inflammation and at the same time stimulate cartilage matrix synthesis, the presence of both anti-inflammatory and growth factor genes from the same species are needed. Currently there is no cure for OA. However, regenerative medicine coupled with gene therapy offers a good alternative in the treatment of arthritis, with more focus on co-expressing two genes to battle the disease processes. This study focuses on the effects of the co-expression of insulin-like growth factor-1 (IGF-1), and the anti-inflammatory cytokine, IL-4, on cartilage degrading and synthesizing factors in an in vitro canine chondrocyte and mesenchymal stem cell (MSC) model.
Regenerative and anti-inflammatory effects of IGF-1 and IGF-1/IL-4 in an in-vitro chondrocyte inflammatory model were analyzed. Co-expression of the transgenes was ascertained by immunoassay. Pro-inflammatory mediators like IL-1β, TNFα, matrix metalleoproteinases (MMPs), inducible nitric oxide synthase (iNOS), as well as IGF-binding proteins (IGFBPs), were analyzed by real-time qRT-PCR. The regeneration of extracellular matrix proteins was demonstrated on the mRNA and protein levels. Canine MSCs were isolated and characterized based on morphological as well as biochemical features utilizing real-time qRT-PCR, FACS, and immunocytochemistry. The stem cells were transfected with IGF-1/IL-4 to test the chondrogenic potential of IGF-1 compared to chondrogenic medium containing TGFβ3. Stable cells with the inflammatory sensitive pCOX2-IL-4 were super-transfected with pVitro2-IGF-1. Co-cultures with stably transfected cells and chondrocytes or MSCs were analyzed on their ability to produce extracellular matrix protein type II collagen and to reduce the expression of the collagen degradative mediator MMP-13.
Results from the chondrocyte pro-inflammatory model show that pro-inflammatory mediators as well as IGFBPs were down-regulated in samples transfected with IGF-1/IL-4 to levels comparable to the non-stimulated, non-transfected control. Also, those samples as well as samples transfected with IGF-1 alone showed signs of regeneration denoted by the expression of aggrecan, type II collagen and SOX9. Canine MSCs were shown to undergo chondrogenesis utilizing chondrogenic medium with TGFβ3 as well as by transfecting them with IGF-1/IL-4. In both situations, chondrogenesis was proven by the expression of cartilage markers, namely aggrecan and type II collagen. Hypertrophy marker, type X collagen, was seen in the 2nd week of cultivation with TGFβ3. Co-cultures with stably transfected cells also demonstrated an up-regulation of type II collagen and a down-regulation of MMP-13 under pro-inflammatory conditions.
Overall, this study shows the ability of the combined expression of IGF-1 and IL 4 to stimulate proteoglycan and type II collagen synthesis and to down-regulate the degradative effects of IL-1β and TNFα, in chondrocyte and co-culture models. Co-expression of therapeutic genes in MSCs offers a dual role in stimulating differentiation of the stem cells and introducing therapeutic genes into the cells to balance catabolic effects in OA tissue. The use of multiple genes in chondrocytes or MSCs could better alleviate the signs and symptoms which are characteristic for the disease process. This study lays foundation for future studies where the use of more than one gene of interest would be necessary.