Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Abstract

The menisci are intraarticular fibrocartilaginous structures essential to the normal function of the knee that lack the ability to self-repair. Human meniscal fibrochondrocytes may respond to beneficial genes like human insulin growth factor-1 (hIGF-1) and the meniscal cell may be a feasible donor for gene therapy. To explore this possibility, we amplified the hIGF-1 gene sequence in full length and cloned it into a bicistronic plasmid. This gene was then transfected into cultured human meniscal fibrochondrocytes by the liposome FuGene 6. Green fluorescence was expressed in part of the cells 6 hours after transfection and increased gradually, with a peak concentration of the hIGF-1 in the supernatants to 22.68 ng/mL 56 hours after transfection. Phenotypes of some cells changed and the proliferation accelerated after transfection. Flow cytometry analysis demonstrated upregulation of cell numbers in the G2 and S stages after hIGF-1 gene introduction. We conclude the hIGF-1 gene can be transfected into the human meniscal cell efficiently by liposome and it causes accelerated proliferation and differentiation. Within 10 days after transfection, the cytokine appears to be secreted into supernatants with the bioactivity and promotes the proliferation of the NIH 3T3 cell line.

Reconstruct large osteochondral defects of the knee with hIGF-1 gene enhanced Mosaicplasty

“…The transgenic efficacy in vitro in the present study is up to 22%, and the cumulated concentration of secreted hIGF-1 reaches up to the 34.75 ng/ml. The data are higher than those of our previously research with same methods, which is transfect the fibrochondrocytes by FuGENE 6, the efficacy and concentration are 16% and 22.68 ng/ml, respectively [44,45]. The reason for such discrepancy may ascribe to the proliferate and differentiate capability of BMSCs and fibrochondrocytes, for that the stromal stem cells split and grow faster than cells in terminal differentiation, so the exogenous gene can be intaken and expressed more effectively.…”

“…Under these conditions, a few weeks of gene expression provided by present generations of liposome may be ideal [14,44,45]. Nonviral gene delivery systems are an alternative that avoids the risk of immunogenicity as adenovirus, the risk of insertional mutagenesis like retrovirus, and the risk of acquiring replication competence [5,7,22].…”

Advances in combining gene therapy with cell and tissue engineering-based approaches to enhance healing of the meniscus

“…Of note, different biomaterials have been tested in experimental settings in vitro , in situ , and in vivo for meniscal repair applications concomitantly with the use of cell- and gene-based approaches, including alginate 49,99 , type-I collagen solutions 74 or type-I collagen/GAG matrices 48 , and PGA scaffolds 45 .…”

Phenotypic Fitness of Primary Endothelial Cells Cultured from Patients with High Cardiovascular Risk or Chronic Kidney Disease for Vascular Tissue Engineering

“…23 Cellular growth was also assessed using a high-throughput proliferation assay. Cells were seeded with a low confluency (5000 cells/cm 2 ) in 96-well plates that were coated with fibronectin and starved overnight before the start of the assay.…”