Synergistic Effects of FGF-2 with Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes

Abstract

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1alpha25-dihydroxy vitamin D3, L-3,3',5'-triodothyronine, interleukine-1 receptor antagonist, 17beta-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10-12-fold increase with 5 microg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 microg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

“…We also found adult meniscal fibrochondrocytes are relatively sensitive to gene transfection in cell proliferation and morphologic changing, with an increased percent of cells in S and M stages, which indicating the vigorous function of synthesizing DNA and protein. The medium from transfected meniscal fibrochondrocyte cultures we tested contained a hIGF-1 level as high as 22.68 ng/mL, and several previous studies suggest hIGF-1 at 50 ng/mL optimally stimulates matrix elaboration and the development of a cartilage-specific matrix structure, whereas as low as 10 ng/mL is sufficient to produce considerable stimulation of the proliferative and metabolic actions of cultured chondrocytes [17,21,26].…”

“…They also contribute to the stability of the knee [3,15,20]. Many reports demonstrate an early onset of degenerative changes in the knee after meniscectomy [1,14,26]. Because there is a greater understanding of the importance of the menisci, most surgeons attempt to preserve as much functional meniscal tissue as possible when treating meniscal injuries [10,13].…”

Collagen scaffolds for nonviral IGF-1 gene delivery in articular cartilage tissue engineering

“…On the other hand, cross-linking these lipid/ plasmid complexes to the collagen scaffold may still have resulted in the association of cells with the lipid/plasmid complexes incorporated into the scaffold, but detachment of cells could have been prevented due to the stronger chemical bond between the lipid/plasmid complexes and the collagen fibrils. The continued presence of the GP/IGF complexes on the scaffold walls that resulted in increased transfection and IGF-1 release may have also stimulated cell proliferation, since IGF-1 has been shown to have a mitogenic effect on both chondrocytes 27 and human mesenchymal stem cells. 28 Future work (viz, cell attachment and proliferation assays) may be needed to further understand the cellular interactions with these GSCG scaffolds.…”

Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

“…The lysate was filtered through a cell strainer with a pore size of 100 μm to remove residues, and then centrifuged at 500 × g for 5 minutes to isolate human chondrocytes. The isolated chondrocytes were seeded in a collagen type I-coat plastic tissue culture dish at a density of 2500 cells/cm 2 , and cultured in chondrocyte growth medium (DMEM/F-12 containing 5% human serum, 100 ng/mL FGF-2, and 5 μg/mL insulin) [11,12] in a 37˚C /5% CO 2 incubator. The medium was changed twice a week.…”