TGF β‐1 administration during Ex vivo expansion of human articular chondrocytes in a serum‐free medium redirects the cell phenotype toward hypertrophy

Abstract

Cell-based cartilage resurfacing requires ex vivo expansion of autologous articular chondrocytes. Defined culture conditions minimize expansion-dependent phenotypic alterations but maintenance of the cells' differentiation potential must be carefully assessed. Transforming growth factor β-1 (TGF β-1) positively regulates the expression of several cartilage proteins, but its therapeutic application in damaged cartilage is controversial. Thus we evaluated the phenotypic outcomes of cultured human articular chondrocytes exposed to TGF β-1 during monolayer expansion in a serum-free medium. After five doublings cells were transferred to micromass cultures to assess their chondrogenic differentiation, or replated in osteogenic medium. Immunocytostainings of micromasses of TGF-expanded cells showed loss of aggrecan and type II collagen. Positivity was evidenced for RAGE, IHH, type X collagen and for apoptotic cells, paralleling a reduction of BCL-2 levels, suggesting hypertrophic differentiation. TGF β-1-exposed cells also evidenced increased mRNA levels for bone sialoprotein, osteopontin, matrix metalloproteinase-13, TIMP-3, VEGF and SMAD7, enhanced alkaline phosphatase activity and pyrophosphate availability. Conversely, SMAD3 mRNA and protein contents were reduced. After osteogenic induction, only TGF-expanded cells strongly mineralized and impaired p38 kinase activity, a contributor of chondrocytes' differentiation. To evaluate possible endochondral ossification progression, we seeded the chondrocytes on hydroxyapatite scaffolds, subsequently implanted in an in vivo ectopic setting, but cells failed to reach overt ossification; nonetheless, constructs seeded with TGF-exposed cells displayed blood vessels of the host vascular supply with enlarged diameters, suggestive of vascular remodeling, as in bone growth. Thus TGF-exposure during articular chondrocytes expansion induces a phenotype switch to hypertrophy, an undesirable effect for cells possibly intended for tissue-engineered cartilage repair.

Human aqueous humor levels of transforming growth factor-β2: Association with matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases

“…The present study has demonstrated that the increase of TIMP-1 and TIMP-3 in the aqueous humor of high myopia patients was positively correlated with the increase of TGF-β2 levels, indicating that TGF-β2 may be the molecule that causes the increase in TIMP expression levels in high myopia. This is consistent with previous reports demonstrating that TGF-β2 increased the levels of TIMP-1 in human RPE cells (52), and the upregulation of TIMP by TGF-βs in human, rat or bovine chondrocytes and fibroblasts (53)(54)(55)(56)(57)(58).…”

No effect of subperiosteal growth factor application on periosteal neo-chondrogenesis in osteoperiosteal bone grafts for osteochondral defect repair

“…Additionally, single dose subperiosteal injection of TGF-β1 has effectively increased cellularity and periosteal cartilage formation in vitro [16] and increased osteochondral repair tissue quality in vivo after subperiosteal injection seven days prior to surgery in a rabbit model [17]. On the other hand it is known that TGF-ß1 might induce a hypertrophic chondrocyte phenotype ex vivo [35] and also might induce arthrofibrosis [36]. In earlier published data looking at the molecular aspects of the GFM treatment we were able to show that hypertrophic markers were found equally in both treatment groups after 52 weeks, indicating no negative effect of GFM on cell hypertrophy in this setting [19].…”

Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA TargetingCol1a1for Hydrogel-Based Tissue-Engineered Cartilage

“…9,10 This feature is important since this ratio is considered as a differentiation index for chondrocytes 11 and is now required for quality control of chondrocytes before grafting. Besides, although TGF-b1 administration has been shown to stimulate proteoglycan and type II collagen production in goat articular chondrocytes amplified in monolayer cultures, 12 a more recent study has revealed that TGF-b1 exposure during expansion of human articular chondrocytes induces a switch to hypertrophy, 13 compromising therefore the application of TGF-b1 for cartilage cell therapy.…”