Rapid phenotypic changes in passaged articular chondrocyte subpopulations

178

149

Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we have investigated the expression profile of 27 chondrocyte-associated genes from both osteoarthritic cartilage tissue and healthy passaged human articular chondrocytes by quantitative real-time PCR. Our results indicate that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture decrease compared with those from cartilage tissue, while monolayer cultured chondrocytes from normal and osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP-3/9/13 and ADAMTS-4/5 decreased with passage, whereas proteinase inhibitors TIMP-2/3 were elevated. The cytokine IL-1 also showed increased expression with monolayer chondrocyte culture, while IGF-1 expression levels were diminished. No significant changes in TGF-b, or the chondrogenic transcription factors Sox-9, c-fos, or c-jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer culture, although the gene expression level of transcription factors necessary for chondrogenesis remains unchanged. This data may prove important for the future development of more specific and efficacious cultivation techniques for human articular chondrocyte-based therapies. ß

Section: Data Normalization and Statistical Analysismentioning

confidence: 99%

mentioning

confidence: 99%

Gene expression profiles of human chondrocytes during passaged monolayer cultivation

Lin

Fitzgerald

et al. 2008

178

149

“…1F). Based on the state of the art on the redifferentiation capacity of culture-expanded chondrocytes from a variety of species, including goats, 27 we would expect that expression of collagen type I persisted in all pellets.…”

Section: Postexpansion Chondrogenic Capacity Of Goat Chondrocytes In mentioning

confidence: 99%

Cartilage tissue engineering by expanded goat articular chondrocytes

Miot

Freitas

Wirz

et al. 2006

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-b1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF 1 -M nonwoven meshes or Polyactive TM foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF 1 -M or Polyactive TM scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF 1 -M-based constructs also increased with time, up to, respectively, 1.3-and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models. ß

“…3 In addition, during ex vivo expansion chondrocytes rapidly lose their chondrogenic state (dedifferentiation) and express a fibroblastic phenotype with decreased expression of chondrocyte proteins, such as aggrecan and type II collagen, and an increase in the production of fibroblast proteins, namely collagen type I. 4,5 The use of microcarriers for chondrocyte expansion has been investigated to overcome some of these effects. [6][7][8][9][10] Microcarrier culture offers a cost-efficient method of propagating large populations of cells due to a high surface area to volume ratio.…”

mentioning

confidence: 99%

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Schrobback

Klein

Schuetz

et al. 2010

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. ß