In Vitro Analysis of the Differentiation Capacity of Postmortally Isolated Human Chondrocytes Influenced by Different Growth Factors and Oxygen Levels

Abstract

Objective In the present in vitro study, we analyzed the chondrogenic differentiation capacity of human chondrocytes postmortally isolated from unaffected knee cartilage by the addition of transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor-1 (IGF-1) and different oxygen levels. Design After 14 and 35 days, DNA concentrations and protein contents of Col1, Col2, aggrecan as well as glycosaminoglycans (GAGs) of chondrocytes cultivated as pellet cultures were analyzed. Additionally, expression rates of mesenchymal stem cell (MSC)-associated differentiation markers were assessed in monolayer cultures. Results All cultivated chondrocytes were found to be CD29/CD44/CD105/CD166. Chondrocytic pellets stimulated with TGF-β1 showed enhanced synthesis rates of hyaline cartilage markers and reduced expression of the non-hyaline cartilage marker Col1 under hypoxic culture conditions. Conclusions Our results underline the substantial chondrogenic potential of human chondrocytes postmortally isolated from unaffected articular knee cartilage especially in case of TGF-β1 administration.

Effect of electric stimulation on human chondrocytes and mesenchymal stem cells under normoxia and hypoxia

“…Using hypoxic culture conditions during cell cultivation creates more physiological conditions due to avascular nature of the cartilage. Jonitz-Heincke et al ( 28 ) had reported that human CHs cultured in spheroid pellets showed superior collagen type II expression under hypoxic culture conditions compared to normoxia. Our results indicated a promoting effect of hypoxia on chondrogenic differentiation of human CHs.…”

“…Human CHs were isolated from the human knee cartilage under sterile conditions as previously described ( 27 ) and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal calf serum (FCS), 1% amphotericin B, 1% penicillin-streptomycin and ascorbic acid (50 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C under 5% CO 2 and 5% O 2 (hypoxia). The reduced oxygen level was used to mimic the low oxygen partial pressure within cartilage tissue ( 28 ). Human CHs have to be cultured up to the 2nd passage to achieve a sufficient cell number for the experiments.…”

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study

“…Dedifferentiation was found to elevate the postoperative failure rate in patients after ACI 11 . Of the strategies employed to improve chondrocyte culture condition is the supplementation of culture medium with cartilaginous growth factors 12 . The selection of these growth factors is based on the understanding of their vital role in the regulation of cartilage developmental processes 13 .…”

Influence of Conditioned Media on the Re-Differentiation Capacity of Human Chondrocytes in 3D Spheroid Cultures

“…As major contributors, growth factors were proven to initiate re-differentiation since the cultivation of chondrocytes in 3D spheroid cultures with IGF-1 and TGF-β1 resulted in an elevated expression of typical ECM components [16]. Thus, as a positive control we stimulated spheroid cultures with TGF-β1 and IGF-1 as previously described [16,18,19] to compare the impact of conditioned media and of sole growth factor supplementation on chondrogenic re-differentiation. In contrast to the spheroids cultivated in conditioned media the positive control showed a tendency for a change towards a hypertrophic phenotype in the histological staining and the induction of the gene expression of hypertrophy marker collagen type X.…”

“…Indeed, our group showed that the surface markers of mesenchymal cells, cluster of differentiation (CD)166, CD105, CD44 and CD29, changed incrementally from passage 0 to passage 4 during in vitro cultivation of isolated human chondrocytes in monolayer cultures [15]. Therefore, research has focused on the optimization of chondrocyte re-differentiation by using essential chondrogenic growth factors, improved three-dimensional culture conditions or physiologically approximated oxygen partial pressures [15][16][17][18][19]. While re-differentiation of chondrocytes was already described after cultivation in various 3D systems [20] we were only able to detect immunostained collagen type II and aggrecan proteins in alginate bead culture after stimulation with chondrogenic growth factors insulin like growth factor (IGF-)1 (50 ng/mL) and transforming growth factor (TGF-) β1 (50 ng/mL) [15].…”