Abstract
Autologous chondrocyte transplantation is a promising option for the repair of isolated osteoarthritic cartilage lesions that requires isolation and expansion of chondrocytes from a small cartilage biopsy prior to implantation. However, when cultured in vitro, chondrocytes lose their stable phenotype and dedifferentiate to fibroblastic-like cells. The study investigated the potential of pentosan polysulfate (PPS) sodium to restore the phenotype of dedifferentiated monolayer articular chondrocytes. Canine articular chondrocytes isolated from four cartilage samples were culture expanded to establish primary culture. First passage chondrocytes were cultured as alginate beads for 18 days under normoxia in PPS concentrations of 0, 1, 5, 15 and 40 μg/mL in 20% DMEM. Effect of PPS on type I, II and X collagen, aggrecan and Runx2 gene expression were evaluated by real-time PCR. Runx2, HIF-1α and HIF-2α protein expression were evaluated by Western blot and proteoglycan deposition was determined by Alcian blue stain. Dedifferentiated chondrocytes fully retained their phenotype as evidenced by increased synthesis of cartilage-specific genes, type II collagen and aggrecan mRNA with complete suppression of type I and X collagen at PPS concentrations of 15 and 40 μg/mL. Compared to the control, type II collagen and aggrecan mRNA were significantly upregulated (P<0.05) at 5, 15 and 40 μg/mL and 5 and 15 μg/mL PPS, respectively. PPS significantly enhanced proteoglycan with peak deposition at 5 μg/mL compared to control. HIF-1α and HIF-2α proteins were detectable at protein level for the first time under normoxia condition in alginate culture. The study demonstrates for the first time the restoration of dedifferentiated canine articular chondrocytes phenotype by combining alginate encapsulation with culture in PPS without the addition of known chondrocytic growth factors. The study confirms PPS as novel chondroinductive factor with potential to offer a solution to the major challenges that exist in cartilage tissue engineering.
Highlights
Autologous chondrocyte transplantation (ACT) which involves in vitro monolayer expansion of chondrocytes prior to implantation is a promising alternative strategy for the repair of isolated osteoarthritic (OA) cartilage lesions
To optimize the redifferentiation of chondrocytes, other studies have combined alginate encapsulation with culture in established chondrocytic growth factors [12-15] and reduced oxygen tension to physiological level [16,17]. While these studies demonstrated restoration of key chondrocyte phenotype markers, the undesirable type I collagen was still detectable and even reported higher in alginate than monolayer chondrocytes cultured in medium supplemented with chondrogenic growth factors (1% insulin-transferrin-sodium selenite medium supplement (ITS), 1% L-ascorbic acid-2-phosphate and 10 ng/ml transforming growth factor beta 3) [12] whereas hypoxia showed no effect on type I collagen [16,17]
Sox-9, Runx2 and parathyroid hormone related protein (PTHrP) mRNA were all Alginate suppresses Sox-9 mRNA expression in Canine articular chondrocytes (CAC); pentosan polysulfate (PPS) selectively promotes a chondrocyte phenotype while suppressing chondrocyte hypertrophy and dedifferentiation
Summary
Autologous chondrocyte transplantation (ACT) which involves in vitro monolayer expansion of chondrocytes prior to implantation is a promising alternative strategy for the repair of isolated osteoarthritic (OA) cartilage lesions. To optimize the redifferentiation of chondrocytes, other studies have combined alginate encapsulation with culture in established chondrocytic growth factors [12-15] and reduced oxygen tension to physiological level [16,17] While these studies demonstrated restoration of key chondrocyte phenotype markers, the undesirable type I collagen was still detectable and even reported higher in alginate than monolayer chondrocytes cultured in medium supplemented with chondrogenic growth factors (1% insulin-transferrin-sodium selenite medium supplement (ITS), 1% L-ascorbic acid-2-phosphate and 10 ng/ml transforming growth factor beta 3) [12] whereas hypoxia showed no effect on type I collagen [16,17]. These findings provoke the desire to improve the current culture strategies or develop novel culture conditions if implantable phenotypically stable chondrocytes are to be established for successful cartilage tissue regeneration and ACT for OA treatment
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