Modulation of gene expression in human subchondral bone cells co-cultured with human articular chondrocytes

Abstract

Purpose: To investigate modulation effects in gene expression in a co-culture model of human bone cells and human condrocytes from osteoarthritic (OA) and normal (N) joint tissue. Methods: Human bone cells from the subchondral plate (hSBCs) and human articular chondrocytes (hCHs) were used. Cells were isolated from knee tissue discarded during joint replacement surgeries using standard procedures. For the co-culture experiments hCHs were cultured on the membrane of BD Falcon culture inserts while hSBCs were cultured in the wells of companion plates. In this way both cells types are co-cultured without physical contact but allowing paracrine interactions. Three different combinations were tested, namely, normal (N) hSBCs co-cultured with N hCHs, N hSBCs co-cultured with OA-hCHs and OA-hSBCs with OA-hCHs. After 7, 14 and 21 days of co-culture, total RNA was extracted using Trizol® following the manufacturer instructions. qRT-PCR analyses were performed to study the effects on gene expression in the hSBCs due to the co-culture with hCHs. In particular, we looked at the expression of alkaline phosphatase (ALP), osteocalcin (OCN), collagen type I (COLI), osteoprotegerin (OPG) and RANKL in the hSBCs. Results: hSBCs monocultures: Differences were found in gene expression between the N and OA hSBCs. ALP expression increased progressively from day 7 to day 21 in both normal and OA hSBCs monocultures up to about 4 fold with respect to the basal cells expression. OCN gene was similarly expressed in normal and OA cells in monoculture at 7 days, but its expression was markedly increased in the OA cells at both 14 and 21 days with respect to the normal cells (15.5 and 16 fold increase, compared to 4.5 and 2.8 fold increase, respectively). Conversely, COLI gene expression was reduced in the OA cells with respect to the normal cells (7.8 vs. 14.9) at 21 days of culture. Additionally, while in the N hSBCs OPG increased up to 22 fold at 14 d and was moderately reduced to 18 fold at 21 d, in the OA hSBCs, OPG increased only to 8 fold at 14 d and was further reduced to 5 fold at 21 d. Finally, both N and OA hSBCs RANKL gene expression was downregulated at 7, 14 and 21 d with respect to the basal cells expression. Co-cultures with normal or OA chondrocytes: ALP, OCN and COLI gene expression in both normal and OA hSBCs was reduced at 21 d when these cells were co-cultured with OA hCHs compared to the expression in N hSBCs co-cultured with N hCHs. OPG gene expression was higher in hSBCs co-cultured with OA hCHs at 14 d but this effect was inversed at 21 d (with respect to the co-culture with normal hCHs). In the case of RANKL, while in N hSBCs co-cultured with N hCHs expression was downregulated for all the time points investigated as was the case in the N hSBCs monoculture, in the hSBCs cultured with OA hCHs RANKL expression was increased in both N and OA cells at 14 d and in the OA hSBCs this effect was also seen at 21 d. Conclusions: Co-culture of hSBCs from either normal or OA tissue with hCHs from OA tissue modulated the expression of ALP, OCN, COLI, OPG and RANKL genes. These results suggest that paracrine signals from chondrocytes in OA might be capable of regulating gene expression of relevant osteoblastic genes involved in bone matrix formation and remodelling in both normal and OA hSBCs cells.