Human osteoclasts/osteoblasts 3D dynamic co‑culture system to study the beneficial effects of glucosamine on bone microenvironment

Abstract

Glucosamine (GlcN) functions as a building block of the cartilage matrix, and its multifaceted roles in promoting joint health have been extensively investigated. However, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. As a result, the benefit of GlcN application in bone disorders remains controversial. In order to further elucidate the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, the effect of GlcN treatment was investigated in human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were cultured with two‑dimensional (2D) traditional methods or co‑cultured in a 3D dynamic system more closely resembling the in vivo bone microenvironment. Under these conditions, osteoclastogenesis was supported by hOBs and sizeable self‑assembling aggregates were obtained. The differentiated hOCs were evaluated using tartrate‑resistant acid phosphatase assays and osteogenic differentiation was monitored by analyzing mineral matrix deposition via Alizarin Red staining, with expression of specific osteogenic markers determined via reverse transcription‑quantitative PCR. It was found that crystalline GlcN sulfate was effective in decreasing osteoclastic cell differentiation and function. hOCs isolated from patients with OA were more sensitive compared with those from healthy donors. Additionally, GlcN exhibited anabolic effects on hOCs both in 2D conventional cell culture and in hOC/hOB 3D dynamic co‑culture. The present study demonstrated for the first time the effectiveness of a 3D dynamic co‑culture system for characterizing the spectrum of action of GlcN on the bone microenvironment, which may pave the way for more fully determining the potential applications of a compound such as GlcN, which is positioned between pharmaceuticals and nutraceuticals. Based on the present findings, it is hypothesized that GlcN may have potential benefits in the treatment of osteopenic diseases such as osteoporosis, as well as in bone maintenance.

Keywords: glucosamine; bone tissue; bone cells; 3D culture system; osteoarthritis.

Figure 1

Experimental set-up. (A) hOCs were obtained after culturing hMCs from the peripheral blood of healthy donors or patients with osteoarthritis for 14 days in osteoclastogenic medium. (B) hMCs were co-cultured with hOBs in a three-dimensional dynamic system to generate self-assembled aggregates in osteogenic medium. In both cases, cell cultures were exposed to GlcN, with medium renewed every 3 days. GlcN, glucosamine; hMC, human primary monocyte; hOB, human primary osteoblast; hOC, human primary osteoclast.

Figure 2

Effect of GlcN on hOC apoptosis. hOCs were incubated with 100 and 200 µg/ml GlcN for 72 h and then subjected to TUNEL staining to detect apoptosis. hOCs were counterstained with hematoxylin. Scale bars, 50 µm. Data are presented as the percentage of TUNEL-positive nuclei (dark brown) when compared with the total number nuclei. Data are presented as the mean ± SD. Healthy donors, n=3; patients with OA, n=7. ^*P<0.05 vs. CTR. CTR, untreated control; GlcN, glucosamine; hOC, human primary osteoclast; OA, osteoarthritis.

Figure 3

Effect of GlcN on hOC actin ring formation. Monocytes were cultured in osteoclastogenic medium in the absence or presence of GlcN (100 and 200 µg/ml) for 14 days. hOC actin rings were analyzed via phalloidin staining; nuclei were counterstained with DAPI. Scale bars, 50 µm. Data are presented as the percentage of actin ring-positive cells relative to total number of cells, and were evaluated by two independent investigators in 10 randomly selected optical fields. Data are presented as the mean ± SD. Healthy donors, n=3; patients with OA, n=7. ^*P<0.05 vs. CTR; ^°P<0.05 vs. 100 µg/ml. CTR, untreated control; GlcN, glucosamine; hOC, human primary osteoclast; OA, osteoarthritis.

Figure 4

Responsiveness of hOCs and hOBs to GlcN in the 3D dynamic co-culture system. Human primary monocytes from healthy donors were co-cultured with hOBs for 14 days in a 3D dynamic system. Cells were cultured in OM or OM/GlcN. GlcN treatment was repeated every 3 days. Representative microphotographs of ARS, TRAP and OPN staining are reported. Scale bars, 50 µm. TRAP activity and ARS were quantified by ImageJ software and expressed as the percentage positive area (mean ± SD, five sections/sample, n=4). OPN levels were quantified by ImageJ software and expressed as the mean pixel intensity/area (mean value ± SD, five sections/sample, n=3). ^*P<0.05 vs. OM. 3D, three-dimensional; ARS, Alizarin Red S; GlcN, glucosamine; hOC, human primary osteoclast; hOB, human primary osteoblast; OM, osteogenic medium; OM/GlcN, OM with 200 µg/ml GlcN; OPN, osteopontin; TRAP, tartrate-resistant acid phosphatase.

Figure 5

Effect of GlcN on hOBs in two-dimensional conventional cell culture. The expression of typical osteogenic markers was analyzed in hOBs cultured in OM or OM/GlcN for 14 days. GlcN treatment was repeated every 3 days. Total RNA was purified, and the mRNA expression levels of Runx2, COL1a1, ALP, OPN, BSP and OCN were evaluated via reverse transcription-quantitative PCR. Relative expression levels were normalized to OM. All reactions were performed in triplicate. Data are presented as the mean ± SD (n=4). ^*P<0.05 vs. OM. ALP, alkaline phosphatase; BSP, bone sialoprotein; COL1a1, collagen type 1α; GlcN, glucosamine; hOB, human primary osteoblast; OCN, osteocalcin; OM, osteogenic medium; OM/GlcN, OM with 200 µg/ml GlcN; OPN, osteopontin; Runx2, runt-related transcription factor 2.