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.

O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study

“…The O-GlcNAcylation changes during osteoblast differentiation have already been reported in many previous studies. 11,13,14,21,22 However, the novel point in the present study is that the time-course pattern and distribution of O-GlcNAcylated proteins with different MWs were quantitatively analyzed in detail using novel bioinformatics analysis. A series of experiments and bioinformatics analyses in this study indicate the role of [Ca 2+ ] i in O-GlcNAcylation-mediated osteoblast differentiation.…”

“…10 Glucosamine has been reported to subtly boost protein O-GlcNAcylation, mucopolysaccharide synthesis, 8 and osteogenic differentiation of human primary osteoblasts. 11 In addition, accumulating evidence suggests that OGT and OGA modulate epigenetic regulators and transcription factors, which are involved in the differentiation of various lineages, including osteoblasts, osteoclasts, chondrocytes, adipocytes, myocytes, neuronal cells, lens fiber cells, keratinocytes, lymphocytes, and embryonic stem cells. These studies also showed that O-GlcNAcylation occurs in a cell lineage-specific manner during cell differentiation.…”

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2

“…The 3-D dynamic culture condition was set up by using the RCCS-4TM bioreactor (Synthecon™, Inc., Houston, TX, USA), with a High Aspect Ratio Vessel (HARV™; Synthecon™, Inc., Houston, TX, USA). hOBs and hMCs isolated from the same patient were combined (2:1 ratio) to create a 3-D culture system [ 27 ]. In detail, 1 × 10 6 hOBs and 0.5 × 10 6 hMCs were inoculated in 2 mL of HARV filled with 10% FCS DMEM high-glucose; all air bubbles were removed from the culture chamber.…”

Liquid chromatography‐tandem mass spectrometry analysis of peptides separated from the insoluble matrix by in‐sample tryptic protein digestion for rapid discrimination of various induced pathological states of human bone models in oral surgery

“…In vitro bone models allow the use of human cells to gain more relevant preclinical research results because these models dispose of the interactions and mechanisms that usually represent a simplified in vivo situation [10]. Until today, many models based on bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts) have been generated to simulate the bone remodeling process and its associated diseases [11][12][13][14][15]. Using these in vitro models based on osteoblast and osteoclasts co-culture systems, the effect of quercetin [16], glucosamine [15], clodronate, alendronate [17,18], miR-21 [19], melatonin, strontium, vitamin D3, and vitamin K2 [14] in bone disorders were investigated.…”