Glucosamine and chondroitin act first as anti-inflammatory agents. A study conducted in rats treated with different dosages of glucosamine (20, 40, 80, or 160 mg/kg/day) showed, by the 6th day, the capability of this substance to reduce the levels of proinflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, also preventing the increase in plasma nitrite levels [35]. Similar results were obtained by Li et al., and Waly et al., on mice with monosodium iodoacetate-induced OA in which glucosamine was shown to downregulate serum pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and C-reactive protein levels and upregulate the anti-inflammatory cytokines IL-2 and IL-10 levels [17,18]. Oral glucosamine administration is able to suppress the early increase in tumor grown factor (TGF)-β levels in monosodium iodoacetate-induced OA rats [17]. Nevertheless, the precise role of TGF-β in different stages of OA is still unclear as well as the effects of glucosamine on this function required further confirmations [17]. Glucosamine appears to also have immune-modulatory activity by inhibiting the expression and/or activity of catabolic enzymes, such as phospholipase A2, MMPs, or aggrecanases [36].

Both chondroitin and glucosamine sulphate were demonstrated to reduce the activation of the nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathway, which represent a pathway of inflammation in OA [37]. In addition, further studies showed that these molecules prevent the cytokine-induced IL-1β expression by suppressing the demethylation of the IL-1β promoter region [36], inhibiting the lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and caspase-1 activation, and upregulation and release of IL-1β [38].

Another important activity of both chondroitin and glucosamine concerns its antioxidant properties, which have been reported in several in vitro models [21]. Even though glucosamine hydrochloride and glucosamine sulphate possess antioxidant activity, glucosamine sulphate shows greater antioxidant and radical scavenging activities compared with glucosamine hydrochloride [39].

The reduction of ROS produced during the early OA stage by mechanical stress, trauma, or chemicals is important in order to reduce the cellular damage on the adjacent cartilage and collagen degradation [40]. Glucosamine was shown to bind directly with malondialdehyde (MDA) and block the subsequent formation of MDA adducts and protein cross-linkages and thus prevent lipoprotein oxidation and inhibit MDA adduct formation in the articular chondrocyte cell matrix [41]. Other in vitro studies reported the ability of these molecules to inhibit hydrogen peroxide (H2O2)-mediated membrane lipid peroxidation, protein and DNA oxidation, as well as the intracellular ROS level in chondrocytes, with the upregulation of glutathione (GSH) and other antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in chondrocytes [42]. However, direct evidence of the antioxidant activity of both glucosamine and chondroitin in OA animal models is limited. In a study conducted in the formalin-induced osteoarthritic rat knee joint, the association of glucosamine hydrochloride, chondroitin sulphate, methylsulfonylmethane, Harpagophytum procumbens root extract, and bromelain administered orally for 30 days resulted in a significant reduction in MDA, NO, and 8-hydroxyguanine levels and an increased total GSH level was observed. These reductions were higher in the combination group (with plants) if compared with the administration of glucosamine or chondroitin alone, probably due to the additional analgesic effects [43].

Glucosamine and chondroitin are also known to improve tissue regeneration as demonstrated by different in vivo studies. In New Zealand rabbits with surgical-induced knee joint cartilage damage, treatment with glucosamine sulphate remarkably improved hyaline cartilage regeneration on autologous chondrocyte implantation repair sites, with upregulation of proteoglycans, type II collagen, and GAGs expression compared with autologous chondrocyte implantation alone [19]. In addition, these nutraceuticals simultaneously suppressed the osteoclastic cell differentiation in MC3T3-E1 osteoblasts, downregulating the receptor activator of NF-κB ligand (RANKL) expression [44] and blocked IL-1β-mediated downregulation of type II collagen and aggrecan gene expression and inhibited the MMP-13 gene expression in both normal and OA chondrocytes [45].

Chondroitin and glucosamine were tested in several clinical trials of OA. However, the results remain at least in part contrasting. A recent meta-analysis of 30 randomized clinical trials including 7127 patients showed that chondroitin could alleviate pain symptoms (−0.071, 95%CI: −0.228 to 0.085) and improve physical function (−0.090, 95%CI: −0.242 to 0.061) compared with the placebo [46]. Similar results were shown in a previous meta-analysis in which treatments with glucosamine and chondroitin were found to significantly reduce pain in VAS [weighted mean difference (WMD) −7.41 mm, 95%CI–14.31, –0.51, p = 0.04 and WMD–8.35 mm, 95% CI–11.84, –4.85, p < 0.001, respectively] [47]. Even the meta-analysis by Ogata et al., including 18 RCTs, demonstrated glucosamine’s potential to alleviate knee OA pain [48]. Moreover, Zhu et al., reported the superiority of the combination of glucosamine and chondroitin for pain reduction and joint stiffness in comparison with acetaminophen, even if celecoxib was most likely the best option for knee or hip OA [49]. In a large meta-analysis of 54 randomized clinical trials including 16,427 patients, the effects of glucosamine and chondroitin were compared with conventional treatments. Although, even in this case, celecoxib was considered the best option for pain reduction, only glucosamine plus chondroitin showed a clinically significant improvement from baseline function. In terms of the structure-modifying effect, both glucosamine alone and chondroitin alone achieved a statistically significant reduction in joint space narrowing [50].

However, other studies reported no significant improvement of the OA condition after both glucosamine and chondroitin supplementation. Kwoh et al., did not observe any significant changes in the joint structure in people with chronic knee pain treated with glucosamine 1500 mg/day [51]. In addition, supplementation with chondroitin or glucosamine (1500 mg/day) for 2 years also did not cause remarkable changes in the joint structure of OA patients (aged 45–75 years) [52]. Similar conclusions were obtained from a study regarding 59 people with temporomandibular OA randomly receiving glucosamine sulphate 1200 mg/day or placebo. At the end of the study (six weeks), no significant differences in all signs and symptoms of OA were observed in both groups [53]. In accordance with this data, in a meta-analysis of 17 clinical studies, only seven showed a statistically significant reduction in pain (−0.35, 95%CI = −0.54 to −0.16) and four studies met the review criteria for joint space narrowing (SMD= −0.10, 95%CI = −0.23 to +0.04) [54]. Moreover, several smaller dosages throughout the day of glucosamine seem to be better in pain reduction effects if compared with a single daily large dose (1500 mg) [38].

Both glucosamine and chondroitin were also demonstrated to reduce joint inflammation and oxidative stress. In an RCT including elderly people with temporomandibular joint OA, treatment with an intra-articular hyaluronic acid injection in combination with glucosamine hydrochloride (oral 720 mg for 3 months) was shown to be greatly beneficial by further reducing the IL-6, IL-1β, and TGF-β levels if compared with people treated with only hyaluronic acid injection [55]. Glucosamine sulphate (1500 mg/day) in addition with celecoxib (200 mg/day) supplemented in patients with knee OA for 8 weeks showed a better redox status expressed as higher serum SOD activity and lower serum MDA levels compared with the control group (celecoxib alone) [56].

In conclusion, data indicates that both glucosamine and chondroitin may have a small to moderate effect in reducing OA-related pain and joint inflammation but little effect on joint-space narrowing. The discrepancies in the effectiveness of chondroitin and glucosamine on osteoarthritis between different studies should be further examined.

The supplementation of both chondroitin and glucosamine in the medium term is considered safe and well tolerated. Different studies reported some mild and transitory effects, such as diarrhea, abdominal pain, nausea, and headache. However, there was no significant difference in the comparison between any options (glucosamine alone, chondroitin alone, glucosamine + chondroitin) versus placebo.