Clodronate may be considered a good therapeutic tool against Osteoarthritis ; its formulation in nanoparticles may represent a promising challenge to counteract cartilage degeneration. 

Osteoarthritis (OA) is a chronic inflammatory disorder and a major cause of morbidity in the elderly, especially in females. Pro-inflammatory cytokines such as TNFα, IL6, IL1β, and metalloproteinase (MMP) released by macrophages are majorly involved in the pathogenesis OA. These pro-inflammatory molecules can bind receptors on chondrocytes surface and alter their metabolism, but they can also reduce mesenchymal stem cells (MSCs) chondrogenic differentiation.

Bisphosphonates are synthetic, non-hydrolyzable analogs of pyrophosphate that contain a PCP core protein and two side chains, named R1 and R2, bound to the central carbon. The chain R2 can be distinguished into two major groups: nitrogen (N-BF) and non-nitrogen (NN-BF) bisphosphonates. These compounds inhibit the farnesyl diphosphonate synthase (FPPS) in osteoclasts, thereby preventing the formation of isoprenoid lipids required for the prenylation of small GTPases, such as Rac, Rho, and Ras. Literature also reported the anti-inflammatory effects of NN-BFs due to their inhibition of macrophagic release of NO and pro-inflammatory mediators. Therefore, in this study NN-BFs have been evaluated for their ability to reduce inflammatory osteoarthritis. Clodronate stimulates cellular differentiation by regulating inflammatory pathways. The gene SOX9 is the master transcription factor for MSC differentiation into chondrocytes, exerting its role along the whole pathway. The expression of SOX9 gene is regulated by Bone morphogenetic proteins (BMPs) and it activates many extracellular matrix (ECM) genes such as COL2A1, COL9A1, COL11A2 and ACAN (aggrecan). Therefore, nanoparticles (NPs) of clodronate considered a better therapeutic option against OA.

Rationale behind the research:

Pro-inflammatory molecules like cytokines (such as TNFα, IL6 and IL1β) and MMP released by macrophages are known to be involved in the pathogenesis of OA.

Therefore, the present study evaluated the effect of non-amino bisphosphonate clodronate on peripheral blood MSCs differentiation.

Objective:

To evaluate the effects of the non-amino bisphosphonate clodronate in OA patients.

Study outcome measure:

Blood sample analysis: M0 (before treatment), M3 and M6 (after 3 and 6 months, respectively, of treatment).

• Circulating progenitor cells (CPCs) were isolated from each blood sample
• Serum blood calcium, PTH, 25 hydroxyvitamin D, CTX (C-terminal peptide of collagen type I) serum creatinin and urinary calcium excretion rate levels in order to exclude secondary osteoporosis causes and to evaluate therapy influences on their expression at the end of study

Time period:  Baseline, 2 months and 6 months 

Study Outcomes:

Patients:

• Average age, height, weight, BMI and menopause age were 71.8 ± 7 years, 153 ± 5.8 cm, 64.2 ± 8.6 kg, 27.4 ± 3.5 kg/m2 and 46.5 ± 7 years, respectively. Among bone metabolism parameters, only CTX values showed a significant reduction at the end of the study (0.25 ± 0.08 ng/mL at M6 vs. 0.39 ± 0.19 ng/mL at M0; p < 0.05)
• Visual analogue pain scale (VAS) showed relevantly decreased scores at the end of treatment in older women. Numerical rating pain scale (NRS) showed a significant decrease of symptoms after three months in the same group (p < 0.05 for both)
• Only NRS pain score reached a significant improvement after drug assumption (4.27 ± 2.06 at M6 vs. 6.00 ± 2.34 at M0; p = 0.01). ISF and ISM indexes of SF36 survey both reached relevant improvement after 6 months (ISF score: 43.04 ± 6.73 at M6 vs. 36.89 ± 12.21 at M0; p < 0.05; ISM score: 45.75 ± 3.86 at M6 vs. 42.54 ± 4.87 at M0; p < 0.05)

Gene Expression in OA Patients’ CPCs:

• CPC cluster differentiation (CD) expression patterns were similar in normal donors (NDs) and Patients (Table 3). Therefore, SOX9 gene expression was analyzed in CPCs from patients and NDs at M0, M3 and M6. Gene expression levels were monitored in all samples. An avrage expression of SOX9 gene increased constantly during the study, matching (M3) and then exceeding (M6) control levels (Figure 1). Interestingly, COL2A1 expression in patients also increased during the study (Figure 2). 

Figure 1: SOX9 fold of expression in CPCs of Normal Donors (NDs) and patients at baseline (M0), after 3 (M3) and 6 (M6) months

Figure 2: COL2A1 fold of expression in CPCs of Normal Donors (NDs) and patients at baseline (M0), after 3 (M3) and 6 (M6) months

Chitosan-Hyaluronic Acid-Clodronate Embedded Nanoparticles:

• Synthesized clodronate nanoparticles were analyzed by dynamic light scattering and showed a single peak at 135.4 nm with a polydispersity index (pdI) of 0.922 and a surface charge of 25.5 mV.

Gene Expression in the In Vitro OA Model:

• All results were reported as normalized values compared to their expression at the end of the differentiation process (in specific mediums) without IL1β and/or clodronate addition to cultures. The in vitro experiments confirmed IL1β inhibition of chondrogenic maturation. This pro-inflammatory cytokine halved MSCs ability to differentiate. On the other hand, clodronate increased MSCs’ potential to undergo chondrogenic differentiation in a dose dependent way. We then added two different combinations of IL1β + clodronate (50 nM and 100 nM) to the cultures. At the lower dose, the drug inhibited cytokine pro-inflammatory action only partially; but at the higher dose, clodronate action exceeded IL1β inhibition, stimulating MSCs maturation (Figure 3A).
• Nanoparticles embedded-clodronate exhibited a stronger effect in counteracting IL1β inhibition of SOX9 (Figure 3B) and COL2A1 (Figure 3C) expression

Figure 3: Effects of clodronate in mesenchymal stem cells (MSCs). SOX9 fold of expression in MSCs treated with and w/o clodronate in chondrogenic medium in the presence or absence of ILβ1 (A). SOX9 (B) and COL2A1 (C) fold of expression in chitosan and hyaluronic acid empty nano particles (NPs) or clodronate embedded nanoparticles in chindrogenic medium with or w/o ILβ1. The synergistic action of NPs and clodronate is noteworthy.

• Notably, MSCs cultured with chondrogenic differentiation medium in the presence of clodronate alone or embedded in nanoparticles, exhibited a strong positive staining with alcian-blue indicating the production of glycosaminoglycan (GAG) and therefore the chondrogenic maturation

The present study evaluated the in vivo and in vitro effects of clodronate on peripheral blood MSCs differentiation. Also, its influence on bone metabolism, osteoarticular pain, mental and physical performance was assessed. The findings revealed that administration of clodronate 200 mg intramuscular weekly stimulates the in vivo MSCs maturation towards the chondrogenic lineage. There was a significant increase in the SOX9 expression after 3 and 6 months of treatment with clodronate. Moreover, after six months of treatment, SOX9 and COL2A1 expressions exceeded than normal donors (NDs). Clodronate also exerts analgesic effects. A significant decrease in the NRS pain scale at the end of treatment in both groups was reported.

After 6 months, a significant decrease in the CTX values was noted that confirmed the bone resorption inhibition. During the study, the level of 25 hydroxyvitamin D did not change and continuously remained above the insufficiency cut-off (20 ng/mL), indicating that the hormone blood levels did not influence SF36, VAS and NRS outcomes. Besides, serum creatinine evaluation confirmed that clodronate therapy does not affect renal function.  The present study cultured the human MSC line with IL1β to analyze the molecular effects of clodronate in an OA in vitro model.  Interestingly, with the help of CPCs, the present data confirmed the chondrogenic differentiation induced by clodronate. The patients treated with clodronate showed significantly increased SOX9 gene expression. This effect was observed even in co-occurrence with IL1β. Clodronate, alone or embedded in nanoparticles, was able to stimulate the chondrogenic maturation, proven by the alcian blue staining data.

Overall. these findings demonstrated that clodronate stimulates chondrogenic differentiation of precursors and may hinder effectively the pathogenesis and progression mechanisms of OA.