Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis

Abstract

Bone defects, including bone loss due to increased osteoclast activity, have become a global health-related issue. Osteoclasts attach to the bone matrix and resorb the same, playing a vital role in bone remodeling. Ca²⁺ homeostasis plays a pivotal role in the differentiation and maturation of osteoclasts. In this work, we examined the role of TRPV1, a nonselective cation channel, in osteoclast function and differentiation. We demonstrate that endogenous TRPV1 is functional and causes Ca²⁺ influx upon activation with pharmacological activators [resiniferatoxin (RTX) and capsaicin] at nanomolar concentration, which enhances the generation of osteoclasts, whereas the TRPV1 inhibitor (5'-IRTX) reduces osteoclast differentiation. Activation of TRPV1 upregulates tartrate-resistant acid phosphatase activity and the expression of cathepsin K and calcitonin receptor genes, whereas TRPV1 inhibition reverses this effect. The slow release of capsaicin or RTX at a nanomolar concentration from a polysaccharide-based hydrogel enhances bone marrow macrophage (BMM) differentiation into osteoclasts whereas release of 5'-IRTX, an inhibitor of TRPV1, prevents macrophage fusion and osteoclast formation. We also characterize several subcellular parameters, including reactive oxygen (ROS) and nitrogen (RNS) species in the cytosol, mitochondrial, and lysosomal profiles in BMMs. ROS were found to be unaltered upon TRPV1 modulation. NO, however, had elevated levels upon RTX-mediated TRPV1 activation. Capsaicin altered mitochondrial membrane potential (ΔΨm) of BMMs but not 5'-IRTX. Channel modulation had no significant impact on cytosolic pH but significantly altered the pH of lysosomes, making these organelles less acidic. Since BMMs are precursors for osteoclasts, our findings of the cellular physiology of these cells may have broad implications in understanding the role of thermosensitive ion channels in bone formation and functions, and the TRPV1 modulator-releasing hydrogel may have application in bone tissue engineering and other biomedical sectors.

Figure 1

Expression and functional analysis of TRPV1 in BMMs and Osteoclasts. (a) BMMs stained for the macrophage marker CD11b (red) and TRPV1 (green) depict the latter’s expression in these cells, both in the absence (upper panel) and presence (lower panel) of RANKL. (b) Expression of TRPV1 (red) in phalloidin-stained osteoclasts (green, upper panel) is confirmed by a peptide segment against anti-TRPV1 antibody (lower panel) that reduces the specific fluorescence signal intensity of the TRPV1 channel.

Figure 2

Functional analysis of TRPV1 in BMMs. (a) BMMs were assessed for intracellular Ca²⁺ levels upon TRPV1 modulation. Representative intensity profiles of Fluo4-AM intensity at different frames are indicated. (b) Time series graphs of intracellular Fluo4-AM intensities across 200 frames of live imaging. The arrow at the x-axis signifies the time of addition of the respective drugs (20th frame). Gray traces are of individual cells, and the black trace represents the average of 50 cells. (c) Compiled average of different treatments of BMMs, individual cell traces omitted.

Figure 3

Functional analysis of TRPV1 in BMMs grown on the CMT:HEMA hydrogel. (a) BMMs grown on hydrogels to check for the endogenous levels of Ca²⁺ using Fluo4-AM Ca²⁺-sensitive dye. TRPV1 activation elevates the intracellular Ca²⁺ levels, as is quantified in (b); n = 100 cells; one-way ANOVA; ns: non-significant, ****p < 0.0001. (c) Correlation representation of the area of cells and per unit area intensity of Fluo4-AM depicts strong positive correlations under basal and TRPV1-activated conditions but not upon inhibition of the channel.

Figure 4

Morphological analysis of BMMs grown on the hydrogel. (a) Representative images of BMMs grown on glass or hydrogel in the presence of RANKL and TRPV1 modulators. Right panels denote marked inset of respective images. Phalloidin intensity (b) and morphometric analyses of BMM’s area (c), perimeter (d), length (e), width (f), and LWR (g). n = 18–51 cells per group; one-way ANOVA; ns: non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 5

Subcellular parameters (cytosolic ROS and NO) of BMMs grown on the hydrogel. (a,c) Intensity profiles of ROS (H₂DCFDA staining) and its quantitation. n = 250 cells per group; one-way ANOVA. (b,d) Intensity profiles of NO (DAF-FM staining) and its quantitation. n = 100 cells per group; one-way ANOVA; ns: non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 6

Mitochondrial profiles of BMMs grown on the hydrogel. (a,c) Representative images of cardiolipin (NAO staining) and its quantitation. n = 200 cells per group; one-way ANOVA. (b,d) Mitochondrial membrane potential as measured by the ratiometric dye JC-1 and its quantitation. n = 250 cells per group; one-way ANOVA; ns: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

Cytosolic and lysosomal pH of BMMs grown on the hydrogel. (a,b) Intensity profiles of cytosolic pH (a; left vertical panels) with the corresponding DIC images (a; right vertical panels) and their quantitation. (c,d) Lysosomal pH as measured using the pH-sensitive dye LysoSensor green (c; left vertical panels) with their corresponding intensity profiles (c; right vertical panels) and their quantitation. n = 200 cells per group; one-way ANOVA; ns: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 8

Differentiation propensities of BMMs into osteoclasts grown on hydrogel. (a) Representative TRAP assay of BMMs grown on the hydrogel in the presence of the TRPV1 activator (RTX) and inhibitor (5′-IRTX) under differentiating conditions (MCSF + RANKL). (b,c) Quantitation of TRAP-positive cells and multinucleated cells in the presence of capsaicin (b) and RTX (c) shows elevated osteoclastogenesis as compared to MCSF and CMT:HEMA control groups. n = 5–10; one-way ANOVA; **p < 0.01, ***p < 0.005, ****p < 0.001.