Impact of Sulfated Hyaluronan on Bone Metabolism in Diabetic Charcot Neuroarthropathy and Degenerative Arthritis

Abstract

Bone in diabetes mellitus is characterized by an altered microarchitecture caused by abnormal metabolism of bone cells. Together with diabetic neuropathy, this is associated with serious complications including impaired bone healing culminating in complicated fractures and dislocations, especially in the lower extremities, so-called Charcot neuroarthropathy (CN). The underlying mechanisms are not yet fully understood, and treatment of CN is challenging. Several in vitro and in vivo investigations have suggested positive effects on bone regeneration by modifying biomaterials with sulfated glycosaminoglycans (sGAG). Recent findings described a beneficial effect of sGAG for bone healing in diabetic animal models compared to healthy animals. We therefore aimed at studying the effects of low- and high-sulfated hyaluronan derivatives on osteoclast markers as well as gene expression patterns of osteoclasts and osteoblasts from patients with diabetic CN compared to non-diabetic patients with arthritis at the foot and ankle. Exposure to sulfated hyaluronan (sHA) derivatives reduced the exaggerated calcium phosphate resorption as well as the expression of genes associated with bone resorption in both groups, but more pronounced in patients with CN. Moreover, sHA derivatives reduced the release of pro-inflammatory cytokines in osteoclasts of patients with CN. The effects of sHA on osteoblasts differed only marginally between patients with CN and non-diabetic patients with arthritis. These results suggest balancing effects of sHA on osteoclastic bone resorption parameters in diabetes.

Keywords: Charcot neuroarthropathy; ankle arthritis; diabetes mellitus; osteoblasts; osteoclasts; sulfated hyaluronan.

Figure 1

TRAP and resorption activity and gene expression of PBMC derived either from arthritis (group A) or Charcot patients (group C), respectively. (A) TRAP activity was determined in PBMC either treated with M-CSF only or with M-CSF and RANKL after 14 days of culture (group A: n = 5; group C: n = 8). (B) Resorption activity was performed by culturing PBMC on a cell-derived calcium phosphate matrix. After 14 days of culture, calcium phosphate was stained black by von Kossa silver staining. Wells were scanned and black (calcium phosphate) and white (resorbed) areas were quantified (group A: n = 7; group C: n = 9). (C) Comparison of initial resorption activity of both patient groups after M-CSF only treatment showing higher initial resorption activity without RANKL in group C than in group A (p = 0.48). ACP5 (D), CTSK (E) and CALCR (F) expression were determined by qPCR after 14 days of culture (group A: n = 4; group C: n = 7). Results are shown relative to GAPDH expression and were normalized to group A M-CSF and were calculated using the CT method. All data are provided as median with interquartile range and min/max values; statistical analyses for (A,B,D–F) were performed using ANOVA and Tukey post-hoc test; statistical analysis for (C) was performed using t test.

Figure 2

Secretion of inflammatory cytokines in PBMC of group A and group C. Cell culture supernatants were collected on day 3 and 7 from cells treated with either M-CSF or M-CSF/RANKL (n = 4). IL-6, IL-10, TGF-β2 and TNF-α were quantified by ELISA. Data are given as median with interquartile range and min/max values, statistical analyses were performed using ANOVA and Tukey post-hoc test.

Figure 3

Gene expression in osteoblasts. Osteoblasts of both patient groups were cultured for 14 days (group A: n = 6; group C: n = 4) before RNA isolation and reverse transcription. Quantitative real-time PCR revealed the gene expressions of ALPL (A), RUNX2 (B) and BGLAP (C). Gene expression was normalized to GAPDH expression and analyzed using the CT method. Data are provided relative to group A control and as median with interquartile range and min/max values; statistical analyses were performed using ANOVA and Tukey post-hoc test.

Figure 4

Effect of sHA on TRAP activity, calcium phosphate resorption and gene expression in osteoclasts. PBMC were cultured on a calcium phosphate matrix. (A–C) After 14 days of culture with M-CSF/RANKL and sHA (200 µg/mL) TRAP activity (A) and resorption activity (B) were analyzed. (C) Representative images of resorption assays in 24-well plates of M-CSF/RANKL-treated PBMC of an arthritis (upper row) and Charcot patient (lower row) after 14 days of culture. Strong resorption of Charcot patients was diminished after sHA administration. Black: silver-stained calcium phosphate; white: resorbed areas. (D–F) After 14 days of culture cells were lysed for RNA isolation followed by reverse transcription and quantitative real-time PCR to evaluate the expression of ACP5 (D), CTSK (E) and CALCR (F). Gene expression was normalized to GAPDH expression and calculated using the CT method. Data are provided as median with interquartile range and min/max values, statistical analyses were performed using ANOVA and Tukey post-hoc test.

Figure 5

Impact of sHA on osteoblasts after 14 days of sHA treatment. (A) TNAP activity was quantified in cells of eight patients per group. The untreated control of group A was set to 1. TNAP activity was reduced significantly after sHA3 treatment. Expression of the osteogenic marker genes ALPL (B), RUNX2 (C) and BGLAP (D) was evaluated by quantitative real-time PCR. Gene expression of the target genes was normalized to GAPDH expression and calculated using the CT method. Data are shown as median with interquartile range and min/max values, statistical analyses were performed using ANOVA and Tukey post-hoc test.

Figure 6

Impact of sHA1 and sHA3 on cytokine concentrations in PBMC of arthritis (group A) and Charcot patients (group C). PBMC were treated with M-CSF/RANKL and sHA derivatives. Cell culture supernatant was collected on day 3 and 7 for cytokine quantification by ELISA (n = 4). Data are given as median with interquartile range and min/max values, statistical analyses were performed using ANOVA and Tukey post-hoc test.

Figure 7

Structural characteristic of sulfated GAG derivatives.