Alendronate release from calcium phosphate cement for bone regeneration in osteoporotic conditions

Abstract

Osteoporosis represents a major health problem in terms of compromising bone strength and increasing the risk of bone fractures. It can be medically treated with bisphosphonates, which act systemically upon oral or venous administration. Further, bone regenerative treatments in osteoporotic conditions present a challenge. Here, we focused on the development of a synthetic bone substitute material with local diminishing effects on osteoporosis. Composites were created using calcium phosphate cement (CPC; 60 wt%) and polylactic-co-glycolic acid (PLGA; 40 wt%), which were loaded with alendronate (ALN). In vitro results showed that ALN-loaded CPC/PLGA composites presented clinically suitable properties, including setting times, appropriate compressive strength, and controlled release of ALN, the latter being dependent on composite degradation. Using a rat femoral condyle bone defect model in osteoporotic animals, ALN-loaded CPC/PLGA composites demonstrated stimulatory effects on bone formation both within and outside the defect region.

Figure 1

(A–E) Characterization analysis for CPC/PLGA-ALN composites. (A) initial and final setting time at different ALN wt%, dashed lines indicate the initial setting time at 5 wt% ALN for CPC and CPC/PLGA, ^ap < 0.05 comparing CPC initial setting time with CPC/PLGA initial setting time; (B) FTIR of Alendronic acid, PLGA, CPC, CPC-highALN and CPC/PLGA-highALN. Dashed lines indicate peaks associated to PLGA (C=O stretching) and CPC (PO₄⁻³ bending) in the materials. (C,D) XRD at days 0 and 7 for the materials, dashed lines indicate HA peaks (26 and 31.8 °2θ); (E) compressive strength, ^bp < 0.05 for CPC-blank compared to CPC/PLGA-highALN. Error bars represent the SD.

Figure 2

(A–F) Micrographs obtained by SEM. (A) CPC-blank. (B) CPC/PLGA-blank. (C) CPC-lowALN. (D) CPC/PLGA-lowALN. (E) CPC-highALN and (F) CPC/PLGA-highALN. Asterisks and arrows indicate CPC and PLGA particles respectively. Magnification of 500×.

Figure 3

(A,B) Cumulative alendronate release from CPC and CPC/PLGA cylinders loaded with the bisphosphonate for up to 148 days. (A) Absolute ALN release (mg). (B) Relative ALN release (% of loaded dose). Error bars represent the SD.

Figure 4

(A–C): (A) Representative micro-CT images of each experimental group. Color settings were adjusted to distinguish between different densities of bone and material, coloring bone yellow and CPC/PLGA purple. Representative histological images of pMMA sections for each experimental group with images at (B) 4 weeks and 12 weeks and (C) corresponding images at high magnification; methylene blue and basic fuchsine staining; red arrows] new bone (pink color); *CPC/PLGA; BM] bone marrow (blue color).

Figure 5

(A–D): (A) Representative fluorochrome microscopy images at 12 weeks, with high magnification images; red box] area of high magnification; green color] calcein green (week 4); red color] alizarine complexone (week 6); yellow color] rolitetracycline (week 8). (B–D): Relative volume areas (mean + SD) of fluorochrome labels in contact with concentric circles for calcein green (week 4), alizarine complexone (week 6) and rolitetracycline (week 8) for both CPC/PLGA-blank and CPC/PLGA-lowALN (B) within ROI (0–2.5 mm) and (C) extended ROI (eROI, 2.5–3 mm); statistical analysis showed significantly more calcein green within ROI for CPC/PLGA-blank compared to CPC/PLGA-lowALN (Student’s t-test, p < 0.05); analysis showed significantly more calcein green in the eROI for CPC/PLGA-lowALN compared to CPC/PLGA-blank (Student’s t-test, p < 0.05); (D) Distance of the first ring that is in contact with a fluorochrome (mean + SD) for both CPC/PLGA-blank and CPC/PLGA-lowALN; statistical analysis showed significantly closer contact with calcein green for CPC/PLGA-blank compared to CPC/PLGA-lowALN (Student’s t-test, p < 0.01); Range] 0 to 55 rings, edge of defect] ring 45 (2.5 mm from centre).

Figure 6

Accumulated volume percentages (mean ± SD) of new bone formation (green bars for eROI and blue bars within ROI) and material remnants (grey bars) at 4 and 12 weeks after implantation for CPC/PLGA-blank and CPC/PLGA-lowALN; statistical analysis showed significant increase of bone formation within ROI from 4 to 12 weeks for both CPC/PLGA-blank and CPC/PLGA-lowALN (***p < 0.001, **p < 0.01); bone formation over time in the eROI significantly decreased for CPC/PLGA-blank (p < 0.001), but significantly increased for CPC/PLGA-lowALN (p < 0.001); material remnants significantly decreased for CPC/PLGA-blank (*p < 0.05) but not significantly for CPC/PLGA-lowALN (ns = p > 0.05); ^aAt 12 weeks there was a significant higher amount of bone in the eROI for CPC/PLGA-lowALN compared to CPC/PLGA-blank (p < 0.05).