The Gradual Release of Alendronate for the Treatment of Critical Bone Defects in Osteoporotic and Control Rats

Abstract

Purpose: Osteoporosis is a severe health problem with social and economic impacts on society. The standard treatment consists of the systemic administration of drugs such as bisphosphonates, with alendronate (ALN) being one of the most common. Nevertheless, complications of systemic administration occur with this drug. Therefore, it is necessary to develop new strategies, such as local administration.

Methods: In this study, emulsion/dispersion scaffolds based on W/O emulsion of PCL and PF68 with ALN, containing hydroxyapatite (HA) nanoparticles as the dispersion phase were prepared using electrospinning. Scaffolds with different release kinetics were tested in vitro on the co-cultures of osteoblasts and osteoclast-like cells, isolated from adult osteoporotic and control rats. Cell viability, proliferation, ALP, TRAP and CA II activity were examined. A scaffold with a gradual release of ALN was tested in vivo in the bone defects of osteoporotic and control rats.

Results: The release kinetics were dependent on the scaffold composition and the used system of the poloxamers. The ALN was released from the scaffolds for more than 22 days. The behavior of cells cultured in vitro on scaffolds with different release kinetics was comparable. The difference was evident between cell co-cultures isolated from osteoporotic and control animals. The PCL/HA scaffold show slow degradation in vivo and residual scaffold limited new bone formation inside the defects. Nevertheless, the released ALN supported bone formation in the areas surrounding the residual scaffold. Interestingly, a positive effect of systemic administration of ALN was not proved.

Conclusion: The prepared scaffolds enabled tunable control release of ALN. The effect of ALN was proved in vitro and in in vivo study supported peri-implant bone formation.

Keywords: alendronate; co-culture; drug delivery system; fibrous scaffold; osteoporosis.

Figure 1

Semiquantitative evaluation of bone defect healing was assessed for: cortical bone healing, trabecular bone healing, bone surrounding former defect and overall evaluation. Healing was scored from 1 (poor) to 3 (excellent) except overall evaluation, which was scored from 1 (poor) to 4 (excellent).

Figure 2

Scanning electron microscopy of prepared scaffolds prior and after performing ALN release study (A), scale bar 20 µm. FTIR spectra of S1 (ALN+, HA-, P31R1-), S2 (ALN+, HA+, P31R1+) and S3 (ALN-, HA+, P31R1+) samples compared to PCL nanofibres without incorporated additives (B). TGA thermographs of samples S1, S2 and S3 expressed as residual weight (%) (C) and weight derivative (%/ °C) (D) curves. Cumulative release of ALN from the scaffolds (E). Sample S3 did not contain ALN; therefore, the release study was not performed.

Figure 3

Visualization of Saos-2 cells adhesion and distribution on scaffolds S1 (ALN+, HA-, P31R1-), S2 (ALN+, HA+, P31R1+) and S3 (ALN-, HA+, P31R1+) using confocal microscopy. Cell nuclei were stained by propidium iodide (red color) and cell internal membranes by DiOC6(3) (green color), scale bar 100 µm. Sample S1, day 1 (A), S2, day 1 (B), S3, day 1 (C), S1, day 3 (D), S2, day 3 (E), S3, day 3 (F), S1, day 7 (G), S2, day 7 (H), S3, day 7 (I).

Figure 4

Metabolic activity of rOBs/rPBMCs cells (A). DNA quantification of rOBs/rPBMCs cells (B). Statistical significance is denoted above the columns (p < 0.05), * means the statistically highest value.

Figure 5

ALP activity of rOBs/rPBMCs (A). CA II activity of rOBs/rPBMCs (B). TRAP activity of rOBs/rPBMCs (C) on day 10. Statistical significance is denoted above the columns (p < 0.05), *means the statistically highest value.

Figure 6

Micro-CT images of bone healing 6 weeks after implantation of the scaffolds.

Figure 7

Semiquantitative evaluation of micro-CT images performed independently by 2 people. An evaluation of overall healing (A), cortical (B) and trabecular (C) bone as well as bone tissue surrounding the former bone defect (D) was performed.

Figure 8

Details of the partially healed defect area in different groups visualized in Masson’s green trichrome-stained histological sections. The formation of new bone is apparent in the margins of the defects in all groups from woven bone of different thickness (black arrow) to the trabecular bone formation expanding from the margin of the defect to its center. The original matured bone (black arrowhead) is colored reddish in contrast to less mineralized new formed bone areas (red arrowhead). In the distinct groups, reddish areas of mineralized matrix (red arrow) were also apparent in the newly formed highly dense areas of woven bone in the margins of the bone defects or directly in the defect area. In the groups with empty defects without scaffold presence, the defect area was almost filled with new bone with remnants of the defect filled with connective tissue rich in blood vessels (asterisk).