Effects of clodronate and alendronate on osteoclast and osteoblast co-cultures on silk-hydroxyapatite films

Abstract

The goal of this study was to explore the effects of osteoporosis-related therapeutics on bone remodeling in vitro. A previously established bone-tissue mimetic system consisting of silk protein biomaterials in combination with hydroxyapatite and human cells was used for the study. Silk-hydroxyapatite films were pre-complexed with the non-nitrogenous bisphosphonate clodronate or the nitrogenous bisphosphonate alendronate and cultured with THP-1 human acute monocytic leukemia cell line-derived osteoclasts, human mesenchymal stem cell derived osteoblasts or a direct co-culture of the two cell types. Metabolic activity, calcium deposition and alkaline phosphatase activity were assessed over 12 weeks, and reconstructed remodeled biomaterial surfaces were also evaluated for quantitative morphological changes. Increased metabolic activity and increased roughness were found on the clodronate-complexed biomaterial substrates remodeled by osteoblasts and co-cultures of osteoblasts with osteoclasts, even at doses high enough to cause a 90% decrease in osteoclast metabolic activity. Films complexed with low doses of alendronate resulted in increased metabolic activity and calcium deposition by osteoblasts, while higher doses were similarly toxic among osteoclasts, osteoblasts and co-cultures. These results point to the utility of these well-defined bone-mimetic in vitro cultures as useful screens for therapeutics for bone-related diseases, particularly with the ability to conduct studies for extended duration (here for 12 weeks) and with pre-complexed drugs to mimic conditions found in vivo.

Keywords: Bisphosphonates; Co-culture; Osteoporosis; Silk.

Figure 1. Schematic of 12 week studies

Top: Films were cast from a dispersion of HA in aqueous silk solution. Following drying and water annealing, films were soaked in solutions of clodronate or alendronate which bound to the HA. Following autoclaving, films were seeded with hMSCs, THP-1s, or a co-culture of the two cell types in equal number. Differentiation was then initiated, and films were remodeled by cells for 4, 8, or 12 weeks. For surface metrology analysis cells were removed from films by soaking in water overnight at 4°C and films were dried and sputter coated. Bottom: Eucentric SEM images were taken with an 8 degree difference in tilt angle. 3D surface models were generated and roughness parameters were calculated. Example surfaces from low and high osteoblast activity (approximately 600 µm²) are shown.

Figure 2. Effects of bisphosphonates on metabolic activity (7 days)

THP-1 osteoclasts, hMSC osteoblasts, or a co-culture of the two cell types were maintained on silk-HA films or silk-HA films loaded with clodronate or alendronate for 7 days, and metabolic activity was measured by Alamar Blue. Metabolic activity of cells grown on drug-loaded films is normalized to metabolic activity of the same cell type(s) grown on control silk-HA films and is expressed as mean ± SD. Top left: Osteoclasts cultured on clodronate-loaded films. Top right: Osteoclasts cultured on alendronate-loaded films. Middle left: Osteoblasts cultured on clodronate-loaded films. Middle right: Osteoblasts cultured on alendronate-loaded films. Bottom left: Co-cultures cultured on clodronate-loaded films. Bottom right: Co-cultures cultured on alendronate-loaded films. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Effects of bisphosphonates on metabolic activity (12 weeks)

THP-1 osteoclasts, hMSC osteoblasts, or a co-culture of the two cell types were maintained on silk-HA films or silk-HA films loaded with clodronate or alendronate for 12 weeks, and metabolic activity was measured by Alamar Blue. Metabolic activity of cells grown on drug-loaded films is normalized to metabolic activity of the same cell type(s) grown on control silk-HA films and is expressed as mean ± SD. Top left: Osteoclasts cultured on clodronate-loaded films. Top right: Osteoclasts cultured on alendronate-loaded films. Middle left: Osteoblasts cultured on clodronate-loaded films. Middle right: Osteoblasts cultured on alendronateloaded films. Bottom left: Co-cultures cultured on clodronate-loaded films. Bottom right: Co-cultures cultured on alendronate-loaded films. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Effects of bisphosphonates on calcium deposition

Calcium content of films was measured after 4, 8, and 12 weeks of culture and expressed as mean ± SD. Top left: Osteoblasts cultured on clodronate-loaded films. Top right: Osteoblasts cultured on alendronate-loaded films. Bottom left: Co-cultures cultured on clodronate-loaded films. Bottom right: Co-cultures cultured on alendronate-loaded films. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Effects of bisphosphonates on developed interfacial ratio

3D surfaces were reconstructed from SEM imaging and the developed interfacial ratio (Sdr) was calculated after 4, 8, and 12 weeks of culture and expressed as mean ± SD. Top left: Osteoclasts cultured on clodronate-loaded films. Top right: Osteoclasts cultured on alendronate-loaded films. Middle left: Osteoblasts cultured on clodronate-loaded films. Middle right: Osteoblasts cultured on alendronate-loaded films. Bottom left: Co-cultures cultured on clodronate-loaded films. Bottom right: Co-cultures cultured on alendronate-loaded films. *p<0.05, **p<0.01, ***p<0.001.