In Vitro and In Vivo Evaluation of Commercially Available Fibrin Gel as a Carrier of Alendronate for Bone Tissue Engineering

Abstract

Alendronate (ALN) is a bisphosphonate drug that is widely used for the treatment of osteoporosis. Furthermore, local delivery of ALN has the potential to improve the bone regeneration. This study was designed to investigate an ALN-containing fibrin (fibrin/ALN) gel and evaluate the effect of this gel on both in vitro cellular behavior using human mesenchymal stem cells (hMSCs) and in vivo bone regenerative capacity. Fibrin hydrogels were fabricated using various ALN concentrations (10^-7-10^-4 M) with fibrin glue and the morphology, mechanical properties, and ALN release kinetics were characterized. Proliferation and osteogenic differentiation of and cytotoxicity in fibrin/ALN gel-embedded hMSCs were examined. In vivo bone formation was evaluated using a rabbit calvarial defect model. The fabricated fibrin/ALN gel was transparent with Young's modulus of ~13 kPa, and these properties were not affected by ALN concentration. The in vitro studies showed sustained release of ALN from the fibrin gel and revealed that hMSCs cultured in fibrin/ALN gel showed significantly increased proliferation and osteogenic differentiation. In addition, microcomputed tomography and histological analysis revealed that the newly formed bone was significantly enhanced by implantation of fibrin/ALN gel in a calvarial defect model. These results suggest that fibrin/ALN has the potential to improve bone regeneration.

Figure 1

Schematic diagrams of the alendronate embedded fibrin hydrogel to improve bone regeneration.

Figure 2

(a) Gross view of fibrin gels containing various ALN concentrations (10⁻⁴M~10⁻⁷M). (b) Effects of ALN concentration on the stiffness of fibrin/ALN gel. The stiffness of the fibrin/ALN gel was not significantly affected by ALN concentration. (c) In vitro cumulative release of ALN from fibrin gel. A slow sustained release was observed. The data shown are the mean ± standard deviation (SD) of three independent experiments.

Figure 3

Effect of ALN concentration in fibrin/ALN gels on human mesenchymal stem cell (hMSC) proliferation. Cells were embedded and cultured in the fibrin gels prepared with ALN at various concentrations. After 3 days of cultivation, cell proliferation was measured. The cells embedded in the fibrin/ALN gels prepared with 10⁻⁷, 10⁻⁶, and 10⁻⁵M ALN exhibited increased proliferation. However, proliferation was significantly inhibited in fibrin/ALN gels prepared with 10⁻⁴M ALN. The data shown are the mean ± standard deviation (SD) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01 compared with the control.

Figure 4

Effect of ALN concentration in fibrin/ALN gels on cell viability. Human mesenchymal stem cells (hMSCs) were embedded in fibrin/ALN gels containing various concentrations of ALN. After 3 days of cultivation, live/dead staining was performed. Most of the hMSCs were viable and retained a fibroblast-like morphology (stained by calcein AM, shown in green) in fibrin gels and 10⁻⁷, 10⁻⁶, and 10⁻⁵M ALN composite fibrin gels. In contrast, when cells were embedded in 10⁻⁴M ALN fibrin/ALN gel, a large number of cells were found to be dead or unhealthy (stained by EthD-1, shown in red) and cells were poorly distributed and had a rounded cellular morphology.

Figure 5

Effect of ALN concentration in fibrin/ALN gels (a) on alkaline phosphatase (ALP) activity (ND, not detected) and (b) calcium accumulation. Human mesenchymal stem cells (hMSCs) were embedded in the fibrin/ALN gels containing various concentrations of ALN. ALP activity assay and alizarin red S staining were performed after 7 and 14 days of cultivation, respectively. (c) In addition, after 7 days of cultivation, real-time PCR was performed for several osteoblast marker genes and cells cultured in fibrin/ALN gels containing 10⁻⁷, 10⁻⁶, and 10⁻⁵ M ALN, and osteoblast differentiation was significantly increased. The data shown are the means ± standard deviation (SD) of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01 compared with control.

Figure 6

Effect of fibrin/ALN gel on new bone formation in a rabbit calvarial defect. Representative three-dimensional microcomputer tomography images (a) and quantification graph (b) of calvarial bone defect regeneration at 2, 4, and 8 weeks after implantation with fibrin gel or fibrin/ALN gel. The new bone formation increased in the fibrin/ALN gel (10⁻⁶M ALN) group compared to that in the fibrin gel-implanted group. Scale bar = 3 mm. The data shown are the mean ± standard deviation (SD). ^∗P < 0.05 compared with the fibrin gel-implanted group.

Figure 7

Hematoxylin and eosin-stained microscopic images of the margin of the defect site at 2, 4, and 8 weeks after implantation of fibrin gel or fibrin/ALN gel (containing 10⁻⁶M ALN). Newly formed bone at the periphery of the defect was observed in both the fibrin gel and fibrin/ALN groups from 2 weeks after implantation. Mature bone was more abundant in the fibrin/ALN group and newly formed bone tended to coalesce with the host bone. Hb, host bone; Nb, new bone. Scale bar: 250 μm.

Figure 8

Histologic morphology of the new bone at 8 weeks after implantation. In the central area, newly formed bone was more mature in the fibrin/ALN gel-implanted group than in the fibrin gel group. The arrow indicates the newly formed bone containing woven bone and lamella. Arrow head indicates the defect marginal site. Hb, host bone; Nb, new bone. Scale bar: 500 μm.

Figure 9

Goldner's Masson trichrome-stained histological images of regenerated bone in the central area of calvarial defects at 2, 4, and 8 weeks after implantation. At 2 weeks, large amounts of fibroblastic connective tissue were observed in the fibrin gel and fibrin/ALN gel-implanted groups. Specifically, a small island-like amount of immature bone (black arrow) was observed in the fibrin/ALN gel-implanted group. Furthermore, mature bone islands (white arrow) were observed at 4 weeks and the newly formed bones were more abundant at 8 weeks in the fibrin/ALN-implanted group than in the fibrin gel-implanted group. Scale bar: 250 μm.