Effects of locally applied BMP-2 and alendronate on bone regeneration

Abstract

Objectives Bone grafting is a way to transplant bone tissue to repair bone fractures. There are four types of bone graft materials are available: autografts, allografts, xenografts, and alloplasts. Alloplastic bone substitutes have been frequently utilized in the clinical application because of no additional surgery for bone harvest and low immunological reaction. However, allografts lack osteoindcutive characteristics. Therefore, osteoinductive growth factor is required for effective new bone formation. Bone morphogenetic protein-2 (BMP-2) has been regarded as a potent bone-inducing factor and has been successfully used in the dental and orthopedic surgery. However, initial burst release of BMP-2 and osteoclast activation by high-dose of BMP-2 resulted in reduced bone forming effect. Recent studies have reported that alendronate (ALN) block cellular activities of osteoclasts and induces osteogenic differentiation. Taken together, combined treatment of BMP-2 with ALN would facilitate effective bone formation by reducing early bone resorption at targeted region. The present study was aimed to investigate sustained release of BMP-2 with and without ALN carried by β-TCP in vitro and to evaluate the effect of this combined application on new bone formation in a critical sized rat calvarial defect model.

Methods 1. Mouse mandible-derived osteoprogenitor cells were treated with BMP-2 (200 ng/mL) and various concentration of ALN (1, 10, and 100 μM). MTT assay and ALP activity were performed on day 1 and 3. Mouse bone marrow macrophages were differentiate into committed osteoclast precursors and stained with leukocyte acid phosphatase staining kit on day 7. 2. BMP-2 release kinetics

In order to assist in adhesion of the ALN onto calcium phosphate ceramics, 30 μg of ALN is deposited on the surface and allowed overnight dry. BMP-2 was loaded directly into β-TCP and ALN coated β-TCP. 3. Rats with a surgically induced 8-mm critical sized defects were seperated into four groups: β-TCP, β-TCP with BMP-2, β-TCP with ALN, and β-TCP with BMP-2 and ALN administered 1, 4, and 8 weeks after surgery. β-TCP with 40 μg BMP-2 and 30 μg ALN were placed in the defect. Early osteogenic process was examined with real time RT-PCR and bone formation was analyzed by μCT, histology, and histomorphometry.

Results The combined use of BMP-2 and ALN in mouse jaw bone derived osteoprogenitor cells showed no cytotoxicity but enhanced ALP activity over control group. However, in higher dose ALN showed decreased cell viability and ostoeblastic differentiation even in presence of BMP-2. The in vitro BMP-2 release in the BMP/ALN exhibited a slower release rate whereas the BMP alone group showed an initial burst phase. In spite of lower release of BMP-2 in presence of ALN both the BMP-2 and BMP/ALN groups showed higher expression of bone forming and resorbing markers within the scaffold at defect after 7 days. Micro-computed tomography (micro-CT) and histology revealed that the BMP/ALN led to better bone quantity and quality at the central defect area over time, whereas the BMP alone exhibited excessive ossification with less bone quality. ALN treatment reduced the expression of adipogenic markers and the amount of bone marrow cavities also decreased.

Conclusion In conclusion, we proved the advantage of β-TCP use in BMP/ALN delivery on the basis that ALN induces a lower burst and subsequent sustained release of BMP-2. Whereas early bone resorption and bone formation induced by high-dose of BMP-2 was not alleviated by ALN. However, new bone formation by ALN was revealed at microCT analysis. Notably, ALN reduced bone marrow cavities, induced by BMP-2, by inhibition of adipocyte markers. These findings suggest that co-delivery of BMP-2 with ALN is effective in terms of quality and quantity of the newly regenerated bone.