The use of RANKL-coated brushite cement to stimulate bone remodelling

doi:10.1016/j.biomaterials.2008.03.035

Biomaterials

Volume 29, Issue 22, August 2008, Pages 3253-3259

https://doi.org/10.1016/j.biomaterials.2008.03.035 Get rights and content

Abstract

Calcium phosphate cements were first proposed as synthetic bone substitutes over two decades ago, however, they are characterised by slow chemical or cellular resorption and a slow osteointegration. In contrast, bone autograft has been shown to stimulate osteoclastogenesis and angiogenesis resulting in active bone remodelling and rapid graft incorporation. Therefore, we aimed to develop a biomaterial able to release a key stimulator of the bone remodelling process, cytokine RANKL. Cylinders of brushite cement, hydroxyapatite cement and sodium alginate were loaded with RANKL either by incorporation into the cement or by coating the material with soluble RANKL. To test the biological activity of these formulations, we assessed their effectiveness in inducing osteoclast formation from RAW 264.7 monocytic cell line. Only brushite and hydroxyapatite cements coated with RANKL allowed for retaining sufficient biological activity to induce osteoclast formation. Most efficient was coating 40 mg cylinder of brushite cement with 800 ng RANKL. We have found that RANKL-coated brushite cement exhibits osteoclastogenic activity for at least 1 month at 37 °C. Thus, we developed a formulation of brushite cement with RANKL – a synthetic bone graft that is similar to autografts in its ability to actively induce osteoclastogenesis.

Introduction

In spite of its ability to regenerate after injury, bone tissue needs guidance to allow sufficient growth after extensive loss or injury (e.g. bone tumours and defects, complex fractures, osteoporosis, osteonecrosis, and other orthopaedic surgeries) [1]. Increased life expectancy in developed countries has led to a steep rise in the number of musculoskeletal disorders, including osteoporosis and osteoarthritis. As the aging population is increasingly active, 2.2 million corrective procedures occur annually, requiring bone substitutes and are expected to increase steadily at a rate of 10% per annum in the coming years [2].

Bone autografts and allografts, including demineralised bone matrix (DBM), remain the clinical standards for the reconstruction of large bone defects because they exhibit osteogenic, osteoinductive and osteoconductive properties [1]. However, these biological grafts are often hampered by complications such as pain at the site of surgery, limited quantity of bone for bone autograft, the perceived risk of disease transmission and the poor osteogenic properties for bone allograft [3], [4], [5], [6].

Calcium phosphate based materials are widely used as synthetic bone substitutes in bone reconstructive surgery because of their osteoconductivity and ability to form chemical bonds with host bone. Depending on their composition and structure, calcium phosphate materials can be either dissolved and gradually replaced by bone or can remain at the site of implantation [7], [8]. Among these materials, calcium phosphate cements (CPCs) were proposed two decades ago by LeGeros et al. [9] and Brown and Chow [10] as synthetic bone substitutes. In general, all CPCs are formed by mixing one or more calcium orthophosphate powders with an aqueous solution to form a paste which is then placed into the bone defect and sets in situ [11]. However, calcium phosphate cements generally lack the osteogenic potential to promote bone healing over large or critical-sized defects and are very slowly resorbed and replaced by new bone tissue [12]. The possibility to use CPCs not only as a “passive” biomaterial for structural repair but also as a carrier for local and controlled supply of drugs that guide and improve the existing tissue regeneration properties is very attractive [13].

Using mathematical modelling, we have previously shown that stimulation of osteoclasts can lead to faster recruitment of more functionally active osteoblasts, resulting in an increase in bone mass [14]. Most convincingly, it was recently demonstrated that a key osteoclastogenic factor, the receptor activator of the nuclear factor κB ligand (RANKL), together with the angiogenic factor, the vascular endothelial growth factor (VEGF), are necessary and sufficient for efficient remodelling of a devitalised allograft [15]. In vitro models of osteoclastogenesis are invaluable for studying the cellular and molecular regulations of osteoclast differentiation and activation. Among different in vitro models, murine monocytic cell line, RAW 264.7, which forms multinucleated TRAP-positive osteoclasts able to resorb calcified matrices, is widely used to study osteoclastogenesis [16], [17].

The objective of the study was to examine the biological efficiency of the combination of brushite cement with RANKL protein in vitro. We tested the effects of brushite cement, hydroxyapatite cement and also a calcium cross-linked alginate hydrogel associated with RANKL. The optimal protocol for RANKL incorporation into brushite cement was established as well as the optimal amount of adsorbed RANKL to induce osteoclastogenesis. Finally, we evaluated the ability of RANKL-coated brushite cement to induce osteoclastogenesis as a function of culture time.

Section snippets

Brushite cement

Brushite cement was prepared as described previously [18]. Briefly, β-tricalcium phosphate (β-TCP) was synthesised by heating a mixture of monetite (DCPA; Mallinckrodt Baker, Germany) and calcium carbonate (CC; Merck, Germany) to 1050 °C for 24 h, followed by quenching at room temperature in a desiccator. The product consisted of a phase pure and highly crystalline β-TCP as verified by X-ray diffraction (XRD). The sintered cake was crushed using a pestle and mortar, then passed through a 355 μm

Characterisation of calcium phosphate cements

Brushite and HA calcium phosphate cements were set in the form of cylinders of 3 mm diameter and 3 mm height. Fig. 1 shows the XRD patterns of both brushite (Fig. 1a) and HA (Fig. 1b) cements which corresponded well with the International Centre for Diffraction Data (ICDD) patterns. Physical properties of brushite and HA cement cylinders were analyzed. After setting, brushite cement showed an average density of 1.37 ± 0.02 g/cm³ with a specific surface area of 5.57 ± 0.01 m²/g and a total porosity of

Discussion

Our goal was to combine a key stimulator of bone remodelling, RANKL and an osteoconductive calcium phosphate material in such a way that osteoclastogenesis potential of this protein was maintained. In this study, by using an original method based on the in vitro induction of osteoclast formation, we developed a RANKL releasing matrix capable of inducing osteoclast formation for 1 month.

Calcium phosphate materials are extensively used as a carrier to deliver drug after in vivo implantation [20]

Conclusion

The possibility of using synthetic bone grafts as a carrier for local and controlled supply of drugs is very attractive and combining biomaterials with factors that enhance the bone remodelling process presents a novel means of accelerating implant osteointegration after surgery. In this study, we developed the formulation to adsorb and release RANKL, a major factor responsible for bone resorption, on calcium phosphate brushite cement. RANKL-coated brushite cement was able to induce the

Acknowledgements

We thank Dr. M.F. Morrison, University of Toronto for providing reagents used in this study. This study was supported financially by the CIHR Training Program in Skeletal Health Research and Canadian Arthritis Network (DLN), Canada Research Chairs (JB) and Québec Ministre des Relations Internationales (Québec-Bavaria Exchange Program), PSR-SIIRI-029 IBI (JB & UG).

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The use of RANKL-coated brushite cement to stimulate bone remodelling

Abstract

Introduction

Section snippets

Brushite cement

Characterisation of calcium phosphate cements

Discussion

Conclusion

Acknowledgements

Orthop Clin North Am

Orthop Clin North Am

Biomaterials

J Arthroplasty

J Controlled Release

Biochem Pharmacol

Biomaterials

Adv Drug Deliv Rev

Biomaterials

Biomaterials

Tissue engineering

Science

The role of the osteoconductive scaffold in synthetic bone graft

Orthopedics

Iliac crest bone graft harvest donor site morbidity. A statistical evaluation

Spine