Bovine BMP osteoinductive potential enhanced by functionalized dextran-derived hydrogels
Introduction
Bone regeneration can be achieved by locally delivering native or recombinant human BMP. The success of growth factor therapy requires the combination of BMP within a carrier matrix [1], [2]. Supra-physiological doses of BMP are generally required to induce bone formation: in the order of milligrams for endogeneously extracted BMPs and micrograms for recombinant BMPs [3]. An optimized delivery system might improve the osteopotency of the device while reducing as much as possible the amount of introduced BMP, which offers double advantage of being safer as well as less expensive.
A number of organic and inorganic, natural and artificial substances have been used to deliver BMP in experimental or preclinical models. Most of them were evaluated in different shape and pattern. Among all tested carriers, collagen sponge appeared to be the most intensively tested matrix in animals’ studies as well as in clinical trials [4], [5]. In clinical practice, absorbable collagen sponge remains the only FDA approved human recombinant BMPs carrier [6], [7]. However, in spite of its good results as BMP carrier and important safety background, collagen sponge demonstrated some disadvantages owing to its xenogeneic origin (mostly from bovine and porcine skin). As with all protein based therapies, an immune response might be induced and clinical trials demonstrated that around 18% of patients treated with rhBMP2/collagen sponge developed anti-Type I collagen antibodies [7]. Still the possibility of xenozoonoses from the animal tissue to the human recipient has to be carefully considered [8].
In this context, to engineer a fully synthetic carrier, free of potential contamination, could be of major interest. Additionally, synthetic polymers present other advantages. First, they allow a better control of physico-chemical properties than natural polymers, leading to more reproducible delivery kinetics of specific molecules. Second, polymers can be easily chemically modified with functional groups making them for example biospecific to a protein. Thus, the interaction of BMP with a polymeric matrix would provide a carrier able to retain the growth factor at the appropriate site, to locally deliver it in sustained fashion and to decrease the BMP amount used in clinical applications.
Taking these factors into consideration, we had tested matrices based on dextran-derived hydrogels as new BMP carriers. Hydrogels have been prepared by the chemical crosslinking of native dextran and a functionalized dextran (FD) with sodium trimetaphosphate. FD is an anionic water-soluble polymer bearing carboxylate, benzylamide and sulfate groups, that exhibits binding capacity to heparin-binding growth factors such as transforming growth factor-β1 (TGF-β1) [9], [10]. TGF-β1 and BMP-2 belong to the same superfamily and share one third of structural homology [11].
Recent studies had shown that these hydrogels were endowed with the ability to retain bioactive TGF-β1 within the polymer matrix depending on their crosslink extend and their content in FD [12]. The capacity of these hydrogels to retain rhBMP-2 was investigated in vitro. Then, the osteoinductive potential of extracted bovine BMP soaked in these carriers was evaluated using a rat ectopic model.
Section snippets
Materials
Recombinant human Bone Morphognetic Protein-2 (rhBMP-2) and kit used for ELISA were purchased from R&D Systems Europe (Lille, France). Collagen sponges (Etik Collagen ®) were purchased from Pierre Roland (Bordeaux, France). Extracted bovine Bone Morphogenetic Protein mixture (BMP) was a kind gift of Sulzer Orthopedics Biologics Inc. (Denver, Co). All chemicals and solvents were of analytical grade and purchased from VWR (Fontenay-sous-bois, France).
Preparation of the hydrogels
The water-soluble functionalized dextran (FD)
Results
SEM views of hydrogel C demonstrated an irregular shape and a rough surface of particles at low magnification (Fig. 1a). At higher magnification (Fig. 1b), the particles exhibited a honey-comb-like structure with a size of the pores on the surface ranging from a few to ten micrometers. Particles of hydrogels A and B presented the same pattern.
Discussion
The main objective of this study was to evaluate in vitro and in vivo synthetic matrices based on dextran-derived hydrogels as BMP carriers. These hydrogels were manufactured as porous particles of 0.5 mm of diameter, which present a double advantage. First, they provide a carrier system with a high exchange surface, which would potentially enhance the growth factor availability. Second, particulate hydrogels provide a surgically convenient injectable material suitable for implantation into
Conclusion
The present in vivo study evaluated dextran-derived hydrogels as potential BMP carrier. The largest bone formation was observed when BMP was combined with hydrogels exhibiting the highest rhTGF-β1 retention in situ. In spite of a weaker potential to retain rhBMP-2 than rhTGFβ1, these hydrogels could allow to decrease the amount of implanted bovine BMP required for bone regeneration. While the hydrogel calcification was variable and still not fully understood, it gives rise both osteoconductive
Acknowledgments
The authors greatly acknowledged Dr. W. Bensaid and F. Anagnostou for surgical procedures, P. Portes (LIMHP, Universite Paris 13) for his technical assistance for SEM and Dr. C. Damien (Sulzer Orthopedics Biologics Inc (Denver, CO, USA)) for its donation in bone proteins. This work was supported by the Centre National de la Recherche Scientifique – Sciences pour l’Ingenieur and by the Institut National de la Santé et de la Recherche Médicale. M. Maire ackowledges the Ministère de l’Education
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