A New Class of Bioactive and Biodegradable Soybean-Based Bone Fillers Matteo Santin,* ,² Christopher Morris, ² Guy Standen, ² Luigi Nicolais, ‡ and Luigi Ambrosio ‡ School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, United Kingdom, and Institute of Composite and Biomedical Materials, Consiglio Nazionale delle Ricerche, Piazzale Tecchio 80, 80130 Naples, Italy Received March 26, 2007; Revised Manuscript Received June 7, 2007 The reconstruction of large bone defects in periodontal, maxillofacial, and orthopedic surgery relies on the implantation of biomaterials able to support the growth of new tissue. None of the materials currently available is able to combine all the properties required, which are (i) easy handling, (ii) biodegradation, (iii) low immunogenicity, and more importantly, (iv) induction of tissue regeneration. A new class of biodegradable biomaterials has been obtained by simple thermosetting of defatted soybean curd. The final material can be processed into films, porous scaffolds, and granules for different surgical needs. When incubated in physiological solutions the material shows water uptake of 80%, elongation at break of 0.9 mm/mm, and 25% (w/w) degradation in 7 days. Soybean-based biomaterial granules are shown to reduce the activity of the monocytes/macrophages and of the osteoclasts and to induce osteoblast differentiation in vitro, thus demonstrating a bone regeneration potential suitable for many clinical applications. Introduction Bone damages and defects can derive from those traumatic events or surgical procedures where bone needs to be removed because of pathological conditions. When the defect reaches a critical size, bone is not able to regenerate spontaneously and bone fillers are required to support its formation. Mineralized and nonmineralized bone grafts derived from the same patient (autograft) and from human (allograft) or animal (xenograft) donors are considered the gold standard in surgery. However, their use presents main drawbacks such as limited availability of autografts, patient’s morbidity, and risks of transmittable diseases associated with allografts. Three main classes of synthetic bone fillers are currently used in surgery as alternatives to bone grafts: (i) ceramics (hydroxyapatite, HA; tricalcium phosphate, TCP; bioglasses), (ii) poly(lactic/glycolic) acid (PLGA), and (iii) collagen. HA, TCP, and bioglass, delivered in forms of porous scaffolds and granules, have excellent osteoconductive properties; 1 they have been shown to support adhesion and proliferation of the bone-producing cells, the osteoblasts in vitro 2 , and to establish strong bonding with the newly deposited bone mineral phase in vivo. 3 However, because of their stiff and brittle mechanical properties, they are difficult to adapt to the bone defect during surgery, and although they can be manufactured with different degrees of crystallinity, their resorption rate cannot be finely tuned to bone regeneration and remodelling. 4 PLGA is a synthetic biodegradable polymer widely used in the biomedical field. 5 This biomaterial is able to support the adhesion of osteoblasts and to undergo complete degradation into CO 2 and water. 6 However, it is recognized that degrading PLGA polymer fragments elicit an inflammatory response, thus impairing bone regeneration. 7,8 Collagen, natu- rally extracted from animal sources or of recombinant origin, is also available in the form of films and sponges, but its clinical performance is debated. 9,10 The presence of amino acid se- quences able to recognize cell receptors such as the integrins makes this material a suitable substrate for colonization by the cells. 11 However, these amino acid sequences are not specific to bone cells, but they can be recognized also by inflammatory cells and by fibroblasts, thus eliciting adverse reactions. 12 As for allografts, the risk of transmittable diseases for materials of animal origin and the purification costs of the recombinant products are additional drawbacks in the clinical use of this natural polymer. Furthermore, true bone induction cannot be obtained by these traditional bone fillers unless growth factors such as the bone morphogenetic protein 2 (BMP-2) are loaded in their structure. 13 Although clinical studies have shown the potential of BMP-2- loaded bone fillers in accelerating bone regeneration, 13 relatively high amounts of BMP-2 are required to achieve satisfactory clinical results, inevitably increasing the costs of the bone filler and raising concerns about the potential carcinogenic effect of the growth factor. 14 Soybean is a natural material made of protein and carbohy- drate fractions (ca. 40% each), an oil fraction (ca. 18%), and minerals (ca. 2%). 15 Soybean also contains isoflavones, phy- toestrogens with an ascertained action on eukaryotic cells. 16 Isoflavones such as genistein and daidzein are considered among the most effective plant estrogens as they are particularly effective in reducing the proliferation of tumor cells and the activity of immunocompetent cells such as lymphocytes and monocytes/macrophages as well as of the bone-resorbing cells, the osteoclasts. 16 Furthermore, these phytoestrogens are able to induce differentiation of the osteoblasts. 17 The reported low incidence of breast and prostate cancer as well as of osteoporosis in eastern populations has been indeed ascribed to the regular dietary intake of soy isoflavones. 18 Genistein and daidzein are also found abundantly in soy as glycosylated forms, genistin and daidzin. When glycosylated, isoflavones are inactive on cells, but it has been demonstrated that they can be readily * Corresponding author: tel +44 (0)1273 642083; fax +44 (0)1273 642674; e-mail m.santin@brighton.ac.uk. ² University of Brighton. ‡ IMCB-CNR. 2706 Biomacromolecules 2007, 8, 2706-2711 10.1021/bm0703362 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/27/2007