Mineralization, Biodegradation, and Drug Release Behavior of Gelatin/Apatite Composite Microspheres for Bone Regeneration Sander C. G. Leeuwenburgh,* ,† Junichiro Jo, ‡ Huanan Wang, † Masaya Yamamoto, ‡ John A. Jansen, † and Yasuhiko Tabata ‡ Department of Biomaterials, Radboud University Nijmegen Medical Center, Philips van Leydenlaan 25, 6525 EX Nijmegen, The Netherlands, and Department of Biomaterials, Kyoto University, 53 Kawara-cho Shoguin, Sakyo-ku, Kyoto 606-8507, Japan Received June 10, 2010; Revised Manuscript Received July 29, 2010 Gelatin microspheres are well-known for their capacity to release growth factors in a controlled manner, but gelatin microspheres do not calcify in the absence of so-called bioactive substances that induce deposition of calcium phosphate (CaP) bone mineral. This study has investigated if CaP nanocrystals can be incorporated into gelatin microspheres to render these inert microspheres bioactive without compromising the drug releasing properties of gelatin microspheres. Incorporation of CaP nanocrystals into gelatin microspheres resulted into reduced biodegradation and drug release rates, whereas their calcifying capacity increased strongly compared to inert gelatin microspheres. The reduced drug release rate was correlated to the reduced degradation rate as caused by a physical cross-linking effect of CaP nanocrystals dispersed in the gelatin matrix. Consequently, these composite microspheres combine beneficial drug-releasing properties of organic gelatin with the calcifying capacity of a dispersed CaP phase. 1. Introduction Although synthetic options for bone-replacement are being researched for more than four decades, the only currently available treatment option that effectively fulfils all requirements needed for the replacement of bone tissue still involves the use of patient’s own (autograft) or donor (allograft) bone. The use of auto- or allografts, however, comes along with major drawbacks such as increasing donor shortages, limited bone volume, risk of disease transfer, and severe pain complications at the site of harvesting, as well as the need for additional surgery at the site of bone harvest, which is accompanied by severe pain complications and donor site morbidity. 1 In view of the aging population, the above-mentioned problems will become increasingly evident, stressing the need for synthetic alternatives to human bone. Typical examples of synthetic bone fillers include calcium phosphate (CaP) ceramics 2 and polymer/CaP composites 3 because CaPs have strong chemical resemblance to the mineral phase of bone and teeth. Generally, however, the clinical success of these synthetic bone fillers is inferior to auto- or allografts for several reasons. First of all, the degradation rate of sintered CaP ceramics does not match with the rate of bone remodelling, which allows these CaP ceramics to be considered as static, permanent implants that hardly participate in the dynamic process of bone remodelling. 4 Second, pure CaP ceramics cannot be molded into the desired defect shape when applied as granules due to the intrinsic brittleness of this class of materials. From a clinical point of view, injectable or moldable bioma- terials for reconstruction of osseous defects offers several clinical and economical advantages as compared to solid, prefabricated implants. 5 Using flowable materials, complete filling of the defect site can be established by means of minimally invasive techniques, thus, avoiding gaps that can lead to fibrous encapsulation or scar formation. Although injectable CaP cements have been developed to overcome this handling problem, these cements are also poorly degradable despite the fact that they are not sintered. Third, attempts to add signaling features to CaP ceramics and composites to increase their functionality were not successful in the past, because control over release and activity of incorporated biomolecules is generally poor due to the very strong interaction between proteins and CaPs. 6 The current study hypothesizes that the above-mentioned drawbacks of a purely ceramic approach toward bone regenera- tion can be overcome by preparing bone fillers based on gelatin- apatite microspheres. In that way, the controllable biodegrad- ability, strong capacity for drug release, and injectability/ moldability of gelatin microspheres can be combined with the excellent osteoconductive properties of CaP ceramics. Gelatin has been extensively studied for pharmaceutical and medical purposes, and its biosafety has been proven through its long clinical usage in several pharmaceutical and medical applica- tions. 7 The biodegradation of gelatin can be tailored by controlling the cross-linking density using a wide variety of chemical and physical cross-linking techniques. 8 Numerous studies have revealed that gelatin-derived scaffolds and micro- spheres are highly suitable for use as drug delivery vehicles for the controlled release of osteogenic and angiogenic growth factors. 9,10 This can be understood from the physiological situation where growth factors are stored in the extracellular matrix (ECM), which consists mainly of fibrillar collagens like type I collagen as well as proteoglycans. Kanematsu et al. have shown that type I collagen can act as a reservoir of growth factors such as basic fibroblast growth factor (bFGF) by complexation and incorporation of bFGF molecules into the collagen fibers, thereby protecting bFGF from premature release by proteolysis until its release following triggering by environ- mental stimuli. 11 From that perspective, incorporation of growth * To whom correspondence should be addressed. Tel.: +31 (0)24 3667305. Fax: +31 (0)24 3614657. E-mail: s.leeuwenburgh@dent.umcn.nl. † Radboud University Nijmegen Medical Center. ‡ Kyoto University. Biomacromolecules 2010, 11, 2653–2659 2653 10.1021/bm1006344  2010 American Chemical Society Published on Web 08/30/2010