Introduction: Recently, some calcium phosphate (CaP) biomaterials have been shown to exhibit an intrinsic osteoinductive potential, that is, the ability to induce mesenchymal stem cells differentiation into mature bone-forming cell lineage, even in the absence of exogenous bone morphogenetic proteins[1]. The underlying mechanism are not well understood, although ionic exchange and protein entrapment are believed to play a role[1],[2] and, therefore, porosity and textural properties of the material become crucial properties. Most commercial CaP bone substitutes are obtained by sintering at high temperatures (1000-1250ºC), which results in absence of nanostructure and low specific surface area (SSA). The possibility to obtain nanostructured CaP scaffolds, with controlled nanoporosity, together with tailored architecture, through low-temperature biomimetic routes opens up new possibilities in the design of osteoinductive bone substitutes with enhanced reactivity and protein entrapment capacity. The objective of this study was to evaluate the effect of the nanostructure and macropore architecture on the intrinsic osteoinductivity of a new family of biomimetic CaP materials.
Materials and Methods: Nanostructured CaP scaffolds were obtained by a biomimetic process based on the hydrolysis of alpha-tricalcium phosphate (alpha-TCP) to calcium deficient hydroxyapatite (CDHA) in physiological conditions. Two different strategies were used to fabricate scaffolds with different architectures: i) Foaming of an alpha-TCP slurry[3] (concave macropores, CDHA-foam); ii) 3D Ink-jet printing using a self-setting alpha-TCP ink[4] (450μm or 250μm prismatic macropores, CDHA-3Dp450 and CDHA-3Dp250 respectively). Two high-temperature sintered CaP ceramics, beta-tricalcium phosphate (beta-TCP) and biphasic calcium phosphate (beta-TCP/hydroxyapatite, BCP) were obtained by sintering the former foamed scaffolds, and used as positive controls. Scaffolds were characterized in terms of composition, porosity, solubility and microstructure. The in vivo study was carried out in a standardized model of intramuscular implantation (epaxial muscles) over 6 and 12 weeks in beagle dog (n = 6 per implant/time point). The presence of newly formed ectopic bone within the macropores was assessed by means of backscattered scanning electron microscopy (BS-SEM) and microscopic computed tomography (µ-CT).
Results and Discussion: Macroporous CaP scaffolds with tailored architectures and distinct macropore geometries were obtained by foaming and 3D-printing. The pore walls in the biomimetic CaP scaffolds consisted of needle-like entangled CDHA crystals, which resulted in interconnected nanoporosity, and a SSA = 40 m2/g, significantly higher than that of sintered BCP and beta-TCP, which were microporous, with SSA = 1 m2/g.
All implants showed tissue infiltration within the macropores at both time points, which demonstrated the open-interconnected macroporosity of the scaffolds. New ectopic lamellar bone formation was observed at the early time point (6 weeks) only in the CDHA-foam (4/6 animals). After 12 weeks, significant new ectopic bone formation was only observed in CDHA-foam (6/6 animals) and BCP (4/6 animals) groups. No ectopic bone formation was found in the 3D-printed scaffolds containing prismatic macropores.
Conclusions: Nanostructured CaP scaffolds obtained through low-temperature biomimetic processes presented higher osteoinductive potential than CaP microporous scaffolds obtained by high temperature sintering. Concave macropores combined with nanoporosity and high SSA fostered osteoinduction.
Spanish Government, Project MAT2012-38438-C03; European Regional Development Funds; Fundació Marató de TV3
References:
[1] Habibovic P, Sees TM, van den Doel M, van Blitterswijk CA, de Groot K: Osteoinduction by biomaterials-physicochemical and structural influences. J Biomed Mater Res 2006;77A:747-62
[2] Zhang J, Luo X, Barbieri D, Barradas AM, de Bruijn JD, van Blitterswijk CA, Yuan H. The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics. Acta Biomater 2014 Jul;10(7):3254-63
[3] Montufar EB, Traykova T, Gil H, Harr I, Almirall A, Aguirrre A, Engel E, Planell JA, Ginebra MP. Foamed Surfactant Solution as a Template for Self-setting Injectable Hydroxyapatite Scaffolds for Bone Regeneration. Acta Biomater 2010; 6:876–885.
[4] Maazouz Y, Montufar EB, Guillem-Marti J, Fleps I, Öhman C, Persson C, Ginebra MP. Robocasting of biomimetic hydroxyapatite scaffolds using self-setting inks. J. Mater. Chem. B 2014;2:5378-5386