Submicron-scale surface architecture of tricalcium phosphate directs osteogenesis in vitro and in vivo

Abstract

A current challenge of synthetic bone graft substitute design is to induce bone formation at a similar rate to its biological resorption, matching bone's intrinsic osteoinductivity and capacity for remodelling. We hypothesise that both osteoinduction and resorption can be achieved by altering surface microstructure of beta-tricalcium phosphate (TCP). To test this, two TCP ceramics are engineered with equivalent chemistry and macrostructure but with either submicron-or micron-scale surface architecture. In vitro, submicron-scale surface architecture differentiates larger, more active osteoclasts -a cell type shown to be important for both TCP resorption and osteogenesis -and enhances their secretion of osteogenic factors to induce osteoblast differentiation of human mesenchymal stem cells. In an intramuscular model, submicrostructured TCP forms 20 % bone in the free space, is resorbed by 24 %, and is densely populated by multinucleated osteoclast-like cells after 12 weeks; however, TCP with micron-scale surface architecture forms no bone, is essentially not resorbed, and contains scarce osteoclast-like cells. Thus, a novel submicron-structured TCP induces substantial bone formation and is resorbed at an equivalent rate, potentially through the control of osteoclast-like cells.

Influence of surface microstructure and chemistry on osteoinduction and osteoclastogenesis by biphasic calcium phosphate discs

“…In support of this theory, it has been reported that osteoclastogenesis precedes osteoinduction by microstructured TCP by several weeks (Akiyama et al, 2011;Kondo et al, 2006), and osteoclast depletion limits (Ripamonti et al, 2010) or completely blocks de novo bone formation by osteoinductive CaPs (Davison et al, 2014a). Recently, we reported a clear link between TCP microstructure, osteoclastogenesis, and subsequent de novo bone formation (Davison et al, 2014a;Davison et al, 2014b). However, (pre-)osteoclast differentiation and activity is influenced by multiple substrate parameters including surface nano-/microroughness (Makihira et al, 2007;Webster et al, 2001), solubility (Benahmed et al, 1996;Yamada et al, 1997), and the accompanied release of nano-/microparticulate (Fellah et al, 2007;Velard et al, 2013), so it is currently unknown if this link also holds true for less resorbable materials like BCP or titanium.…”

“…Crystal chemistry of the materials was analysed by X-ray diffraction (Rigaku Miniflex II) scanning the range 2θ = 25-45° (step size = 0.01°, rate = 1° min -1 ) as previously described (Davison et al, 2014b). The surface reactivity of the discs was analysed in simulated physiologic solution (SPS) (50 mM HEPES, 140 mM NaCl, and 0.4 mM NaN 3 for sterility; all from Sigma Aldrich, Saint Louis, MO, USA) at pH 3 and pH 7.…”

“…Disc constructs were implanted in the dorsal muscle of dogs, the classical model for evaluating osteoinduction, and the formation of de novo bone and multinucleated osteoclast-like cells was analysed by histology. The effects of surface structure and chemistry on osteoclastogenesis were further evaluated in vitro using the RAW264.7 pre-osteoclast cell line, as previously described (Davison et al, 2014b). Osteoclast differentiation, survival and morphology were measured and quantitatively compared using several biochemical, histological and morphological techniques.…”

“…RAW264.7 cells were cultured for 5 d with medium refreshment (basic medium + RANKL) after 1 d. In our previous experience with this culture model (Davison et al, 2014b), cells begin to fuse and differentiate into osteoclasts by day 3, continue fusing through day 4-5, and undergo apoptosis by day 6-7 (Collin- Osdoby et al, 2003;Takahashi et al, 2007). Therefore, biochemical assays focused on day 3-5 as the relevant period of osteoclastogenesis.…”

“…Similarly, microstructured biphasic calcium phosphate (BCP) -a mixture of HA and tricalcium phosphate (TCP) -induced de novo bone formation in the muscle of sheep (Le Nihouannen et al, 2005), goats (Habibovic et al, 2005b;Yuan et al, 2002), and dogs (Yuan et al, 2010); however, BCP with larger grains and fewer micropores induced less bone formation (Yuan et al, 2010) or in other cases none at all (Habibovic et al, 2006b). More recently, the dimensions of surface microstructure have also been shown to be important for osteoinduction -for instance, TCP with submicron-scale surface structure consistently stimulated de novo bone formation in dog muscle, while TCP with micron-scale surface structure was not at all osteoinductive (Davison et al, 2014b;Zhang et al, 2014). Surface microstructure may also be critical in triggering osteoinduction by other biomaterials such as titanium (Fujibayashi et al, 2004;Fukuda et al, 2011).…”