**1. Introduction**

Bone regeneration is a natural event, but there are certain clinical situations where this physiological process is impaired. For instance, when either the bone defect to be repaired is too large or bone has lost its regenerative capacity as occurs in osteoporosis conditions. In these cases, bone regeneration needs to be stimulated by using bone tissue engineering approaches [1,2]. Such approaches use constructs formed by 3-D porous scaffolds decorated with biological signals and/or bone-forming cells. In the last decade, SiO2–CaO–P2O5 mesoporous bioactive glasses (MBGs) were proposed as optimum candidates for these scaffolds. These glasses exhibit bone regenerative properties and highly ordered mesoporous structures enabling binding and release bone promoting agents [1,3]. Moreover, the huge surface area and pore volumes of MBGs yield quicker in vitro responses when compared with other bioactive materials [4,5]. The behaviour of these glasses in a biological medium can be improved by incorporating bioactive metal ions in the glass network. This is the case of Zn2+ ions which exhibit osteogenic and angiogenic features, as well as antioxidant, cancer preventive, and antimicrobial activities [6–10]. In this regard, since bacterial infection [11] is an important problem after bone implant surgery [12,13], the combination of the regenerative properties of MBGs with the beneficial effects of Zn2+ ions has generated potential interest in bioengineering applications [14].

Following the bone tissue engineering principles, the bioactivity of a scaffold can be improved by loading it with osteogenic agents, such as parathyroid hormone (PTH)-related protein (PTHrP), which is emerging as an interesting promoter of bone regeneration. PTHrP contains an N-terminal 1–37 region homologous to PTH and a C-terminal PTH-unlike region containing the highly conserved 107–111 sequence osteostatin [15]. N-terminal PTHrP analogues have been shown to induce bone anabolism in rodents and humans upon systemic intermittent administration [16,17]. On the other hand, osteostatin has anti-resorptive activity [18], but also exhibits osteogenic features in vitro and in vivo [19–24]. Moreover, it has recently been shown that osteostatin coating onto various types of ceramic implants accelerates healing of critical and noncritical bone defects in the long bones of adult normal and osteoporotic rabbits and in rats [25–29]. Therefore, recent findings point to osteostatin as an attractive small peptide for consideration in a bone tissue engineering scenario.

In this study, the biological consequences of the concurrent inclusion of ZnO and osteostatin impregnation in MBGs were investigated. Three MBGs were synthesized, all with a basic composition of 80% SiO2–15% CaO–5% P2O5 (mol%), containing or not (Blank, BL) 4% or 5% ZnO, respectively (4ZN and 5ZN). These compositions were selected based on our previous studies [14], which were consistent with those reported for other glass systems showing 5% as the maximum content of ZnO enhancing osteoblast cell development without being cytotoxic [30–32]. MBG powders were processed as disk-shape pieces for several in vitro studies: uptake and release of osteostatin; assays in simulated body fluid (SBF); release of the inorganic ions, calcium, phosphate, and zinc from disks to the surrounding medium; and bioactivity in mouse pre-osteoblastic MC3T3-E1 cell cultures. This approach allowed us to evaluate the putative advantage of loading osteostatin onto ZnO-containing glasses to produce an optimal biomaterial for bone regeneration.
