New 3D glass-ceramic scaffolds functionalised with bioartificial blend to model the normal and osteoporotic bone

In the last few decades, as a consequence of the increase in average life expectancy, we witness an increased occurrence of age-related diseases such as osteoporosis, which is predominant in humans older than 50 [1]. Osteoporotic bone is characterised by loss of bone mass and micro-architectural skeletal deterioration, with higher porosity and bigger pores compared to the normal bone, resulting in increased fragility and a higher risk of bone fracture when a fall occurs. Osteoporosis is the result of an imbalance of the bone tissue homeostasis, which is normally regulated by the synergic action of osteoblasts and osteoclasts [2]. Nevertheless, all the molecular mechanisms driving osteoporosis onset and progression have not been fully clarified yet. To study the basic mechanisms driving to osteoporosis, two approaches are usually employed: animal models and cell culture on classic plastic two-dimensional (2D) supports. The animal models have shown limits in the reproduction of human conditions, implying at the same time high costs, significant level of complexity, and a limited control of the local environment. On the other hand, traditional 2D cultures lack in the imitation of the three-dimensional (3D) environment of the native bone, which is fundamental for the regulation of the cell-cell and cell-extracellular matrix (ECM) interactions. In this context, the tissue engineering (TE) approach represents a straightforward alternative, which offers suitable tools for the in vitro replication of bone environment. This research work developed tissue engineered in vitro 3D models of normal and osteoporotic bone, which were subsequently employed to study the influence of the pathologic architecture on cell response. These models can help to gain new knowledge on the effect of the environment on osteoporosis progression, as well as to support the development of new therapeutic approaches. The first step in the design of bone in vitro models was the biomaterials selection for the scaffolds fabrication. Bone inorganic phase was mimed through a bioactive glass-ceramic deriving from a SiO2–P2O5–CaO–MgO–Na2O–K2O parent glass (CEL2). CEL2 glass-ceramics (CEL2-GC) substrates are bioactive, leading to the deposition of a hydroxyapatite layer, similar to the mineral phase of bone. To mimic the bone organic phase, the CEL2-GC substrate was surface functionalised with a type I collagen-based coating. Type I collagen was isolated from rabbit bone in the native triple helix conformation, with a high degree of purity, and it was blended with a newly synthesised water-soluble polyurethane (PUR) in order to improve the long-term stability of the coating. In fact, PURs bring the advantages of tuneable chemical and mechanical properties, and biocompatibility [3]. The PUR was synthesised from poly(ethylene glycol) (PEG), 1,6-hexamethylene diisocyanate (HDI) and N-BOC-serinol, and it was designed to expose amino groups on the polymeric chain, which can be exploited for the blend stabilisation through crosslinking. Consecutive steps of functionalisation were performed to link the polymeric coating on the CEL2-GC surface, which was thoroughly characterised at each stage, in order to select the optimal conditions. Firstly, the surface was cleaned for both eliminating eventual contaminants and exposing –OH groups, without negatively influencing the bioactivity of the glass-ceramic substrate. Consecutively, a uniform silanisation on the surface was obtained and the bioartificial blend was linked to the surface using genipin as a bridge between the silane and the polymer amino groups. A further addition of genipin to stabilise the blend through crosslinking showed to improve the cell response. In vitro tests were performed on the glass-ceramic 2D substrate functionalised with the bioartificial blend in order to evaluate the ionic release, the coating stability and the bioactivity. Thanks to the presence of the PUR, the bioartificial coating allowed incorporating a higher amount of collagen on the surface of the CEL2-GC, and its stabilisation for long-term incubation (up to 28 days). Biological test performed using human periosteal derived precursor cells (PDPCs) demonstrated that the proposed material was a good substrate for bone cell adhesion and growth, thus maintaining the biological and structural integrity of the ECM. The selected biomaterials were then employed for the scaffold fabrication. 3D porous glass-ceramic scaffolds were produced through the sponge replication technique. The processing parameters were systematically varied, allowing a good control over the morphological and microstructural characteristics of the scaffold, which closely mimicked the ones of normal and osteoporotic trabecular bone. In particular, the porosity, pore size, and structure thickness were very close to the ones of normal and osteoporotic bones harvested from femoral head [4, 5]. The developed scaffolds were functionalised by the collagen-PUR bioartificial blend, allowing to obtain bone-like model substrates for advanced in vitro investigations. Even though to overcome the limits of the animal models human primary cells must be included in the in vitro model, in these initial stages of the model development rat derived primary cells were employed as a cheaper and easier accessible cell sources for a preliminary biological characterisation. Thus, the effect of 3D porous CEL2-GC scaffolds mimicking the normal and osteoporotic bone architectures on the osteogenic differentiation of rat mesenchymal stem cells (MSCs) was evaluated. CEL2-GC scaffolds functionalised with the bioartificial blend demonstrated to be a suitable support for MSCs osteogenic differentiation, which showed a good adhesion and morphology, alkaline phosphatase activity, and the synthesis of proteins specific of bone ECM (osteocalcin and osteopontin). The MSCs were cultured on the 3D scaffolds with and without the addition of osteogenic factors to the medium. Osteogenic factors have shown to improve the osteogenic effect imparted by the scaffold. The architecture of the osteoporotic-like scaffold affected MSCs differentiation, since cells synthetized slightly lower amount of osteopontin compared to the cells cultured on the scaffold mimicking the osteoporotic bone tissue. These results suggest the hypothesis that, in an osteoporotic bone, MSCs may have a reduced differentiation ability due to the altered bone architecture induced by this pathology. This research opens the door to new approaches in osteoporosis studies, highlighting i) the importance of considering the 3D environment in in vitro studies due to the influence of architecture on cells response; ii) the need of well-timed screening of this pathology, since the results suggest that the architecture of the osteoporotic bone enhances the disease progression, iii) the possibility to investigate therapeutic approaches that address the MSCs, e.g. by inducing their differentiation. Finally, a preliminary evaluation on the scaffolds ability to support Human Umbilical Vein Endothelial Cells (HUVEC) viability in co-culture with osteoblast-like cell was performed, constituting the proof of concept for a future long-term experiment for the evaluation of the influence of endothelial cells on the remodelling process in healthy and pathological conditions. 1 Chen, H., Zhou, X., Shoumura, S., Emura, S. & Bunai, Y. Osteoporosis international 21, 627-636 (2010). 2 Raisz, L. G. The Journal of clinical investigation 115, 3318-3325 (2005). 3 Lamba, N. M., Woodhouse, K. A. & Cooper, S. L. (CRC press, 1997). 4 Hildebrand, T., Laib, A., Müller, R., Dequeker, J. & Rüegsegger, P. Journal of bone and mineral research 14, 1167-1174 (1999). 5 Chiba, K., Burghardt, A. J., Osaki, M. & Majumdar, S. Bone 56, 139-146 (2013).