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Bone-tissue regeneration induced by biomimetic bioactive materials is the most promising approach alternative to the clinical ones used to treat bone loss caused by trauma or diseases such as osteoporosis. The goal is to design nanostructured bioactive constructs able to reproduce the physiological environment: By mimicking the natural features of bone tissue, the cell behavior during the regeneration process may be addressed. At present, 3D-printing technologies are the only techniques able to design complex structures avoiding constraints of final shape and porosity. However, this type of biofabrication requires complex optimization of biomaterial formulations in terms of specific rheological and mechanical properties while preserving high biocompatibility. In this work, we combined nano-sized mesoporous bioactive glasses enriched with strontium ions with type I collagen, to formulate a bioactive ink for 3D-printing technologies. Moreover, to avoid the premature release of strontium ions within the crosslinking medium and to significantly increase the material mechanical and thermal stability, we applied an optimized chemical treatment using ethanol-dissolved genipin solutions. The high biocompatibility of the hybrid system was confirmed by using MG-63 and Saos-2 osteoblast-like cell lines, further highlighting the great potential of the innovative nanocomposite for the design of bone-like scaffolds.

In the last years bone tissue engineering has been increasingly indicated as a valid solution to meet the challenging requirements for a healthy bone regeneration in case of bone loss or fracture. In such a context, bioactive glasses have already proved their great potential in promoting the regeneration of new bone tissue due to their high bioactivity. In addition, their composition and structure enable us to incorporate and subsequently release therapeutic ions such as strontium, enhancing the osteogenic properties of the material. The incorporation of these inorganic systems in polymeric matrices enables the formulation of composite systems suitable for the design of bone scaffolds or delivery platforms. Among the natural polymers, type I collagen represents the main organic phase of bone and thus is a good candidate to develop biomimetic bioactive systems for bone tissue regeneration. However, alongside the specific composition and structure, the key factor in the design of new biosystems is creating a suitable interaction with cells and the host tissue. In this scenario, the presented study aimed at combining nano-sized mesoporous bioactive glasses produced by means of a sol–gel route with type I collagen in order to develop a bioactive hybrid formulation suitable for bone tissue engineering applications. The designed system has been fully characterized in terms of physico-chemical and morphological analyses and the ability to release Sr2+ ions has been studied observing a more sustained profile in presence of the collagenous matrix. With the aim to improve the mechanical and thermal stability of the resulting hybrid system, a chemical crosslinking approach using 4-star poly (ethylene glycol) ether tetrasuccinimidyl glutarate (4-StarPEG) has been explored. The biocompatibility of both non-crosslinked and 4-StarPEG crosslinked systems was evaluated by in vitro tests with human osteoblast-like MG-63 cells. Collected results confirmed the high biocompatibility of composites, showing a good viability and adhesion of cells when cultured onto the biomaterial samples.

Strontium (Sr) is a trace element taken with nutrition and found in bone in close connection to native hydroxyapatite. Sr is involved in a dual mechanism of coupling the stimulation of bone formation with the inhibition of bone resorption, as reported in the literature. Interest in studying Sr has increased in the last decades due to the development of strontium ranelate (SrRan), an orally active agent acting as an anti-osteoporosis drug. However, the use of SrRan was subjected to some limitations starting from 2014 due to its negative side effects on the cardiac safety of patients. In this scenario, an interesting perspective for the administration of Sr is the introduction of Sr ions in biomaterials for bone tissue engineering (BTE) applications. This strategy has attracted attention thanks to its positive effects on bone formation, alongside the reduction of osteoclast activity, proven by in vitro and in vivo studies. The purpose of this review is to go through the classes of biomaterials most commonly used in BTE and functionalized with Sr, i.e., calcium phosphate ceramics, bioactive glasses, metal-based materials, and polymers. The works discussed in this review were selected as representative for each type of the above-mentioned categories, and the biological evaluation in vitro and/or in vivo was the main criterion for selection. The encouraging results collected from the in vitro and in vivo biological evaluations are outlined to highlight the potential applications of materials’ functionalization with Sr as an osteopromoting dopant in BTE.

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and other factors, is considered a potential alternative to conventional treatments. Innovative scaffolds need to be tested in in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs), the two main players of bone remodeling, is required to mimic their crosstalk and molecular cooperation. To this aim, two composite materials were developed, based on type I collagen, and containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. The developed nanostructured systems underwent genipin chemical crosslinking and were then tested with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors, for 2–3 weeks. The favorable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of cells, encouraging a further investigation of the developed bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

No abstract

Reproducing in vitro a model of the bone microenvironment is a current need. Preclinical in vitro screening, drug discovery, as well as pathophysiology studies may benefit from in vitro three-dimensional (3D) bone models, which permit high-throughput screening, low costs, and high reproducibility, overcoming the limitations of the conventional two-dimensional cell cultures. In order to obtain these models, 3D bioprinting offers new perspectives by allowing a combination of advanced techniques and inks. In this context, we propose the use of hydroxyapatite nanoparticles, assimilated to the mineral component of bone, as a route to tune the printability and the characteristics of the scaffold and to guide cell behavior. To this aim, both stoichiometric and Sr-substituted hydroxyapatite nanocrystals are used, so as to obtain different particle shapes and solubility. Our findings show that the nanoparticles have the desired shape and composition and that they can be embedded in the inks without loss of cell viability. Both Sr-containing and stoichiometric hydroxyapatite crystals permit enhancing the printing fidelity of the scaffolds in a particle-dependent fashion and control the swelling behavior and ion release of the scaffolds. Once Saos-2 cells are encapsulated in the scaffolds, high cell viability is detected until late time points, with a good cellular distribution throughout the material. We also show that even minor modifications in the hydroxyapatite particle characteristics result in a significantly different behavior of the scaffolds. This indicates that the use of calcium phosphate nanocrystals and structural ion-substitution is a promising approach to tune the behavior of 3D bioprinted constructs.