New strontium-based coatings show activity against pathogenic bacteria in spine infection.

Infections of implants and prostheses represent relevant complications associated with the implantation of biomedical devices in spine surgery. Indeed, due to the length of the surgical procedures and the need to implant invasive devices, infections have high incidence, interfere with osseointegration, and are becoming increasingly difficult to threat with common therapies due to the acquisition of antibiotic resistance genes by pathogenic bacteria. The application of metal-substituted tricalcium phosphate coatings onto the biomedical devices is a promising strategy to simultaneously prevent bacterial infections and promote osseointegration/osseoinduction. Strontium-substituted tricalcium phosphate (Sr-TCP) is known to be an encouraging formulation with osseoinductive properties, but its antimicrobial potential is still unexplored. To this end, novel Sr-TCP coatings were manufactured by Ionized Jet Deposition technology and characterized for their physiochemical and morphological properties, cytotoxicity, and bioactivity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538P human pathogenic strains. The coatings are nanostructured, as they are composed by aggregates with diameters from 90 nm up to 1 µm, and their morphology depends significantly on the deposition time. The Sr-TCP coatings did not exhibit any cytotoxic effects on human cell lines and provided an inhibitory effect on the planktonic growth of E. coli and S. aureus strains after 8 h of incubation. Furthermore, bacterial adhesion (after 4 h of exposure) and biofilm formation (after 24 h of cell growth) were significantly reduced when the strains were cultured on Sr-TCP compared to tricalcium phosphate only coatings. On Sr-TCP coatings, E. coli and S. aureus cells lost their organization in a biofilm-like structure and showed morphological alterations due to the toxic effect of the metal. These results demonstrate the stability and anti-adhesion/antibiofilm properties of IJD-manufactured Sr-TCP coatings, which represent potential candidates for future applications to prevent prostheses infections and to promote osteointegration/osteoinduction.

Advantages and limitations of using cell viability assays for 3D bioprinted constructs.

Bioprinting shows promise for bioengineered scaffolds and three-dimensional (3D) disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumor cells, with or without normal bone cells). Despite demonstrating 80%-90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell-cell interactions through thez-axis. In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques.

A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings.

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

Incorporation/Enrichment of 3D Bioprinted Constructs by Biomimetic Nanoparticles: Tuning Printability and Cell Behavior in Bone Models.

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.

Ionized Jet Deposition of Calcium Phosphates-Based Nanocoatings: Tuning Coating Properties and Cell Behavior by Target Composition and Substrate Heating.

Calcium phosphate-based coatings are widely studied in orthopedics and dentistry for their similarity to the mineral component of bone and their capability to promote osseointegration. Different calcium phosphates have tunable properties that result in different behaviors in vitro, but the majority of studies focus only on hydroxyapatite. Here, different calcium phosphate-based nanostructured coatings are obtained by ionized jet deposition, starting with hydroxyapatite, brushite and beta-tricalcium phosphate targets. The properties of the coatings obtained from different precursors are systematically compared by assessing their composition, morphology, physical and mechanical properties, dissolution, and in vitro behavior. In addition, for the first time, depositions at high temperature are investigated for the further tuning of the coatings mechanical properties and stability. Results show that different phosphates can be deposited with good composition fidelity even if not in a crystalline phase. All coatings are nanostructured and non-cytotoxic and display variable surface roughness and wettability. Upon heating, higher adhesion and hydrophilicity are obtained as well as higher stability, resulting in better cell viability. Interestingly, different phosphates show very different in vitro behavior, with brushite being the most suitable for promoting cell viability and beta-tricalcium phosphate having a higher impact on cell morphology at the early timepoints.

Strontium Functionalization of Biomaterials for Bone Tissue Engineering Purposes: A Biological Point of View.

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.

Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering.

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.

Assessment of Collagen-Based Nanostructured Biomimetic Systems with a Co-Culture of Human Bone-Derived Cells.

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.

Collagen Hybrid Formulations for the 3D Printing of Nanostructured Bone Scaffolds: An Optimized Genipin-Crosslinking Strategy.

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.

Co-culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches.

Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a "smart" scaffold, as a support to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooperation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they allow cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE. STATEMENT OF SIGNIFICANCE: Bone is a dynamic tissue under continuous remodeling, but spontaneous healing may fail in the case of excessive bone loss which often requires valid alternatives to conventional treatments to restore bone integrity, like bone tissue engineering (BTE). Pre-clinical evaluation of scaffolds for BTE requires in vitro testing where co-cultures combining innovative materials with osteoblasts (OBs) and osteoclasts (OCs) closely mimic the in vivo repair process. This review considers the direct and indirect OB/OC co-cultures relevant to BTE, from the early mouse-cell models to the recent bone regenerative systems. The co-culture modeling of bone microenvironment provides reliable information on bone cell cross-talk. Starting from improved knowledge on bone remodeling, bone disease mechanisms may be understood and new BTE solutions are designed.

Development and Biocompatibility of Collagen-Based Composites Enriched with Nanoparticles of Strontium Containing Mesoporous Glass.

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.