Osteosarcoma cell death induced by innovative scaffolds doped with chemotherapeutics.

Osteosarcoma (OS) cancer treatments include systemic chemotherapy and surgical resection. In the last years, novel treatment approaches have been proposed, which employ a drug-delivery system to prevent offside effects and improves treatment efficacy. Locally delivering anticancer compounds improves on high local concentrations with more efficient tumour-killing effect, reduced drugs resistance and confined systemic effects. Here, the synthesis of injectable strontium-doped calcium phosphate (SrCPC) scaffold was proposed as drug delivery system to combine bone tissue regeneration and anticancer treatment by controlled release of methotrexate (MTX) and doxorubicin (DOX), coded as SrCPC-MTX and SrCPC-DOX, respectively. The drug-loaded cements were tested in an in vitro model of human OS cell line SAOS-2, engineered OS cell line (SAOS-2-eGFP) and U2-OS. The ability of doped scaffolds to induce OS cell death and apoptosis was assessed analysing cell proliferation and Caspase-3/7 activities, respectively. To determine if OS cells grown on doped-scaffolds change their migratory ability and invasiveness, a wound-healing assay was performed. In addition, the osteogenic potential of SrCPC material was evaluated using human adipose derived-mesenchymal stem cells. Osteogenic markers such as (i) the mineral matrix deposition was analysed by alizarin red staining; (ii) the osteocalcin (OCN) protein expression was investigated by enzyme-linked immunosorbent assay test, and (iii) the osteogenic process was studied by real-time polymerase chain reaction array. The delivery system induced cell-killing cytotoxic effects and apoptosis in OS cell lines up to Day 7. SrCPC demonstrates a good cytocompatibility and it induced upregulation of osteogenic genes involved in the skeletal development pathway, together with OCN protein expression and mineral matrix deposition. The proposed approach, based on the local, sustained release of anticancer drugs from nanostructured biomimetic drug-loaded cements is promising for future therapies aiming to combine bone regeneration and anticancer local therapy.

Marine-Inspired Approaches as a Smart Tool to Face Osteochondral Regeneration.

The degeneration of osteochondral tissue represents one of the major causes of disability in modern society and it is expected to fuel the demand for new solutions to repair and regenerate the damaged articular joints. In particular, osteoarthritis (OA) is the most common complication in articular diseases and a leading cause of chronic disability affecting a steady increasing number of people. The regeneration of osteochondral (OC) defects is one of the most challenging tasks in orthopedics since this anatomical region is composed of different tissues, characterized by antithetic features and functionalities, in tight connection to work together as a joint. The altered structural and mechanical joint environment impairs the natural tissue metabolism, thus making OC regeneration even more challenging. In this scenario, marine-derived ingredients elicit ever-increased interest for biomedical applications as a result of their outstanding mechanical and multiple biologic properties. The review highlights the possibility to exploit such unique features using a combination of bio-inspired synthesis process and 3D manufacturing technologies, relevant to generate compositionally and structurally graded hybrid constructs reproducing the smart architecture and biomechanical functions of natural OC regions.

Strontium-doped apatitic bone cements with tunable antibacterial and antibiofilm ability.

Injectable calcium phosphate cements (CPCs) represent promising candidates for the regeneration of complex-shape bone defects, thanks to self-hardening ability, bioactive composition and nanostructure offering high specific surface area for cell attachment and conduction. Such features make CPCs also interesting for functionalization with various biomolecules, towards the generation of multifunctional devices with enhanced therapeutic ability. In particular, strontium-doped CPCs have been studied in the last years due to the intrinsic antiosteoporotic character of strontium. In this work, a SrCPC previously reported as osteointegrative and capable to modulate the fate of bone cells was enriched with hydroxyapatite nanoparticles (HA-NPs) functionalized with tetracycline (TC) to provide antibacterial activity. We found that HA-NPs functionalized with TC (NP-TC) can act as modulator of the drug release profile when embedded in SrCPCs, thus providing a sustained and tunable TC release. In vitro microbiological tests on Escherichia coli and Staphylococcus aureus strains proved effective bacteriostatic and bactericidal properties, especially for the NP-TC loaded SrCPC formulations. Overall, our results indicate that the addition of NP-TC on CPC acted as effective modulator towards a tunable drug release control in the treatment of bone infections or cancers.

Design Strategies and Biomimetic Approaches for Calcium Phosphate Scaffolds in Bone Tissue Regeneration.

Bone is a complex biologic tissue, which is extremely relevant for various physiological functions, in addition to movement, organ protection, and weight bearing. The repair of critical size bone defects is a still unmet clinical need, and over the past decades, material scientists have been expending efforts to find effective technological solutions, based on the use of scaffolds. In this context, biomimetics which is intended as the ability of a scaffold to reproduce compositional and structural features of the host tissues, is increasingly considered as a guide for this purpose. However, the achievement of implants that mimic the very complex bone composition, multi-scale structure, and mechanics is still an open challenge. Indeed, despite the fact that calcium phosphates are widely recognized as elective biomaterials to fabricate regenerative bone scaffolds, their processing into 3D devices with suitable cell-instructing features is still prevented by insurmountable drawbacks. With respect to biomaterials science, new approaches maybe conceived to gain ground and promise for a substantial leap forward in this field. The present review provides an overview of physicochemical and structural features of bone tissue that are responsible for its biologic behavior. Moreover, relevant and recent technological approaches, also inspired by natural processes and structures, are described, which can be considered as a leverage for future development of next generation bioactive medical devices.

Nature-Inspired Unconventional Approaches to Develop 3D Bioceramic Scaffolds with Enhanced Regenerative Ability.

Material science is a relevant discipline in support of regenerative medicine. Indeed, tissue regeneration requires the use of scaffolds able to guide and sustain the natural cell metabolism towards tissue regrowth. This need is particularly important in musculoskeletal regeneration, such as in the case of diseased bone or osteocartilaginous regions for which calcium phosphate-based scaffolds are considered as the golden solution. However, various technological barriers related to conventional ceramic processing have thus far hampered the achievement of biomimetic and bioactive scaffolds as effective solutions for still unmet clinical needs in orthopaedics. Driven by such highly impacting socioeconomic needs, new nature-inspired approaches promise to make a technological leap forward in the development of advanced biomaterials. The present review illustrates ion-doped apatites as biomimetic materials whose bioactivity resides in their unstable chemical composition and nanocrystallinity, both of which are, however, destroyed by the classical sintering treatment. In the following, recent nature-inspired methods preventing the use of high-temperature treatments, based on (i) chemically hardening bioceramics, (ii) biomineralisation process, and (iii) biomorphic transformations, are illustrated. These methods can generate products with advanced biofunctional properties, particularly biomorphic transformations represent an emerging approach that could pave the way to a technological leap forward in medicine and also in various other application fields.

Nanotechnological approach and bio-inspired materials to face degenerative diseases in aging.

The aging of the world population is increasingly claimed as an alarming situation, since an ever-raising number of persons in advanced age but still physically active is expected to suffer from invalidating and degenerative diseases. The impairment of the endogenous healing potential provoked by the aging requires the development of more effective and personalized therapies, based on new biomaterials and devices able to direct the cell fate to stimulate and sustain the regrowth of damaged or diseased tissues. To obtain satisfactory results, also in cases where the cell senescence, typical of the elderly, makes the regeneration process harder and longer, the new solutions have to possess excellent ability to mimic the physiological extracellular environment and thus exert biomimetic stimuli on stem cells. To this purpose, the "biomimetic concept" is today recognized as elective to fabricate bioactive and bioresorbable devices such as hybrid osteochondral scaffolds and bioactive bone cements closely resembling the natural hard tissues and with enhanced regenerative ability. The review will illustrate some recent results related to these new biomimetic materials developed for application in different districts of the musculoskeletal system, namely bony, osteochondral and periodontal regions, and the spine. Further, it will be shown how new bioactive and superparamagnetic calcium phosphate nanoparticles can give enhanced results in cardiac regeneration and cancer therapy. Since tissue regeneration will be a major demand in the incoming decades, the high potential of biomimetic materials and devices is promising to significantly increase the healing rate and improve the clinical outcomes even in aged patients.

Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche.

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

Mussel Shell-Derived Macroporous 3D Scaffold: Characterization and Optimization Study of a Bioceramic from the Circular Economy.

Fish industry by-products constitute an interesting platform for the extraction and recovery of valuable compounds in a circular economy approach. Among them, mussel shells could provide a calcium-rich source for the synthesis of hydroxyapatite (HA) bioceramics. In this work, HA nanoparticles have been successfully synthesized starting from mussel shells (Mytilus edulis) with a two steps process based on thermal treatment to convert CaCO3 in CaO and subsequent wet precipitation with a phosphorus source. Several parameters were studied, such as the temperature and gaseous atmosphere of the thermal treatment as well as the use of two different phosphorus-containing reagents in the wet precipitation. Data have revealed that the characteristics of the powders can be tailored, changing the conditions of the process. In particular, the use of (NH4)2HPO4 as the phosphorus source led to HA nanoparticles with a high crystallinity degree, while smaller nanoparticles with a higher surface area were obtained when H3PO4 was employed. Further, a selected HA sample was synthesized at the pilot scale; then, it was employed to fabricate porous 3D scaffolds using the direct foaming method. A highly porous scaffold with open and interconnected porosity associated with good mechanical properties (i.e., porosity in the range 87-89%, pore size in the range 50-300 µm, and a compressive strength σ = 0.51 ± 0.14 MPa) suitable for bone replacement was achieved. These results suggest that mussel shell by-products are effectively usable for the development of compounds of high added value in the biomedical field.

Evaluation of different crosslinking agents on hybrid biomimetic collagen-hydroxyapatite composites for regenerative medicine.

This study focuses on the development of novel bone-like scaffolds by bio-inspired, pH-driven, mineralization of type I collagen matrix with magnesium-doped hydroxyapatite nanophase (MgHA/Coll). To this aim, this study evaluates the altered modifications in the obtained composite due to different crosslinkers such as dehydrothermal treatment (DHT), 1,4-butanediol diglycidyl ether (BDDGE) and ribose in terms of morphological, physical-chemical and biological properties. The physical-chemical properties of the composites evaluated by XRD, FTIR, ICP and TGA demonstrated that the chemical mimesis of bone was effectively achieved using the in-lab biomineralization process. Furthermore, the presence of various crosslinkers greatly promoted beneficial enzymatic resistivity and swelling ability. The morphological results revealed highly porous and fibrous micro-architecture with total porosity above 85% with anisotropic pore size within the range of 50-200µm in all the analysed composites. The mechanical behaviour in response to compressive forces demonstrated enhanced compressive modulus in all crosslinked composites, suggesting that mechanical behaviour is largely dependent on the type of crosslinker used. The biomimetic compositional and morphological features of the composites elicited strong cell-material interaction. Therefore, the results showed that by activating specific crosslinking mechanisms, hybrid composites can be designed and tailored to develop tissue-specific biomimetic biomaterials for hard tissue engineering.

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering.

Sr-substituted bone cements direct mesenchymal stem cells, osteoblasts and osteoclasts fate.

Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

Novel Osteointegrative Sr-Substituted Apatitic Cements Enriched with Alginate.

The present work describes the synthesis of novel injectable, self-setting bone cements made of strontium-substituted hydroxyapatite (Sr-HA), obtained by single-phase calcium phosphate precursors doped with different amounts of strontium and enriched with alginate. The addition of alginate improved the injectability, cohesion, and compression strength of the cements, without affecting the hardening process. A Sr-HA cement exhibiting adequate hardening times and mechanical strength for clinical applications was further tested in vivo in a rabbit model, in comparison with a commercial calcium phosphate cement, revealing the maintenance of biomimetic composition and porous microstructure even after one month in vivo, as well as enhanced ability to induce new bone formation and penetration.

Synthesis and mechanical behavior of ß-tricalcium phosphate/titania composites addressed to regeneration of long bone segments.

Bioactive tricalcium phosphate/titania ceramic composites were synthesized by pressureless air sintering of mixed hydroxyapatite and titania (TiO2) powders. The sintering process was optimized to achieve dense ceramic bodies consisting in a bioactive/bioresorbable matrix (ß-tricalcium phosphate) reinforced with defined amounts of sub-micron sized titania particles. Extensive chemico-physical and mechanical characterization was carried out on the resulting composites, which displayed values of flexural strength, fracture toughness and elastic modulus in the range or above the typical ranges of values manifested by human cortical bone. It was shown that titania particles provided a toughening effect to the calcium-phosphate matrix and a reinforcement in fracture strength, in comparison with sintered hydroxyapatite bodies characterized by similar relative density. The characteristics of the resulting composites, i.e. bioactivity/bioresorbability and ability of manifesting biomimetic mechanical behavior, are features that can promote processes of bone regeneration in load-bearing sites. Hence, in the perspective of developing porous bone scaffolds with high bioactivity and improved biomechanical behavior, TCP/TiO2 composites with controlled composition can be considered as very promising biomaterials for application in a field of orthopedics where no acceptable clinical solutions still exist.

High biocompatibility and improved osteogenic potential of novel Ca-P/titania composite scaffolds designed for regeneration of load-bearing segmental bone defects.

Regeneration of load-bearing bone segments is still an open challenge due to the lack of biomaterials mimicking natural bone with a suitable chemicophysical and mechanical performance. This study proposes ceramic bone scaffolds made of ß-tricalcium phosphate (ß-TCP) and titania (TiO2 ), developed from hydroxyapatite (HA) and TiO2 starting nanopowders, which exhibit high and interconnected macroporosity (>70 vol %). The scaffold composition was designed to achieve a synergistic effect of bioactivity/resorbability and mechanical properties suitable for load-bearing regenerative applications. The analysis of the morphology, structure, and mechanical strength of the scaffolds resulted in compression strength nearly twice that of commercially available HA bone grafts with similar structure (Engipore(®)). Biological characterization was carried out for human MG-63 osteoblast-like cells proliferation, activity, attachment, and viability. ß-TCP/TiO2 scaffolds show high proliferation rate, high viability, and high colonization rates. Moreover, an increased activity of the osteogenic marker alkaline phosphatase (ALP) was found. These results demonstrate that ß-TCP/TiO2 scaffolds have good potential as osteogenically active load-bearing scaffolds; moreover, given the high and interconnected macroporosity as well as the resorbability properties of ß-TCP, these scaffolds may enhance in vivo osteointegration and promote the formation of new organized bone, thus resulting in very promising biomimetic scaffolds for long bone regeneration.