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

3d printing in alginic acid bath of in-situ crosslinked collagen composite scaffolds

Bone-tissue regeneration is a growing field, where nanostructured-bioactive materials are designed to replicate the natural properties of the target tissue, and then are processed with technolo-gies such as 3D printing, into constructs that mimic its natural architecture. Type I bovine collagen formulations, containing functional nanoparticles (enriched with therapeutic ions or biomolecules) or nanohydroxyapatite, are considered highly promising, and can be printed using support baths. These baths ensure an accurate deposition of the material, nonetheless their full removal post-printing can be difficult, in addition to undesired reactions with the crosslinking agents often used to improve the final structural integrity of the scaffolds. Such issues lead to partial collapse of the printed constructs and loss of geometrical definition. To overcome these limitations, this work presents a new alternative approach, which consists of adding a suitable concentration of crosslinking agent to the printing formulations to promote the in-situ crosslinking of the constructs prior to the removal of the support bath. To this aim, genipin, chosen as crosslinking agent, was added (0.1 wt.%) to collagen-based biomaterial inks (containing either 38 wt.% mesoporous bioactive glasses or 65 wt.% nanohydroxyapatite), to trigger the crosslinking of collagen and improve the stability of the 3D printed scaffolds in the post-processing step. Moreover, to support the material deposition, a 15 wt.% alginic acid solution was used as a bath, which proved to sustain the printed structures and was also easily removable, allowing for the stable processing of high-resolution geometries

SURFACE FUNCTIONALIZATION OF 3D GLASS-CERAMIC POROUS SCAFFOLDS FOR ENHANCED MINERALIZATION IN VITRO

Bone reconstruction after tissue loosening due to traumatic, pathological or surgical causes is in increasing demand. 3D scaffolds are a widely studied solution for supporting new bone growth. Bioactive glass–ceramic porous materials can offer a three-dimensional structure that is able to chemically bond to bone. The ability to surface modify these devices by grafting biologically active molecules represents a challenge, with the aim of stimulating physiological bone regeneration with both inorganic and organic signals. In this research work glass ceramic scaffolds with very high mechanical properties and moderate bioactivity have been functionalized with the enzyme alkaline phosphatase (ALP). The material surface was activated in order to expose hydroxyl groups. The activated surface was further grafted with ALP both via silanization and also via direct grafting to the surface active hydroxyl groups. Enzymatic activity of grafted samples were measured by means of UV–vis spectroscopy before and after ultrasonic washing in TRIS–HCl buffer solution. In vitro inorganic bioactivity was investigated by soaking the scaffolds after the different steps of functionalization in a simulated body fluid (SBF). SEM observations allowed the monitoring of the scaffold morphology and surface chemical composition after soaking in SBF. The presence of ALP enhanced the in vitro inorganic bioactivity of the tested material

PEG-coated large mesoporous silicas as smart platform for protein delivery and their use in a collagen-based formulation for 3d printing

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6–8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140◦C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds

Electrophoretic deposition of Sr-containing mesoporous bioactive glass particles produced by spray-drying

Introduction Mesoporous bioactive glasses (MBGs) are gaining increasing interest in the biomedical field thanks to their exceptional textural characteristics (high surface area, high pore volume and highly ordered mesoporosity). These properties lead to an improved apatite kinetics formation, which allow these glasses to be successfully applied in bone tissue regeneration [1]. In this work we adopted an aerosol-based spray drying process in order to have high control and reproducibility over the morphology of particles. In order to increase their regenerative potential, the particles have been doped with strontium, element known for its osteogenic and bone antiresorptive properties [2]. Later the particles have been deposed by electrophoretic deposition (EPD) on glass-ceramic scaffolds fabricated by the polymer sponge replication method. EPD is a versatile technique which allows an easy control of the thickness of the deposited film through simple adjustment of the applied voltage and the deposition time. The scaffolds, based on a quaternary silicate glass (SCNA, SiO2–CaO–Na2O–Al2O3 oxide system), have good mechanical properties but low bioactivity [3]. Thanks to MBG particle deposition, they acquire a pronounced bioactive behaviour, thus becoming an excellent solution for bone tissue regeneration. Results and Discussion MBGs synthesized with the aerosol-based spray-drying process have a basic composition on the SiO2-CaO system and have been doped with the 1% molar of strontium (SD_Sr1). FESEM image of particles shows micro-sized spherical particles, with size mostly ranging between 500 nm and 5 µm. N2 adsorption analysis gives back a high specific surface area value, 160 m2/g, and a pore size distribution between 5 and 9 nm, which confirms the mesoporosity of the sample. Strontium incorporation inside the binary composition does not modify the bioactive behaviour of the glass: after 14 days in SBF nanoparticles are completely covered by a layer of hydroxyapatite.The EDS quantitative analysis shows that the amount of strontium effectively incorporated in the microparticles was 70% of the theoretical one, probably because of the high dimension of the ion which hinders its entrance into the glass network. Nevertheless, most of the Sr incorporated has been released after 14 days of immersion in SBF, as the coupled plasma-atomic emission spectrometry (ICP-AES) reveals. On the basis of literature data, the released concentrations are suitable for inducing osteogenesis [4]. EPD has been performed in ethanol, applying a voltage of 120 V for 5 minutes. The scaffolds, being not conductive, have been suspended between two stainless steel electrodes through a clamp. A dispersant (TEA, triethanolamine) has been used to keep the particles in suspension during the whole deposition time. The deposited layer was abundant but not uniform on the scaffold surface. After immersion for 7 days in SBF, hydroxyapatite formation has been observed on the surface of the microparticles deposited on the scaffold struts. This demonstrates that MBGs not only maintain their bioactivity after deposition but also transfer this property to scaffolds. Conclusions MBGs synthetized with aerosol-based spray-drying process and doped with strontium have excellent textural properties and a bioactive behaviour. After electrophoretic deposition, they maintain these properties and consequently they improve the bioactivity of SCNA scaffolds, which initially are almost biologically inert. In this way we demonstrate that it is possible to obtain a successful construct for bone tissue engineering with both excellent regenerative and mechanical properties