Yassine Maazouz

Self-hardening and thermoresponsive alpha tricalcium phosphate/pluronic pastes.

Although calcium phosphate cements (CPCs) are used for bone regeneration in a wide range of clinical applications, various physicochemical phenomena are known to hinder their potential use in minimally invasive surgery or in highly vascularized surgical sites, mainly because of their lack of injectability or their low washout resistance. The present work shows that the combination of CPCs with an inverse-thermoresponsive hydrogel is a good strategy for finely tuning the cohesive and rheological properties of CPCs to achieve clinical acceptable injectability to prevent phase separation during implantation and cohesion to avoid washout of the paste. The thermoresponsive CPC developed combines alpha-tricalcium phosphate with an aqueous solution of pluronic F127, which exhibits an inverse thermoresponsive behaviour, with a gelling transformation at around body temperature. These novel CPCs exhibited temperature-dependent properties. Addition of the polymer enhanced the injectability of the paste, even at a low liquid-to-powder ratio, and allowed the rheological properties of the cement to be tuned, with the injection force decreasing with the temperature of the paste. Moreover, the cohesion of the paste was also temperature-dependent and increased as the temperature of the host medium increased due to gelling induced in the paste. The thermoresponsive cement exhibited excellent cohesion and clinically acceptable setting times at 37°C, irrespective of the initial temperature of the paste. The addition of pluronic F127 slightly delayed the setting reaction in the early stages but did not hinder the full transformation to calcium-deficient hydroxyapatite. Moreover, the frozen storage of premixed thermoresponsive cement pastes was explored, the main physicochemical properties of the cements being maintained upon thawing, even after 18months of frozen storage. This avoids the need to mix the cement in the operating theatre and allows its use off-the-shelf. The reverse thermoresponsive cements studied herein open up new perspectives in the surgical field, where the sequential gelling/hardening of these novel cements could allow for a better and safer clinical application. STATEMENT OF SIGNIFICANCE Calcium phosphate cements are attractive bone substitutes due to their similarity to the bone mineral phase. Although they can be injectable, cohesion and stability of the paste are crucial in terms of performance and safety. A common strategy is the combination with hydrogels. However, this often results in a decrease of viscosity with increasing temperature, which can lead to extravasation and particle leakage from the bone defect. The preferred evolution would be the opposite: a low viscosity would enhance mixing and injection, and an instantaneous increase of viscosity after injection would ensure washout resistance to the blood flow. Here we develop for the first time a calcium phosphate cement exhibiting reverse thermoresponsive properties using a poloxamer featuring inverse thermal gelling.

Osteoinduction by foamed and 3D-printed calcium phosphate scaffolds: effect of nanostructure and pore architecture

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and β-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

Evaluation of a porosity measurement method for wet calcium phosphate cements

The porosity of a calcium phosphate cement is a key parameter as it affects several important properties of the cement. However, a successful, non-destructive porosity measurement method that does not include drying has not yet been reported for calcium phosphate cements. The aim of this study was to evaluate isopropanol solvent exchange as such a method. Two different types of calcium phosphate cements were used, one basic (hydroxyapatite) and one acidic (brushite). The cements were allowed to set in an aqueous environment and then immersed in isopropanol and stored under three different conditions: at room temperature, at room temperature under vacuum (300 mbar) or at 37℃. The specimen mass was monitored regularly. Solvent exchange took much longer time to reach steady state in hydroxyapatite cements compared to brushite cements, 350 and 18 h, respectively. Furthermore, the immersion affected the quasi-static compressive strength of the hydroxyapatite cements. However, the strength and phase composition of the brushite cements were not affected by isopropanol immersion, suggesting that isopropanol solvent exchange can be used for brushite calcium phosphate cements. The main advantages with this method are that it is non-destructive, fast, easy and the porosity can be evaluated while the cements remain wet, allowing for further analysis on the same specimen.

Measuring wettability of biosurfaces at the microscale

Determining the contact angle of a liquid on a solid surface is a simple method to assess the surface wettability. The most common method to measure the contact angle of a liquid consists of capturing the profile of a sessile drop of a few microliters on the surface using an optical system. Currently, this is a widely used technique to analyze wettability both in researched materials and in products of multiple technological fields. However, the drop dispensed by a traditional macroscopic contact angle meter is too big to assess the wettability properties of individual topographical features and/or chemical patterns at the micro/nanoscale. Recently, contact angle meters that can discharge drops that are microscopic, with volumes in the range of 1 × 10(-3) to 10(-5) μL have been developed. The novel microscopic contact angle meter uses a pneumatic injection system to discharge the drop of the liquid through a capillary of a few micrometers of internal diameter and a high-resolution ultrafast digital camera. We have tested different biosurfaces - microimprinted polymers for biosensors, calcium-phosphate cements with different topographical microfeatures, orthodontic wires - and assessed the potential applicability in the field in comparison with the conventional macroscopic contact angle meters. This protocol describes the basic tasks needed to test wettability on biosurfaces with a microscopic contact angle meter. The focus of the protocol is on the challenging methodological steps and those that differentiate the use of this equipment to the use of a traditional macroscopic contact angle meter.

Bioceramics and bone healing

Calcium phosphates have long been used as synthetic bone grafts. Recent studies have shown that the modulation of composition and textural properties, such as nano-, micro- and macro-porosity, is a powerful strategy to control and synchronize material resorption and bone formation. Biomimetic calcium phosphates, which closely mimic the composition and structure of bone mineral, can be produced using low-temperature processing routes, and offer the possibility to modulate the material properties to a larger extent than conventional high temperature sintering processes. Advanced technologies open up new possibilities in the design of bioceramics for bone regeneration; 3D-printing technologies, in combination with the development of hybrid materials with enhanced mechanical properties, supported by finite element modelling tools, are expected to enable the design and fabrication of mechanically competent patient-specific bone grafts. The association of ions, drugs and cells allows leveraging of the osteogenic potential of bioceramic scaffolds in compromised clinical situations, where the intrinsic bone regeneration potential is impaired. Cite this article: EFORT Open Rev 2018;3 DOI: 10.1302/2058-5241.3.170056

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.