Hydroxyapatite 3D-printed scaffolds with Gyroid-Triply periodic minimal surface porous structure: Fabrication and an in vivo pilot study in sheep.

Bone repair is a major challenge in regenerative medicine, e.g. for large defects. There is a need for bioactive, highly percolating bone substitutes favoring bone ingrowth and tissue healing. Here, a modern 3D printing approach (VAT photopolymerization) was exploited to fabricate hydroxyapatite (HA) scaffolds with a Gyroid-"Triply periodic minimal surface" (TPMS) porous structure (65% porosity, 90.5% HA densification) inspired from trabecular bone. Percolation and absorption capacities were analyzed in gaseous and liquid conditions. Mechanical properties relevant to guided bone regeneration in non-load bearing sites, as for maxillofacial contour reconstruction, were evidenced from 3-point bending tests and macrospherical indentation. Scaffolds were implanted in a clinically-relevant large animal model (sheep femur), over 6 months, enabling thorough analyses at short (4 weeks) and long (26 weeks) time points. In vivo performances were systematically compared to the bovine bone-derived Bio-OssⓇ standard. The local tissue response was examined thoroughly by semi-quantitative histopathology. Results demonstrated the absence of toxicity. Bone healing was assessed by bone dynamics analysis through epifluorescence using various fluorochromes and quantitative histomorphometry. Performant bone regeneration was evidenced with similar overall performances to the control, although the Gyroid biomaterial slightly outperformed Bio-OssⓇ at early healing time in terms of osteointegration and appositional mineralization. This work is considered a pilot study on the in vivo evaluation of TPMS-based 3D porous scaffolds in a large animal model, for an extended period of time, and in comparison to a clinical standard. Our results confirm the relevance of such scaffolds for bone regeneration in view of clinical practice. STATEMENT OF SIGNIFICANCE: Bone repair, e.g. for large bone defects or patients with defective vascularization is still a major challenge. Highly percolating TPMS porous structures have recently emerged, but no in vivo data were reported on a large animal model of clinical relevance and comparing to an international standard. Here, we fabricated TPMS scaffolds of HA, determined their chemical, percolation and mechanical features, and ran an in-depth pilot study in the sheep with a systematic comparison to the Bio-OssⓇ reference. Our results clearly show the high bone-forming capability of such scaffolds, with outcomes even better than Bio-OssⓇ at short implantation time. This preclinical work provides quantitative data validating the relevance of such TMPS porous scaffolds for bone regeneration in view of clinical evaluation.

A Novel Approach for Powder Bed Fusion of Ceramics Using Two Laser Systems.

The one-step AM process is considered the goal many researchers seek in the field of Additive Manufacturing (AM) of high-technology ceramics. Among the several AM techniques, only Powder Bed Fusion (PBF) can directly print high-technology ceramics using one step. However, the PBF technique faces numerous challenges to efficiently be employed in the PBF of ceramics. These challenges include the formation of cracks, generated thermal stress, effective laser-powder interaction, and low acquired relative density. This study developed a new preheating mechanism for ceramic materials using two laser systems to surpass beyond these challenges and successfully print ceramics with a single-step AM method. One laser is used to preheat the powder particles before the second laser is utilised to complete the melting/sintering process. Both lasers travel along the same scanning path. There is a slight delay (0.0001 s) between the preheating laser and the melting/sintering laser to guarantee that the melting/sintering laser scans a properly preheated powder. To further facilitate testing of the preheating system, a numerical model has been developed to simulate the preheating and melting process and to acquire proper process parameters. The developed numerical model was shown to determine the correct process parameters without needing costly and time-consuming experiments. Alumina samples (10 × 10 × 6 mm3) were successfully printed using alumina powder as feedstock. The surface of the samples was nearly defect-free. The samples' relative densities exceeded 80%, the highest reported relative density for alumina produced by a single-step AM method. This discovery can significantly accelerate the transition to a one-step AM process of ceramics.

Toward Smart Biomimetic Apatite-Based Bone Scaffolds with Spatially Controlled Ion Substitutions.

Biomimetic apatites exhibit a high reactivity allowing ion substitutions to modulate their in vivo response. We developed a novel approach combining several bioactive ions in a spatially controlled way in view of subsequent releases to address the sequence of events occurring after implantation, including potential microorganisms' colonization. Innovative micron-sized core-shell particles were designed with an external shell enriched with an antibacterial ion and an internal core substituted with a pro-angiogenic or osteogenic ion. After developing the proof of concept, two ions were particularly considered, Ag+ in the outer shell and Cu2+ in the inner core. In vitro evaluations confirmed the cytocompatibility through Ag-/Cu-substituting and the antibacterial properties provided by Ag+. Then, these multifunctional "smart" particles were embedded in a polymeric matrix by freeze-casting to prepare 3D porous scaffolds for bone engineering. This approach envisions the development of a new generation of scaffolds with tailored sequential properties for optimal bone regeneration.

Composite Drug Delivery System Based on Amorphous Calcium Phosphate-Chitosan: An Efficient Antimicrobial Platform for Extended Release of Tetracycline.

One major warning emerging during the first worldwide combat against healthcare-associated infections concerns the key role of the surface in the storage and transfer of the virus. Our study is based on the laser coating of surfaces with an inorganic/organic composite mixture of amorphous calcium phosphate-chitosan-tetracycline that is able to fight against infectious agents, but also capable of preserving its activity for a prolonged time, up to several days. The extended release in simulated fluids of the composite mixture containing the drug (tetracycline) was demonstrated by mass loss and UV-VIS investigations. The drug release profile from our composite coatings proceeds via two stages: an initial burst release (during the first hours), followed by a slower evolution active for the next 72 h, and probably more. Optimized coatings strongly inhibit the growth of tested bacteria (Enterococcus faecalis and Escherichia coli), while the drug incorporation has no impact on the in vitro composite's cytotoxicity, the coatings proving an excellent biocompatibility sustaining the normal development of MG63 bone-like cells. One may, therefore, consider that the proposed coatings' composition can open the prospective of a new generation of antimicrobial coatings for implants, but also for nosocomial and other large area contamination prevention.

First successful stabilization of consolidated amorphous calcium phosphate (ACP) by cold sintering: toward highly-resorbable reactive bioceramics.

In the field of bone regeneration, some clinical conditions require highly-resorbable, reactive bone substitutes to rapidly initiate tissue neo-formation. In this view, Amorphous Calcium Phosphates (ACP) appear as well suited bioceramics taking into account their high metastability. However, the metastability also leads to difficulties of sintering without transformation into crystalline compounds. In this work, various calcium phosphate samples (co)doped with carbonate (CO32-) and magnesium ions were synthesized by the double decomposition method in alkaline media using ammonium and potassium hydroxide solutions. The obtained amorphous powders possess an exceptionally-high carbonate content up to 18.3 wt%. Spark Plasma Sintering (SPS) at very low temperature (150 °C) was then utilized to consolidate initial powders with the view to preserve their amorphous character. The influence of the introduction of different apatite growth inhibitors such as carbonate (CO32-) and magnesium ions was studied. XRD and FTIR analyses revealed that sintered ceramics generally consisted in highly carbonated low-crystallinity apatites, which are expected to have higher solubility than conventional apatite-based systems. However, most interestingly, modulation of the doping conditions allowed us to retain, for the first time, the amorphous character of ACP powders after SPS. Such consolidated ACP compounds may now be considered as a new family of bioceramics with high metastability allowing the fast release of bioactive ions upon resorption.

Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides.

Surface modifications are usually performed on titanium alloys to improve osteo-integration and surface bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. In this work, functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium peptide binder (KKLPDA) and hydroxyapatite peptide binder (EEEEEEEE). Homogeneous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30wt% aqueous solution of H2O2 for 24 and 48h. The treated titanium surfaces presented an average roughness of Sa=197nm (24h) and Sa=128nm (48h); an untreated mirror polished sample exhibited an Sa of 13nm. The advancing water contact angle of the titanium oxide layer after 1h of exposure to 30wt% aqueous solution of H2O2 was around 65°, decreasing gradually with time until it reached 35° after a 48h exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) amino acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16% higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium peptide binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminals (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The combined Ti-Hap peptide which exhibited the strongest intensity after the titanium surface dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-HAp4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness of Sa=1.42µm, showed a higher fluorescence for peptides with a higher negative net charge.

Biomineralization of a titanium-modified hydroxyapatite semiconductor on conductive wool fibers.

Metal ions are frequently incorporated into crystalline materials to improve their electrochemical properties and to confer new physicochemical properties. Naturally-occurring phosphate apatite, which is formed geologically and in biomineralization processes, has extensive potential applications and is therefore an attractive functional material. In this study, we generate a novel building block for flexible optoelectronics using bio-inspired methods to deposit a layer of photoactive titanium-modified hydroxyapatite (TiHA) nanoparticles (NPs) on conductive polypyrrole(PPy)-coated wool yarns. The titanium concentration in the reaction solution was varied between 8-50 mol% with respect to the phosphorous, which led to titanate ions replacing phosphate in the hydroxyapatite lattice at levels up to 17 mol%. PPy was separately deposited on wool yarns by oxidative polymerization, using two dopants: (i) anthraquinone-2,6-disulfonic acid to increase the conductivity of the PPy layer and (ii) pyroglutamic acid, to reduce the resistivity of the wool yarns and to promote the heterogeneous nucleation of the TiHA NPs. A specific titanium concentration (25 mol% wrt P) was used to endow the TiHA NPs on the PPy-coated fibers with a desirable band gap value of 3.68 eV, and a specific surface area of 146 m2 g-1. This is the first time that a thin film of a wide-band gap semiconductor has been deposited on natural fibers to create a fiber-based building block that can be used to manufacture flexible electronic devices.

Electrodeposition and Characterization of Hydroxyapatite on TiN/316LSS.

The deposition of TiN on stainless steel substrates may improve the stability and compatibility of this material with bone, which may be advantageously exploited for the elaboration of advanced pros- thetic devices. In this work, TiN-coated 316LSS (by way of DC magnetron sputtering) was used as a starting material for investigating the electrochemical post-deposition of hydroxyapatite (HAp) which has a composition close to that of bone. Electrodeposition was carried out starting from an aqueous medium containing solubilized Ca(NO3)2 and NH4H2PO4 in the presence of H2O2. We report the influence of experimental conditions on the morphology of the obtained HAp coating on TiN/316LSS. The effect of applied potential, temperature, H2O2 concentration, pH and duration of reaction were thoroughly discussed on the basis of X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy and Energy Dispersive X-ray Spectroscopy (EDX) results. This method appears advantageous for producing HAp-coated implant materials.

Biomimetic apatite-based composite materials obtained by spark plasma sintering (SPS): physicochemical and mechanical characterizations.

Nanocrystalline calcium phosphate apatites are biomimetic compounds analogous to bone mineral and are at the origin of the bioactivity of most biomaterials used as bone substitutes. Their unique surface reactivity originates from the presence of a hydrated layer containing labile ions (mostly divalent ones). So the setup of 3D biocompatible apatite-based bioceramics exhibiting a high reactivity requests the development of «low¼ temperature consolidation processes such as spark plasma sintering (SPS), in order to preserve the characteristics of the hydrated nanocrystals. However, mechanical performances may still need to be improved for such nanocrystalline apatite bioceramics, especially in view of load-bearing applications. The reinforcement by association with biopolymers represents an appealing approach, while preserving the advantageous biological properties of biomimetic apatites. Herein, we report the preparation of composites based on biomimetic apatite associated with various quantities of microcrystalline cellulose (MCC, 1-20 wt%), a natural fibrous polymer. The SPS-consolidated composites were analyzed from both physicochemical (X-ray diffraction, Fourier transform infrared, solid state NMR) and mechanical (Brazilian test) viewpoints. The preservation of the physicochemical characteristics of apatite and cellulose in the final material was observed. Mechanical properties of the composite materials were found to be directly related to the polymer/apatite ratios and a maximum crushing strength was reached for 10 wt% of MCC.

Comparison of Physical-chemical and Mechanical Properties of Chlorapatite and Hydroxyapatite Plasma Sprayed Coatings.

Chlorapatite can be considered a potential biomaterial for orthopaedic applications. Its use as plasma-sprayed coating could be of interest considering its thermal properties and particularly its ability to melt without decomposition unlike hydroxyapatite. Chlorapatite (ClA) was synthesized by a high-temperature ion exchange reaction starting from commercial stoichiometric hydroxyapatites (HA). The ClA powder showed similar characteristics as the original industrial HA powder, and was obtained in the monoclinic form. The HA and ClA powders were plasma-sprayed using a low-energy plasma spraying system with identical processing parameters. The coatings were characterized by physical-chemical methods, i.e. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy, including distribution mapping of the main phases detected such as amorphous calcium phosphate (ACP), oxyapatite (OA), and HA or ClA. The unexpected formation of oxyapatite in ClA coatings was assigned to a side reaction with contaminating oxygenated species (O2, H2O). ClA coatings exhibited characteristics different from HA, showing a lower content of oxyapatite and amorphous phase. Although their adhesion strength was found to be lower than that of HA coatings, their application could be an interesting alternative, offering, in particular, a larger range of spraying conditions without formation of massive impurities.

Biomimetic apatite sintered at very low temperature by spark plasma sintering: physico-chemistry and microstructure aspects.

Nanocrystalline apatites analogous to bone mineral are very promising materials for the preparation of highly bioactive ceramics due to their unique intrinsic physico-chemical characteristics. Their surface reactivity is indeed linked to the presence of a metastable hydrated layer on the surface of the nanocrystals. Yet the sintering of such apatites by conventional techniques, at high temperature, strongly alters their physico-chemical characteristics and biological properties, which points out the need for "softer" sintering processes limiting such alterations. In the present work a non-conventional technique, spark plasma sintering, was used to consolidate such nanocrystalline apatites at non-conventional, very low temperatures (T<300 degrees C) so as to preserve the surface hydrated layer present on the nanocrystals. The bioceramics obtained were then thoroughly characterized by way of complementary techniques. In particular, microstructural, nanostructural and other major physico-chemical features were investigated and commented on. This work adds to the current international concern aiming at improving the capacities of present bioceramics, in view of elaborating a new generation of resorbable and highly bioactive ceramics for bone tissue engineering.