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.</p

Calcium phosphate 3D-printed Triply Periodic Minimal Surfaces (TPMS) scaffolds for bone subtitution : design, manufacturing, post process, mechanical properties and biological performances

Les premières traces de comblement de défauts osseux avec des matériaux synthétiques remontent au 13e siècle, où les Incas ont tenté de remplacer l'os par de l'or. Au cours des dernières décennies, cette approche s'est significativement développée, en grande partie grâce à l'émergence de substituts osseux tels que les phosphates de calcium, présentant une composition chimique et cristalline proche de celle de l'os naturel. L'utilisation de ces matériaux s'est avérée très prometteuse, notamment en raison de leur biocompatibilité et de leur capacité d'ostéointégration, suscitant ainsi de nombreuses questions et perspectives d'amélioration qui ont alimenté la recherche dans ce domaine. Un axe de recherche important se concentre sur la porosité de ces matériaux, une propriété nécessaire à la migration et à la prolifération cellulaire, mais pouvant réduire leurs propriétés mécaniques. En effet, un biomatériau idéal pour la substitution osseuse doit présenter une porosité élevée, comprenant des pores concaves entièrement interconnectés, ainsi qu'une résistance mécanique se rapprochant, voire équivalente, à celle de l'os naturel. Le développement de l'impression 3D ces dernières décennies offre une réelle opportunité pour faciliter la création de structures poreuses répondant à ces critères. Cependant, l'impression 3D de céramiques, bien que prometteuse, n'en est encore qu'à ses débuts par rapport à l'impression de polymères ou de métaux. Pour résoudre ces problématiques, le projet DOC-3D-Printing a été financé dans le cadre du programme Horizon Europe 2020. Cette thèse s'inscrit dans ce projet et vise à sélectionner et de concevoir des structures poreuses conformes aux critères de porosité, tout en optimisant le processus d'impression 3D pour faciliter leur production et leur étude. Pour ce faire, nous avons sélectionné trois types de structures poreuses TPMS:Schwartz Diamonds, Schwartz Primitives et des Gyroids, et étudié l'ensemble du processus de fabrication, du choix des matières premières, notamment les poudres d'hydroxyapatite, à la configuration des paramètres d'impression 3D et au post-traitement des structures, y compris le nettoyage et le traitement thermique. Ces études ont permis de sélectionner une méthode de synthèse de poudre préservant leur pureté cristalline après frittage à des températures relativement élevées, d'optimiser les paramètres d'impression pour contrôler la taille des parois et des pores des structures, ainsi que de mettre en place des méthodes de nettoyage et de traitement thermique améliorées. L'optimisation du processus de fabrication a rendu possible l'impression des trois structures TPMS sélectionnées . Des tests d’indentation macro-sphérique (recommandés par la norme ISO13175-3:2012) et de flexion ont été réalisés pour comprendre l'impact de la taille des parois et des pores sur les propriétés mécaniques. Cela a permis de mieux comprendre les facteurs influençant les propriétés mécaniques et de sélectionner une porosité pour des tests ultérieurs. Des tests de compression ont été effectués pour comparer leurs résultats à ceux de l'indentation macro-sphérique, pour mieux évaluer les avantages et les inconvénients de chaque méthode d'analyse. La porosité sélectionnée a ensuite été soumise à des tests in vitro avec des cellules ostéoblastes immortalisées. Les résultats de ces tests, combinés aux résultats des tests mécaniques, ont permis de choisir la structure en forme de Gyroids à haute porosité pour des tests in vivo sur des moutons, qui se sont révélés concluants. Ces structures ont ensuite été utilisées pour réaliser une rhinopoïèse, une première mondiale ou un substitut nasal, composé de structures Gyroids fabriquées à partir d'hydroxyapatite, a été prévascularisé dans l'avant-bras de la patiente avant d'être utilisé pour reconstruire son nez. Cette intervention a significativement amélioré la qualité de vie, la confiance en soi de la patiente, favorisant ainsi une meilleure intégration sociale.Filling bone defects with synthetic materials has been a practice for centuries, dating back to the 13th century when the Incas attempted to replace bone with gold. Over the past few decades, significant progress has been made in this field, largely due to the emergence of bone substitutes like calcium phosphates, which closely mimic the chemical and crystalline composition of natural bone. The use of these materials has shown tremendous promise, driven by their biocompatibility and osteointegration capabilities, prompting extensive research and improvement efforts. A significant research focus revolves around the porosity of these materials, which is crucial for cell migration and proliferation but can potentially compromise mechanical properties. Ideally, an ideal biomaterial for bone substitution should exhibit high porosity with interconnected concave pores, all while maintaining mechanical strength comparable to natural bone. The advent of 3D printing in recent decades offers a genuine opportunity to create porous structures that meet these criteria. However, 3D printing of ceramics, although promising, is still in its early stages compared to polymer or metal printing. To address these challenges, the DOC-3D-Printing project received funding under the Horizon Europe 2020 program. This thesis is an integral part of this project and aims to select and design porous structures that meet porosity criteria while optimizing the 3D printing process for their production and study. To accomplish this, three types of porous TPMS structures were chosen, and the entire fabrication process was scrutinized, starting from the selection of raw materials, including hydroxyapatite powders, to configuring 3D printing parameters and post-processing structures, such as cleaning and thermal treatment. These studies led to the selection of a powder synthesis method that preserves their crystalline purity after sintering at relatively high temperatures, optimization of printing parameters to control wall and pore size, and the implementation of improved cleaning and thermal treatment methods. Process optimization enabled the printing of the three selected TPMS structures (Schwartz Diamonds, Schwartz Primitives and gyroids). Spherical macro-indentation tests (recommended by ISO 13175-3:2012) and bending tests were conducted to gain insights into the impact of wall thickness and pore size on mechanical properties. This improved understanding of factors influencing mechanical properties led to the selection of porosity levels for further testing. Additional compression tests were conducted to compare their results with spherical indentation, providing a more comprehensive evaluation of the advantages and disadvantages of each analysis method. The selected porosity was subsequently subjected to in vitro tests with immortalized osteoblast cells, analyzed at 10 and 21 days. The results of these tests, combined with mechanical test results, led to the choice of high-porosity Gyroids structures for in vivo testing in sheep, which proved successful. These structures were then employed for a world premiere, a custom-made bone substitute rhinopoiesis surgery in a human patient. A nasal substitute composed of Gyroids structures made from hydroxyapatite was prevascularized in the patient's forearm before being used to reconstruct her nose. This intervention significantly enhanced the patient's quality of life and self-confidence, thereby promoting improved social integration

3D printed triply periodic minimal surfaces calcium phosphate bone substitute: The effect of porosity design on mechanical properties

peer reviewedTriply periodic minimal surfaces (TPMSs) are relevant structures for building synthetic bone grafts due to their tortuosity and interconnected pores. In order to investigate their porosity and mechanical properties, three different hydroxyapatite-based TPMSs were printed by vat polymerization: the Schwartz diamond, Schwartz primitive, and gyroid scaffolds. Each structure was designed with three levels of porosity, two different pore sizes, and two different wall thicknesses so as to gain an understanding of the effect of pore size and wall thickness on the mechanical properties. The results highlighted the importance of cleaning in the manufacturing process and its impact on the final porosity, especially for small pore sizes. This article intends to be among the first to discuss mechanical testing with macro spherical indentation. Bending and macro spherical indentation resistance tests revealed differences in the mechanical properties between the different structures, with a strong sensitivity of bending strength to the presence of cracks after thermal treatment. Notably, increasing the wall thickness was shown to increase the risk of damage to the solid parts of the scaffolds, therefore lowering the bending strength

Ultrasound imaging modalities

After a short introduction on generation and propagation of ultrasound waves, this chapter presents and discusses currently available and emerging ultrasound imaging modalities, such as standard modes in one, two, and three dimensions; Doppler and nonlinear imaging, including also the use of ultrasound contrast agents; and quantitative imaging, ranging from tissue characterization by analysis of speckle, strain, and elastic properties of tissue to perfusion imaging quantification. Emerging imaging modalities, such as molecular imaging (and treatment) by targeted agents and hybrid (photo- and magnetoacoustics) imaging, are also presented. Each technique is discussed with the aim of bridging basic principles with implementation and clinical application and relevance