Publications

Bone regeneration often fails due to implants/grafts lacking vascular supply, causing necrotic tissue and poor integration. Microsurgical techniques are used to overcome this issue, allowing the graft to anastomose. These techniques have limitations, including severe patient morbidity and current research focuses on stimulating angiogenesis in situ using growth factors, presenting limitations, such as a lack of control and increased costs. Non-biological stimuli are necessary to promote angiogenesis for successful bone constructs. Recent studies have reported that bioactive glass dissolution products, such as calcium-releasing nanoparticles, stimulate hMSCs to promote angiogenesis and new vasculature. Moreover, the effect of 3D microporosity has also been reported to be important for vascularisation in vivo. . Therefore, we used room-temperature extrusion 3D printing with polylactic acid (PLA) and calcium phosphate (CaP) based glass scaffolds, focusing on geometry and solvent displacement for scaffold recovery. Combining both methods enabled reproducible control of 3D structure, porosity, and surface topography. Scaffolds maintained calcium ion release at physiological levels and supported human mesenchymal stem cell proliferation. Scaffolds stimulated the secretion of vascular endothelial growth factor (VEGF) after 3 days of culture. Subcutaneous implantation in vivo indicated good scaffold integration and blood vessel infiltration as early as one week after. PLA-CaP scaffolds showed increased vessel maturation 4 weeks after implantation without vascular regression. Results show PLA/CaP-based glass scaffolds, made via controlled 3D printing, support angiogenesis and vessel maturation, promising improved vascularization for bone regeneration.

JTD Keywords: 3d-printed porosity, Angiogenic growth-factors, Bioceramic scaffolds, Bone angiogenesis, Calcium phosphat, Calcium-phosphate, Cells, Composite, Extracellular calcium, Functional-role, In-vitro, Inorganic trace-elements, Matri, Polylactic acid, Regeneration

Human mesenchymal stromal cells (hMSCs) seeded on calcium phosphate (CaP) bioceramics are extensively explored in bone tissue engineering and have recently shown effective clinical outcomes. In previous pre-clinical studies, hMSCs-CaP-mediated bone formation was preceded by osteoclastogenesis at the implantation site. The current study evaluates to what extent phase composition of CaPs affects the osteoclast response and ultimately influence bone formation. To this end, four different CaP bioceramics were used, hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two biphasic composites of HA/beta- TCP ratios of 60/40 and 20/80 respectively, for in vitro osteoclast differentiation and correlation with in vivo osteoclastogenesis and bone formation. All ceramics allowed osteoclast formation in vitro from mouse and human precursors, except for pure HA, which significantly impaired their maturation. Ectopic implantation alongside hMSCs in subcutis sites of nude mice revealed new bone formation at 8 weeks in all conditions with relative amounts for beta-TCP > biphasic CaPs > HA. Surprisingly, while hMSCs were essential for osteoinduction, their survival did not correlate with bone formation. By contrast, the degree of early osteoclastogenesis (2 weeks) seemed to define the extent of subsequent bone formation. Together, our findings suggest that the osteoclastic response could be used as a predictive marker in hMSC-CaPbased bone regeneration and strengthens the need to understand the underlying mechanisms for future biomaterial development. Statement of significance The combination of mesenchymal stromal cells (MSCs) and calcium phosphate (CaP) materials has demonstrated its safety and efficacy for bone regeneration in clinical trials, despite our insufficient understanding of the underlying biological mechanisms. Osteoclasts were previously suggested as key mediators between the early inflammatory phase following biomaterial implantation and the subsequent bone formation. Here we compared the affinity of osteoclasts for various CaP materials with different ratios of hydroxyapatite to beta-tricalcium phosphate. We found that osteoclast formation, both in vitro and at early stages in vivo, correlates with bone formation when the materials were implanted alongside MSCs in mice. Surprisingly, MSC survival did not correlate with bone formation, suggesting that the number or phenotype of osteoclasts formed was more important. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

JTD Keywords: Acid phosphatase tartrate resistant isoenzyme, Animal, Animal cell, Animal experiment, Animal tissue, Animals, Article, Beta-tricalcium phosphate, Bioceramics, Biocompatible materials, Biomaterial, Bone, Bone development, Bone formation, Bone regeneration, Calcium phosphate, Calcium phosphate materials, Calcium phosphates, Cd14 antigen, Cell differentiation, Cell engineering, Cell maturation, Cell survival, Ceramics, Chemical composition, Controlled study, Correlation analysis, Correlation coefficient, Data correlation, Durapatite, Engraftment, Flowcharting, Human, Human cell, Human mesenchymal stromal cell, Human mesenchymal stromal cells, Humans, Hydroxyapatite, Hydroxyapatites, In vitro study, In vivo study, In-vitro, In-vivo, Mammals, Marrow stromal cells, Material composition, Material compositions, Mesenchymal stroma cell, Mesenchymal stromal cells, Mice, Mice, nude, Monocyte, Mouse, Nonhuman, Nude mouse, Ossification, Osteoclast, Osteoclastogenesis, Osteoclasts, Osteogenesis, Osteoinduction, Phase composition, Regeneration strategies, Resorption, Scaffolds, Stem-cells, Subcutaneous tissue, Tissue engineering, Transmission control protocol, Tri-calcium phosphates, Vimentin

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.

JTD Keywords: Bioceramics, Bone, Bone regeneration, Composites, Degradation, Fabrication, Hydroxyapatite, Hydroxyapatite scaffolds, Injectability, Porosity, Recombinant spider silk, Rgd motifs, Silk, Stem-cells

Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds. Statement of significance: Recent studies have stressed the role that concave surfaces play in tissue regeneration and, more specifically, in osteoinduction and osteogenesis. Direct ink writing enables the production of patient-specific bone grafts with controlled architecture. However, besides many advantages, it has the serious limitation that the surfaces obtained are convex. In this article, for the first time we develop a strategy to introduce concave pores in the printed filaments of biomimetic hydroxyapatite by incorporation and partial dissolution of gelatin microspheres. The retention of part of the gelatin results in a more elastic behavior compared to the brittleness of hydroxyapatite scaffolds, while the needle-shaped nanostructure of biomimetic hydroxyapatite is maintained and gelatin-coated concave pores on the surface of the filaments enhance cell spreading. © 2021 The Authors

JTD Keywords: 3d printing, bioceramics, biomimetic, bone, bone regeneration, concavity, concavity, bone regeneration, gelatin, hydrogel, hydroxyapatite, microspheres, osteoinduction, porosity, porous filament, substitutes, tissue-growth, 3d printing, Biomimetic, Biomimetics, Calcium-phosphate scaffolds, Concavity, bone regeneration, Durapatite, Gelatin, Humans, Hydroxyapatite, Porosity, Porous filament, Printing, three-dimensional, Tissue engineering, Tissue scaffolds

Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.

JTD Keywords: Antibiotic, Bioceramic, Biomaterial, Bone regeneration, Calcium phosphate cement, Ceramic matrix, Growth factor, Hydroxyapatite, Ions, Protein