Publications

The efficiency of synthetic bone grafts can be evaluated either in osseous sites, to analyze osteoconduction or ectopically, in intramuscular or subcutaneous sites, to assess osteoinduction. Bone regeneration is usually evaluated in terms of the presence and quantity of newly formed bone, but little information is normally provided on the quality of this bone. Here, we propose a novel approach to evaluate bone quality by the combined use of spectroscopy techniques and nanoindentation. Calcium phosphate scaffolds with different architectures, either foamed or 3D-printed, that were implanted in osseous or intramuscular defects in Beagle dogs for 6 or 12 weeks were analyzed. ATR-FTIR and Raman spectroscopy were performed, and mineral-to-matrix ratio, crystallinity, and mineral and collagen maturity were calculated and mapped for the newly regenerated bone and the mature cortical bone from the same specimen. For all the parameters studied, the newly-formed bone showed lower values than the mature host bone. Hardness and elastic modulus were determined by nanoindentation and, in line with what was observed by spectroscopy, lower values were observed in the regenerated bone than in the cortical bone. While, as expected, all techniques pointed to an increase in the maturity of the newly-formed bone between 6 and 12 weeks, the bone found in the intramuscular samples after 12 weeks presented lower mineralization than the intraosseous counterparts. Moreover, scaffold architecture also played a role in bone maturity, with the foamed scaffolds showing higher mineralization and crystallinity than the 3D-printed scaffolds after 12 weeks.

JTD Keywords: Atr-ftir, Bone regeneration, Calcium-phosphate, Ectopic implantation, Implant interface, In-vivo, Indentation, Mechanical-properties, Micromechanical properties, Nanoindentation, Orthotropic implantation, Raman spectroscop, Raman-spectroscopy, Strengt, Substitutes

Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-beta 1) is bound. rLTBP1 facilitates the interaction of LAP with integrin beta 1 and the subsequent mechanically driven release of TGF-beta 1 to stimulate canonical TGF-beta 1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo. An osteogenic platform that functions by capturing inactive growth factor molecules is engineered to overcome conventional challenges associated with the use of active growth factors. The platform triggers capture of inactive transforming growth factor beta-1 for its subsequent integrin-mediated activation which activates osteogenic downstream signaling in vitro and fully repairs critical-sized bone defect in vivo. image

JTD Keywords: Animals, Bone, Bone defect, Bone regeneration, Cell proliferation, Cells, Chemical activation, Defects, Differentiation, Fibronectin, Fibronectins, Growth factor, Growth factors, Humans, Integrin beta1, Integrins, Latency associated peptides, Latent tgf-beta binding proteins, Ltbp1, Osteogenesis, Osteogenic, Protein binding, Recombinant proteins, Release, Repair, Signal transduction, Surface properties, Tgf-beta, Tgf-β1, Transforming growth factor beta1, Transforming growth factors

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

JTD Keywords: 3d printing, Biodegradable, Guided bone regeneration, Implant

Fibrillar collagen structures mineralized with hydroxyapatite using the polymer-induced liquid precursor (PILP) process have been explored as synthetic models for studying biomineralization of human hard tissues and have also been applied in the fabrication of scaffolds for hard tissue regeneration. Strontium has important biological functions in bone and has been used as a therapeutic agent for treating diseases that result in bone defects, such as osteoporosis. Here, we developed a strategy to mineralize collagen with Sr-doped hydroxyapatite (HA) using the PILP process. Doping with Sr altered the crystal lattice of HA and inhibited the degree of mineralization in a concentration-dependent manner, but did not affect the unique formation of intrafibrillar minerals using the PILP. The Sr-doped HA nanocrystals were aligned in the [001] direction but did not recapitulate the parallel alignment of the c-axis of pure Ca HA in relation to the collagen fiber long axis. The mimicry of doping Sr in PILP-mineralized collagen can help understand the doping of Sr in natural hard tissues and during treatment. The fibrillary mineralized collagen with Sr-doped HA will be explored in future work as biomimetic and bioactive scaffolds for regeneration of bone and tooth dentin.

JTD Keywords: bone regeneration, osteoblast differentiation, osteoporosis, ranelate, risk, scaffolds, women, Intrafibrillar mineralization

Lately, there has been an increasing demand for materials that could improve tissue regenerative therapies and provide antimicrobial effects. Similarly, there is a growing need to develop or modify biomaterials for the diagnosis and treatment of different pathologies. In this scenario, hydroxyapatite (HAp) appears as a bioceramic with extended functionalities. Nevertheless, there are certain disadvantages related to the mechanical properties and lack of antimicrobial capacity. To circumvent them, the doping of HAp with a variety of cationic ions is emerging as a good alterative due to the different biological roles of each ion. Among many elements, lanthanides are understudied despite their great potential in the biomedical field. For this reason, the present review focuses on the biological benefits of lanthanides and how their incorporation into HAp can alter its morphology and physical properties. A comprehensive section of the applications of lanthanides-substituted HAp nanoparticles (HAp NPs) is presented to unveil the potential biomedical uses of these systems. Finally, the need to study the tolerable and non-toxic percentages of substitution with these elements is highlighted.

JTD Keywords: biolabeling, biomedicine, biosensors, bone regeneration, calcium, cancer treatment, cationic ions, cell imaging, cerium, doped hap, hydroxyapatite, implants, in-vitro bioactivity, lanthanides-substitutions, lanthanidessubstitutions, nanoparticles, radiation synovectomy, sm-153 particulate hydroxyapatite, structural-characterization, theragnostics, theranostic nanoplatforms, Europium-doped hydroxyapatite, Hydroxyapatite, Theragnostics

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

Most of the conventional in vitro models to test biomaterial-driven vascularization are too simplistic to recapitulate the complex interactions taking place in the actual cell microenvironment, which results in a poor prediction of the in vivo performance of the material. However, during the last decade, cell culture models based on microfluidic technology have allowed attaining unprecedented levels of tissue biomimicry. In this work, we propose a microfluidic-based 3D model to evaluate the effect of bioactive biomaterials capable of releasing signalling cues (such as ions or proteins) in the recruitment of endogenous endothelial progenitor cells, a key step in the vascularization process. The usability of the platform is demonstrated using experimentally-validated finite element models and migration and proliferation studies with rat endothelial progenitor cells (rEPCs) and bone marrow-derived rat mesenchymal stromal cells (BM-rMSCs). As a proof of concept of biomaterial evaluation, the response of rEPCs to an electrospun composite made of polylactic acid with calcium phosphates nanoparticles (PLA+CaP) was compared in a co-culture microenvironment with BM-rMSC to a regular PLA control. Our results show a significantly higher rEPCs migration and the upregulation of several pro-inflammatory and proangiogenic proteins in the case of the PLA+CaP. The effects of osteopontin (OPN) on the rEPCs migratory response were also studied using this platform, suggesting its important role in mediating their recruitment to a calcium-rich microenvironment. This new tool could be applied to screen the capacity of a variety of bioactive scaffolds to induce vascularization and accelerate the preclinical testing of biomaterials. STATEMENT OF SIGNIFICANCE: : For many years researchers have used neovascularization models to evaluate bioactive biomaterials both in vitro, with low predictive results due to their poor biomimicry and minimal control over cell cues such as spatiotemporal biomolecule signaling, and in vivo models, presenting drawbacks such as being highly costly, time-consuming, poor human extrapolation, and ethically controversial. We describe a compact microphysiological platform designed for the evaluation of proangiogenesis in biomaterials through the quantification of the level of sprouting in a mimicked endothelium able to react to gradients of biomaterial-released signals in a fibrin-based extracellular matrix. This model is a useful tool to perform preclinical trustworthy studies in tissue regeneration and to better understand the different elements involved in the complex process of vascularization.Copyright © 2022. Published by Elsevier Ltd.

JTD Keywords: angiogenesis, bioactive materials, bone regeneration, bone-formation, calcium-phosphate, extracellular calcium, in-vitro, interstitial flow, ion release, microfluidic model, signalling gradient, substitutes, tissue engineering, vascularization, vegf, Ion release, Mesenchymal stem-cells, Tissue engineering, Vascularization

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: adhesion formation, bmp-2, cell adhesions, in-vivo, integrin, mesenchymal stem-cells, morphogenetic protein-2, multifunctionality, osteoblastic differentiation, osteogenic differentiation, rgd-dwiva, rgd-peptides, titanium biofunctionalization, titanium surfaces, Animals, Biocompatible materials, Biomimetic peptides, Bone morphogenetic protein 2, Bone regeneration, Cell adhesions, Cell differentiation, Epitopes, Extracellular matrix, Integrins, Marrow stromal cells, Multifunctionality, Osteogenesis, Osteogenic differentiation, Peptides, Rats, Rgd-dwiva, Titanium, Titanium biofunctionalization

Concave surfaces have shown to promote bone regeneration in vivo. However, bone scaffolds obtained by direct ink writing, one of the most promising approaches for the fabrication of personalized bone grafts, consist mostly of convex surfaces, since they are obtained by microextrusion of cylindrical strands. By modifying the geometry of the nozzle, it is possible to print 3D structures composed of non-cylindrical strands and favor the presence of concave surfaces. In this work, we compare the in vivo performance of 3D-printed calcium phosphate scaffolds with either conventional cylindrical strands or star-shaped strands, in a rabbit femoral condyle model. Mono cortical defects, drilled in contralateral positions, are randomly grafted with the two scaffold configurations, with identical composition. The samples are explanted eight weeks post-surgery and assessed by ??-CT and resin embedded histological observations. The results reveal that the scaffolds containing star-shaped strands have better osteoconductive properties, guiding the newly formed bone faster towards the core of the scaffolds, and enhance bone regeneration, although the increase is not statistically significant (p > 0.05). This new approach represents a turning point towards the optimization of pore shape in 3D-printed bone grafts, further boosting the possibilities that direct ink writing technology offers for patient-specific applications.

JTD Keywords: 3d printing, biomimetic calcium phosphate, bone regeneration, in vivo, pore architecture, 3d printing, Architecture, Biomimetic calcium phosphate, Bone regeneration, Calcium-phosphate scaffolds, Geometry, Growth, Implants, In vivo, Induction, Microporosity, Osteoinduction, Pore architecture, Scaffold, Surfaces, Tissue

JTD Keywords: augmentation, calcium-phosphate, expansion, fabrication, hydroxyapatite, made artificial bones, osteogenesis, reconstruction, regeneration, 3-d printing, 3d printers, 3d printing, 3d-printing, Abstracting, Additives, Biomaterial, Bone, Bone regeneration, Bone substitution, Bone tissue engineering, Ceramic, Ceramics, Clinic, Controlled drug delivery, Personalized medicines, Surgical planning, Targeted drug delivery, Three-dimensional-printing, Tricalcium phosphate scaffolds

Objectives This study aimed to compare the performance of a xenograft (XG) and a biomimetic synthetic graft (SG) in three-wall alveolar defects in minipigs by means of 3D computerised tomography and histology. Materials and methods Eight minipigs were used. A total of eight defects were created in the jaw of each animal, three of which were grafted with XGs, three with SGs, and two were left empty as a negative control. The allocation of the different grafts was randomised. Four animals were euthanised at 6 weeks and four at 12 weeks. The grafted volume was then measured by spiral computed tomography to assess volume preservation. Additionally, a histological analysis was performed in undecalcified samples by backscattered scanning electron microscopy and optical microscopy after Masson's trichrome staining. Results A linear mixed-effects model was applied considering four fixed factors (bone graft type, regeneration time, anatomic position, and maxilla/mandible) and one random factor (animal). The SG exhibited significantly larger grafted volume (19%) than the XG. The anterior sites preserved better the grafted volume than the posterior ones. Finally, regeneration time had a positive effect on the grafted volume. Histological observations revealed excellent osseointegration and osteoconductive properties for both biomaterials. Some concavities found in the spheroidal morphologies of SGs were associated with osteoclastic resorption. Conclusions Both biomaterials met the requirements for bone grafting, i.e. biocompatibility, osseointegration, and osteoconduction. Granule morphology was identified as an important factor to ensure a good volume preservation.

JTD Keywords: bone graft, bone regeneration, in vivo, miniature swine, synthetic graft, 3-dimensional changes, Anorganic bovine bone, Autogenous bone, Bio-oss, Biomaterials, Bone graft, Bone regeneration, Calcium-phosphate, Hydroxyapatite, In vivo, Miniature swine, Sinus floor augmentation, Substitute, Synthetic graft, Volume, Xenograft

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

JTD Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Animals, Beta-tricalcium phosphate, Biomimetics, Bone regeneration, Calcium deficient hydroxyapatite, Calcium phosphate, Calcium phosphates, Cell differentiation, Engraftment, Human bone marrow mesenchymal stromal cells, Humans, Mesenchymal stem cells, Mice, Mice, nude, Osteogenesis, Tissue scaffolds

Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% β-tricalcium phosphate (β-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. Statement of significance: The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.

JTD Keywords: 3d printing, behavior, biomimetic, bone scaffolds, calcium phosphate, deficient hydroxyapatite, design, graft, hydrothermal, in vivo, morbidity, osteoinduction, porosity, standard, tricalcium phosphate, 3d printing, Animals, Biomimetic, Biomimetics, Bone regeneration, Bone scaffolds, Calcium phosphate, Calcium phosphates, Fibula free-flap, Humans, Hydrothermal, In vivo, Osteogenesis, Printing, three-dimensional, Rabbits, Tissue scaffolds

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

Direct ink writing (DIW) techniques open up new possibilities for the fabrication of patient-specific bone grafts. Self-setting calcium phosphate inks, which harden at low temperature, allow obtaining nanostructured scaffolds with biomimetic properties and enhanced bioactivity. However, the slow hardening kinetics hampers the translation to the clinics. Different hydrothermal treatments for the consolidation of DIW scaffolds fabricated with an α-tricalcium phosphate /pluronic F127 ink were explored, comparing them with a biomimetic treatment. Three different scaffold architectures were analysed. The hardening process, associated to the conversion of α-tricalcium phosphate to hydroxyapatite was drastically accelerated by the hydrothermal treatments, reducing the time for complete reaction from 7 days to 30 minutes, while preserving the scaffold architectural integrity and retaining the nanostructured features. β-tricalcium phosphate was formed as a secondary phase, and a change of morphology from plate-like to needle-like crystals in the hydroxyapatite phase was observed. The binder was largely released during the treatment. The hydrothermal treatment resulted in a 30% reduction of the compressive strength, associated to the residual presence of β-tricalcium phosphate. Biomimetic and hydrothermally treated scaffolds supported the adhesion and proliferation of rat mesenchymal stem cells, indicating a good suitability for bone tissue engineering applications. Statement of Significance: 3D plotting has opened up new perspectives in the bone regeneration field allowing the customisation of synthetic bone grafts able to fit patient-specific bone defects. Moreover, this technique allows the control of the scaffolds’ architecture and porosity. The present work introduces a new method to harden biomimetic hydroxyapatite 3D-plotted scaffolds which avoids high-temperature sintering. It has two main advantages: i) it is fast and simple, reducing the whole fabrication process from the several days required for the biomimetic processing to a few hours; and ii) it retains the nanostructured character of biomimetic hydroxyapatite and allows controlling the porosity from the nano- to the macroscale. Moreover, the good in vitro cytocompatibility results support its suitability for cell-based bone regeneration therapies.

JTD Keywords: Calcium phosphate, Hydroxyapatite, Biomimetic, Bone regeneration, 3D plotting, Direct ink writing, Bone graft

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca2+ release rate, with the faster Ca2+ release composite gel showing improved bone repair at 3 weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6 weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. Statement of Significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

JTD Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglasses

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low-and high-Temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-Temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.

JTD Keywords: Bone Regeneration, Calcium Phosphate, Focus Ion Beam, Osteoclast, Resorption, Scanning Electron Microscopy

Abstract In current bone tissue engineering strategies the achievement of sufficient angiogenesis during tissue regeneration is still a major limitation in order to attain full functionality. Several strategies have been described to tackle this problem, mainly by the use of angiogenic factors or endothelial progenitor cells. However, when facing a clinical scenario these approaches are inherently complex and present a high cost. As such, more cost effective alternatives are awaited. Here, we demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate ormoglass (CaP) particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. We show that the current approach elicited the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial. As both PLA and CaP are currently accepted for clinical application these off-the-shelf novel membranes have great potential for guided bone regeneration applications. Statement of significance In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, our group has found that calcium ions released by the degradation of calcium phosphate ormoglasses (CaP) are effective angiogenic promoters. Based on this, in this work we successfully produced hybrid fibrous mats with different contents of CaP nanoparticles and thus with different calcium ion release rates, using an ormoglass – poly(lactic acid) blend approach. We show that these matrices, upon implantation in a subcutaneous site, could elicit the local expression of angiogenic factors, associated to a chemotactic effect on macrophages, and sustained angiogenesis into the biomaterial, in a CaP dose dependent manner. This off-the-shelf cost effective approach presents great potential to translate to the clinics.

JTD Keywords: Angiogenesis, Bone regeneration, Calcium phosphate ormoglass

Major limitations of calcium phosphate cements (CPCs) are their relatively slow degradation rate and the lack of macropores allowing the ingrowth of bone tissue. The development of self-setting cement foams has been proposed as a suitable strategy to overcome these limitations. In previous work we developed a gelatine-based hydroxyapatite foam (G-foam), which exhibited good injectability and cohesion, interconnected porosity and good biocompatibility in vitro. In the present study we evaluated the in vivo performance of the G-foam. Furthermore, we investigated whether enrichment of the foam with soybean extract (SG-foam) increased its bioactivity. G-foam, SG-foam and non-foamed CPC were implanted in a critical-size bone defect in the distal femoral condyle of New Zealand white rabbits. Bone formation and degradation of the materials were investigated after 4, 12 and 20 weeks using histological and biomechanical methods. The foams maintained their macroporosity after injection and setting in vivo. Compared to non-foamed CPC, cellular degradation of the foams was considerably increased and accompanied by new bone formation. The additional functionalization with soybean extract in the SG-foam slightly reduced the degradation rate and positively influenced bone formation in the defect. Furthermore, both foams exhibited excellent biocompatibility, implying that these novel materials may be promising for clinical application in non-loaded bone defects.

JTD Keywords: Bone regeneration, Calcium phosphate cement, Gelatine, Rabbit model, Soybean

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.

JTD Keywords: Angiogenesis, Bioluminescence imaging, Bone regeneration, Fibrin, Mesenchymal stem cell

Novel hydroxyapatite (HA)-collagen microcarriers (MCs) with different micro/nanostructures were developed for bone tissue-engineering applications. The MCs were fabricated via calcium phosphate cement (CPC) emulsion in oil. Collagen incorporation in the liquid phase of the CPC resulted in higher MC sphericity. The MCs consisted of a porous network of entangled hydroxyapatite crystals, formed as a result of the CPC setting reaction. The addition of collagen to the MCs, even in an amount as small as 0.8wt%, resulted in an improved interaction with osteoblast-like Saos-2 cells. The micro/nanostructure and the surface texture of the MCs were further tailored by modifying the initial particle size of the CPC. A synergistic effect between the presence of collagen and the nanosized HA crystals was found, resulting in significantly enhanced alkaline phosphatase activity on the collagen-containing nanosized HA MCs.

JTD Keywords: Bone regeneration, Calcium phosphate cement, Cell response, Collagen, Hydroxyapatite, Microcarrier

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

Hydroxyapatite foams are potential synthetic bone grafting materials or scaffolds for bone tissue engineering. A novel method to obtain injectable hydroxyapatite foams consists in foaming the liquid phase of a calcium phosphate cement. In this process, the cement powder is incorporated into a liquid foam, which acts as a template for macroporosity. After setting, the cement hardens maintaining the macroporous structure of the foam. In this study a low molecular weight surfactant, Polysorbate 80, and a protein, gelatine, were compared as foaming agents of a calcium phosphate cement. The foamability of Polysorbate 80 was greater than that of gelatine, resulting in higher macroporosity in the set hydroxyapatite foam and higher macropore interconnectivity. Gelatine produced less interconnected foams, especially at high concentrations, due to a higher liquid foam stability. However it increased the injectability and cohesion of the foamed paste, and enhanced osteoblastic-like cell adhesion, all of them important properties for bone grafting materials.

JTD Keywords: Hydroxyapatite, Porosity, Calcium phosphate cement, Scaffolds, Foaming, Bone regeneration

In this study, a three-dimensional (3D) computational simulation of bone regeneration was performed in a human tibia under realistic muscle loading. The simulation was achieved using a discrete lattice modeling approach combined with a mechanoregulation algorithm to describe the cellular processes involved in the healing process namely proliferation, migration, apoptosis, and differentiation of cells. The main phases of fracture healing were predicted by the simulation, including the bone resorption phase, and there was a qualitative agreement between the temporal changes in interfragmentary strain and bending stiffness by comparison to experimental data and clinical results. Bone healing was simulated beyond the reparative phase by modeling the transition of woven bone into lamellar bone. Because the simulation has been shown to work with realistic anatomical 3D geometry and muscle loading, it demonstrates the potential of simulation tools for patient-specific pre-operative treatment planning.

JTD Keywords: Tissue differentiation, Computational analysis, Mechanical conditions, Bone regeneration, Weight-bearing, Proliferation, Osteoblast, Stiffness, Ingrowth, Scaffold

The key feature of calcium phosphate cements (CPCs) lies in the setting reaction triggered by mixing one or more solid calcium phosphate salts with an aqueous solution. Upon mixture, the reaction takes place through a dissolution-precipitation process which is macroscopically observed by a gradual hardening of the cement paste. The precipitation of hydroxyapatite nanocrystals at body or room temperature, and the fact that those materials can be used as self-setting pastes, have for many years been the most attractive features of CPCs. However, the need to develop materials able to sustain bone tissue ingrowth and be capable of delivering drugs and bioactive molecules, together with the continuous requirement from surgeons to develop more easily handling cements, has pushed the development of new processing routes that can accommodate all these requirements, taking advantage of the possibility of manipulating the self-setting CPC paste. It is the goal of this paper to provide a brief overview of the new processing developments in the area of CPCs and to identify the most significant achievements.

JTD Keywords: Bone regeneration, Calcium phosphate cements, Granules, Microcarriers, Scaffolds

The use of injectable self-setting calcium phosphate cements or soluble glass granules represent two different strategies for bone regeneration, each with distinct advantages and potential applications. This study compares the in vivo behavior of two calcium phosphate cements and two phosphate glasses with different composition, microstructure and solubility, using autologous bone as a control, in a rabbit model. The implanted materials were alpha-tricalcium phosphate cement (cement H), calcium sodium potassium phosphate cement (cement R), and two phosphate glasses in the P2O5-CaO-Na2O and P2O5-CaO-Na2O-TiO2 systems. The four materials were osteoconductive, biocompatible and biodegradable. Radiological and histological studies demonstrated correct osteointegration and substitution of the implants by new bone. The reactivity of the different materials, which depends on their solubility, porosity and specific surface area, affected the resorption rate and bone formation mainly during the early stages of implantation, although this effect was weak. Thus, at 4 weeks the degradation was slightly higher in cements than in glasses, especially for cement R. However, after 12 weeks of implantation all materials showed a similar degradation degree and promoted bone neoformation equivalent to that of the control group.

JTD Keywords: Calcium phosphates, Calcium phosphate cements, Phosphate glasses, Bone grafts, Bone regenerations