Selection and Optimization of a Bioink Based on PANC-1- Plasma/Alginate/Methylcellulose for Pancreatic Tumour Modelling.

3D bioprinting involves using bioinks that combine biological and synthetic materials. The selection of the most appropriate cell-material combination for a specific application is complex, and there is a lack of consensus on the optimal conditions required. Plasma-loaded alginate and alginate/methylcellulose (Alg/MC) inks were chosen to study their viscoelastic behaviour, degree of recovery, gelation kinetics, and cell survival after printing. Selected inks showed a shear thinning behavior from shear rates as low as 0.2 s-1, and the ink composed of 3% w/v SA and 9% w/v MC was the only one showing a successful stacking and 96% recovery capacity. A 0.5 × 106 PANC-1 cell-laden bioink was extruded with an Inkredible 3D printer (Cellink) through a D = 410 µm tip conical nozzle into 6-well culture plates. Cylindrical constructs were printed and crosslinked with CaCl2. Bioinks suffered a 1.845 Pa maximum pressure at the tip that was not deleterious for cellular viability. Cell aggregates can be appreciated for the cut total length observed in confocal microscopy, indicating a good proliferation rate at different heights of the construct, and suggesting the viability of the selected bioink PANC-1/P-Alg3/MC9 for building up three-dimensional bioprinted pancreatic tumor constructs.

Preparation of covalently bonded silica-alginate hybrid hydrogels by SCHIFF base and sol-gel reactions.

Organic-inorganic hybrid materials overcome drawbacks associated with alginate hydrogels. In this work, covalently coupled silica-alginate hybrids were prepared by Schiff base formation and sol-gel reaction using alginate dialdehyde (ADA), (3-Aminopropyl) triethoxysilane (APTES), and APTES/tetraethylorthosilicate (TEOS) precursors. The influence of the polysaccharide/inorganic ratio, the nature of the inorganic precursor and the ionic crosslinking ability are studied. Prepared hybrids were characterized by FT-IR, 13C and 29Si NMR spectroscopies, SEM, and rheology. For ADA/APTES hybrids, at higher ADA content, Schiff base formation is predominant, but at lower ADA content, the sol-gel reaction is prevalent. However, the progress of the sol-gel reactions for ADA/(APTES+TEOS), is favored with higher ADA compositions. Introducing a posterior ionic crosslinking treatment was possible, increasing the moduli in ADA/(APTES+TEOS) hybrids from 86,207 Pa for 1.5 ADA/Si to 362,171 Pa for 1.5 ADA/Si-Ca. In-situ ADA-Silica hybrid hydrogels containing both ionic and covalent crosslinking can be successfully synthesized with the proposed method. CARBPOL-D-21-01042.

Assessment of a PCL-3D Printing-Dental Pulp Stem Cells Triplet for Bone Engineering: An In Vitro Study.

The search of suitable combinations of stem cells, biomaterials and scaffolds manufacturing methods have become a major focus of research for bone engineering. The aim of this study was to test the potential of dental pulp stem cells to attach, proliferate, mineralize and differentiate on 3D printed polycaprolactone (PCL) scaffolds. A 100% pure Mw: 84,500 ± 1000 PCL was selected. 5 × 10 × 5 mm3 parallelepiped scaffolds were designed as a wood-pilled structure composed of 20 layers of 250 µm in height, in a non-alternate order ([0,0,0,90,90,90°]). 3D printing was made at 170 °C. Swine dental pulp stem cells (DPSCs) were extracted from lower lateral incisors of swine and cultivated until the cells reached 80% confluence. The third passage was used for seeding on the scaffolds. Phenotype of cells was determined by flow Cytometry. Live and dead, Alamar blue™, von Kossa and alizarin red staining assays were performed. Scaffolds with 290 + 30 µm strand diameter, 938 ± 80 µm pores in the axial direction and 689 ± 13 µm pores in the lateral direction were manufactured. Together, cell viability tests, von Kossa and Alizarin red staining indicate the ability of the printed scaffolds to support DPSCs attachment, proliferation and enable differentiation followed by mineralization. The selected material-processing technique-cell line (PCL-3D printing-DPSCs) triplet can be though to be used for further modelling and preclinical experiments in bone engineering studies.

Design of Thermoplastic 3D-Printed Scaffolds for Bone Tissue Engineering: Influence of Parameters of "Hidden" Importance in the Physical Properties of Scaffolds.

Additive manufacturing (AM) techniques are becoming the approaches of choice for the construction of scaffolds in tissue engineering. However, the development of 3D printing in this field brings unique challenges, which must be accounted for in the design of experiments. The common printing process parameters must be considered as important factors in the design and quality of final 3D-printed products. In this work, we study the influence of some parameters in the design and fabrication of PCL scaffolds, such as the number and orientation of layers, but also others of "hidden" importance, such as the cooling down rate while printing, or the position of the starting point in each layer. These factors can have an important impact oin the final porosity and mechanical performance of the scaffolds. A pure polycaprolactone filament was used. Three different configurations were selected for the design of the internal structure of the scaffolds: a solid one with alternate layers (solid) (0°, 90°), a porous one with 30% infill and alternate layers (ALT) (0°, 90°) and a non-alternated configuration consisting in printing three piled layers before changing the orientation (n-ALT) (0°, 0°, 0°, 90°, 90°, 90°). The nozzle temperature was set to 172 °C for printing and the build plate to 40 °C. Strand diameters of 361 ± 26 µm for room temperature cooling down and of 290 ± 30 µm for forced cooling down, were obtained. A compression elastic modulus of 2.12 ± 0.31 MPa for n-ALT and 8.58 ± 0.14 MPa for ALT scaffolds were obtained. The cooling down rate has been observed as an important parameter for the final characteristics of the scaffold.

Alginate hydrogels for bone tissue engineering, from injectables to bioprinting: A review.

This review focuses on recently developed alginate injectable hydrogels and alginate composites for applications in bone tissue regeneration, and it evaluates the alternatives to overcome the problems that avoid their utilization in the field. Section 2 covers the properties of alginates that have made them useful for medical applications, in particular their ionic gelling ability for preparing injectable compositions used as delivery drugs systems. The advantages and shortcomings of these preparations are revised together with the chemical modifications assayed. Section 3 describes how it has been taken advantage of alginates into the new field of biofabrication and the developments in bone engineering. The state of the art of this field is reviewed. Finally in Section 4, new developments and approaches that in opinion of the authors can lead to a breakthrough in bone tissue engineering using alginates are introduced.

Synthesis and in Vitro Cytocompatibility of Segmented Poly(Ester-Urethane)s and Poly(Ester-Urea-Urethane)s for Bone Tissue Engineering.

Two series of segmented polyurethanes were obtained and their mechanical and thermal properties as well as their biodegradability and cytotoxicity were evaluated. The chemical nature of the polyurethanes was varied by using either 1,4 butanediol (poly-ester-urethanes, PEUs) or l-lysine ethyl ester dihydrochloride (poly-ester-urea-urethanes, PEUUs) as chain extenders. Results showed that varying the hard segment influenced the thermal and mechanical properties of the obtained polymers. PEUs showed strain and hardness values of about 10⁻20 MPa and 10⁻65 MPa, respectively. These values were higher than the obtained values for the PEUUs due to the phase segregation and the higher crystallinity observed for the polyester-urethanes (PEUs); phase segregation was also observed and analyzed by XRD and DSC. Moreover, both series of polymers showed hydrolytic degradation when they were submerged in PBS until 90 days with 20% of weight loss. In vitro tests using a Human Osteoblastic cell line (Hob) showed an average of 80% of cell viability and good adhesion for both series of polymers.

Bioceramic nanocomposite thiol-acrylate polyHIPE scaffolds for enhanced osteoblastic cell culture in 3D.

Emulsion-templated (polyHIPE) scaffolds for bone tissue engineering were produced by photopolymerisation of a mixture of trimethylolpropane tris(3-mercaptopropionate) and dipentaerythritol penta-/hexa-acrylate in the presence of hydroxyapatite (HA) or strontium-modified hydroxyapatite (SrHA) nanoparticles. Porous and permeable polyHIPE materials were produced regardless of the type or incorporation level of the bioceramic, although higher loadings resulted in a larger average pore diameter. Inclusion of HA and SrHA into the scaffolds was confirmed by EDX-SEM, FTIR and XPS and quantified by thermogravimetry. Addition of HA to polyHIPE scaffolds significantly enhanced compressive strength (148-216 kPa) without affecting compressive modulus (2.34-2.58 MPa). The resulting materials were evaluated in vitro as scaffolds for the 3D culture of MG63 osteoblastic cells vs. a commercial 3D cell culture scaffold (Alvetex®). Cells were able to migrate throughout all scaffolds, achieving a high density by the end of the culture period (21 days). The presence of HA and in particular SrHA gave greatly enhanced cell proliferation, as determined by staining of histological sections and total protein assay (Bradford). Furthermore, Von Kossa and Alizarin Red staining demonstrated significant mineralisation from inclusion of bioceramics, even at the earliest time point (day 7). Production of alkaline phosphatase (ALP), an early osteogenic marker, was used to investigate the influence of HA and SrHA on cell function. ALP levels were significantly reduced on HA- and SrHA-modified scaffolds by day 7, which agrees with the observed early onset of mineralisation in the presence of the bioceramics. The presented data support our conclusions that HA and SrHA enhance osteoblastic cell proliferation on polyHIPE scaffolds and promote early mineralisation.

Novel non-cytotoxic, bioactive and biodegradable hybrid materials based on polyurethanes/TiO2 for biomedical applications.

Titanium compounds have demonstrated great interfacial properties with biological tissues whereas a wide variety of polyurethanes have also been successfully probed in medical applications. However, studies about hybrids based on polyurethanes/TiO2 for medical applications are scarce. The aim of this work is to design novel biodegradable hybrid materials based on polyurethanes/TiO2 (80% organic-20% inorganic) and to perform a preliminary study of the potential applications in bone regeneration. The hybrids have been prepared by a sol-gel reaction using titanium isopropoxide as precursor of the inorganic component and polyurethane as the organic one. A series of polyurethanes has been prepared using different polyesters glycol succinate as soft segment, and 1,6-diisocyanatohexane (HDI) and butanediol (BD) as linear hard segment. The spectroscopy techniques used allow to confirm the formation of the required polyurethanes by the identification of bands related to carboxylic groups (COOH), and the amine groups (NH), and also the TiOH bonds and the bonds related to the interconnected network between the inorganic and the organic components from hybrids. The results from SEM/EDS show a homogeneous distribution of the inorganic component into the organic matrix. The nontoxic character of the hybrid (H400) was probed using MG-63 cell line with over 90% of cell viability. Finally, the formation of a hydroxyapatite layer in the material surface after 21days of soaking in SBF shows the bioactive character.

Application of calcium phosphates and fibronectin as complementary treatment for osteoporotic bone fractures.

INTRODUCTION: The gradual aging of the population results in increased incidence of osteoporotic bone fractures. In a good quality bone, the fixation with the usual methods is adequate, but not in osteoporotic bone, in which consolidation delays and other complications are common, with failure rates for screws up to 25%. OBJECTIVE: To test fibronectin loaded hydroxyapatite as a complementary treatment for osteoporotic fractures. MATERIAL AND METHODS: This study was performed in a vivo model; 42 female osteoporotic adult rabbits 4-5kg (White New Zealand) were used. Two groups (hydroxyapatite and fibronectin loaded hydroxyapatite) and a control group were tested. 3 time points 24h, 48h and 5days were studied. Defects were created in both femurs, in one of them, a cannulated screw (4mm) and a biocompatible material were placed; in the other femur a screw was inserted without supplemented material forming the control group. Osteoporosis was induced from models already known throughout administration of steroids. Samples were analyzed histologically and through imaging (micro Ct). RESULTS: Basal levels of BMD are observed below to normal when compared to other studies (0.25/0.3 instead of 0.4). Global and dependent of time analysis of samples, show no significant differences for samples analyzed. However, an important trend was noted for variables that define the trabecular bone microarchitecture. Indices that define trabecular microarchitecture in the comparative analysis found to have statistical differences (p<0.01). DISCUSSION: Osteosynthesis in an osteoporotic bone is a challenge for the surgeon, due to a reduced bone mineral density and different bone architecture. The main finding was the verification of the hypothesis that the trabecular bone parameters increases with our augmentation material in weak rabbit bone quality. Also, the histological analyses of samples show an increase of non inflammatory cells in protein samples (OHAp-Fn) from the first 24hours. CONCLUSION: An early response of rabbit osteroporotic bone to a complementary treatment with fibronectin loaded hydroxyapatite has been observed. This response is reflected in greater values for indices that define the trabecular bone microarchitecture, thickness and separation, a greater non-inflammatory cellularity after only 24hours and an increased amount of connective tissue observed at 48hours.

Adsorption and conformational modification of fibronectin and fibrinogen adsorbed on hydroxyapatite. A QCM-D study.

Hydroxyapatite is a bioactive ceramic frequently used for bone engineering/replacement. One of the parameters that influence the biological response to implanted materials is the conformation of the first adsorbed protein layer. In this work, the adsorption and conformational changes of two fibroid serum proteins; fibronectin and fibrinogen adsorbed onto four different hydroxyapatite powders are studied with a Quartz Crystal Microbalance with Dissipation (QCM-D). Each of the calcined apatites adsorbs less protein than their corresponding synthesized samples. Adsorption on synthesized samples yields always an extended conformation whereas a reorganization of the layer is observed for the calcined samples. Fg acquires a "Side on" conformation in all the samples at the beginning of the experiment except for one of the synthesized samples where an "End-on" conformation is obtained during the whole experiment. The Extended conformation is the active conformation for Fn. This conformation is favored by apatites with large specific surface area (SSA) and on highly concentrated media. Apatite surface features should be considered in the selection or design of materials for bone regeneration, since it is possible to control the conformation mode of attachment of Fn and Fg by an appropriate selection of them. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2585-2594, 2016.

Influence of surface features of hydroxyapatite on the adsorption of proteins relevant to bone regeneration.

Protein-surface interaction may determine the success or failure of an implanted device. Not much attention have been paid to the specific surface parametes of hydroxyapatite (OHAp) that modulates and determines the formation and potential activity of the layer of proteins that is first formed when the material get in contact with the host tissue. the influence of specific surface area (SSA), crystallite size (CS) and particle size (PS) of OHAp on the adsorption of proteins relevant for bone regeneration is evaluated in this article. OHAp have been prepared by a wet chemical reaction of Ca(OH)2 with H3PO4. One set of reactions included poly acrylic acid in the reactant solution to modify the properties of the powder. Fibrinogen (Fg) Fraction I, type I: from Human plasma, (67% Protein), and Fibronectin (Fn) from Human plasma were selected to perform the adsorption experiments. The analysis of protein adsorption was carried out by UV/Vis spectrometry. A lower SSA and a different aspect ratio are obtained when the acrylic acid is included in the reaction badge. The deconvolution of the amide I band on the Raman spectra of free and adsorbed proteins reveals that the interaction apatite-protein happens through the carboxylate groups of the proteins. The combined analysis of CS, SSA and PS should be considered on the design of OHAp materials intended to interact with proteins.

Feasibility of ceramic-polymer composite cryogels as scaffolds for bone tissue engineering.

The purpose of the current study was to investigate whether the cryopolymerization technique is capable of producing suitable scaffolds for bone tissue engineering. Cryopolymers made of 2-hydroxyethyl methacrylate and acrylic acid with (W1 and W20) and without (W0) wollastonite particles were prepared. The elastic modulus of the specimens rose one order of magnitude from W1 to W20. Total porosity reached 56% for W0, 72% for W1 and 36% for W20, with pore sizes of up to 2 mm, large interconnection sizes of up to 1 mm and small interconnection sizes of 50-80 µm on dry specimens. Cryogels swell up to 224 ± 17% for W0, 315 ± 18% for W1 and 231 ± 27% for W20 specimens, while maintaining the integrity of the bodies. Pore sizes > 5 mm can be observed for swollen specimens. The biocompatibility of the samples was tested using human mesenchymal stem cells isolated from bone marrow and adipose tissues. Both types of cells attached and grew on the three tested substrates, colonized their inner regions and organized an extracellular cell matrix. Fibronectin and osteopontin levels decreased in the media from cells cultured on W20 samples, likely due to increased binding on the ECM deposited by cells. The osteoprotegerin-to-receptor activator of nuclear factor-κB ligand secretion ratios increased with increasing wollastonite content. Altogether, these results indicate that an appropriate balance of surface properties and structure that favours stromal cell colonization in the porous cryogels can be achieved by modulating the amount of wollastonite.

Effects of calcination temperature on the drug delivery behaviour of Ibuprofen from hydroxyapatite powders.

The effects of heat treatment time and temperature on the delivery behaviour of Ibuprofen from hydroxyapatite particles were investigated in this study. The drug release was seen to follow Fickian diffusion for the initial period of release for all heat treatment conditions. The gradient of Fickian release increased with (1) increasing crystallite size, attributed to the decreasing amount of boundary area, and (2) with decreasing surface area, due to the reduction in porosity and hence tortuosity within the apatite particles. This study has shown that altering the heat treatment conditions used to calcine hydroxyapatite may alter its drug delivery abilities, whereby calcination temperature was noted to influence the drug release behaviour to a greater extent than calcination time.

Development of wollastonite-poly(ethylmethacrylate co-vinylpyrrolidone) based materials for multifunctional devices.

The manufacturing of a composite made of a synthetic bioactive ceramic, pseudowollastonite (psW), and a bioresorbable copolymer ethylmethacrylate-vinylpyrrolidone (EMA/VP) is presented in this article. psW porous blocks were produced by dipping an open porous polyurethane foam in a psW containing slurry. A 40/60 wt % EMA/VP monomers mixture was poured on the blocks, and free radical polymerization initiated by azobis(isobutyronitrile) at 50 degrees C. Disks of 1 mm height were obtained by cutting the composite with a diamond saw, and bioresorption and bioactivity of the specimens were tested by immersion of the disks into SBF. A ceramic/polymer weight ratio of 72/28, greater than the usually achievable ratio by polymeric solidification of slurries of monomers charged with a powdered solid component, has been obtained. The system is bioactive and does not change the pH of the medium during the degradation test.

Rietveld refinements and spectroscopic studies of the structure of Ca-deficient apatite.

Nine samples of Ca-deficient apatite (Ca-def Ap) were prepared from suspensions of CaHPO4 (monetite) at 90 degrees C by raising the pH from approximately 4 through release of NH3 produced by the hydrolysis of urea. Products were dried at 100 degrees C for 24h and studied by chemical analyses, X-ray powder diffraction (XRPD) (and Rietveld analysis of this data), Ca/P ratio determination (quantitative phase analysis of samples after heating to 900 degrees C from Rietveld analysis of XRPD data), scanning electron microscopy, He pycknometry, 1H and 31P MAS NMR spectrometry and Fourier transform infrared and Raman spectroscopy. All samples contained apatite, but three also contained monetite. Infrared and Raman spectroscopy confirmed the presence of HPO4(2-) and absence of carbonate ions in the six monetite-free samples. Mean results for the six samples were: a = 9.4320(40), c = 6.8751(31) A; unit cell formula from chemical analysis neglecting protonation of phosphate ion, Ca(9.303(50))(PO4)6(OH)(0.606(99)).1.97(12)H2O; theoretical density 3.10 g cm(-3); experimental density (mean for three samples) 3.15 g cm(-3); and Ca/P mole ratio from chemical analysis and phase analysis after heating to 900 degrees C, 1.550(8) and 1.550(2), respectively. An earlier assignment of a line at 6 ppm in the 1H NMR spectrum of similar samples to HPO4(2-) ions could not be confirmed; hence no information about the HPO4(2-) ion content could be derived, in disagreement with the previous NMR study. A shoulder at approximately 0.9 ppm relative to 85 wt% H3PO4 in the 31P NMR spectrum was assigned to HPO4(2-) ions. Occupancies from the Rietveld structure refinements indicated preferential loss of Ca from Ca2 sites compared with Ca1, but the loss was substantially smaller than expected from chemical analyses. It is suggested that imperfect modelling of the structure in the refinement, particularly disorder associated with the Ca2 site, resulted in errors in Ca2 occupancies. The P-O bonds were slightly shorter than those in stoichiometric hydroxyapatite, rather than longer as might be expected from protonation of phosphate tetrahedra. However, consideration of known acid phosphate structures indicated that it was unlikely that the increase in P-O lengths would be sufficient to be detected. The observed decrease was tentatively assigned to the presence of Ca2+ ion vacancies.

Biodegradable composite scaffolds with an interconnected spherical network for bone tissue engineering.

Tissue engineering scaffolds are highly engineered structures that accommodate cells, facilitate their expression, and resorb to facilitate regeneration of tissue. A new technique for producing controlled pore shape and pore size interconnectivity offers promise for application as a tissue engineering scaffold. Salt particles were spheroidized in a flame and sintered to provide an interconnecting salt template. The salt template was filled with a carbonated fluorapatite powder and a polylactic polymer to produce a composite scaffold. It was found that a higher pore space is possible with the use of spherical and larger salt particle sizes. This technique can produce scaffolds with good interconnectivity and be suitable for producing pore size graded bodies.

Sintered hydroxyfluorapatites. Part I: sintering ability of precipitated solid solution powders.

The presence of fluoride within apatites occurring naturally within the body provides the basis for investigating the sintering ability of fluoride containing hydroxyapatites for use as biomaterials. Hydroxyfluorapatites were synthesized and then calcined to produce a 0%, 20%, 40%, 60%, 80% and 100% replacement of the hydroxyl ions with fluoride in the hydroxyapatite structure. Fluoride ion occupancy within the apatites was found to be about 90% of the anticipated value. Pycnometry results revealed a constant true density for powders of low to medium fluoride content followed by a rapid increase to the fluorapatite composition. Powders were uniaxially pressed, cold-isostatically pressed and sintered at 1150 degrees C, 1200 degrees C and 1250 degrees C. All hydroxyfluorapatite powders displayed a comparable ability for densification except when hydroxyl groups and fluoride ions were present at a comparable concentration. The grain size revealed that this composition also exhibited the smallest grain size and displayed the highest activation energy for grain growth. The lower diffusion created by similar amounts of fluoride and hydroxide within the lattice decreases grain growth and densification.

Sintered hydroxyfluorapatites. Part II: mechanical properties of solid solutions determined by microindentation.

Fluoride substitution within hydroxyapatite is an important occurrence for biological apatites and is a promising approach for the chemical modification of synthetic hydroxyapatite. Limited information on the influence of fluoride substitution for hydroxyl groups on the mechanical properties has provided the rationale for this study. Hydroxyfluorapatites with 0%, 20%, 40%, 60%, 80% and 100% replacement of hydroxyl groups with fluoride ions were assessed for hardness, elastic modulus, fracture toughness and brittleness using microindentation of sintered pellets. The production of samples with a similar grain size and density allowed the influence of fluoride on mechanical properties to be determined. It was found that the hardness remains unaffected until 80% replacement of hydroxyl groups with fluoride, after which the hardness rapidly increases. The elastic modulus increases linearly with fluoride content. Fracture toughness is improved with fluoride incorporation into the lattice and reaches a peak of 1.8 for a 95% dense sintered pellet with a 60% fluoride replacement, followed by a rapid decrease at higher fluoride concentrations. The brittleness index is lowered to a minimum at 60%, after which a rapid increase occurs. High fluoride levels are unfavourable from a mechanical perspective, are not recommended for biomaterials, and can lead to a higher incidence of fracture where sodium fluoride, for treatment of osteoporosis, may produce a highly fluoridated hydroxyapatite.