Physicochemical Characterization of Pectin-Gelatin Biomaterial Formulations for 3D Bioprinting.

AbstractDeveloping biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3‐glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion‐based 3D bioprinting. Self‐supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion‐based bioprinting and freeze‐drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue‐like scaffolds for tissue regeneration

Orbital floor repair using patient specific osteoinductive implant made by stereolithography

The orbital floor (OF) is an anatomical location in the craniomaxillofacial (CMF) region known to be highly variable in shape and size. When fractured, implants commonly consisting of titanium meshes are customized by plying and crude hand-shaping. Nevertheless, more precise customized synthetic grafts are needed to meticulously reconstruct the patients’ OF anatomy with better fidelity. As alternative to titanium mesh implants dedicated to OF repair, we propose a flexible patient-specific implant (PSI) made by stereolithography (SLA), offering a high degree of control over its geometry and architecture. The PSI is made of biodegradable poly(trimethylene carbonate) (PTMC) loaded with 40 wt % of hydroxyapatite (called Osteo-PTMC). In this work, we developed a complete work-flow for the additive manufacturing of PSIs to be used to repair the fractured OF, which is clinically relevant for individualized medicine. This work-flow consists of (i) the surgical planning, (ii) the design of virtual PSIs and (iii) their fabrication by SLA, (iv) the monitoring and (v) the biological evaluation in a preclinical large-animal model. We have found that once implanted, titanium meshes resulted in fibrous tissue encapsulation, whereas Osteo-PMTC resulted in rapid neovascularization and bone morphogenesis, both ectopically and in the OF region, and without the need of additional biotherapeutics such as bone morphogenic proteins. Our study supports the hypothesis that the composite osteoinductive Osteo-PTMC brings advantages compared to standard titanium mesh, by stimulating bone neoformation in the OF defects. PSIs made of Osteo-PTMC represent a significant advancement for patients whereby the anatomical characteristics of the OF defect restrict the utilization of traditional hand-shaped titanium mesh

Additive manufacturing of bone-forming composite implants using photo-curable poly(trimethylene carbonate)-based resins

Bone tissue is generally able to perfectly restore and remodel itself after fracture or the formation of a defect due to traumatic injury or tumor removal. In some cases however, critical size bone defects are formed. These are of such size that the surrounding bone tissue is unable to restore the damaged site. Implantable and resorbable structures are then needed, to act as a temporary support and to induce de novo bone formation in the defect. Natural bone grafts currently outperform most synthetic implants. The use of natural grafts comes with several disadvantages however, such as risk for disease transmittance, graft rejection and limited options for shaping. Thus, novel synthetic implants are required which do not possess the disadvantages of natural grafts and perform similarly well. This thesis describes the preparation and characterization of a novel composite material of photo-crosslinked, methacrylate end-group functionalized poly(trimethylene carbonate) (PTMC-MA) and nano-hydroxyapatite (nHA) particles. These composites were prepared by two additive manufacturing techniques, in order to fabricate a material with a designed external as well as internal structure. Stereolithography was used to prepare well-fitting implants for defects with complex shapes. A low-temperature extrusion-based additive manufacturing technique was used to prepare composite structures with designed macro-scale porosity (several hundreds of micrometers) and micro-scale porosity (in the range of ten micrometer). The macro-scale porosity may allow for bone ingrowth, whereas the micro-scale porosity can allow for nutrient diffusion throughout the structure once implanted. Composites produced by both techniques are cytocompatible and allow for osteogenic differentiation of human bone marrow mesenchymal stem cells on their surface. Furthermore, the nHA in the composites allows for an enhancement of their stiffness and toughness, the hydrophilicity and the resorbability. Composite structures prepared by stereolithography show a strong surface enrichment with nHA particles as the particle content is increased. This ultimately results in enhanced bone defect restoration and a better osseointegration of the structures in newly formed bone, as shown in small animal experiments. Overall, the work in this thesis shows that composites of photo-crosslinked PTMC-MA and nHA may indeed be useful in bone tissue engineering applications

Hydrogels by supramolecular crosslinking of terpyridine end group functionalized 8-arm poly(ethylene glycol)

Metallo supramolecular assemblies of an 8-arm poly(ethylene glycol) partially substituted with terpyridyl end-groups and the transition metal ions Ni2+, Fe2+, Co2+ and Zn2+ were studied for their nano-particle formation at dilute conditions and gelation at higher concentrations. The large differences in dissociation rate constants of the metal ligand complexes largely determine the assembly behavior. Thermodynamically stable complexes are generated with Ni2+ and Fe2+ chlorides, which lead to distinct particle sizes of 200 nm in dilute conditions. The Co2+ and Zn2+ chlorides provide multiple size distributions revealing that mono and bis-complexes are present at equilibrium. Upon complexation, terpyridyl groups move to the outer sphere giving aggregates with a charged surface. At polymer concentrations above 5 wt%, crosslinking upon addition of transition metal ions provides hydrogels. Elastic hydrogels were obtained with Ni2+, Fe2+ and Co2+ having storage moduli in excess of 20 kPa, whereas Zn2+ gels are relatively viscous. Only Zn2+ gels show a thermoreversible sol to gel transition at a temperature of 25 °C independent of polymer concentratio

Cavitation-Assisted Micromixing for Polymeric Nanoparticle Generation

The high-throughput generation of polymeric nanoparticles (PNPs) with tailored size and narrow size distribution is key for applications as relevant as sensing and nanomedicine. Here we show how cavitation bubbles in a microfluidic channel can induce rapid nanoprecipitation of PNPs with user-selectable control. Specifically, we used two tip-electrodes perpendicular to the flow to induce electrical breakdown of a polymer solution and a miscible non-solvent. As a result, a plasma is formed causing cavitation and rapid mixing of the fluids, yielding nanoprecipitates of polymer. We demonstrated mL/min generation of PNPs with a diameter as low as 150 nm and polydispersity below 0.15

Osteogenic differentiation of hBMSCs on porous photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite composites

Large bone defects are challenging to repair and novel implantable materials are needed to aid in their reconstruction. Research in the past years has proven the beneficial effect of porosity in an implant on osteogenesis in vivo. Building on this research we report here on porous composites based on photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite. These composites were prepared by a temperature induced phase separation of poly(trimethylene carbonate) macromers from solution in ethylene carbonate. By controlling the ethylene carbonate content in viscous dispersions of nano-hydroxyapatite in poly(trimethylene carbonate) macromer solutions, composites with 40 wt% nano-hydroxyapatite and 27 to 71% porosity were prepared. The surface structure of these porous composites was affected by their porosity and their topography became dominated by deep micro-pore channels with the majority of pore widths below 20 µm and rougher surfaces on the nano-scale. The stiffness and toughness of the composites decreased with increasing porosity from 67 to 3.5 MPa and 263 to 2.2 N/mm2, respectively. In cell culture experiments, human bone marrow mesenchymal stem cells proliferated well on the composites irrespective of their porosity. Furthermore, differentiation of the cells was demonstrated by determination of ALP activity and calcium production. The extent of differentiation was affected by the porosity of the films, offering a reduced mechanical incentive for osteogenic differentiation at higher porosities with topographies likely offering a reduced possibility for cells to aggregate and to elongate into morphologies favourable for osteogenic differentiation. This ultimately resulted in a 3-fold reduction of calcium production of the differentiated cells on composites with 71% porosity compared to those on composites with 27% porosity

Thermoresponsive copolymer brushes possessing quaternary amine groups for strong anion-exchange chromatographic matrices

A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices

Engineering 3D parallelized microfluidic droplet generators with equal flow profiles by computational fluid dynamics and stereolithographic printing.

International audienceMicrofluidic droplet generators excel in generating monodisperse micrometer-sized droplets and particles. However, the low throughput of conventional droplet generators hinders their clinical and industrial translation. Current approaches to parallelize microdevices are challenged by the two-dimensional nature of the standard fabrication methods. Here, we report the facile production of three-dimensionally (3D) parallelized microfluidic droplet generators consisting of stacked and radially multiplexed channel designs. Computational fluid dynamics simulations form the design basis for a microflow distributor that ensures similar flow rates through all droplet generators. Stereolithography is the selected technique to fabricate microdevices, which enables the manufacturing of hollow channels with dimensions as small as 50 μm. The microdevices could be operated up to 4 bars without structural damage, including deformation of channels, or leakage of the on-chip printed Luer-Lok type connectors. The printed microdevices readily enable the production of water-in-oil emulsions, as well as polymer containing droplets that act as templates for both solid and core-shell hydrogel microparticles. The cytocompatibility of the 3D printed device is demonstrated by encapsulating mesenchymal stem cells in hydrogel microcapsules, which results in the controllable formation of stem cell spheroids that remain viable and metabolically active for at least 21 days. Thus, the unique features of stereolithography fabricated microfluidic devices allow for the parallelization of droplet generators in a simple yet effective manner by enabling the realization of (complex) 3D designs

Engineering 3D parallelized microfluidic droplet generators with equal flow profiles by computational fluid dynamics and stereolithographic printing

Microfluidic droplet generators excel in generating monodisperse micrometer-sized droplets and particles. However, the low throughput of conventional droplet generators hinders their clinical and industrial translation. Current approaches to parallelize microdevices are challenged by the two-dimensional nature of the standard fabrication methods. Here, we report the facile production of three-dimensionally (3D) parallelized microfluidic droplet generators consisting of stacked and radially multiplexed channel designs. Computational fluid dynamics simulations form the design basis for a microflow distributor that ensures similar flow rates through all droplet generators. Stereolithography is the selected technique to fabricate microdevices, which enables the manufacturing of hollow channels with dimensions as small as 50 μm. The microdevices could be operated up to 4 bars without structural damage, including deformation of channels, or leakage of the on-chip printed Luer-Lok type connectors. The printed microdevices readily enable the production of water-in-oil emulsions, as well as polymer containing droplets that act as templates for both solid and core-shell hydrogel microparticles. The cytocompatibility of the 3D printed device is demonstrated by encapsulating mesenchymal stem cells in hydrogel microcapsules, which results in the controllable formation of stem cell spheroids that remain viable and metabolically active for at least 21 days. Thus, the unique features of stereolithography fabricated microfluidic devices allow for the parallelization of droplet generators in a simple yet effective manner by enabling the realization of (complex) 3D designs.status: publishe