3D Bioplotter Research Papers

Objective. The objective of this study was to compare the host inflammatory response of our previously described hyperelastic, 3D-printed (3DP) hydroxyapatite (HA)-demineralized bone matrix (DBM) composite scaffold to the response elicited with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a preclinical rat posterolateral lumbar fusion model. Summary of Background Data. Our group previously found that this 3D-printed HA-DBM composite material shows promise as a bone graft substitute in a preclinical rodent model, but its safety profile had yet to be assessed. Methods. Sixty female Sprague-Dawley rats underwent bilateral posterolateral intertransverse lumbar spinal fusion using with the following…

3D ink-extrusion of powders followed by sintering is an emerging additive manufacturing method capable of creating metallic microlattices. Here, we study the creation of hierarchically porous Fe or Ni scaffolds by 3D extrusion of 0/90° lattices from inks consisting of fine oxide powders (Fe2O3 or NiO, < 3 µm), coarse space-holder particles (CuSO4, < 45 µm) and a polymer binder within a solvent. After space-holder leaching and debinding of the lattices, a sintering step densifies the metallic Fe or Ni powders created by oxide reduction with H2, while maintaining the larger pores templated by the space-holder particles within the printed…

Cobalt-based superalloy microlattices were created via (i) three-dimensional-extrusion printing of inks containing a suspension of Co-, Ni- and W-oxide particles, (ii) H2-reduction of the oxides and sintering to a homogenous Co-Ni-W alloy, (iii) Al pack-cementation to deposit Al on the microlattice struts, followed by Al-homogenization. The resulting Co-(18–20)Ni-(5–6)W-(10–13)Al (at.%) microlattices, with 27–30% relative density and 350 μm diameter struts, display a peak in yield strength at 750°C, consistent with their γ/γ′ aged microstructure. Oxidation resistance is strongly improved compared to Al-free printed Co-Ni-W lattices, via the formation of an Al2O3 surface layer. However, the resulting Al depletion within the struts…

We recently developed a recombinant growth factor-free bone regenerative scaffold composed of stoichiometric hydroxyapatite (HA) ceramic particles and human demineralized bone matrix (DBM) particles (HA-DBM). Here, we performed the first pre-clinical comparative evaluation of HA-DBM relative to the industry standard and established positive control, recombinant human bone morphogenetic protein-2 (rhBMP-2), using a rat posterolateral spinal fusion model (PLF). Female Sprague–Dawley rats underwent bilateral L4-L5 PLF with implantation of the HA-DBM scaffold or rhBMP-2. Fusion was evaluated using radiography and blinded manual palpation, while biomechanical testing quantified the segmental flexion-extension range-of-motion (ROM) and stiffness of the fused segments at 8-weeks postoperatively.…

3D ink-extrusion of powders followed by sintering is an emerging alternative to beam-based additive manufacturing, capable of creating 3D metallic objects from 1D-extruded microfilaments. Here, in situ synchrotron X-ray diffraction and tomography are combined to study the phase evolution, alloy formation and sinter-densification of Fe-20Ni-5Mo (at.%) microfilaments. The filaments are

Although numerous spinal biologics are commercially available, a cost-effective and safe bone graft substitute material for spine fusion has yet to be proven. In this study, “3D-Paints” containing varying volumetric ratios of hydroxyapatite (HA) and human demineralized bone matrix (DBM) in a poly(lactide-co-glycolide) elastomer were three-dimensional (3D) printed into scaffolds to promote osteointegration in rats, with an end goal of spine fusion without the need for recombinant growth factor. Spine fusion was evaluated by manual palpation, and osteointegration and de novo bone formation within scaffold struts were evaluated by laboratory and synchrotron microcomputed tomography and histology. The 3:1 HA:DBM composite…

The field of 3D bioprinting has rapidly grown, yet the fundamental ability to manipulate material properties has been challenging with current bioink methods. Here, we change bioink properties using our PEG cross-linking (PEGX) bioink method with the objective of optimizing cell viability while retaining control of mechanical properties of the final bioprinted construct. First, we investigate cytocompatible, covalent cross-linking chemistries for bioink synthesis (e.g. Thiol Michael type addition and bioorthogonal inverse electron demand Diels-Alder reaction). We demonstrate these reactions are compatible with the bioink method, which results in high cell viability. The PEGX method is then exploited to optimize extruded…

Recent advances have demonstrated the ability to 3D-print, via extrusion, solvent-based liquid materials (previously named 3D-Paints) which solidify nearly instantaneously upon deposition and contain a majority by volume of solid particulate material. In prior work, the dissolved polymer binder which enables this process is a high molecular weight biocompatible elastomer, poly(lactic-co-glycolic) acid (PLGA). We demonstrate in this study an expansion of this solvent-based 3D-Paint system to two additional, less-expensive, and less-specialized polymers, polystyrene (PS) and polyethylene oxide (PEO). The polymer binder used within the 3D-Paint was shown to significantly affect the as-printed and thermal postprocessing behavior of printed structures. This…

Soft biological tissues such as skeletal muscle and brain white matter can be inhomogeneous and anisotropic due to the presence of fibers. Unlike biological tissue, phantoms with known microstructure and defined mechanical properties enable a quantitative assessment and systematic investigation of the influence of inhomogeneities on the nature of shear wave propagation. This study introduces a mathematical measure for the wave shape, which the authors call as the 1-Norm, to determine the conditions under which homogenization may be a valid approach. This is achieved through experimentation using the Magnetic Resonance Elastography technique on 3D printed inhomogeneous fiber phantoms as well…

Ni-29Mn-21.5Ga (at. %) wires are fabricated via a combination of (i) extrusion of liquid inks containing a binder, solvents, and elemental Ni, Mn, and Ga powders and (ii) heat treatments to remove the polymer binder and to interdiffuse and sinter the powders. To study the microstructural evolution, sintering mechanisms, and grain growth in these wires, both ex situ metallography and in situ X-Ray tomography were conducted while sintering at 800–1050 °C for up to 4 h. After debinding, Ga-rich regions melt and induce transient liquid phase sintering of the surrounding Ni and Mn powders, resulting in localized swelling of the wires and…

Three-dimensional (3D) printing of decellularized extracellular matrix (dECM) hydrogels is a promising technique for regenerative engineering. 3D-printing enables the reproducible and precise patterning of multiple cells and biomaterials in 3D, while dECM has high organ-specific bioactivity. However, dECM hydrogels often display poor printability on their own and necessitate additives or support materials to enable true 3D structures. In this study, we used a sacrificial material, 3D-printed Pluronic F-127, to serve as a platform into which dECM hydrogel can be incorporated to create specifically designed structures made entirely up of dECM. The effects of 3D dECM are studied in the context…

Soft tissue fixation of implant and bioelectrodes relies on mechanical means (e.g., sutures, staples, and screws), with associated complications of tissue perforation, scarring, and interfacial stress concentrations. Adhesive bioelectrodes address these shortcomings with voltage cured carbene‐based bioadhesives, locally energized through graphene interdigitated electrodes. Electrorheometry and adhesion structure activity relationships are explored with respect to voltage and electrolyte on bioelectrodes synthesized from graphene 3D‐printed onto resorbable polyester substrates. Adhesive leachates effects on in vitro metabolism and human‐derived platelet‐rich plasma response serves to qualitatively assess biological response. The voltage activated bioadhesives are found to have gelation times of 60 s or less…

Vascularization of engineered bone tissue is critical for ensuring its survival after implantation. In vitro pre-vascularization of bone grafts with endothelial cells is a promising strategy to improve implant survival. In this study, we pre-cultured human smooth muscle cells (hSMCs) on bone scaffolds for 3 weeks followed by seeding of human umbilical vein endothelial cells (HUVECs), which produced a desirable environment for microvasculature formation. The sequential cell-seeding protocol was successfully applied to both natural (decellularized native bone, or DB) and synthetic (3D-printed Hyperelastic “Bone” scaffolds, or HB) scaffolds, demonstrating a comprehensive platform for developing natural and synthetic-based in vitro vascularized…

ungsten is of industrial relevance due its outstanding intrinsic properties (e.g., highest melting‐point of all elements) and therefore difficult to 3D‐print by conventional methods. Here, tungsten micro‐lattices are produced by room‐temperature extrusion‐based 3D‐printing of an ink comprising WO3–0.5%NiO submicron powders, followed by H2‐reduction and Ni‐activated sintering. The green bodies underwent isotropic linear shrinkage of ≈50% during the thermal treatment resulting in micro‐lattices, with overall 35–60% open‐porosity, consisting of 95–100% dense W–0.5%Ni struts having ≈80–300 μm diameter. Ball‐milling the powders and inks reduced the sintering temperature needed to achieve full densification from 1400 to 1200 °C and enabled the ink to be extruded…

Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries.…

We introduce a new process that enables the ability to 3D-print high porosity materials and structures by combining the newly introduced 3D-Painting process with traditional salt-leaching. The synthesis and resulting properties of three 3D-printable inks comprised of varying volume ratios (25:75, 50:50, 70:30) of CuSO4 salt and polylactide-co-glycolide (PLGA), as well as their as-printed and salt-leached counterparts, are discussed. The resulting materials are comprised entirely of PLGA (F-PLGA), but exhibit porosities proportional to the original CuSO4 content. The three distinct F-PLGA materials exhibit average porosities of 66.6–94.4%, elastic moduli of 112.6-2.7 MPa, and absorbency of 195.7–742.2%. Studies with adult human mesenchymal…

The development of in situ fabrication methods for the infrastructure required to support human life on the Moon is necessary due to the prohibitive cost of transporting large quantities of materials from the Earth. Cellular structures, consisting of a regular network (truss) of micro-struts with ∼500 μm diameters, suitable for bricks, blocks, panels, and other load-bearing structural elements for habitats and other infrastructure are created by direct-extrusion 3D-printing of liquid inks containing JSC-1A lunar regolith simulant powders, followed by sintering. The effects of sintering time, temperature, and atmosphere (air or hydrogen) on the microstructures, mechanical properties, and magnetic properties of…

Ni-Mn-Ga magnetic shape memory alloy (SMA) micro-trusses, suitable for high magnetic field induced strains and/or a large magnetocaloric effect, are created via a new additive manufacturing method combining (i) 3D-printing ∼400 μm struts with an ink containing a polymer binder and elemental Ni, Mn, and Ga powders, (ii) binder burn-out and metallic powder interdiffusion and homogenization to create the final alloy, and (iii) further sintering to increase strut density. Controlled amounts of hierarchical porosity, desirable to enable twinning in this polycrystalline alloy, are achieved: (i) continuous ∼450 μm channels between the printed Ni-Mn-Ga ∼300 μm diameter struts (after sintering) and…

Using an innovative, tissue-independent approach to decellularized tissue processing and biomaterial fabrication, the development of a series of “tissue papers” derived from native porcine tissues/organs (heart, kidney, liver, muscle), native bovine tissue/organ (ovary and uterus), and purified bovine Achilles tendon collagen as a control from decellularized extracellular matrix particle ink suspensions cast into molds is described. Each tissue paper type has distinct microstructural characteristics as well as physical and mechanical properties, is capable of absorbing up to 300% of its own weight in liquid, and remains mechanically robust (E = 1–18 MPa) when hydrated; permitting it to be cut, rolled,…

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover,…

Here, we present a comprehensive approach for creating robust, elastic, designer Lunar and Martian regolith simulant (LRS and MRS, respectively) architectures using ambient condition, extrusion-based 3D-printing of regolith simulant inks. The LRS and MRS powders are characterized by distinct, highly inhomogeneous morphologies and sizes, where LRS powder particles are highly irregular and jagged and MRS powder particles are rough, but primarily rounded. The inks are synthesized via simple mixing of evaporant, surfactant, and plasticizer solvents, polylactic-co-glycolic acid (30% by solids volume), and regolith simulant powders (70% by solids volume). Both LRS and MRS inks exhibit similar rheological and 3D-printing characteristics,…

Inks comprised of metallic Fe or Ni powders, an elastomeric binder, and graded volatility solvents are 3D-printed via syringe extrusion and sintered to form metallic cellular structures. Similar structures are created from Fe2O3 and NiO particle-based inks, with an additional hydrogen reduction step before sintering. All sintered structures exhibit 92–98% relative density within their struts, with neither cracking nor visible warping despite extensive volumetric shrinkage (≈70–80%) associated with reduction (for oxide powders) and sintering (for both metal and oxide powders). The cellular architectures, with overall relative densities of 32–49%, exhibit low stiffness (1–6 GPa, due to the particular architecture used), high…

Despite substantial attention given to the development of osteoregenerative biomaterials, severe deficiencies remain in current products. These limitations include an inability to adequately, rapidly, and reproducibly regenerate new bone; high costs and limited manufacturing capacity; and lack of surgical ease of handling. To address these shortcomings, we generated a new, synthetic osteoregenerative biomaterial, hyperelastic “bone” (HB). HB, which is composed of 90 weight % (wt %) hydroxyapatite and 10 wt % polycaprolactone or poly(lactic-co-glycolic acid), could be rapidly three-dimensionally (3D) printed (up to 275 cm3/hour) from room temperature extruded liquid inks. The resulting 3D-printed HB exhibited elastic mechanical properties (~32…

A model sample consisting of two different hydroxyapatite (hAp) powders was used as a bone phantom to investigate the extent to which X-ray diffraction tomography could map differences in hAp lattice constants and crystallite size. The diffraction data were collected at beamline 1-ID, the Advanced Photon Source, using monochromatic 65 keV X-radiation, a 25 × 25 µm pinhole beam and translation/rotation data collection. The diffraction pattern was reconstructed for each volume element (voxel) in the sample, and Rietveld refinement was used to determine the hAp lattice constants. The crystallite size for each voxel was also determined from the 00.2 hAp…

With the emergence of 3D-printing (3DP) as a vital tool in tissue engineering and medicine, there is an ever growing need to develop new biomaterials that can be 3D-printed and also emulate the compositional, structural, and functional complexities of human tissues and organs. In this work, we probe the 3D-printable biomaterials spectrum by combining two recently established functional 3D-printable particle-laden biomaterial inks: one that contains hydroxyapatite microspheres (Hyperelastic Bone, HB) and another that contains graphene nanoflakes (3D-Graphene, 3DG). We demonstrate that not only can these distinct, osteogenic and neurogenic inks be co-3D-printed to create complex, multi-material constructs, but that composite…

The exceptional properties of graphene enable applications in electronics, optoelectronics, energy storage, and structural composites. Here we demonstrate a 3D printable graphene (3DG) composite consisting of majority graphene and minority polylactide-co-glycolide, a biocompatible elastomer, 3D-printed from a liquid ink. This ink can be utilized under ambient conditions via extrusion-based 3D printing to create graphene structures with features as small as 100 μm composed of as few as two layers (10 cm thick object). The resulting 3DG material is mechanically robust and flexible while retaining electrical conductivities greater than 800 S/m, an order of magnitude increase over previously reported 3D-printed carbon…

A multimaterial bio-ink method using polyethylene glycol crosslinking is presented for expanding the biomaterial palette required for 3D bioprinting of more mimetic and customizable tissue and organ constructs. Lightly crosslinked, soft hydrogels are produced from precursor solutions of various materials and 3D printed. Rheological and biological characterizations are presented, and the promise of this new bio-ink synthesis strategy is discussed.

A new method for complex metallic architecture fabrication is presented, through synthesis and 3D-printing of a new class of 3D-inks into green-body structures followed by thermochemical transformation into sintered metallic counterparts. Small and large volumes of metal-oxide, metal, and metal compound 3D-printable inks are synthesized through simple mixing of solvent, powder, and the biomedical elastomer, polylactic-co-glycolic acid (PLGA). These inks can be 3D-printed under ambient conditions via simple extrusion at speeds upwards of 150 mm s–1 into millimeter- and centimeter-scale thin, thick, high aspect ratio, hollow and enclosed, and multi-material architectures. The resulting 3D-printed green-bodies can be handled immediately, are…

Bioactive, in situ forming materials have the potential to complement minimally invasive surgical procedures and enhance tissue healing. For such biomaterials to be adopted in the clinic, they must be cost-effective, easily handled by the surgeon and have a history of biocompatibility. To this end, we report a novel and facile self-assembling strategy to create membranes and encapsulating structures using collagen and hyaluronic acid (HA). Unlike membranes built by layer-by-layer deposition of oppositely charged biomolecules, the collagen–HA membranes described here form a diffusion barrier upon electrostatic interaction of the oppositely charged biomolecules, which is further driven by osmotic pressure imbalances.…

Fabricating three-dimensional (3D) porous scaffolds with controlled structure and geometry is crucial for tissue regeneration. To date, exploration in printing 3D natural protein scaffolds is limited. In this study, soy protein slurry was successfully printed using the 3D Bioplotter to form scaffolds. A method to verify the structural integrity of resulting scaffolds during printing was developed. This process involved measuring the mass extrusion flow rate of the slurry from the instrument, which was directly affected by the extrusion pressure and the soy protein slurry properties. The optimal mass flow rate for printing soy slurry at 27°C was 0.0072±0.0002 g/s. The addition…

Increasing interest in using soy biomaterials for tissue engineering applications has prompted investigation into the in vivo biocompatibility of soy implants. In this study, the biocompatibility of soy protein scaffolds fabricated using freeze-drying and 3-D printing was assessed using a subcutaneous implant model in BALB/c mice. The main objectives of this study were: (1) to compare soy protein with bovine collagen, a well-characterized natural protein implant, by implanting scaffolds of the same protein weight, and (2) to observe the effects of soy scaffold microstructure and amount of protein loading, which also alters the degradation properties, on the acute and humoral…