Preclinical Safety of a 3D-Printed Hydroxyapatite-Demineralized Bone Matrix Scaffold for Spinal Fusion.

STUDY DESIGN: Prospective, randomized, controlled preclinical study. 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 implants: 1) type I absorbable collagen sponge (ACS) alone; 2) 10 µg rhBMP-2/ACS; or 3) the 3DP HA-DBM composite scaffold (n = 20). The host inflammatory response was assessed using magnetic resonance imaging, while the local and circulating cytokine expression levels were evaluated by enzyme-linked immunosorbent assays at subsequent postoperative time points (N = 5/time point). RESULTS: At both 2 and 5 days postoperatively, treatment with the HA-DBM scaffold produced significantly less soft tissue edema at the fusion bed site relative to rhBMP-2-treated animals as quantified on magnetic resonance imaging. At every postoperative time point evaluated, the level of soft tissue edema in HA-DBM-treated animals was comparable to that of the ACS control group. At 2 days postoperatively, serum concentrations of tumor necrosis factor-α and macrophage chemoattractant protein-1 were significantly elevated in the rhBMP-2 treatment group relative to ACS controls, whereas these cytokines were not elevated in the HA-DBM-treated animals. CONCLUSION: The 3D-printed HA-DBM composite induces a significantly reduced host inflammatory response in a preclinical spinal fusion model relative to rhBMP-2.Level of Evidence: N/A.

Regional gene therapy for bone healing using a 3D printed scaffold in a rat femoral defect model.

At the present time there are no consistently satisfactory treatment options for some challenging bone loss scenarios. We have previously reported on the properties of a novel 3D-printed hydroxyapatite-composite material in a pilot study, which demonstrated osteoconductive properties but was not tested in a rigorous, clinically relevant model. We therefore utilized a rat critical-sized femoral defect model with a scaffold designed to match the dimensions of the bone defect. The scaffolds were implanted in the bone defect after being loaded with cultured rat bone marrow cells (rBMC) transduced with a lentiviral vector carrying the cDNA for BMP-2. This experimental group was compared against 3 negative and positive control groups. The experimental group and positive control group loaded with rhBMP-2 demonstrated statistically equivalent radiographic and histologic healing of the defect site (p > 0.9), and significantly superior to all three negative control groups (p < 0.01). However, the healed defects remained biomechanically inferior to the unoperated, contralateral femurs (p < 0.01). When combined with osteoinductive signals, the scaffolds facilitate new bone formation in the defect. However, the scaffold alone was not sufficient to promote adequate healing, suggesting that it is not substantially osteoinductive as currently structured. The combination of gene therapy with 3D-printed scaffolds is quite promising, but additional work is required to optimize scaffold geometry, cell dosage and delivery.

Osteoinductivity and biomechanical assessment of a 3D printed demineralized bone matrix-ceramic composite in a rat spine fusion model.

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. For mechanistic studies, pro-osteogenic gene and protein expression at 2-days and 1-, 2-, and 8-weeks postoperatively was assessed with another cohort. Unilateral fusion rates did not differ between the HA-DBM (93%) and rhBMP-2 (100%) groups; however, fusion scores were higher with rhBMP-2 (p = 0.008). Both treatments resulted in significantly reduced segmental ROM (p < 0.001) and greater stiffness (p = 0.009) when compared with non-operated controls; however, the degree of stabilization was significantly higher with rhBMP-2 treatment relative to the HA-DBM scaffold. In the mechanistic studies, PLGA and HA scaffolds were used as negative controls. Both rhBMP-2 and HA-DBM treatments resulted in significant elevations of several osteogenesis-associated genes, including Runx2, Osx, and Alp. The rhBMP-2 treatment led to significantly greater early, mid, and late osteogenic markers, which may be the mechanism in which early clinical complications are seen. The HA-DBM scaffold also induced osteogenic gene expression, but primarily at the 2-week postoperative timepoint. Overall, our findings show promise for this 3D-printed composite as a recombinant growth factor-free bone graft substitute for spinal fusion. STATEMENT OF SIGNIFICANCE: Despite current developments in bone graft technology, there remains a significant void in adequate materials for bone regeneration in clinical applications. Two of the most efficacious bone graft options are the gold-standard iliac crest bone graft and recombinant human-derived bone morphogenetic protein-2 (rhBMP-2), available commercially as Infuse™. Although efficacious, autologous graft is associated with donor-site morbidity, and Infuse™ has known side effects related to its substantial host inflammatory response, possibly associated with a immediate, robust osteoinductive response. Hence, there is a need for a bone graft substitute that provides adequate osteogenesis without associated adverse events. This study represents a significant step in the design of off-the-shelf growth factor-free devices for spine fusion.

Influence of Geometry and Architecture on the In Vivo Success of 3D-Printed Scaffolds for Spinal Fusion.

We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed material comprising hydroxyapatite (HA) and demineralized bone matrix (DBM) for bone regeneration. This material has demonstrated the capacity to promote re-mineralization of the DBM particles within the scaffold struts and shows potential to promote successful spine fusion. Here, we investigate the role of geometry and architecture in osteointegration, vascularization, and facilitation of spine fusion in a preclinical model. Inks containing HA and DBM particles in a poly(lactide-co-glycolide) elastomer were 3D-printed into scaffolds with varying relative strut angles (90° vs. 45° advancing angle), macropore size (0 µm vs. 500 µm vs. 1000 µm), and strut alignment (aligned vs. offset). The following configurations were compared with scaffolds containing no macropores: 90°/500 µm/aligned, 45°/500 µm/aligned, 90°/1000 µm/aligned, 45°/1000 µm/aligned, 90°/1000 µm/offset, and 45°/1000 µm/offset. Eighty-four female Sprague-Dawley rats underwent spine fusion with bilateral placement of the various scaffold configurations (n = 12/configuration). Osteointegration and vascularization were assessed by using microComputed Tomography and histology, and spine fusion was assessed via blinded manual palpation. The 45°/1000 µm scaffolds with aligned struts achieved the highest average fusion score (1.61/2) as well as the highest osteointegration score. Both the 45°/1000 µm/aligned and 90°/1000 µm/aligned scaffolds elicited fusion rates of 100%, which was significantly greater than the 45°/500 µm/aligned iteration (p < 0.05). All porous scaffolds were fully vascularized, with blood vessels present in every macropore. Vessels were also observed extending from the native transverse process bone, through the protrusions of new bone, and into the macropores of the scaffolds. When viewed independently, scaffolds printed with relative strut angles of 45° and 90° each allowed for osteointegration sufficient to stabilize the spine at L4-L5. Within those parameters, a pore size of 500 µm or greater was generally sufficient to achieve unilateral fusion. However, our results suggest that scaffolds printed with the larger pore size and with aligned struts at an advancing angle of 45° may represent the optimal configuration to maximize osteointegration and fusion capacity. Overall, this work suggests that the HA/DBM composite scaffolds provide a conducive environment for bone regeneration as well as vascular infiltration. This technology, therefore, represents a novel, growth-factor-free biomaterial with significant potential as a bone graft substitute for use in spinal surgery. Impact statement We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed composite material comprising hydroxyapatite and demineralized bone matrix for bone regeneration. Here, we identify a range of 3D geometric and architectural parameters that support the preclinical success of the scaffold, including efficient vascularization, osteointegration, and, ultimately, spinal fusion. Our results suggest that this material holds great promise as a clinically translatable biomaterial for use as a bone graft substitute in orthopedic procedures requiring bone regeneration.

Inclusion of a 3D-printed Hyperelastic Bone mesh improves mechanical and osteogenic performance of a mineralized collagen scaffold.

Regenerative repair of craniomaxillofacial bone injuries is challenging due to both the large size and irregular shape of many defects. Mineralized collagen scaffolds have previously been shown to be a promising biomaterial implant to accelerate craniofacial bone regeneration in vivo. Here we describe inclusion of a 3D-printed polymer or ceramic-based mesh into a mineralized collagen scaffold to improve mechanical and biological activity. Mineralized collagen scaffolds were reinforced with 3D-printed Fluffy-PLG (ultraporous polylactide-co-glycolide co-polymer) or Hyperelastic Bone (90wt% calcium phosphate in PLG) meshes. We show degradation byproducts and acidic release from the printed structures have limited negative impact on the viability of mesenchymal stem cells. Further, inclusion of a mesh formed from Hyperelastic Bone generates a reinforced composite with significantly improved mechanical performance (elastic modulus, push-out strength). Composites formed from the mineralized collagen scaffold and either Hyperelastic Bone or Fluffy-PLG reinforcement both supported human bone-marrow derived mesenchymal stem cell osteogenesis and new bone formation. This was observed by increased mineral formation in Fluffy-PLG composites and increased cell viability and upregulation of RUNX2, Osterix, and COL1A2 genes in both composites. Strikingly, composites reinforced with Hyperelastic Bone mesh elicited significantly increased secretion of osteoprotegerin, a soluble glycoprotein and endogenous inhibitor of osteoclast activity. These results suggest that architectured meshes can be integrated into collagen scaffolds to boost mechanical performance and actively instruct cell processes that aid osteogenicity; specifically, secretion of a factor crucial to inhibiting osteoclast-mediated bone resorption. Future work will focus on further adapting the polymer mesh architecture to confer improved shape-fitting capacity as well as to investigate the role of polymer reinforcement on MSC-osteoclast interactions as a means to increase regenerative potential.

3D-Printed Ceramic-Demineralized Bone Matrix Hyperelastic Bone Composite Scaffolds for Spinal Fusion.

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 achieved the highest mean fusion score and fusion rate (92%), which was significantly greater than the 3D printed DBM-only scaffold (42%). New bone was identified extending from the host transverse processes into the scaffold macropores, and osteointegration scores correlated with successful fusion. Strikingly, the combination of HA and DBM resulted in the growth of bone-like spicules within the DBM particles inside scaffold struts. These spicules were not observed in DBM-only scaffolds, suggesting that de novo spicule formation requires both HA and DBM. Collectively, our work suggests that this recombinant growth factor-free composite shows promise to overcome the limitations of currently used bone graft substitutes for spine fusion. Impact Statement Currently, there exists a no safe, yet highly effective, bone graft substitute that is well accepted for use in spine fusion procedures. With this work, we show that a three-dimensional printed scaffold containing osteoconductive hydroxyapatite and osteoinductive demineralized bone matrix that promotes new bone spicule formation, osteointegration, and successful fusion (stabilization) when implemented in a preclinical model of spine fusion. Our study suggests that this material shows promise as a recombinant growth factor-free bone graft substitute that could safely promote high rates of successful fusion and improve patient care.

Employing PEG crosslinkers to optimize cell viability in gel phase bioinks and tailor post printing mechanical properties.

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 cell viability by manipulating bioink gel robustness, characterized by mass flow rate. Below a critical point, cell viability linearly decreases with decreasing flow rates, but above this point, high viability is achieved. This work underscores the importance of building a foundational understanding of the relationships between extrudable bioink properties and cell health post-printing to more efficiently tune material properties for a variety of tissue and organ engineering applications. Finally, we also develop a post-printing, cell-friendly cross-linking strategy utilizing the same reactions used for synthesis. This secondary cross-linking leads to a range of mechanical properties relevant to soft tissue engineering as well as highly viable cell-laden gels stable for over one month in culture. STATEMENT OF SIGNIFICANCE: We demonstrate that a PEG crosslinking bioink method can be used with various cytocompatible, covalent cross-linking reactions: Thiol Michael type addition and tetrazine-norbornene click. The ability to vary bioink chemistry expands candidate polymers, and therefore can expedite development of new bioinks from unique polymers. We confirm post-printed cell viability and are the first to probe, in covalently cross-linked inks, how cell viability is impacted by different flow properties (mass flow rate). Finally, we also present PEG cross-linking as a new method of post-printing cross-linking that improves mechanical properties and stability while maintaining cell viability. By varying the cross-linking reaction, this method can be applicable to many types of polymers/inks for easy adoption by others investigating bioinks and hydrogels.

Effect of Polymer Binder on the Synthesis and Properties of 3D-Printable Particle-Based Liquid Materials and Resulting Structures.

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 development enables users to select one of several polymers to impart the most desirable properties for a given application. Additionally, 3D-Paints based on these new binders are not adversely affected by classes of particles that can chemically degrade PLGA, notably particles containing large quantities of alkali ions. This study demonstrates the ability to successfully use PS and PEO as binders in the 3D-Paint system and compares the rheological, mechanical, microstructural, and thermal properties of the modified 3D-Paints and resulting as-printed and thermally post-processed objects. These objects include, for the first time, structures resulting from 3D-Painting which mostly contain soda-lime glass and 45S5 bioactive glass.

An investigation into the relationship between inhomogeneity and wave shapes in phantoms and ex vivo skeletal muscle using Magnetic Resonance Elastography and finite element analysis.

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 as on ex-vivo porcine lumbus muscle. In addition, Finite Element Analysis is used as a tool to decouple the effects of directional anisotropy from those of inhomogeneity. A correlation is then established between the values of 1-Norm derived from the wave front geometry, and the spacing (d) between neighboring inhomogeneities (spherical inclusions or fibers and fiber intersections in phantoms and muscle). Smaller values of 1-Norm indicate less wave scattering at the locations of fiber intersections, which implies that the wave propagation may be approximated to that of a homogeneous medium; homogenization may not be a valid approximation when significant scattering occurs at the locations of inhomogeneities. In conclusion, the current study proposes 1-Norm as a quantitative measure of the magnitude of wave scattering in a medium, which can potentially be used as a homogeneity index of a biological tissue.

Three-Dimensionally Printed Hyperelastic Bone Scaffolds Accelerate Bone Regeneration in Critical-Size Calvarial Bone Defects.

BACKGROUND: Autologous bone grafts remain the gold standard for craniofacial reconstruction despite limitations of donor-site availability and morbidity. A myriad of commercial bone substitutes and allografts are available, yet no product has gained widespread use because of inferior clinical outcomes. The ideal bone substitute is both osteoconductive and osteoinductive. Craniofacial reconstruction often involves irregular three-dimensional defects, which may benefit from malleable or customizable substrates. "Hyperelastic Bone" is a three-dimensionally printed synthetic scaffold, composed of 90% by weight hydroxyapatite and 10% by weight poly(lactic-co-glycolic acid), with inherent bioactivity and porosity to allow for tissue integration. This study examines the capacity of Hyperelastic Bone for bone regeneration in a critical-size calvarial defect. METHODS: Eight-millimeter calvarial defects in adult male Sprague-Dawley rats were treated with three-dimensionally printed Hyperelastic Bone, three-dimensionally printed Fluffy-poly(lactic-co-glycolic acid) without hydroxyapatite, autologous bone (positive control), or left untreated (negative control). Animals were euthanized at 8 or 12 weeks postoperatively and specimens were analyzed for new bone formation by cone beam computed tomography, micro-computed tomography, and histology. RESULTS: The mineralized bone volume-to-total tissue volume fractions for the Hyperelastic Bone cohort at 8 and 12 weeks were 74.2 percent and 64.5 percent of positive control bone volume/total tissue, respectively (p = 0.04). Fluffy-poly(lactic-co-glycolic acid) demonstrated little bone formation, similar to the negative control. Histologic analysis of Hyperelastic Bone scaffolds revealed fibrous tissue at 8 weeks, and new bone formation surrounding the scaffold struts by 12 weeks. CONCLUSION: Findings from our study suggest that Hyperelastic Bone grafts are effective for bone regeneration, with significant potential for clinical translation.

Poly(ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides influence endothelial cell behavior.

The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.

Anisotropic composite material phantom to improve skeletal muscle characterization using magnetic resonance elastography.

The presence and progression of neuromuscular pathology, including spasticity, Duchenne's muscular dystrophy and hyperthyroidism, has been correlated with changes in the intrinsic mechanical properties of skeletal muscle tissue. Tools for noninvasively measuring and monitoring these properties, such as Magnetic Resonance Elastography (MRE), could benefit basic research into understanding neuromuscular pathologies, as well as translational research to develop therapies, by providing a means of assessing and tracking their efficacy. Dynamic elastography methods for noninvasive measurement of tissue mechanical properties have been under development for nearly three decades. Much of the technological development to date, for both Ultrasound (US)-based and Magnetic Resonance Imaging (MRI)-based strategies, has been grounded in assumptions of local homogeneity and isotropy. Striated skeletal and cardiac muscle, as well as brain white matter and soft tissue in some other organ regions, exhibit a fibrous microstructure which entails heterogeneity and anisotropic response; as one seeks to improve the accuracy and resolution in mechanical property assessment, heterogeneity and anisotropy need to be accounted for in order to optimize both the dynamic elastography experimental protocol and the interpretation of the measurements. Advances in elastography methodology at every step have been aided by the use of tissue-mimicking phantoms. The aim of the present study was to develop and characterize a heterogeneous composite phantom design with uniform controllable anisotropic properties meant to be comparable to the frequency-dependent anisotropic properties of skeletal muscle. MRE experiments and computational finite element (FE) studies were conducted on a novel 3D-printed composite phantom design. The displacement maps obtained from simulation and experiment show the same elliptical shaped wavefronts elongated in the plane where the structure presents higher shear modulus. The model exhibits a degree of anisotropy in line with literature data from skeletal muscle tissue MRE experiments. FE simulations of the MRE experiments provide insight into proper interpretation of experimental measurements, and help to quantify the importance of heterogeneity in the anisotropic material at different scales.

Directing the growth and alignment of biliary epithelium within extracellular matrix hydrogels.

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 of engineering the intrahepatic biliary tree, an often-understudied topic in liver tissue engineering. Encapsulating biliary epithelial cells (cholangiocytes) within liver dECM has been shown to lead to the formation of complex biliary trees in vitro. By varying several aspects of the dECM structures' geometry, such as width and angle, we show that we can guide the directional formation of biliary trees. This is confirmed by computational 3D image analysis of duct alignment. This system also enables fabrication of a true multi-layer dECM structure and the formation of 3D biliary trees into which other cell types can be seeded. For example, we show that hepatocyte spheroids can be easily incorporated within this system, and that the seeding sequence influences the resulting structures after seven days in culture. STATEMENT OF SIGNIFICANCE: The field of liver tissue engineering has progressed significantly within the past several years, however engineering the intrahepatic biliary tree has remained a significant challenge. In this study, we utilize the inherent bioactivity of decellularized extracellular matrix (dECM) hydrogels and 3D-printing of a sacrificial biomaterial to create spatially defined, 3D biliary trees. The creation of patterned, 3D dECM hydrogels in the past has only been possible with additives to the gel that may stifle its bioactivity, or with rigid and permanent support structures that may present issues upon implantation. Additionally, the biological effect of 3D spatially patterned liver dECM has not been demonstrated independent of the effects of dECM bioactivity alone. This study demonstrates that sacrificial materials can be used to create pure, multi-layer dECM structures, and that strut width and angle can be changed to influence the formation and alignment of biliary trees encapsulated within. Furthermore, this strategy allows co-culture of other cells such as hepatocytes. We demonstrate that not only does this system show promise for tissue engineering the intrahepatic biliary tree, but it also aids in the study of duct formation and cell-cell interactions.

Voltaglue Bioadhesives Energized with Interdigitated 3D-Graphene Electrodes.

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 with maximum shear storage modulus (G') of 3 kPa. Shear modulus mimics reported values for human soft tissues (0.1-10 kPa). The maximum adhesion strength achieved for the ≈50 mg bioelectrode films is 170 g cm-2 (17 kPa), which exceeds the force required for tethering of electrodes on dynamic soft tissues. The method provides the groundwork for implantable bio/electrodes that may be permanently incorporated into soft tissues, vis-à-vis graphene backscattering wireless electronics since all components are bioresorbable.

Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.

The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce the in vitro formation of complex biliary networks using encapsulated immortalized mouse small biliary epithelial cells (cholangiocytes). This phenomenon is not observed using immortalized mouse large cholangiocytes, or with purified collagen 1 gels or Matrigel. We also show phenotypic stability via immunostaining for specific cholangiocyte markers. Moreover, tight junction formation and maturation was observed to occur between cholangiocytes, exhibiting polarization and transporter activity. To better define the mechanism of duct formation, we utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, radiating from a single nucleation point, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.

Vascularization of Natural and Synthetic Bone Scaffolds.

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 bone grafts. Using this sequential cell-seeding process, the HUVECs formed lumen structures throughout the DB scaffolds as well as vascular tissue bridging 3D-printed fibers within the HB. The pre-cultured hSMCs were essential for endothelial cell (EC) lumen formation within DB scaffolds, as well as for upregulating EC-specific gene expression of HUVECs grown on HB scaffolds. We further applied this co-culture protocol to DB scaffolds using a perfusion bioreactor, to overcome the limitations of diffusive mass transport into the interiors of the scaffolds. Compared with static culture, panoramic histological sections of DB scaffolds cultured in bioreactors showed improved cellular density, as well as a nominal increase in the number of lumen structures formed by ECs in the interior regions of the scaffolds. In conclusion, we have demonstrated that the sequential seeding of hSMCs and HUVECs can serve to generate early microvascular networks that could further support the in vitro tissue engineering of naturally or synthetically derived bone grafts and in both random (DB) and ordered (HB) pore networks. Combined with the preliminary bioreactor study, this process also shows potential to generate clinically sized, vascularized bone scaffolds for tissue and regenerative engineering.

NiTi-Nb micro-trusses fabricated via extrusion-based 3D-printing of powders and transient-liquid-phase sintering.

We present a novel additive manufacturing method for NiTi-Nb micro-trusses combining (i) extrusion-based 3D-printing of liquid inks containing NiTi and Nb powders, solvents, and a polymer binder into micro-trusses with 0/90° ABAB layers of parallel, ∼600 µm struts spaced 1 mm apart and (ii) subsequent heat-treatment to remove the binder and solvents, and then bond the NiTi powders using liquid phase sintering via the formation of a transient NiTi-Nb eutectic phase. We investigate the effects of Nb concentration (0, 1.5, 3.1, 6.7 at.% Nb) on the porosity, microstructure, and phase transformations of the printed NiTi-Nb micro-trusses. Micro-trusses with the highest Nb content exhibit long channels (from 3D-printing) and struts with smaller interconnected porosity (from partial sintering), resulting in overall porosities of ∼75% and low compressive stiffnesses of 1-1.6 GPa, similar to those of trabecular bone and in agreement with analytical and finite element modeling predictions. Diffusion of Nb into the NiTi particles from the bond regions results in a Ni-rich composition as the Nb replaces Ti atoms, leading to decreased martensite/austenite transformation temperatures. Adult human mesenchymal stem cells seeded on these micro-trusses showed excellent viability, proliferation, and extracellular matrix deposition over 14 days in culture. STATEMENT OF SIGNIFICANCE: Near-equiatomic NiTi micro-trusses are attractive for biomedical applications such as stents, actuators, and bone implants because of their combination of biocompatibility, low compressive stiffness, high surface area, and shape-memory or superelasticity. Extrusion-based 3D-printing of NiTi powder-based inks into micro-trusses is feasible, but the subsequent sintering of the powders into dense struts is unachievable due to low diffusivity, large particle size, and low packing density of the NiTi powders. We present a solution, whereby Nb powders are added to the NiTi inks, thus forming during sintering a eutectic NiTi-Nb liquid phase which bonds the solid NiTi powders and improves densification of the struts. This study investigates the microstructure, porosity, phase transformation behavior, compressive stiffness, and cytocompatibility of these printed NiTi-Nb micro-trusses.

Three-Dimensional Printing of Cytocompatible, Thermally Conductive Hexagonal Boron Nitride Nanocomposites.

Hexagonal boron nitride (hBN) is a thermally conductive yet electrically insulating two-dimensional layered nanomaterial that has attracted significant attention as a dielectric for high-performance electronics in addition to playing a central role in thermal management applications. Here, we report a high-content hBN-polymer nanocomposite ink, which can be 3D printed to form mechanically robust, self-supporting constructs. In particular, hBN is dispersed in poly(lactic- co-glycolic acid) and 3D printed at room temperature through an extrusion process to form complex architectures. These constructs can be 3D printed with a composition of up to 60% vol hBN (solids content) while maintaining high mechanical flexibility and stretchability. The presence of hBN within the matrix results in enhanced thermal conductivity (up to 2.1 W K-1 m-1) directly after 3D printing with minimal postprocessing steps, suggesting utility in thermal management applications. Furthermore, the constructs show high levels of cytocompatibility, making them suitable for use in the field of printed bioelectronics.

Kidney decellularized extracellular matrix hydrogels: Rheological characterization and human glomerular endothelial cell response to encapsulation.

Hydrogels, highly-hydrated crosslinked polymer networks, closely mimic the microenvironment of native extracellular matrix (ECM) and thus present as ideal platforms for three-dimensional cell culture. Hydrogels derived from tissue- and organ-specific decellularized ECM (dECM) may retain bioactive signaling cues from the native tissue or organ that could in turn modulate cell-material interactions and response. In this study, we demonstrate that porcine kidney dECM can be processed to form hydrogels suitable for cell culture and encapsulation studies. Scanning electron micrographs of hydrogels demonstrated a fibrous ultrastructure with interconnected pores, and rheological analysis revealed rapid gelation times with shear moduli dependent upon the protein concentration of the hydrogels. Conditionally-immortalized human glomerular endothelial cells (GEnCs) cultured on top of or encapsulated within hydrogels exhibited high cell viability and proliferation over a one-week culture period. However, gene expression analysis of GEnCs encapsulated within kidney dECM hydrogels revealed significantly lower expression of several relevant genes of interest compared to those encapsulated within hydrogels composed of only purified collagen I. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2448-2462, 2018.