Osseointegration of 3D-printed titanium implants with surface and structure modifications.

BACKGROUND: The purpose of this study was to establish a mechanical and histological basis for the development of biocompatible maxillofacial reconstruction implants by combining 3D-printed porous titanium structures and surface treatment. Improved osseointegration of 3D-printed titanium implants for reconstruction of maxillofacial segmental bone defect could be advantageous in not only quick osseointegration into the bone tissue but also in stabilizing the reconstruction. METHODS: Various macro-mesh titanium scaffolds were fabricated by 3D-printing. Human mesenchymal stem cells were used for cell attachment and proliferation assays. Osteogenic differentiation was confirmed by quantitative polymerase chain reaction analysis. The osseointegration rate was measured using micro computed tomography imaging and histological analysis. RESULTS: In three dimensional-printed scaffold, globular microparticle shape was observed regardless of structure or surface modification. Cell attachment and proliferation rates increased according to the internal mesh structure and surface modification. However, osteogenic differentiation in vitro and osseointegration in vivo revealed that non-mesh structure/non-surface modified scaffolds showed the most appropriate treatment effect. CONCLUSION: 3D-printed solid structure is the most suitable option for maxillofacial reconstruction. Various mesh structures reduced osteogenesis of the mesenchymal stem cells and osseointegration compared with that by the solid structure. Surface modification by microarc oxidation induced cell proliferation and increased the expression of some osteogenic genes partially; however, most of the markers revealed that the non-anodized solid scaffold was the most suitable for maxillofacial reconstruction.

Bone Fracture-Treatment Method: Fixing 3D-Printed Polycaprolactone Scaffolds with Hydrogel Type Bone-Derived Extracellular Matrix and ß-Tricalcium Phosphate as an Osteogenic Promoter.

Bone formation and growth are crucial for treating bone fractures. Improving bone-reconstruction methods using autologous bone and synthetic implants can reduce the recovery time. Here, we investigated three treatments using two different materials, a bone-derived decellularized extracellular matrix (bdECM) and ß-tricalcium phosphate (ß-TCP), individually and in combination, as osteogenic promoter between bone and 3D-printed polycaprolactone scaffold (6-mm diameter) in rat calvarial defects (8-mm critical diameter). The materials were tested with a human pre-osteoblast cell line (MG63) to determine the effects of the osteogenic promoter on bone formation in vitro. A polycaprolactone (PCL) scaffold with a porous structure was placed at the center of the in vivo rat calvarial defects. The gap between the defective bone and PCL scaffold was filled with each material. Animals were sacrificed four weeks post-implantation, and skull samples were preserved for analysis. The preserved samples were scanned by micro-computed tomography and analyzed histologically to examine the clinical benefits of the materials. The bdECM-ß-TCP mixture showed faster bone formation and a lower inflammatory response in the rats. Therefore, our results imply that a bdECM-ß-TCP mixture is an ideal osteogenic promoter for treating fractures.

Production of Multiple Cell-Laden Microtissue Spheroids with a Biomimetic Hepatic-Lobule-Like Structure.

The construction of an in vitro 3D cellular model to mimic the human liver is highly desired for drug discovery and clinical applications, such as patient-specific treatment and cell-based therapy in regenerative medicine. However, current bioprinting strategies are limited in their ability to generate multiple cell-laden microtissues with biomimetic structures. This study presents a method for producing hepatic-lobule-like microtissue spheroids using a bioprinting system incorporating a precursor cartridge and microfluidic emulsification system. The multiple cell-laden microtissue spheroids can be successfully generated at a speed of approximately 45 spheroids min-1 and with a uniform diameter. Hepatic and endothelial cells are patterned in a microtissue spheroid with the biomimetic structure of a liver lobule. The spheroids allow long-term culture with high cell viability, and the structural integrity is maintained longer than that of non-structured spheroids. Furthermore, structured spheroids show high MRP2, albumin, and CD31 expression levels. In addition, the in vivo study reveals that structured microtissue spheroids are stably engrafted. These results demonstrate that the method provides a valuable 3D structured microtissue spheroid model with lobule-like constructs and liver functions.

Bioprinting on 3D Printed Titanium Scaffolds for Periodontal Ligament Regeneration.

The three-dimensional (3D) cell-printing technique has been identified as a new biofabrication platform because of its ability to locate living cells in pre-defined spatial locations with scaffolds and various growth factors. Osseointegrated dental implants have been regarded as very reliable and have long-term reliability. However, host defense mechanisms against infections and micro-movements have been known to be impaired around a dental implant because of the lack of a periodontal ligament. In this study, we fabricated a hybrid artificial organ with a periodontal ligament on the surface of titanium using 3D printing technology. CEMP-1, a known cementogenic factor, was enhanced in vitro. In animal experiments, when the hybrid artificial organ was transplanted to the calvarial defect model, it was observed that the amount of connective tissue increased. 3D-printed hybrid artificial organs can be used with dental implants, establishing physiological tooth functions, including the ability to react to mechanical stimuli and the ability to resist infections.

3D-Printed Collagen Scaffolds Promote Maintenance of Cryopreserved Patients-Derived Melanoma Explants.

The development of an in vitro three-dimensional (3D) culture system with cryopreserved biospecimens could accelerate experimental research screening anticancer drugs, potentially reducing costs and time bench-to-beside. However, minimal research has explored the application of 3D bioprinting-based in vitro cancer models to cryopreserved biospecimens derived from patients with advanced melanoma. We investigated whether 3D-printed collagen scaffolds enable the propagation and maintenance of patient-derived melanoma explants (PDMEs). 3D-printed collagen scaffolds were fabricated with a 3DX bioprinter. After thawing, fragments from cryopreserved PDMEs (approximately 1-2 mm) were seeded onto the 3D-printed collagen scaffolds, and incubated for 7 to 21 days. The survival rate was determined with MTT and live and dead assays. Western blot analysis and immunohistochemistry staining was used to express the function of cryopreserved PDMEs. The results show that 3D-printed collagen scaffolds could improve the maintenance and survival rate of cryopreserved PDME more than 2D culture. MITF, Mel A, and S100 are well-known melanoma biomarkers. In agreement with these observations, 3D-printed collagen scaffolds retained the expression of melanoma biomarkers in cryopreserved PDME for 21 days. Our findings provide insight into the application of 3D-printed collagen scaffolds for closely mimicking the 3D architecture of melanoma and its microenvironment using cryopreserved biospecimens.

Coherent many-body exciton in van der Waals antiferromagnet NiPS3.

An exciton is the bosonic quasiparticle of electron-hole pairs bound by the Coulomb interaction1. Bose-Einstein condensation of this exciton state has long been the subject of speculation in various model systems2,3, and examples have been found more recently in optical lattices and two-dimensional materials4-9. Unlike these conventional excitons formed from extended Bloch states4-9, excitonic bound states from intrinsically many-body localized states are rare. Here we show that a spin-orbit-entangled exciton state appears below the Néel temperature of 150 kelvin in NiPS3, an antiferromagnetic van der Waals material. It arises intrinsically from the archetypal many-body states of the Zhang-Rice singlet10,11, and reaches a coherent state assisted by the antiferromagnetic order. Using configuration-interaction theory, we determine the origin of the coherent excitonic excitation to be a transition from a Zhang-Rice triplet to a Zhang-Rice singlet. We combine three spectroscopic tools-resonant inelastic X-ray scattering, photoluminescence and optical absorption-to characterize the exciton and to demonstrate an extremely narrow excitonic linewidth below 50 kelvin. The discovery of the spin-orbit-entangled exciton in antiferromagnetic NiPS3 introduces van der Waals magnets as a platform to study coherent many-body excitons.

Pre-set extrusion bioprinting for multiscale heterogeneous tissue structure fabrication.

Recent advances in three-dimensional bioprinting technology have led to various attempts in fabricating human tissue-like structures. However, current bioprinting technologies have limitations for creating native tissue-like structures. To resolve these issues, we developed a new pre-set extrusion bioprinting technique that can create heterogeneous, multicellular, and multimaterial structures simultaneously. The key to this ability lies in the use of a precursor cartridge that can stably preserve a multimaterial with a pre-defined configuration that can be simply embedded in a syringe-based printer head. The multimaterial can be printed and miniaturized through a micro-nozzle without conspicuous deformation according to the pre-defined configuration of the precursor cartridge. Using this system, we fabricated heterogeneous tissue-like structures such as spinal cords, hepatic lobule, blood vessels, and capillaries. We further obtained a heterogeneous patterned model that embeds HepG2 cells with endothelial cells in a hepatic lobule-like structure. In comparison with homogeneous and heterogeneous cell printing, the heterogeneous patterned model showed a well-organized hepatic lobule structure and higher enzyme activity of CYP3A4. Therefore, this pre-set extrusion bioprinting method could be widely used in the fabrication of a variety of artificial and functional tissues or organs.

Spectroscopic Studies on the Metal-Insulator Transition Mechanism in Correlated Materials.

The metal-insulator transition (MIT) in correlated materials is a novel phenomenon that accompanies a large change in resistivity, often many orders of magnitude. It is important in its own right but its switching behavior in resistivity can be useful for device applications. From the material physics point of view, the starting point of the research on the MIT should be to understand the microscopic mechanism. Here, an overview of recent efforts to unravel the microscopic mechanisms for various types of MITs in correlated materials is provided. Research has focused on transition metal oxides (TMOs), but transition metal chalcogenides have also been studied. Along the way, a new class of MIT materials is discovered, the so-called relativistic Mott insulators in 5d TMOs. Distortions in the MO6 (M = transition metal) octahedron are found to have a large and peculiar effect on the band structure in an orbital dependent way, possibly paving a way to the orbital selective Mott transition. In the final section, the character of the materials suitable for applications is summarized, followed by a brief discussion of some of the efforts to control MITs in correlated materials, including a dynamical approach using light.

Design of Magnetically Labeled Cells (Mag-Cells) for in Vivo Control of Stem Cell Migration and Differentiation.

Cell-based therapies are attractive for treating various degenerative disorders and cancer but delivering functional cells to the region of interest in vivo remains difficult. The problem is exacerbated in dense biological matrices such as solid tissues because these environments impose significant steric hindrances for cell movement. Here, we show that neural stem cells transfected with zinc-doped ferrite magnetic nanoparticles (ZnMNPs) can be pulled by an external magnet to migrate to the desired location in the brain. These magnetically labeled cells (Mag-Cells) can migrate because ZnMNPs generate sufficiently strong mechanical forces to overcome steric hindrances in the brain tissues. Once at the site of lesion, Mag-Cells show enhanced neuronal differentiation and greater secretion of neurotrophic factors than unlabeled control stem cells. Our study shows that ZnMNPs activate zinc-mediated Wnt signaling to facilitate neuronal differentiation. When implemented in a rodent brain stroke model, Mag-Cells led to significant recovery of locomotor performance in the impaired limbs of the animals. Our findings provide a simple magnetic method for controlling migration of stem cells with high therapeutic functions, offering a valuable tool for other cell-based therapies.

Three-Dimensional Hepatocellular Carcinoma/Fibroblast Model on a Nanofibrous Membrane Mimics Tumor Cell Phenotypic Changes and Anticancer Drug Resistance.

Three-dimensional (3D) in vitro tissue or organ models can effectively mimic the complex microenvironment of many types of human tissues for medical applications. Unfortunately, development of 3D cancer models, which involve cancer/stromal cells in a 3D environment, has remained elusive due to the extreme complexity of the tumor microenvironment (TME) and the stepwise progression of human cancer. Here, we developed hepatocellular carcinoma (HCC) models, which consist of fibroblasts as stromal cells, HCC cells, and a nanofibrous membrane to mimic the complex TME. The 3D HCC models were fabricated using three distinct culture methods: cancer cells grown directly on the nanofibrous membrane (mono model), fibroblasts covering the nanofibrous membrane (layer model), and both cancer cells and fibroblasts grown on the nanofibrous membrane (mixed model). Interestingly, the mono model and layer model showed similar tissue structures, whereas the mixed model resulted in phenotypic changes to the cancer cells. Further analysis demonstrated that the mixed models promoted the expression of fibronectin and vimentin, and showed higher resistance to anticancer drugs compared with the other models. Thus, our 3D HCC model could be utilized for testing efficient anticancer therapies at various stages of cancer, with potential application to different tumor types.

Spectral and magnetic properties of Na2RuO3.

We present measurements of resistivity, x-ray absorption (XAS) and emission (XES) spectroscopy together with ab initio band structure calculations for quasi two dimensional ruthenate Na2RuO3. Density function calculations (DFT) and XAS and XES spectra both show that Na2RuO3 is a semiconductor with an activation energy of ∼80 meV. Our DFT calculations reveal large magneto-elastic coupling in Na2RuO3 and predict that the ground state of Na2RuO3 should be antiferromagnetic zig-zag.

Neurogenin-2-transduced human neural progenitor cells attenuate neonatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic (HI) brain injury leads to high mortality and neurodevelopmental disabilities. Multipotent neural progenitor cells (NPCs) with self-renewing capacity have the potential to reduce neuronal loss and improve the compromised environment in the HI brain injury. However, the therapeutic efficacy of neuronal-committed progenitor cells and the underlying mechanisms of recovery are not yet fully understood. Therefore, this study investigated the regenerative ability and action mechanisms of neuronally committed human NPCs (hNPCs) transduced with neurogenin-2 (NEUROG2) in neonatal HI brain injury. NEUROG2- or green fluorescent protein (GFP)-encoding adenoviral vector-transduced hNPCs (NEUROG2- or GFP-NPCs) were transplanted into neonatal mouse brains with HI injury. Grafted NEUROG2-NPCs showed robust dispersion and engraftment, prolonged survival, and neuronal differentiation in HI brain injury. NEUROG2-NPCs significantly improved neurological behaviors, decreased cellular apoptosis, and increased the neurite outgrowth and axonal sprouting in HI brain injury. In contrast, GFP-NPC grafts moderately enhanced axonal extension with limited behavioral recovery. Notably, NEUROG2-NPCs showed increased secretion of multiple factors, such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 (NTF3), fibroblast growth factor 9 (FGF9), ciliary neurotrophic factor (CNTF), and thrombospondins 1 and 2 (THBS 1/2), which promoted SH-SY5Y neuroblastoma cell survival and neurite outgrowth. Thus, we postulate that NEUROG2-expressing human NPCs facilitate functional recovery after neonatal HI brain injury via their ability to secrete multiple factors that enhance neuronal survival and neuroplasticity.

Sliding Fibers: Slidable, Injectable, and Gel-like Electrospun Nanofibers as Versatile Cell Carriers.

Designing biomaterial systems that can mimic fibrous, natural extracellular matrix is crucial for enhancing the efficacy of various therapeutic tools. Herein, a smart technology of three-dimensional electrospun fibers that can be injected in a minimally invasive manner was developed. Open surgery is currently the only route of administration of conventional electrospun fibers into the body. Coordinating electrospun fibers with a lubricating hydrogel produced fibrous constructs referred to as slidable, injectable, and gel-like (SLIDING) fibers. These SLIDING fibers could pass smoothly through a catheter and fill any cavity while maintaining their fibrous morphology. Their injectable features were derived from their distinctive rheological characteristics, which were presumably caused by the combinatorial effects of mobile electrospun fibers and lubricating hydrogels. The resulting injectable fibers fostered a highly favorable environment for human neural stem cell (hNSC) proliferation and neurosphere formation within the fibrous structures without compromising hNSC viability. SLIDING fibers demonstrated superior performance as cell carriers in animal stroke models subjected to the middle cerebral artery occlusion (MCAO) stroke model. In this model, SLIDING fiber application extended the survival rate of administered hNSCs by blocking microglial infiltration at the early, acute inflammatory stage. The development of SLIDING fibers will increase the clinical significance of fiber-based scaffolds in many biomedical fields and will broaden their applicability.

Clinical Trial of Human Fetal Brain-Derived Neural Stem/Progenitor Cell Transplantation in Patients with Traumatic Cervical Spinal Cord Injury.

In a phase I/IIa open-label and nonrandomized controlled clinical trial, we sought to assess the safety and neurological effects of human neural stem/progenitor cells (hNSPCs) transplanted into the injured cord after traumatic cervical spinal cord injury (SCI). Of 19 treated subjects, 17 were sensorimotor complete and 2 were motor complete and sensory incomplete. hNSPCs derived from the fetal telencephalon were grown as neurospheres and transplanted into the cord. In the control group, who did not receive cell implantation but were otherwise closely matched with the transplantation group, 15 patients with traumatic cervical SCI were included. At 1 year after cell transplantation, there was no evidence of cord damage, syrinx or tumor formation, neurological deterioration, and exacerbating neuropathic pain or spasticity. The American Spinal Injury Association Impairment Scale (AIS) grade improved in 5 of 19 transplanted patients, 2 (A → C), 1 (A → B), and 2 (B → D), whereas only one patient in the control group showed improvement (A → B). Improvements included increased motor scores, recovery of motor levels, and responses to electrophysiological studies in the transplantation group. Therefore, the transplantation of hNSPCs into cervical SCI is safe and well-tolerated and is of modest neurological benefit up to 1 year after transplants. This trial is registered with Clinical Research Information Service (CRIS), Registration Number: KCT0000879.

Human neural stem cells alleviate Alzheimer-like pathology in a mouse model.

BACKGROUND: Alzheimer's disease (AD) is an inexorable neurodegenerative disease that commonly occurs in the elderly. The cognitive impairment caused by AD is associated with abnormal accumulation of amyloid-ß (Aß) and hyperphosphorylated tau, which are accompanied by inflammation. Neural stem cells (NSCs) are self-renewing, multipotential cells that differentiate into distinct neural cells. When transplanted into a diseased brain, NSCs repair and replace injured tissues after migration toward and engraftment within lesions. We investigated the therapeutic effects in an AD mouse model of human NSCs (hNSCs) that derived from an aborted human fetal telencephalon at 13 weeks of gestation. Cells were transplanted into the cerebral lateral ventricles of neuron-specific enolase promoter-controlled APPsw-expressing (NSE/APPsw) transgenic mice at 13 months of age. RESULTS: Implanted cells extensively migrated and engrafted, and some differentiated into neuronal and glial cells, although most hNSCs remained immature. The hNSC transplantation improved spatial memory in these mice, which also showed decreased tau phosphorylation and Aß42 levels and attenuated microgliosis and astrogliosis. The hNSC transplantation reduced tau phosphorylation via Trk-dependent Akt/GSK3ß signaling, down-regulated Aß production through an Akt/GSK3ß signaling-mediated decrease in BACE1, and decreased expression of inflammatory mediators through deactivation of microglia that was mediated by cell-to-cell contact, secretion of anti-inflammatory factors generated from hNSCs, or both. The hNSC transplantation also facilitated synaptic plasticity and anti-apoptotic function via trophic supplies. Furthermore, the safety and feasibility of hNSC transplantation are supported. CONCLUSIONS: These findings demonstrate the hNSC transplantation modulates diverse AD pathologies and rescue impaired memory via multiple mechanisms in an AD model. Thus, our data provide tangible preclinical evidence that human NSC transplantation could be a safe and versatile approach for treating AD patients.

Real-time discrimination between proliferation and neuronal and astroglial differentiation of human neural stem cells.

Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling.

Human fetal brain-derived neural stem/progenitor cells grafted into the adult epileptic brain restrain seizures in rat models of temporal lobe epilepsy.

Cell transplantation has been suggested as an alternative therapy for temporal lobe epilepsy (TLE) because this can suppress spontaneous recurrent seizures in animal models. To evaluate the therapeutic potential of human neural stem/progenitor cells (huNSPCs) for treating TLE, we transplanted huNSPCs, derived from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres over a long time period, into the epileptic hippocampus of fully kindled and pilocarpine-treated adult rats exhibiting TLE. In vitro, huNSPCs not only produced all three central nervous system neural cell types, but also differentiated into ganglionic eminences-derived γ-aminobutyric acid (GABA)-ergic interneurons and released GABA in response to the depolarization induced by a high K+ medium. NSPC grafting reduced behavioral seizure duration, afterdischarge duration on electroencephalograms, and seizure stage in the kindling model, as well as the frequency and the duration of spontaneous recurrent motor seizures in pilocarpine-induced animals. However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals. Following transplantation, grafted cells showed extensive migration around the injection site, robust engraftment, and long-term survival, along with differentiation into ß-tubulin III+ neurons (∼34%), APC-CC1+ oligodendrocytes (∼28%), and GFAP+ astrocytes (∼8%). Furthermore, among donor-derived cells, ∼24% produced GABA. Additionally, to explain the effect of seizure suppression after NSPC grafting, we examined the anticonvulsant glial cell-derived neurotrophic factor (GDNF) levels in host hippocampal astrocytes and mossy fiber sprouting into the supragranular layer of the dentate gyrus in the epileptic brain. Grafted cells restored the expression of GDNF in host astrocytes but did not reverse the mossy fiber sprouting, eliminating the latter as potential mechanism. These results suggest that human fetal brain-derived NSPCs possess some therapeutic effect for TLE treatments although further studies to both increase the yield of NSPC grafts-derived functionally integrated GABAergic neurons and improve cognitive deficits are still needed.

T1 and T2 dual-mode MRI contrast agent for enhancing accuracy by engineered nanomaterials.

One of the holy grails in biomedical imaging technology is to achieve accurate imaging of biological targets. The development of sophisticated instrumentation and the use of contrast agents have improved the accuracy of biomedical imaging. However, the issue of false imaging remains a problem. Here, we developed a dual-mode artifact filtering nanoparticle imaging agent (AFIA) that comprises a combination of paramagnetic and superparamagnetic nanomaterials. This AFIA has the ability to perform "AND logic gate" algorithm to eliminate false errors (artifacts) from the raw images to enhance accuracy of the MRI. We confirm the artifact filtering capability of AFIA in MRI phantoms and further demonstrate that artifact-free imaging of stem cell migration is possible in vivo.