Biofabrication, biochemical profiling, and in vitro applications of salivary gland decellularized matrices via magnetic bioassembly platforms.

Trending three-dimensional tissue engineering platforms developed via biofabrication and bioprinting of exocrine glands are on the rise due to a commitment to organogenesis principles. Nevertheless, a proper extracellular matrix (ECM) microarchitecture to harbor primary cells is yet to be established towards human salivary gland (SG) organogenesis. By using porcine submandibular gland (SMG) biopsies as a proof-of-concept to mimic the human SG, a new decellularized ECM bioassembly platform was developed herein with varying perfusions of sodium dodecyl sulfate (SDS) to limit denaturing events and ensure proper preservation of the native ECM biochemical niche. Porcine SMG biopsies were perfused with 0.01%, 0.1%, and 1% SDS and bio-assembled magnetically in porous polycarbonate track-etched (PCTE) membrane. Double-stranded DNA (dsDNA), cell removal efficiency, and ECM biochemical contents were analyzed. SDS at 0.1% and 1% efficiently removed dsDNA (< 50 ng/mg) and preserved key matrix components (sulfated glycosaminoglycans, collagens, elastin) and the microarchitecture of native SMG ECM. Bio-assembled SMG decellularized ECM (dECM) perfused with 0.1-1% SDS enhanced cell viability, proliferation, expansion confluency rates, and tethering of primary SMG cells during 7 culture days. Perfusion with 1% SDS promoted greater cell proliferation rates while 0.1% SDS supported higher acinar epithelial expression when compared to basement membrane extract and other substrates. Thus, this dECM magnetic bioassembly strategy was effective for decellularization while retaining the original ECM biochemical niche and promoting SMG cell proliferation, expansion, differentiation, and tethering. Altogether, these outcomes pave the way towards the recellularization of this novel SMG dECM in future in vitro and in vivo applications.

The Human Cytomegalovirus Protein UL116 Interacts with the Viral Endoplasmic-Reticulum-Resident Glycoprotein UL148 and Promotes the Incorporation of gH/gL Complexes into Virions.

Heterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), the glycoprotein UL116 assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Kifunensine, a mannosidase inhibitor that interferes with endoplasmic reticulum (ER)-associated degradation (ERAD) of terminally misfolded glycoproteins, restored levels of gH, gL, and gO in UL116-null-infected cells, indicating that constituents of HCMV gH complexes are unstable in the absence of UL116. Further, we find that gH/UL116 complexes are abundant in virions, since a major gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is detected from wild-type but not UL116-null virions. Interestingly, UL116 coimmunoprecipitates with UL148, a viral ER-resident glycoprotein that attenuates ERAD of gO, and we observe elevated levels of UL116 in UL148-null virions. Collectively, our findings argue that UL116 is a chaperone for gH that supports the assembly, maturation, and incorporation of gH/gL complexes into virions. IMPORTANCE HCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH)/glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding HCMV cell tropism as well as for the development of vaccines against the virus.

Chiral Photomelting of DNA-Nanocrystal Assemblies Utilizing Plasmonic Photoheating.

Chiral plasmonic nanostructures exhibit anomalously strong chiroptical signals and offer the possibility to realize asymmetric photophysical and photochemical processes controlled by circularly polarized light. Here, we use a chiral DNA-assembled nanorod pair as a model system for chiral plasmonic photomelting. We show that both the enantiomeric excess and consequent circular dichroism can be controlled with chiral light. The nonlinear chiroptical response of our plasmonic system results from the chiral photothermal effect leading to selective melting of the DNA linker strands. Our study describes both the single-complex and collective heating regimes, which should be treated with different models. The chiral asymmetry factors of the calculated photothermal and photomelting effects exceed the values typical for the chiral molecular photochemistry at least 10-fold. Our proposed mechanism can be used to develop chiral photoresponsive systems controllable with circularly polarized light.

The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

Towards an in vitro model of the glomerular barrier unit with an innovative bioassembly method.

BACKGROUND: The development of an artificial glomerular unit may be pivotal for renal pathophysiology studies at a multicellular scale. Using a tissue engineering approach, we aimed to reproduce in part the specific glomerular barrier architecture by manufacturing a glomerular microfibre (Mf). METHODS: Immortalized human glomerular cell lines of endothelial cells (GEnCs) and podocytes were used. Cells and a three-dimensional (3D) matrix were characterized by immunofluorescence with confocal analysis, Western blot and polymerase chain reaction. Optical and electron microscopy were used to study Mf and cell shapes. We also analysed cell viability and cell metabolism within the 3D construct at 14 days. RESULTS: Using the Mf manufacturing method, we repeatedly obtained a cellularized Mf sorting human glomerular cells in 3D. Around a central structure made of collagen I, we obtained an internal layer composed of GEnC, a newly formed glomerular basement membrane rich in α5 collagen IV and an external layer of podocytes. The cell concentration, optimal seeding time and role of physical stresses were modulated to obtain the Mf. Cell viability and expression of specific proteins (nephrin, synaptopodin, vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrandt factor (vWF)) were maintained for 19 days in the Mf system. Mf ultrastructure, observed with EM, had similarities with the human glomerular barrier. CONCLUSION: In summary, with our 3D bio-engineered glomerular fibre, GEnC and podocytes produced a glomerular basement membrane. In the future, this glomerular Mf will allow us to study cell interactions in a 3D system and increase our knowledge of glomerular pathophysiology.

Microbial diversity and co-occurrence patterns in deep soils contaminated by polycyclic aromatic hydrocarbons (PAHs).

Numerous studies have enriched our knowledge of the microbial community composition and metabolic versatility of contaminated soil. However, there remains a substantial gap regarding the bioassembly patterns of the indigenous microbial community distribution in contaminated deep soils. Herein, the indigenous microbial community structure diversity, function, and co-occurrence relationships in aged PAH-contaminated deep soil collected from an abandoned chemical facility were investigated using high-throughput sequencing. The results showed that the dominant phyla in all samples were responsible for PAH degradation and included Proteobacteria (20.86%-81.37%), Chloroflexi (2.03%-28.44%), Firmicutes (3.06%-31.16%), Actinobacteria (2.92%-11.91%), Acidobacteria (0.41%-12.68%), and Nitrospirae (0.81%-9.21%). Eighty biomarkers were obtained by linear discriminant analysis of effect size (LEfSe), and most of these biomarkers were PAH degraders. Functional predictions using Tax4Fun indicated that the aged contaminated soil has the potential for PAH degradation. Statistical analysis showed that in contrast with the PAH concentration, edaphic properties (nutrients and pH) were significantly correlated (r > 0.25, P < 0.01) with the bacterial community and functional composition. Co-occurrence network analysis (modularity index of 0.781) revealed non-random assembly patterns of the bacterial communities in the PAH-contaminated soils. The modules in the network were mainly involved in carbon and nitrogen cycles, organic substance degradation, and biological electron transfer processes. Microbes from the same module had strong ecological linkages. Additionally, SAR202 clade, Thermoanaerobaculum, Nitrospira, and Xanthomonadales, which were identified as keystone species, played an irreplaceable role in the network. Overall, our results suggested that environmental factors such as nutrients and pH, together with ecological function, are the main factors driving the assembly of microbial communities in aged PAH-contaminated deep soils.

Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.

Scaffolded spheroids as building blocks for bottom-up cartilage tissue engineering show enhanced bioassembly dynamics.

Due to the capability of cell spheroids (SPH) to assemble into large high cell density constructs, their use as building blocks attracted a lot of attention in the field of biofabrication. Nevertheless, upon maturation, the composition along with the size of such building blocks change, affecting their fusiogenic ability to form a cohesive tissue construct of controllable size. This natural phenomenon remains a limitation for the standardization of spheroid-based therapies in the clinical setting. We recently showed that scaffolded spheroids (S-SPH) can be produced by forming spheroids directly within porous PCL-based microscaffolds fabricated using multiphoton lithography (MPL). In this new study, we compare the bioassembly potential of conventional SPHs versus S-SPHs depending on their degree of maturation. Doublets of both types of building blocks were cultured and their fusiogenicity was compared by measuring the intersphere angle, the length of the fusing spheroid pairs (referred to as doublet length) as well as their spreading behaviour. Finally, the possibility to fabricate macro-sized tissue constructs (i.e. cartilage-like) from both chondrogenic S-SPHs and SPHs was analyzed. This study revealed that, in contrast to conventional SPHs, S-SPHs exhibit robust and stable fusiogenicity, independently from their degree of maturation. In order to understand this behavior, we further analyze the intersection area of doublets, looking at the kinetic of cell migration and at the mechanical stability of the formed tissue using dissection measurements. Our findings indicate that the presence of microscaffolds enhances the ability of spheroids to be used as building blocks for bottom-up tissue engineering, which is an important advantage compared to conventional spheroid-based therapy approaches. STATEMENT OF SIGNIFICANCE: The approach of using SPHs as building blocks for bottom-up tissue engineering offers a variety of advantages. At the same time the self-assembly of large tissues remains challenging due to several intrinsic properties of SPHs, such as for instance the shrinkage of tissues assembled from SPHs, or the reduced fusiogenicity commonly observed with mature SPHs. In this work, we demonstrate the capability of scaffolded spheroids (S-SPH) to fuse and recreate cartilage-like tissue constructs despite their advanced maturation stage. In this regard, the presence of microscaffolds compensates for some of the intrinsic limitations of SPHs and can help to overcome current limitations of spheroid-based tissue engineering.

Biofabrication of Tissue-Engineered Cartilage Constructs Through Faraday Wave Bioassembly of Cell-Laden Gelatin Microcarriers.

Acoustic biofabrication is an emerging strategy in tissue engineering due to its mild and fast manufacturing process. Herein, tissue-engineered cartilage constructs with high cell viability are fabricated from cell-laden gelatin microcarriers (GMs) through Faraday wave bioassembly, a typical acoustic "bottom-up" manufacturing process. Assembly modules are first prepared by incorporating cartilage precursor cells, the chondrogenic cell line ATDC5, or bone marrow-derived mesenchymal stem cells (BMSCs), into GMs. Patterned structures are formed by Faraday wave bioassembly of the cell-laden GMs. Due to the gentle and efficient assembly process and the protective effects of microcarriers, cells in the patterned structures maintain high activity. Subsequently, tissue-engineered cartilage constructs are obtained by inducing cell differentiation of the patterned structures. Comprehensive evaluations are conducted to verify chondrocyte differentiation and the formation of cartilage tissue constructs in terms of cell viability, morphological analysis, gene expression, and matrix production. Finally, implantation studies with a rat cartilage defect model demonstrate that these tissue-engineered cartilage constructs are beneficial for the repair of articular cartilage damage in vivo. This study provides the first biofabrication of cartilage tissue constructs using Faraday wave bioassembly, extending its application to engineering tissues with a low cell density.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death.

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

Biofabrication's Contribution to the Evolution of Cultured Meat.

Cultured Meat (CM) is a growing field in cellular agriculture, driven by the environmental impact of conventional meat production, which contributes to climate change and occupies ≈70% of arable land. As demand for meat alternatives rises, research in this area expands. CM production relies on tissue engineering techniques, where a limited number of animal cells are cultured in vitro and processed to create meat-like tissue comprising muscle and adipose components. Currently, CM is primarily produced on a small scale in pilot facilities. Producing a large cell mass based on suitable cell sources and bioreactors remains challenging. Advanced manufacturing methods and innovative materials are required to subsequently process this cell mass into CM products on a large scale. Consequently, CM is closely linked with biofabrication, a suite of technologies for precisely arranging cellular aggregates and cell-material composites to construct specific structures, often using robotics. This review provides insights into contemporary biomedical biofabrication technologies, focusing on significant advancements in muscle and adipose tissue biofabrication for CM production. Novel materials for biofabricating CM are also discussed, emphasizing their edibility and incorporation of healthful components. Finally, initial studies on biofabricated CM are examined, addressing current limitations and future challenges for large-scale production.

Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement.

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures. Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators. Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.

Modular Microgel-Based Bioassembly Scaffold Induced Chondrogenic and Osteogenic Differentiation of BMSCs.

Bioactive scaffolds capable of simultaneously repairing osteochondral defects remain a big challenge due to the heterogeneity of bone and cartilage. Currently modular microgel-based bioassembly scaffolds are emerged as potential solution to this challenge. Here, microgels based on methacrylic anhydride (MA) and dopamine modified gelatin (GelMA-DA) are loaded with chondroitin sulfate (CS) (the obtained microgel named GC Ms) or bioactive glass (BG) (the obtained microgel named GB Ms), respectively. GC Ms and GB Ms show good biocompatibility with BMSCs, which suggested by the adhesion and proliferation of BMSCs on their surfaces. Specially, GC Ms promote chondrogenic differentiation of BMSCs, while GB Ms promote osteogenic differentiation. Furthermore, the injectable GC Ms and GB Ms are assembled integrally by bottom-up in situ cross-linking to obtain modular microgel-based bioassembly scaffold (GC-GB/HM), which show a distinct bilayer structure and good porous properties and swelling properties. Particularly, the results of in vivo and in vitro experiments show that GC-GB/HM can simultaneously regulate the expression levels of chondrogenic- and osteogenesis-related genes and proteins. Therefore, modular microgel-based assembly scaffold in this work with the ability to promote bidirectional differentiation of BMSCs and has great potential for application in the minimally invasive treatment of osteochondral tissue defects.

Fabricating vascularized, anatomically accurate bone grafts using 3D bioprinted sectional bone modules, in-situ angiogenesis, BMP-2 controlled release, and bioassembly.

Bone grafting is the most common treatment for repairing bone defects. However, current bone grafting methods have several drawbacks. Bone tissue engineering emerges as a promising solution to these problems. An ideal engineered bone graft should exhibit high mechanical strength, osteogenic properties, and pre-vascularization. Both top-down (using bulk scaffold) and bottom-up (using granular modules) approaches face challenges in fulfilling these requirements. In this paper, we propose a novel sectional modular bone approach to construct osteogenic, pre-vascularized bone grafts in anatomical shapes. We 3D-printed a series of rigid, thin, sectional, porous scaffolds from a biodegradable polymer, tailored to the dimensions of a femur bone shaft. These thin sectional modules promote efficient nutrition and waste removal due to a shorter diffusion distance. The modules were pre-vascularized viain-situangiogenesis, achieved through endothelial cell sprouting from the scaffold struts. Angiogenesis was further enhanced through co-culture with bioprinted fibroblast microtissues, which secreted pre-angiogenic growth factors. Sectional modules were assembled around a porous rod incorporated with Bone Morphogenetic Protein-2 (BMP-2), which released over 3 weeks, demonstrating sustained osteogenic activity. The assembled scaffold, in the anatomical shape of a human femur shaft, was pre-vascularized, osteogenic, and possessed high mechanical strength, supporting 12 times the average body weight. The feasibility of implanting the assembled bone graft was demonstrated using a 3D-printed femur bone defect model. Our method provides a novel modular engineering approach for regenerating tissues that require high mechanical strength and vascularization.

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids.

Introduction: Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue. Methods: dROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects. Results: Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01). Discussion: With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

Rational Design and Acoustic Assembly of Human Cerebral Cortex-Like Microtissues from hiPSC-Derived Neural Progenitors and Neurons.

Development of biologically relevant and clinically relevant human cerebral cortex models is demanded by mechanistic studies of human cerebral cortex-associated neurological diseases and discovery of preclinical neurological drug candidates. Here, rational design of human-sourced brain-like cortical tissue models is demonstrated by reverse engineering and bionic design. To implement this design, the acoustic assembly technique is employed to assemble hiPSC-derived neural progenitors and neurons separately in a label-free and contact-free manner followed by subsequent neural differentiation and culture. The generated microtissues encapsulate the neuronal microanatomy of human cerebral-cortex tissue that contains six-layered neuronal architecture, a 400-µm interlayer distance, synaptic connections between interlayers, and neuroelectrophysiological transmission. Furthermore, these microtissues are infected with herpes simplex virus type I (HSV-1) virus, and the HSV-induced pathogenesis associated with Alzheimer's disease is determined, including neuron loss and the expression of Aß. Overall, a high-fidelity human-relevant in vitro histotypic model is provided for the cerebral cortex, which will facilitate wide applications in probing the mechanisms of neurodegenerative diseases and screening the candidates for neuroprotective agents.

Research Dynamics of Tissue Spheroids as Building Blocks: A Scientometric Analysis.

Tissue spheroids represent an innovative solution for tissue engineering and regenerative medicine. They constitute an in vitro three-dimensional cell culture model capable of mimicking the complex composition of a native tissue on a micro-scale; this model can function as a building block and be assembled into larger tissue constructs. Due to the potential tissue spheroids have for the evolution of the health industry, there is a need to assess the research dynamics of this field. Thus far, there have been no studies on their use as building blocks. To fill this gap, a study was performed to characterize the evolution of research where tissue spheroids were used as building blocks to generate tissue constructs. A scientometric analysis of the literature regarding tissue spheroid technologies was developed by quantification of bibliometric performance indicators. For this purpose, articles published during the period January 1, 2015 - December 31, 2021, from the Scopus database were organized and analyzed. The main subject areas, countries, cities, journals, institutions, and top-cited articles as well as the types of techniques, cells, culture time, and principal applications were identified. This research supports the definition and growth of research and development strategies for new technologies such as tissue spheroids.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Articular Cartilage Regeneration through Bioassembling Spherical Micro-Cartilage Building Blocks.

Articular cartilage lesions are prevalent and affect one out of seven American adults and many young patients. Cartilage is not capable of regeneration on its own. Existing therapeutic approaches for articular cartilage lesions have limitations. Cartilage tissue engineering is a promising approach for regenerating articular neocartilage. Bioassembly is an emerging technology that uses microtissues or micro-precursor tissues as building blocks to construct a macro-tissue. We summarize and highlight the application of bioassembly technology in regenerating articular cartilage. We discuss the advantages of bioassembly and present two types of building blocks: multiple cellular scaffold-free spheroids and cell-laden polymer or hydrogel microspheres. We present techniques for generating building blocks and bioassembly methods, including bioprinting and non-bioprinting techniques. Using a data set of 5069 articles from the last 28 years of literature, we analyzed seven categories of related research, and the year trends are presented. The limitations and future directions of this technology are also discussed.

In-situ bio-assembled specific Au NCs-Aptamer-Pyro conjugates nanoprobe for tumor imaging and mitochondria-targeted photodynamic therapy.

Mitochondrion has emerged as a promising drug target for photodynamic therapy (PDT), due to its significant role in supporting life activities and being reactive oxygen species (ROS)-sensitive. Herein, we establish a new strategy that in-situ bio-synthesized Au NCs combine with mitochondria-targeted aptamer-Pyro conjugates (ApPCs) for specific tumor imaging and PDT. The prepared ApPCs can serve as template for the in-situ bio-synthesis of Au NCs, thereby facilitating the generation of Au NCs-ApPCs assemblies in unique tumor microenvironment. Compared with highly negatively charged ApPCs, bio-synthesized nanoscale Au NCs-ApPCs assemblies are conducive to cell uptake, which consequently benefits the delivery of ApPCs. After dissociated from Au NCs-ApPCs, internalized ApPCs can selectively accumulate in mitochondria and generate excess ROS to disrupt the mitochondrial membrane upon irradiation, thus inducing efficient cell killing. In vitro assays demonstrated that the fluorescent Au NCs-ApPCs assemblies could be specifically produced in cancerous cells, indicating the specific tumor imaging ability, while intracellular ApPCs co-localized well with mitochondria. CCK-8 results revealed over 80% cell death after PDT. In vivo study showed that fluorescent Au NCs-ApPCs assemblies were exclusively generated in tumor and achieved long-term retention; tumor growth was significantly inhibited after 15-day PDT treatment. All these evidences suggest that in-situ bio-synthesized Au NCs-ApPCs assembly is a potent mitochondria-targeted nanoprobe to boost the PDT efficacy of cancers.