Thyrotropin inhibits osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) are derived from the periodontal ligament and have the characteristics of pluripotent differentiation, including osteogenesis, and are one of the important seed cells in oral tissue engineering. Thyrotropin (TSH) has been shown to regulate bone metabolism independently of thyroid hormone, including the fate of osteoblasts and osteoclasts, but whether it affects osteogenic differentiation of PDLSCs is unknown. MATERIALS AND METHODS: PDLSCs were isolated and cultured from human periodontal ligament and grown in osteogenic medium (containing sodium ß-glycerophosphate, ascorbic acid, and dexamethasone). Recombinant human TSH was added to the culture medium. Osteogenic differentiation of PDLSCs was assessed after 14 days by staining with alkaline phosphatase and alizarin red and by detection of osteogenic differentiation genes. Differentially expressed genes (DEGs) in PDLSCs under TSH were detected by high-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzed the biological functions and signaling pathways involved in DEGs. RESULTS: We found that osteogenic differentiation of PDLSCs was significantly inhibited in the presence of TSH: including decreased calcium nodule formation, decreased alkaline phosphatase levels, and decreased collagen synthesis. Using high-throughput sequencing, we found changes in the expression of some osteogenesis-related genes, which may be the reason that TSH inhibits osteogenic differentiation of PDLSCs. CONCLUSION: Unless TSH is ≥10 mU/L, patients with subclinical hypothyroidism usually do not undergo thyroxine supplementation therapy. However, in this work, we found that elevated TSH inhibited the osteogenic differentiation of PDLSCs. Therefore, correction of TSH levels in patients with subclinical hypothyroidism may be beneficial to improve orthodontic, implant, and periodontitis outcomes in these patients.

Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

A comparative experimental study of the anaerobic treatment of food wastes using an anaerobic digester with a polyamide stirring rake or a stainless-steel stirring rake.

A low treatment capacity and unstable operation are the main drawbacks of the anaerobic digestion of food wastes. The present work improved the efficiency and stabilization of the anaerobic digestion of food wastes using digesters with a polyamide stirring rake (DPSR) and compared it to a traditional digester with a stainless-steel stirring rake (DSSSR). The DPSR had a higher reliability and produced 3.97 times the methane yield of DSSSR in batch experiments at high loading rates (105 VS/L). Uniform design experiments were applied to investigate the relationship between methane yield and the stirring factors of the DPSR. A regression analysis of the uniform design indicated that stirring factors synergistically affect methane yield. The experiment verifying the optimal conditions showed that in the DPSR with 82 r/min stirring intensity and 10 min/d stirring time, the first 20 days of methane yield (392.1 mL/g VS) achieved to 85.26% of the theoretically derived methane yield. In brief, in the anaerobic digestion of food wastes for high methane production and stable operation, the DPSR was more beneficial for the anaerobic digestion of food wastes than the DSSSR.

Glycerol-mediated nanostructure modification leading to improved transparency of porous polymeric scaffolds for high performance 3D cell imaging.

Glycerol is among the most commonly used optical clearing agents for tissues clearance largely due to refractive index (RI) matching between glycerol and the submerged tissues. Here we applied glycerol as structure modifier at both macroscopic (as porogen) and nanoscopic (as nanostructure ameliorant) scales to fabricate transparent porous scaffolds made from poly(ethylene glycol) (PEG) as well as other widely used biomaterials (e.g., PLGA, PA, or gelatin), whose nanostructures, in the scale of light wavelength, dominantly improved the optical transmittance of the scaffolds even when immersed in RI mismatched medium (e.g., culture medium or water). We further exploited the clearing mechanisms based on Mie scattering theory, illustrating that conformational changes of polymer chains induced by solvent effects of glycerol enhanced the anisotropy (i.e., directional alignment) of the nanostructures, leading to reduced crystallinity and scattering of the resulted PEG scaffolds. Our findings represent the first and systematic demonstration with both experimental and theoretical evidence in effectively clearing porous polymeric scaffolds by mechanisms other than RI matching, which could tackle the limitations of current optical imaging of cells cultured within three-dimensional (3D) opaque porous scaffolds, such as poor visibility, low spatial resolution, and small penetration depth.

Formation and characterization of konjac glucomannan/ethyl cellulose films by using ethanol and water as the solvents.

Hydrophilic konjac glucomannan (KGM)/hydrophobic ethyl cellulose (EC) film was prepared in the ethanol/water environment. The film-forming solution and film properties were both characterized to analyze the molecular interaction changes. Although higher ethanol usage enhanced the stability of the film-forming solution, it did not benefit the film property improvement. The SEM images showed some fibrous structure on the air surface of the films, consistent with the XRD results. The changing trend of mechanical properties and the FTIR results suggested that both ethanol content and ethanol evaporation impacted the molecular interaction during the film formation. The surface hydrophobicity results indicated that the ethanol content could cause significant EC aggregation changes on the film surface only with high EC contents. The water vapor permeability results suggested that higher ethanol usage decreased the compactness of the films. Considering all results, the 20 % ethanol content and the weight ratio of KGM: EC = 7:3 were suggested for the film preparation due to the superior properties in most properties. This study contributed to the understanding of polysaccharide interaction in the ethanol/water environment and offered an alternative biodegradable packaging film.

Cell membrane inspired nano-shell enabling long-acting Glucose Oxidase for Melanoma starvation therapy via microneedles-based percutaneous delivery.

Rationale: Glucose oxidase (GOx) has gained tremendous research interest recently as a glucose-consuming enzyme for tumor starvation therapy, while its in vivo applications are strictly limited by rapid deactivation, as well as side effects of non-specific catalysis. Methods: To address these issues, here we report a protective nano-shell to encapsule GOx for localized melanoma therapy delivered by dissolving microneedles (MNs). Inspired by cell membrane that separates and protects cell organelles and components from outside environment while selectively ingesting nutrition sources, we designed polydopamine (PDA)-structured nano-shell to allow free transportation of glucose for catalytic reaction, while impede the penetration of GOx, proteinase, and other GOx-deactivating macromolecules across the shell membrane. Results: GOx was well protected in core layer with persistent catalytic activity for at least 6 d under various biological matrixes (e.g., PBS, serum, and cell lysate) and surviving different harsh conditions (e.g., acid/base treatments, and proteinase-induced degradation). Such long-acting nano-catalyst can be easily integrated into MNs as topical delivery carrier for effective glucose consumption in melanoma tissue, achieving significant tumor growth inhibition via starvation therapy with minimized side effects as compared to systemic administration. Conclusion: This work provides an elegant platform for in vivo delivery of GOx, and our cell-mimicking nano-system can also be applied for other enzyme-based therapeutics.

Preconditioning of mesenchymal stromal cells toward nucleus pulposus-like cells by microcryogels-based 3D cell culture and syringe-based pressure loading system.

To precondition mesenchymal stromal/stem cells (MSCs) with mechanical stimulation may enhance cell survival and functions following implantation in load bearing environment such as nucleus pulposus (NP) in intervertebral disc (IVD). In this study, preconditioning of MSCs toward NP-like cells was achieved in previously developed poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PMs) within a syringe-based three-dimensional (3D) culture system which provided a facile and cost-effective pressure loading approach. PMs loaded with alginate and MSCs could be incubated in a sealable syringe which could be air-compressed to apply pressure loading through a programmable syringe pump. Expression levels of chondrogenic marker genes SOX9, COL II, and ACAN were significantly upregulated in MSCs when pressure loading of 0.2 MPa or 0.8 MPa was implemented. Expression levels of COL I and COL X were downregulated when pressure loading was applied. In a nude mouse model, MSCs loaded in PMs mechanically stimulated for three days were subcutaneously injected using the same culture syringe. Three weeks postinjection, more proteoglycans (PGs) were deposited and more SOX9 and COL II but less COL I and COL X were stained in 0.2 MPa group. Furthermore, injectable MSCs-loaded PMs were utilized in an ex vivo rabbit IVD organ culture model that demonstrated the leak-proof function and enhanced cell retention of PMs assisted cell delivery to a load bearing environment for potential NP regeneration. This microcryogels-based 3D cell culture and syringe-based pressure loading system represents a novel method for 3D cell culture with mechanical stimulation for better function. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 507-520, 2017.

Fluoride and arsenic exposure affects spatial memory and activates the ERK/CREB signaling pathway in offspring rats.

Fluoride and arsenic are inorganic contaminants that occur in the natural environment. Chronic fluoride and/or arsenic exposure can induce developmental neurotoxicity and negatively influence intelligence in children, although the underlying molecular mechanisms are poorly understood. This study explored the effects of fluoride and arsenic exposure in drinking water on spatial learning, memory and key protein expression in the ERK/CREB signaling pathway in hippocampal and cerebral cortex tissue in rat offspring. Pregnant rats were divided into four groups. Control rats drank tap water, while rats in the three exposure groups drank water with sodium fluoride (100mg/L), sodium arsenite (75mg/L), and a sodium fluoride (100mg/L) and sodium arsenite (75mg/L) combination during gestation and lactation. After weaning, rat pups drank the same solution as their mothers. Spatial learning and memory ability of pups at postnatal day 21 (PND21) and postnatal day 42 (PND42) were measured using a Morris water maze. ERK, phospho-ERK (p-ERK), CREB and phospho-CREB (p-CREB) protein expression in the hippocampus and cerebral cortex was detected using Western blot. Compared with the control pups, escape latencies increased in PND42 pups exposed to arsenic and co-exposed to fluoride and arsenic, and the short-term and long-term spatial memory ability declined in pups exposed to fluoride and arsenic, both alone and in combination. Compared with controls, ERK and p-ERK levels decreased in the hippocampus and cerebral cortex in pups exposed to combined fluoride and arsenic. CREB protein expression in the cerebral cortex decreased in pups exposed to fluoride, arsenic, and the fluoride and arsenic combination. p-CREB protein expression in both the hippocampus and cerebral cortex was decreased in pups exposed to fluoride and arsenic in combination compared to the control group. There were negative correlation between the proteins expression and escape latency periods in pups. These data indicate that exposure to fluoride and arsenic in early life stage changes ERK, p-ERK, CREB and p-CREB protein expression in the hippocampus and cerebral cortex of rat offspring at PND21 and PND 42, which may contribute to impaired neurodevelopment following exposure.

Comparison of Calcium and Barium Microcapsules as Scaffolds in the Development of Artificial Dermal Papillae.

This study aimed to develop and evaluate barium and calcium microcapsules as candidates for scaffolding in artificial dermal papilla. Dermal papilla cells (DPCs) were isolated and cultured by one-step collagenase treatment. The DPC-Ba and DPC-Ca microcapsules were prepared by using a specially designed, high-voltage, electric-field droplet generator. Selected microcapsules were assessed for long-term inductive properties with xenotransplantation into Sprague-Dawley rat ears. Both barium and calcium microcapsules maintained xenogenic dermal papilla cells in an immunoisolated environment and induced the formation of hair follicle structures. Calcium microcapsules showed better biocompatibility, permeability, and cell viability in comparison with barium microcapsules. Before 18 weeks, calcium microcapsules gathered together, with no substantial immune response. After 32 weeks, some microcapsules were near inflammatory cells and wrapped with fiber. A few large hair follicles were found. Control samples showed no marked changes at the implantation site. Barium microcapsules were superior to calcium microcapsules in structural and mechanical stability. The cells encapsulated in hydrogel barium microcapsules exhibited higher short-term viability. This study established a model to culture DPCs in 3D culture conditions. Barium microcapsules may be useful in short-term transplantation study. Calcium microcapsules may provide an effective scaffold for the development of artificial dermal papilla.

Effects of Polyamidoamine Dendrimers on a 3-D Neurosphere System Using Human Neural Progenitor Cells.

The practical application of engineered nanomaterials or nanoparticles like polyamidoamine (PAMAM) dendrimers has been promoted in medical devices or industrial uses. The safety of PAMAM dendrimers needs to be assessed when used as a drug carrier to treat brain disease. However, the effects of PAMAM on the human nervous system remain unknown. In this study, human neural progenitor cells cultured as a 3D neurosphere model were used to study the effects of PAMAM dendrimers on the nervous system. Neurospheres were exposed to different G4-PAMAM dendrimers for 72 h at concentrations of 0.3, 1, 3, and 10 µg/ml. The biodistribution was investigated using fluorescence-labeled PAMAM dendrimers, and gene expression was evaluated using microarray analysis followed by pathway and network analysis. Results showed that PAMAM dendrimer nanoparticles can penetrate into neurospheres via superficial cells on them. PAMAM-NH2 but not PAMAM-SC can inhibit neurosphere growth. A reduced number of MAP2-positive cells in flare regions were inhibited after 10 days of differentiation, indicating an inhibitory effect of PAMAM-NH2 on cell proliferation and neuronal migration. A microarray assay showed 32 dendrimer toxicity-related genes, with network analysis showing 3 independent networks of the selected gene targets. Inducible immediate early gene early growth response gene 1 (Egr1), insulin-like growth factor-binding protein 3 (IGFBP3), tissue factor pathway inhibitor (TFPI2), and adrenomedullin (ADM) were the key genes in each network, and the expression of these genes was significantly down regulated. These findings suggest that exposure of neurospheres to PAMAM-NH2 dendrimers affects cell proliferation and migration through pathways regulated by Egr1, IGFBP3, TFPI2, and ADM.

Preformed gelatin microcryogels as injectable cell carriers for enhanced skin wound healing.

Wound dressings of cell-laden bulk hydrogel or scaffold were mainly applied for enhanced cell engraftment in contrast to free cell injection. However, dressing of cells laden in biomaterials on wound surface might not effectively and timely exert functions on deep or chronic wounds where insufficient blood supply exists. Previously, we developed injectable gelatin microcryogels (GMs) which could load cells for enhanced cell delivery and cell therapy. In this study, biological changes of human adipose-derived stem cells (hASCs) laden in GMs were compared in varied aspects with traditional two dimensional (2D) cell culture, such as cell phenotype markers, stemness genes, differentiation, secretion of growth factors, cell apoptosis and cell memory by FACS, QRT-PCR and ELISA, that demonstrated the priming effects of GMs on upregulation of stemness genes and improved secretion of growth factors of hASCs for potential augmented wound healing. In a full-thickness skin wound model in nude mice, multisite injection and dressing of hASCs-laden GMs could significantly accelerate the healing compared to free cell injection. Bioluminescence imaging and protein analysis indicated improved cell retention and secretion of multiple growth factors. Our study suggests that GMs as primed injectable 3D micro-niches represent a new cell delivery methodology for skin wound healing which could not only benefit on the recovery of wound bed but also play direct effects on wound basal layer for healing enhancement. Injectable GMs as facile multisite cell delivery approach potentially provide new minimally-invasive therapeutic strategy for refractory wounds such as diabetic ulcer or radiative skin wound. STATEMENT OF SIGNIFICANCE: This work applied a type of elastic micro-scaffold (GMs) to load and prime hMSCs for skin wound healing. Due to the injectability of GMs, the 3D cellular micro-niches could simply realize minimally-invasive and multisite cell delivery approach for accelerating the wound healing process superior to free cell injection. The biological features of MSCs has been thoroughly characterized during 3D culture in GMs (i.e. cell proliferation, characterization of cell surface markers, stemness of MSCs in GMs, differentiation of MSCs in GMs, secretion of MSCs in GMs, induced apoptosis of MSCs in GMs). Multiple methods such as bioluminescent imaging, immunohistochemistry, immunofluorescence, qRT-PCR, ELSA and western blot were used to assess the in vivo results between groups.

Microcryogels as injectable 3-D cellular microniches for site-directed and augmented cell delivery.

The success of cell therapy for tissue repair and regeneration demands efficient and reliable cell delivery methods. Here we established a novel microengineered cryogel (microcryogel) array chip containing microcryogels with predefined size and shape as injectable cell delivery vehicles. The microscale macroporous cryogels enabled automatic and homogeneous loading of tailored cellular niches (e.g. cells, matrices, bioactive factors) and could be easily harvested from the ready-to-use array chip. In contrast to microscale hydrogels, microcryogels exhibited excellent elasticity and could retain their shape and integrity after injection through the microsyringe routinely used for cell therapy. Human mesenchymal stromal cells loaded within microcryogels could be shielded from the mechanical insult and necrosis caused by during direct cell injection. After subcutaneous injection to the mice, cell-loaded microcryogels exhibited concentrated localization and enhanced retention at the injection site compared to dissociated cells. To demonstrate the potential therapeutic application for ischemic diseases, site-directed induction of angiogenesis was achieved subcutaneously in mice 2weeks after injection of NIH/3T3 fibroblast-loaded microcryogels, indicating long-term engraftment, accumulative paracrine stimulation and augmented host tissue integration. Our results convincingly showed the great promise of microcryogels as 3-D cellular microniches and injectable cell delivery vehicles to tackle major challenges faced by cell therapy-based regenerative medicine including shear-induced damages, uncontrolled localization, poor retention, limited cellular survival and functionalities in vivo.