P3HT:Bebq2-Based Photovoltaic Device Enhances Differentiation of hiPSC-Derived Retinal Ganglion Cells.

Electric field stimulation is known to affect various cellular processes, including cell fate specification and differentiation, particularly towards neuronal lineages. This makes it a promising therapeutic strategy to stimulate regeneration of neuronal tissues. Retinal ganglion cells (RGCs) is a type of neural cells of the retina responsible for transduction of visual signals from the retina to the brain cortex, and is often degenerated in various blindness-causing retinal diseases. The organic photovoltaic materials such as poly-3-hexylthiophene (P3HT) can generate electric current upon illumination with light of the visible spectrum, and possesses several advantageous properties, including light weight, flexibility and high biocompatibility, which makes them a highly promising tool for electric stimulation of cells in vitro and in vivo. In this study, we tested the ability to generate photocurrent by several formulations of blend (bulk heterojunction) of P3HT (which is electron donor material) with several electron acceptor materials, including Alq3 and bis(10-hydroxybenzo[h]quinolinato)beryllium (Bebq2). We found that the photovoltaic device based on bulk heterojunction of P3HT with Bebq2 could generate photocurrent when illuminated by both green laser and visible spectrum light. We tested the growth and differentiation capacity of human induced pluripotent stem cells (hiPSC)-derived RGCs when grown in interface with such photostimulated device, and found that they were significantly increased. The application of P3HT:Bebq2-formulation of photovoltaic device has a great potential for developments in retinal transplantation, nerve repair and tissue engineering approaches of treatment of retinal degeneration.

Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization.

Age-related macular degeneration (AMD) is the eye disease with the highest epidemic incidence, and has great impact on the aged population. Wet-type AMD commonly has the feature of neovascularization, which destroys the normal retinal structure and visual function. So far, effective therapy options for rescuing visual function in advanced AMD patients are highly limited, especially in wet-type AMD, in which the retinal pigmented epithelium and Bruch's membrane structure (RPE-BM) are destroyed by abnormal angiogenesis. Anti-VEGF treatment is an effective remedy for the latter type of AMD; however, it is not a curative therapy. Therefore, reconstruction of the complex structure of RPE-BM and controlled release of angiogenesis inhibitors are strongly required for sustained therapy. The major purpose of this study was to develop a dual function biomimetic material, which could mimic the RPE-BM structure and ensure slow release of angiogenesis inhibitor as a novel therapeutic strategy for wet AMD. We herein utilized plasma-modified polydimethylsiloxane (PDMS) sheet to create a biomimetic scaffold mimicking subretinal BM. This dual-surface biomimetic scaffold was coated with laminin and dexamethasone-loaded liposomes. The top surface of PDMS was covalently grafted with laminin and used for cultivation of the retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSC-RPE). To reach the objective of inhibiting angiogenesis required for treatment of wet AMD, the bottom surface of modified PDMS membrane was further loaded with dexamethasone-containing liposomes via biotin-streptavidin linkage. We demonstrated that hiPSC-RPE cells could proliferate, express normal RPE-specific genes and maintain their phenotype on laminin-coated PDMS membrane, including phagocytosis ability, and secretion of anti-angiogenesis factor PEDF. By using in vitro HUVEC angiogenesis assay, we showed that application of our membrane could suppress oxidative stress-induced angiogenesis, which was manifested in decreased secretion of VEGF by RPE cells and suppression of vascularization. In conclusion, we propose modified biomimetic material for dual delivery of RPE cells and liposome-enveloped dexamethasone, which can be potentially applied for AMD therapy.

Development of a Graphene Oxide-Incorporated Polydimethylsiloxane Membrane with Hexagonal Micropillars.

Several efforts have been made on the development of bioscaffolds including the polydimethylsiloxane (PDMS) elastomer for supporting cell growth into stable sheets. However, PDMS has several disadvantages, such as intrinsic surface hydrophobicity and mechanical strength. Herein, we generated a novel PDMS-based biomimetic membrane by sequential modifications of the PMDS elastomer with graphene oxide (GO) and addition of a hexagonal micropillar structure at the bottom of the biomembrane. GO was initially homogenously mixed with pure PDMS and then was further coated onto the upper surface of the resultant PDMS. The elastic modulus and hydrophilicity were significantly improved by such modifications. In addition, the development of hexagonal micropillars with smaller diameters largely improved the ion permeability and increased the motion resistance. We further cultured retinal pigment epithelial (RPE) cells on the surface of this modified PDMS biomembrane and assayed its biocompatibility. Remarkably, the GO incorporation and coating exhibited beneficial effect on the cell growth and the new formation of tight junctions in RPE cells. Taken together, this GO-modified PDMS scaffold with polyhexagonal micropillars may be utilized as an ideal cell sheet and adaptor for cell cultivation and can be used in vivo for the transplantation of cells such as RPE cells.

Using cationic polyurethane-short branch PEI as microRNA-driven nano-delivery system for stem cell differentiation.

BACKGROUND: Non-viral gene delivery, such as using biodegradable polyurethane short-branch polyethylenimine (PU-PEI), has been considered a potentially safer gene delivery system in comparison to conventional virus systems. METHODS: The polycationization of DNA complexes protects DNA from nuclease degradation, and these DNA complexes are nanoscale in size to enter the cell through endocytosis. RESULTS: Due to the net positive surface charge of the cell, these polyplexes efficiently bind to the cell through electrostatic interactions with negatively charged membrane components. Cationic PU-PEI has been shown to be non-cytotoxic and has a high transfection efficiency, making it a practical gene delivery material in diseases. CONCLUSION: We developed a PU-PEI nanomedicine-based platform to efficiently deliver microRNA in promoting differentiation capacity of stem cells, especially on induced pluripotent stem cells.

Development of polydimethylsiloxane-based biomimetic scaffolds with cylinder micropillars for retinal pigment epithelial cell cultivation.

BACKGROUND: Age-related macular degeneration (AMD) is one of the leading causes of vision loss. Once the retinal pigment epithelium (RPE) layers are destroyed, the poor visual acuity and recognition are generally irreversible. Cell therapy that possesses enormous potential in regenerative medicine may provide an alternative treatment for several incurable diseases such as AMD. In this study, we developed an innovative polydimethylsiloxane (PDMS)-based biomimetic scaffolds with cylinder micropillars for the cultivation of induced pluripotent stem cell-derived RPEs (iPSC-RPEs). RPEs were cultured on the PDMS-based biomimetic scaffolds and validated the cells gene expression. METHODS: The biomimetic PDMS scaffold was fabricated through spin coating and lithography method. It was further modified on surface with biomolecules to improve cell affinity and stability. The iPSC-RPEs were seeded on the scaffold and analyzed with characteristic gene expression. RESULTS: PDMS biomimetic scaffold was analyzed with Fourier transform infrared spectroscopy and proved its chemical composition. iPSC-RPEs demonstrated confluent cell monolayer on the scaffold and maintained RPE-specific gene expression, which proved the PDMS-based biomimetic scaffold to be supportive for iPSC-RPEs growth. CONCLUSION: The PDMS interface allowed regular growth of iPSC-RPEs and the design of cylinder micropillars further provided the bioscaffold high motion resistance may improve the engraftment stability of iPSC-RPEs after transplantation. Taken together, this innovative PDMS-based biomimetic scaffold may serve as an ideal interface for in vitro iPSC-RPE cultivation and subsequent transplantation in vivo. This novel device exhibits better bioavailability than conventional injection of donor cells and may be an alternative option for the treatment of AMD.

Nanomedicine-based Curcumin Approach Improved ROS Damage in Best Dystrophy-specific Induced Pluripotent Stem Cells.

Best dystrophy (BD), also termed best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and can cause central visual loss. Unfortunately, there is no clear definite therapy for BD or improving the visual function on this progressive disease. The human induced pluripotent stem cell (iPSC) system has been recently applied as an effective tool for genetic consultation and chemical drug screening. In this study, we developed patient-specific induced pluripotent stem cells (BD-iPSCs) from BD patient-derived dental pulp stromal cells and then differentiated BD-iPSCs into retinal pigment epithelial cells (BD-RPEs). BD-RPEs were used as an expandable platform for in vitro candidate drug screening. Compared with unaffected sibling-derived iPSC-derived RPE cells (Ctrl-RPEs), BD-RPEs exhibited typical RPE-specific markers with a lower expression of the tight junction protein ZO-1 and Bestrophin-1 (BEST1), as well as reduced phagocytic capabilities. Notably, among all candidate drugs, curcumin was the most effective for upregulating both the BEST1 and ZO-1 genes in BD-RPEs. Using the iPSC-based drug-screening platform, we further found that curcumin can significantly improve the mRNA expression levels of Best gene in BD-iPSC-derived RPEs. Importantly, we demonstrated that curcumin-loaded PLGA nanoparticles (Cur-NPs) were efficiently internalized by BD-RPEs. The Cur-NPs-based controlled release formulation further increased the expression of ZO-1 and Bestrophin-1, and promoted the function of phagocytosis and voltage-dependent calcium channels in BD-iPSC-derived RPEs. We further demonstrated that Cur-NPs enhanced the expression of antioxidant enzymes with a decrease in intracellular ROS production and hydrogen peroxide-induced oxidative stress. Collectively, these data supported that Cur-NPs provide a potential cytoprotective effect by regulating the anti-oxidative abilities of degenerated RPEs. In addition, the application of patient-specific iPSCs provides an effective platform for drug screening and personalized medicine in incurable diseases.

Nuclear Localization Signal-Enhanced Polyurethane-Short Branch Polyethylenimine-Mediated Delivery of Let-7a Inhibited Cancer Stem-Like Properties by Targeting the 3'-UTR of HMGA2 in Anaplastic Astrocytoma.

Anaplastic astrocytoma (AA) is a grade III glioma that often occurs in middle-aged patients and presents a uniformly poor prognosis. A small subpopulation of cancer stem cells (CSCs) possessing a self-renewing capacity is reported to be responsible for tumor recurrence and therapeutic resistance. An accumulating amount of microRNAs (miRNA) were found aberrantly expressed in human cancers and regulate CSCs. Efforts have been made to couple miRNAs with nonviral gene delivery approaches to target specific genes in cancer cells. However, the efficiency of delivery of miRNAs to AA-derived CSCs is still an applicability hurdle. The present study aimed to investigate the effectiveness and applicability of nonviral vector-mediated delivery of Let-7a with regard to eradication of AA and AA-derived CSC cells. Herein, our miRNA/mRNA microarray and RT-PCR analysis showed that the expression of Let-7a, a tumor-suppressive miRNA, is inversely correlated with the levels of HMGA2 and Sox2 in the AA side population (SP(+)) cells. Luciferase reporter assay showed that Let-7a directly targets the 3'-UTRs of HMGA2 in AA-SP(+) cells. Knockdown of HMGA2 significantly suppressed the protein expression of Sox2 in AA-SP(+) cells, whereas overexpression of HMGA2 upregulated Sox2 expression in AA-SP(-). Nuclear localization signal (NLS) peptides can facilitate nuclear targeting of DNA and are used to improve gene delivery. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic delivery vehicle, we conjugated NLS with Let-7 and successfully delivered it to AA-SP(+) cells, resulting in significantly suppressed expression of HMGA2 and Sox2, tumorigenicity, and CSC-like abilities. This treatment facilitated the differentiation of AA-SP(+) cells into non-SP CSCs. Furthermore, PU-PEI-mediated delivery of NLS-conjugated Let-7a in AA-SP(+) cells suppressed the expression of drug-resistant and antiapoptotic genes, and increased cell sensitivity to radiation. Finally, the in vivo delivery of PU-PEI-NLS-Let-7a significantly suppressed the tumorigenesis of AA-SP(+) cells and synergistically improved the survival rate of orthotopically AA-SP(+)-transplanted immunocompromised mice when combined with radiotherapy. Therefore, PU-PEI-NLS-Let-7a is a potential novel therapeutic approach for AA.

Bovine lactoferrin and piroxicam as an adjunct treatment for lymphocytic-plasmacytic gingivitis stomatitis in cats.

Feline lymphocytic-plasmacytic gingivitis/stomatitis (LPGS) or caudal stomatitis is an inflammatory disease that causes painfully erosive lesions and proliferations of the oral mucosa. The disease is difficult to cure and can affect cats at an early age, resulting in lifetime therapy. In this study, a new treatment using a combination of bovine lactoferrin (bLf) oral spray and oral piroxicam was investigated using a randomized double-blinded clinical trial in 13 cats with caudal stomatitis. Oral lesion grading and scoring of clinical signs were conducted during and after the trial to assess treatment outcome. Oral mucosal biopsies were used to evaluate histological changes during and after treatment. Clinical signs were significantly improved in 77% of the cats. In a 4-week study, clinical signs were considerably ameliorated by oral piroxicam during the first 2 weeks. In a 12-week study, the combined bLf oral spray and piroxicam, when compared with piroxicam alone, exhibited an enhanced effect that reduced the severity of the oral lesions (P = 0.059), while also significantly improving clinical signs (P <0.05), quality of life (P <0.05), and weight gain (P <0.05). The remission of oral inflammation was closely correlated with the decreased number of macrophages (OR = 4.719, P < 0.05). There was no detectable influence on liver or kidney function during a 12-week assessment. It was concluded that combining oral bLf spray and piroxicam was safe and might be used to decrease the clinical signs of caudal stomatitis in cats.

Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microRNA145 with cationic polyurethane-short branch PEI.

Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133(+) cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133(+). Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133(+) significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133(-)-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133(+) effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133(+)-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.

[Pulp revascularization of immature anterior teeth with apical periodontitis].

OBJECTIVE: To compare the therapeutic efficacy both apexification and revascularization in the immature anterior teeth of animal model with apical periodontitis, and observe the histological situation of revascularization in the root canal. METHODS: Six immature anterior teeth of one animal model (dog) aged approximately 4.5 months was selected. Afterwards, periapical periodontitis pattern were established, the samples were randomly divided into the experimental group (revascularization, 3 teeth) and the control group (apexification, 3 teeth). To compare the development of root and the healing of periapical inflammation, the involved teeth were respectively radiographed 1, 4, 8 weeks after surgery. The animals were sacrificed after 8 weeks, and the closure of apical foramen and the content of root canal were observed by hematine-eosine (HE) staining. RESULTS: The postoperative radiography after 1 week and 4 weeks, the apical foramen size and the periapical radiolucency of the samples was shown no perceptual change. After 8 weeks, the experimental group periapical radiolucency area was obviously more narrowing, and had a apical closure tendency whereas the thickness of the root canal walls had imperceptible changed. While the control group periapical radiolucency change varied. The granulation tissue could be seen within the lumen of the experimental group, which contained a large number of irregular calcification, the calcification was obvious in the apical and adjacent the root canal wall. A small quantity of hard tissue was deposited in the apical of the control group. CONCLUSION: Revascularization may increase the recovery of immature anterior teeth with chronic periapical inflammation, the vital regenerative tissue within root canal is the granulation tissue contained calcification.

Culturing adult human bone marrow stem cells on gelatin scaffold with pNIPAAm as transplanted grafts for skin regeneration.

Skin tissue engineering is a possible solution for the treatment of extensive skin defect. The ultimate goal of skin tissue engineering is to restore the complete functions of native skin, but until now the structures and functions of skins are only partially restored. By negative immunoselection (CD45 and glycophorin A), we isolated and cultivated adult human bone marrow stem cells (hBMSCs) that are of multilineage differentiation potential. In this study, we first demonstrated that by using gelatin/thermo-sensitive poly N-isopropylacrylamide (pNIPAAm) and the immunocompromised mice model, the hBMSCs possess the differentiation potential of epidermis and the capability of healing skin wounds. The in vitro observations and the results of the scanning electron microscope showed that the hBMSCs can attach and proliferate in the gelatin/thermo-sensitive pNIPAAm. To further monitor the in vivo growth effect of the hBMSCs in the skin-defected nude mice, the green fluorescence protein (GFP) gene was transduced into the hBMSCs by the murine stem cell viral vector. The results showed that the rates of cell growth and wound recovery in the hBMSC-treated group were significantly higher than those in the control group, which was only treated with the gelatin/pNIPAAm (p < 0.01). More importantly, the re-epithelialization markers of human pan-cytokeratin and E-cadherin were significantly increased on day 7, day 14, and day 21 after the hBMSC-scaffold with the pNIPAAM in the mice with skin defects (p < 0.05). Moreover, the stem cell markers of human CD13 and CD105 were gradually decreased during the period of wound healing. In sum, this novel method provides a transferring system for cell therapies and maintains its temperature-sensitive property of easy-peeling by lower-temperature treatment. In addition, the in vitro and in vivo GFP imaging systems provide a new imaging modality for understanding the differentiation process and the effective expression of stem cells in wound healing.

[The effect of overdose fluoride on the expression of TGF-beta1 in rat's dental pulps].

PURPOSE: To observe the effect of overdose fluoride on the expression of TGF-beta1 in rat's dental pulps. METHODS: 20 wister rats were divided into two groups. In the control group, equal dose distilled water were given to the rats. In the experimental group, 20 mg.kg(-1).d(-1) NaF were given. After 8 weeks of treatment, immunohistochemical staining was adopted for detection of the expression of TGF-beta1 in dental pulps of the rats. SPSS10.0 software package was used for Student's t test. RESULTS: Image analysis results showed that the expression of TGF-beta1 in the dental pulp and inner dentin were inhibited in the experimental group as compared with the control group (P<0.01). CONCLUSION: The overdose fluoride will inhibit the secretion of TGF-beta1, which leads to abnormal development of the teeth.

[The effect of dog bilateral distraction osteogensis in temporomandibular joint].

OBJECTIVE: To investigate the effect of bilateral mandibular distraction osteogenesis in the condyles. METHODS: 16 adult hybrid dogs were randomly divided into normal control group and experiment group. Experimental dogs underwent bilateral mandibular osteodistraction at a rate of 1 min/day. 4 dogs were killed respectively in distraction period, 2 and 8 weeks after completion of 10 days distraction. The bilateral condyles specimens were harvested and examined with histological and immunohistochemical methods. RESULTS: Compared with normal control group, various degrees of irregularities and erosion were found in fibrocartilage of condyle in experiment group, including damage in fibrous layer, hyperplasia layer and proliferative layer and osteogenic activity in cartilage layer. A significant increase of TGF-beta1 expression was also found in experiment groups. TGF-beta1 positive staining was noted in hypertrophic cell, matrix and chondroblast, osteoblast and matrix in osteogenic activity areas. These changes were the most obvious in 2 weeks after completion of distraction. CONCLUSION: Gradual bilateral mandibular distraction at a rate of 1 mm/day brought degenerative changes of condyle, but the changes are reversible.

Gradient conducting polymer surfaces with netrin-1-conjugation promote axon guidance and neuron transmission of human iPSC-derived retinal ganglion cells.

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.