SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Identification of the lncRNA-miRNA‒mRNA regulatory network for middle cerebral artery occlusion-induced ischemic stroke.

Stroke known as a neurological disease has significant rates of disability and mortality. Middle cerebral artery occlusion (MCAO) models in rodents is crucial in stroke research to mimic human stroke. Building the mRNA and non-conding RNA network is essential for preventing MCAO-induced ischemic stroke occurrence. Herein, genome-wide mRNA, miRNA, and lncRNA expression profiles among the MCAO group at 3 h, 6 h, and 12 h after surgery and controls using high-throughput RNA sequencing. We detected differentially expressed mRNAs (DE-mRNAs), miRNAs (DE-miRNAs), and lncRNAs (DE-lncRNAs) between the MCAO and control groups. In addition, biological functional analyses were conducted, including GO/KEGG enrichment analysis, and protein-protein interaction analysis (PPI). GO analysis indicated that the DE-mRNAs were mainly enriched in several important biological processes as lipopolysaccharide, inflammatory response, and response to biotic stimulus. The PPI network analysis revealed that the 12 DE-mRNA target proteins showed more than 30° with other proteins, and the top three proteins with the highest node degree were Alb, IL-6, and TNF. In the DE-mRNAs, we found the mRNA of Gp6 and Elane interacting with two miRNAs (novel_miR_879 and novel_miR_528) and two lncRNAs (MSTRG.348134.3 and MSTRG.258402.19). As a result of this study, a new perspective can be gained into the molecular pathophysiology leading to the formation of MCAO. The mRNA-miRNA‒lncRNA regulatory networks play an important role in MCAO-induced ischemic stroke pathogenesis and could be applied to the treatment and prevention of ischemic stroke in the future.

Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults.

BACKGROUND: Cytomegalovirus (CMV) infection leads to effector memory CD8+ T cell expansion and is associated with immune dysfunction in older adults. However, the molecular alterations of CMV-specific CD8+ T cells in CMV infected healthy young and middle-aged adults has not been fully characterized. RESULTS: We compared CD8+ T cells specific for a CMV epitope (pp65495-503, NLV) and an influenza A virus (IAV) epitope (M158-66, GIL) from the same young and middle-aged healthy adults with serum positive for anti-CMV IgG. Compared to the IAV-specific CD8+ T cells, CMV-specific CD8+ T cells contained more differentiated effector memory (TEM and TEMRA) cells. Isolated CMV-specific central memory (TCM) but not naïve (TN) cells had a significant reduced activation-induced expansion in vitro compared to their IAV-specific counterparts. Furthermore, we found that CD70 expression was reduced in CMV-specific CD28+CD8+ TCM and that CD70+ TCM had better expansion in vitro than did CD70- TCM. Mechanistically, we showed that CD70 directly enhanced MAPK phosphorylation and CMV-specific CD8+ TCM cells had a reduced MAPK signaling upon activation. Lastly, we showed that age did not exacerbate reduced CD70 expression in CMV- specific CD8+ TCM cells. CONCLUSION: Our findings showed that CMV infection causes mild expansion of CMV-NLV-specific CD8+ T cells, reduced CD70 expression and signaling, and proliferation of CMV-NLV-specific CD8+ TCM cells in young and middle-aged healthy adults and revealed an age-independent and CMV infection-specific impact on CD8+ memory T cells.

Laboratory-Scale Production of Sterile Targeted Microbubbles.

Perfluorocarbon gas-filled microbubbles are clinically used as ultrasound contrast agents. We have been developing targeted microbubbles based BACS (buoyancy activated cell sorting) or BUBLES (buoyancy enabled separation) for ex vivo cell isolation from bloods for circulating tumor cell (CTC) detection and hematopoietic cell isolation. Recently, we further applied targeted microbubbles for multimarker cell sorting, and as artificial antigen presenting cells (aAPC) for T cell activation and expansion by taking advantage of a number of interesting properties of lipid-shelled microbubbles. This chapter will describe the process of manufacturing sterile targeted microbubbles for research applications.

Efficacy of tripterygium glycosides for diabetic nephropathy: a meta-analysis of randomized controlled trials.

BACKGROUNDS: Diabetic nephropathy (DN) is one of the most important clinical complications of diabetes mellitus (DM) and is the most common cause of end-stage renal disease. Currently, there is no highly effective medicine that can prevent, halt, or reverse the progressive course of DN. Initial clinical data showed that Tripterygium glycosides (TGs), a traditional Chinese medicine, can decrease proteinuria in patients with DN. OBJECTIVES: The objective of the present study is to investigate the efficacy and safety of TGs for the treatment of DN through meta-analysis of randomized controlled trials (RCTs). METHODS: All RCTs of TGs for DN were collected from The China National Knowledge Infrastructure (CNKI), PubMed, Web of Science, Wanfang Data, Chinese Biomedical Literature Database (CBM), China Science and Technology Journal Database (VIP) by setting the study inclusion and elimination standards. Two reviewers evaluated the quality of the trials and extracted the data independently. RevMan 5.4 software was used for meta-analyses. The primary outcome was a change in 24-hours urinary total protein (24 h TUP). RESULTS: 26 RCTs with 1824 participants were identified. Studies were assessed using the Cochrane risk of bias tool. The overall effects showed that TGs was compared with the controls, TGs showed significant effects in reducing 24 h TUP [WMD = -0.84, 95 % CI (-1.09, -0.59)], elevating serum albumin [WMD = 2.88, 95 % CI (1.87, 3.90)], and the total efficiency [OR = 4.08, 95 % CI (2.37, 7.04)]. This effect was consistent across the subgroups of period of intervention. CONCLUSIONS: The present research showed that TGs was significantly associated with improvement of renal function in patients with DN. TGs offers a novel approach to the treatment of DN, more high-quality RCTs are needed for a better understanding of the role of TGs in DN therapy.

Lipid Microbubble-Conjugated Anti-CD3 and Anti-CD28 Antibodies (Microbubble-Based Human T Cell Activator) Offer Superior Long-Term Expansion of Human Naive T Cells In Vitro.

Stimulation of human primary T cells with immobilized anti-CD3 and anti-CD28 Abs in vitro provide a system to study T cell activation and proliferation and an avenue for expanding T cells for immunotherapy. Magnetic beads conjugated with anti-CD3 and anti-CD28 Abs (Dynabeads Human T-Activator [D-TCA]) have been a golden standard for stimulating human primary T cells in vitro. In this study, we report that an application using anti-CD3 and anti-CD28 Abs conjugated on lipid microbubbles (microbubble-based human T cell activator [MB-TCA]) to stimulate primary human naive T cells resulted in expansion superior to D-TCA. In 56-d cultures with three repeated stimulation cycles (14 d per stimulation), we found that 1) MB-TCA induced significantly better expansion (20- and 10-fold increase) of naive CD4+ and CD8+ T cells than did D-TCA; 2) MB-TCA- and D-TCA-stimulated T cells had a similar number of initial cell divisions, but MB-TCA had significantly lower activation-induced cell death than D-TCA; 3) MB-TCA-stimulated T cells produced less TNF-α than did D-TCA; and 4) blocking TNF-α action via adding an Ab against TNF-αR (TNFRSF1A) significantly improved expansion of T cells activated by D-TCA in vitro. Together, we demonstrated that the MB-TCA induces a better expansion of human naive T cells in vitro and offers advantages in both basic and clinical applications in which the outcome depends on the number of T cells.

Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles.

Circulating tumor cells (CTCs) are extremely rare cells found in blood of metastatic cancer patients. There is a need for inexpensive technologies for fast enrichment of CTCs from large blood volumes. Previous data showed that antibody-conjugated lipid shell immuno-microbubbles (MBs) bind and isolate cells from biological fluids by flotation. Here, blood-stable MBs targeted to several surface markers for isolation of breast tumor cells were developed. MBs coated with anti-human EpCAM antibodies showed efficient binding of EpCAM+ breast cancer cell lines SKBR-3, MCF-7, and MDA-MB-453, whereas anti-human EGFR MBs showed binding of EpCAMLOW/NEGATIVE cell lines MDA-MB-231 and BT-549. Multitargeted anti-human EpCAM/EGFR MBs bound all cell lines with over 95% efficiency. Highly concentrated MB-bound tumor cells were collected in a microliter volume via an inverted vacuum-assisted harvesting setup. Using anti-EpCAM and/or anti-EpCAM/EGFR MBs, an efficient (70-90%) recovery and fast (30min) isolation of the above-mentioned cells and cell clusters was achieved from 7.5mL of spiked human blood. Using anti-EpCAM MBs and anti-EpCAM/EGFR MBs, cytokeratin-positive, CD45-negative CTCs were detected in 62.5% (10/16) of patients with metastatic breast cancer and CTC clusters were detected in 41.7% (5/12) of CTC-positive samples. Moreover, in some samples MBs isolated cytokeratin positive, CD45 negative tumor-derived microparticles. None of these structures were detected in blood from non-epithelial malignancies. The fast and inexpensive multitargeted platform for batch isolation of CTCs can promote research and clinical applications involving primary tumors and metastases.

Removal of ligand-bound liposomes from cell surfaces by microbubbles exposed to ultrasound.

Gas-filled microbubbles attached to cell surfaces can interact with focused ultrasound to create microstreaming of nearby fluid. We directly observed the ultrasound/microbubble interaction and documented that under certain conditions fluorescent particles that were attached to the surface of live cells could be removed. Fluorescently labeled liposomes that were larger than 500 nm in diameter were attached to the surface of endothelial cells using cRGD targeting to αvß3 integrin. Microbubbles were attached to the surface of the cells through electrostatic interactions. Images taken before and after the ultrasound exposure were compared to document the effects on the liposomes. When exposed to ultrasound with peak negative pressure of 0.8 MPa, single microbubbles and groups of isolated microbubbles were observed to remove targeted liposomes from the cell surface. Liposomes were removed from a region on the cell surface that averaged 33.1 µm in diameter. The maximum distance between a single microbubble and a detached liposome was 34.5 µm. Single microbubbles were shown to be able to remove liposomes from over half the surface of a cell. The distance over which liposomes were removed was significantly dependent on the resting diameter of the microbubble. Clusters of adjoining microbubbles were not seen to remove liposomes. These observations demonstrate that the fluid shear forces generated by the ultrasound/microbubble interaction can remove liposomes from the surfaces of cells over distances that are greater than the diameter of the microbubble.

Individual construction of freeform-fabricated polycaprolactone scaffolds for osteogenesis.

The construction of engineered bone mostly focuses on simulating the extracellular matrix (ECM) for proper biological activity. However, the complexity of architecture and the variability of the mechanical properties of natural bones are related to individual differences in age, nutritional state, mechanical loading and disease status. Defect substitutions should be normed with the host natural bone, balancing architectural and mechanical adaption, as well as biological activity. Using a freeform fabrication (FFF) method, we prepared polycaprolactone (PCL) scaffolds with different architectures. With simulation of structural and mechanical parameters of rabbit femur cancellous bone, individual defect substitution with the characteristics of the rabbit femur was obtained with high porosity and connectivity. Biological adaption in vitro was examined and osteoid formation in vivo was assessed by implantation in situ. Simulating the femur cancellous bone, 300-µm FFF PCL scaffolds had better architectural and mechanical properties. The protocol produced an architecturally, mechanically and biologically adaptive construction of an individual model for rapid-prototype PCL scaffolds. A guide system was developed to accurately reproduce virtually individual defect substitutions of the bone.

[Vena Cava Filter Products Review].

Vena cava filter (VCF)has been increasingly applied in clinical to efficiently prevent the pulmonary embolism (PE) with the rapid development of VCF. This article summarized the development of VCF, analyzed the relationship between structure and function, described the clinical behaviour of VCF, and final y forecasted the development trend of VCF products.

The behavior of lipid debris left on cell surfaces from microbubble based ultrasound molecular imaging.

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition.

Targeting and depletion of circulating leukocytes and cancer cells by lipophilic antibody-modified erythrocytes.

There is a great interest in targeting and selective ablation of populations of circulating cells for research or therapeutic purposes. Red blood cells (RBCs) are readily available and fully biocompatible long-circulating intravascular carriers (natural life is 120days) that are amenable to chemical modifications, drug loading and reinjection. Here we demonstrate that using our previously described lipophilic ligand painting strategy, red blood cells (RBCs) could be in one step converted into targeted entities that selectively seek and bind various cells in vitro and in vivo. In vitro, RBCs modified with lipophilic anti-EpCAM or anti-CD45 antibodies efficiently bound to cancer cells and leukocytes, forming characteristic rosettes. In vivo, intravenously injected RBCs painted with anti-CD45 antibody immediately associated with CD45 positive cells in blood, forming RBC-leukocyte rosettes. Moreover, anti-CD45-modified RBCs, but not the same amount of anti-CD45 antibody or anti-CD45-lipid conjugate (1-2µg/mouse), depleted over 50% of CD45+ leukocytes from circulation, with main clearance organs of leukocytes being liver and spleen with no visible deposition in kidneys and lungs. Anti-CD20 (Rituximab)-painted RBCs efficiently (over 90%) depleted CD19+/CD20+/CD45+ human lymphoma cells in mantle cell lymphoma (MCL) JeKo-1 model, while the same amount of rituximab-lipid (2µg/mouse) was much less efficient in lymphoma cell depletion. Treatment of MCL mice with rituximab-modified RBCs carrying only 2µg of the antibody resulted in a significant prolongation of survival as compared to the same amount of antibody-lipid control. Lipophilic ligand-painted RBCs is a novel tool that can be utilized for targeting blood borne cells for experimental immunology and drug delivery applications.

Distearoyl anchor-painted erythrocytes with prolonged ligand retention and circulation properties in vivo.

Red blood cells (RBCs) attract significant interest as carriers of biomolecules, drugs, and nanoparticles. In this regard, versatile technologies to attach molecules and ligands to the RBC surface are of great importance. Reported here is a fast and efficient surface painting strategy to attach ligands to the surface of RBCs, and the factors that control the stability and circulation properties of the modified RBCs in vivo. Distearoyl phosphatidylethanolamine anchor-conjugated immunoglobulin (IgG) efficiently incorporates in the RBC membrane following 15-30 min incubation. The optimized RBCs show prolonged circulation in vivo (70% of the injected dose after 48 h) and efficient retention of IgG in the membrane with terminal half-life of 73 h. The IgG construct is gradually lost from the RBCs mainly due to the transfer to plasma components, liver endothelial cells, and Kupffer cells. The ligand retention efficiency is partially dictated by ligand type, anchor type, and ligand concentration in the membrane, while RBC half-life is determined by initial concentration of the ligand in the membrane and presence of PEG linker between the ligand and the anchor. This work provides important guidance for non-covalent surface painting of RBCs as well as other types of blood borne cells for in vivo therapeutic and targeting applications.

Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles.

With the emerging interest in personalized medicine, there is strong demand for new technologies for clinical sample interrogation. Exfoliated tumor cells in variety of pathological samples (e.g., blood, bone marrow, urine) could provide invaluable information for diagnosis and prognosis of cancers. Here we describe a detailed method for capture and isolation of tumor cells in medium, blood, or large issue buffy coat using EpCAM-targeted buoyant microbubbles (MBs). Perflorohexane gas lipid shell MBs were prepared with emulsification method and conjugated with antibody as described by us before [25]. The binding of EpCAM-targeted MBs to A549 (human lung carcinoma) and 4T1 (mouse breast carcinoma) cells spiked into BSA/PBS or blood was more than 90%, which was comparable with commercial anti-EpCAM immunomagnetic beads (DynaBeads). Anti-EpCAM MBs efficiently (75-82%) isolated BxPC3 pancreatic tumor cells spiked into medium, blood or a buffy coat, within 15-30 min of incubation. We discuss MB parameters and experimental conditions critical to achieve efficient cells binding and isolation. In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples.

Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles.

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

Bioactivating electrically conducting polypyrrole with fibronectin and bovine serum albumin.

Electrically conducting polypyrrole (PPy) and its composite materials are useful in interfacing electrical components and cells or living tissue. In recent years, significant efforts have been made to bioactivate PPy by incorporating biomolecules. The main objective of this work was to chemically bioactivate PPy particules by incorporating fibronectin (FN) and bovine serum albumin (BSA). Modified PPy particles were synthesized through a water-in-oil emulsion polymerization. XPS and FTIR confirmed the presence of biomolecules on the PPy particles, and the surface morphology was observed by SEM. A four-point probe was used to measure the conductivity of the newly synthesized PPy particles, which was in the range of 10(-1) S cm(-1). Conductive biodegradable membranes were prepared with 5 and 10% (wt/wt) PPy to poly(L,L-lactide) (PPy/PLLA). The contact angles of each synthesized membrane were approximately 75 degrees , supporting their usefulness for cell culture. The cultured human skin fibroblasts demonstrated normal morphology and significantly higher adhesion and spreading on the PPy/PLLA approximately FN membrane than on the unmodified PPy/PLLA membrane. On the other hand, the PPy/PLLA approximately BSA membranes showed decreased cell adhesion. Bioactivated PPy may be useful in tissue engineering to fabricate conducting biodegradable scaffolds with either improved or reduced cell adhesion properties for various cell culture and in vivo applications.

The regulation of cell functions electrically using biodegradable polypyrrole-polylactide conductors.

Electricity has a long history of being used as an alternative clinical treatment and as an effective approach to modifying cellular behaviours in vitro. It has been difficult, however, to take advantage of this modality in tissue generation because of the lack of suitable conductive, biocompatible scaffolding materials. In this study, in order to electrically regulate cell activities, a largely biodegradable conductor made of 5% conductive polypyrrole and 95% biodegradable poly(L-lactide) (PPy/PLLA) was prepared. Human cutaneous fibroblasts were cultured on the conductors in the presence or absence of a direct current (DC) electrical field (EF) of 50 mV/mm. The growth of the cells was characterized using fluorescent staining, SEM, and a MTT assay. The RNA expressions of interleukin-6 (IL-6) and interleukin-8 (IL-8) were assayed by RT-PCR. The amounts of IL-6 and IL-8 secreted by the fibroblasts were quantified by ELISA. The results showed that the PPy/PLLA conductors supported cell adhesion, spreading, and proliferation in both the presence and absence of the EF. Electrical stimulation (ES) applied through PPy/PLLA conductors dramatically enhanced cytokine secretion approximately 10-fold when compared to the non-ES controls. This effect lasted several days after the end of the ES. These findings highlight for the first time the possibility of a potent, effective approach to regulating tissue regeneration in conductive scaffolds through ES-modulated cytokine secretion, and to increasing cytokine productivity for biotechnological applications.

Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,L-lactide) composites.

Polypyrrole (PPy) is a promising conductive polymer for tissue engineering and bioelectrical applications. However, its electrical conductivity deteriorates easily in aqueous conditions. Cell adhesion to PPy is also relatively poor. The goal of this study was to simultaneously improve the electrical stability of and cell adhesion to PPy by using heparin (HE) as dopant, for HE is both a polyanion and an important glycosaminoglycan in cell membranes and extracellular matrix. PPy particles doped with HE were synthesized through emulsion polymerization using Fenton's reagent as an oxidant. X-ray photoelectron spectroscopy (XPS), infrared and scanning electron microscopy (SEM) were used to investigate the PPy particles. Conductive biodegradable membranes of 10(2) to 10(3) Omega/square were prepared from 5% (w) PPy with various amounts of HE and 95% (w) poly(L,L-lactide) (PPy/PLLA). Azure A staining was employed to quantify the HE exposed on the surface of the PPy particles and PPy/PLLA membranes. The distribution of HE on membranes was demonstrated by DAPI staining. Results showed that HE was incorporated into the PPy particles as counterions and presented on particle surface. A unique "filament"-like morphology of the PPy preparation was observed at high-HE content. The electrical stability of the PPy/PLLA membranes was tested in saline at 37 degrees C for 500 h. Human skin fibroblasts were used to test the cell adhesion capacity. The conductive membranes containing HE-doped PPy particles recorded significantly increased electrical stability, cell adhesion, and growth. The electrically more stable and cell adhesive conductive biodegradable membrane may act as a platform for various biomedical applications.

Electrical stimulation enhances viability of human cutaneous fibroblasts on conductive biodegradable substrates.

Electrically conductive biodegradable polymer membranes were prepared by mixing conductive polypyrrole particles with poly(L-lactic acid) solution followed by solution casting and solvent evaporation. Multi-well electrical cell culture plates were fabricated to host electrically stimulated cell culture and monitor parameters. Human cutaneous fibroblasts were cultured on conductive membranes with or without electrical stimulation (ES). Cell count, MTT, Hoechst staining, and SEM were performed to characterize the cells. The membranes supported the adhesion and proliferation of the fibroblasts in both the presence and absence of ES. In the presence of direct electrical field strength of 100 mV/mm, cell viability on the PPy/PLLA membranes at 2 and 24 h was 2.2- and 4.0-fold (p < 0.05) respectively of that on the same membranes without ES. Direct electrical current ranging from 2.5 to 250 microA/mm had no effect on the viability of cells cultured on the gold-coated Petri dish. Electrical field applied to conductive biodegradable polymer surfaces is therefore an effective approach to upregulate the mitochondrial activity of human skin fibroblasts.

Electrically conductive biodegradable polymer composite for nerve regeneration: electricity-stimulated neurite outgrowth and axon regeneration.

Normal and electrically stimulated PC12 cell cultures and the implantation of nerve guidance channels were performed to evaluate newly developed electrically conductive biodegradable polymer composites. Polypyrrole (PPy) doped by butane sulfonic acid showed a significantly higher number of viable cells compared with PPy doped by polystyrenesulfonate after a 6-day culture. The PC12 cells were left to proliferate for 6 days, and the PPy-coated membranes, showing less initial cell adherence, recorded the same proliferation rate as did the noncoated membranes. Direct current electricity at various intensities was applied to the PC12 cell-cultured conductive membranes. After 7 days, the greatest number of neurites appeared on the membranes with a current intensity approximating 1.7-8.4 microA/cm. Nerve guidance channels made of conductive biodegradable composite were implanted into rats to replace 8 mm of sciatic nerve. The implants were harvested after 2 months and analyzed with immunohistochemistry and transmission electron microscopy. The regenerated nerve tissue displayed myelinated axons and Schwann cells that were similar to those in the native nerve. Electrical stimulation applied through the electrically conductive biodegradable polymers therefore enhanced neurite outgrowth in a current-dependent fashion. The conductive polymers also supported sciatic nerve regeneration in rats.