Tissue Sheet Engineered Using Human Umbilical Cord-Derived Mesenchymal Stem Cells Improves Diabetic Wound Healing.

Diabetic foot ulceration is a common chronic diabetic complication. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been widely used in regenerative medicine owing to their multipotency and easy availability. We developed poly(lactic-co-glycolic acid) (PLGA)-based scaffold to create hUC-MSC tissue sheets. In vitro immunostaining showed that hUC-MSC tissue sheets formed thick and solid tissue sheets with an abundance of extracellular matrix (ECM). Diabetic wounds in mice treated with or without either the hUC-MSC tissue sheet, hUC-MSC injection, or fiber only revealed that hUC-MSC tissue sheet transplantation promoted diabetic wound healing with improved re-epithelialization, collagen deposition, blood vessel formation and maturation, and alleviated inflammation compared to that observed in other groups. Taken collectively, our findings suggest that hUC-MSCs cultured on PLGA scaffolds improve diabetic wound healing, collagen deposition, and angiogenesis, and provide a novel and effective method for cell transplantation, and a promising alternative for diabetic skin wound treatment.

Metagenomic analysis of the Rhinopithecus bieti fecal microbiome reveals a broad diversity of bacterial and glycoside hydrolase profiles related to lignocellulose degradation.

BACKGROUND: The animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host and the diet adopted by the host. Although the importance of gut microbiota of humans has been well demonstrated, there is a paucity of research regarding non-human primates (NHPs), especially herbivorous NHPs. RESULTS: In this study, an analysis of 97,942 pyrosequencing reads generated from Rhinopithecus bieti fecal DNA extracts was performed to help better understanding of the microbial diversity and functional capacity of the R. bieti gut microbiome. The taxonomic analysis of the metagenomic reads indicated that R. bieti fecal microbiomes were dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria phyla. The comparative analysis of taxonomic classification revealed that the metagenome of R. bieti was characterized by an overrepresentation of bacteria of phylum Fibrobacteres and Spirochaetes as compared with other animals. Primary functional categories were associated mainly with protein, carbohydrates, amino acids, DNA and RNA metabolism, cofactors, cell wall and capsule and membrane transport. Comparing glycoside hydrolase profiles of R. bieti with those of other animal revealed that the R. bieti microbiome was most closely related to cow rumen. CONCLUSIONS: Metagenomic and functional analysis demonstrated that R. bieti possesses a broad diversity of bacteria and numerous glycoside hydrolases responsible for lignocellulosic biomass degradation which might reflect the adaptations associated with a diet rich in fibrous matter. These results would contribute to the limited body of NHPs metagenome studies and provide a unique genetic resource of plant cell wall degrading microbial enzymes. However, future studies on the metagenome sequencing of R. bieti regarding the effects of age, genetics, diet and environment on the composition and activity of the metagenomes are required.

3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients.

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening.

Sulfoxide-containing polymers conjugated prodrug micelles with enhanced anticancer activity and reduced intestinal toxicity.

Poly(ethylene glycol) (PEG) is widely utilized as a hydrophilic coating to extend the circulation time and improve the tumor accumulation of polymeric micelles. Nonetheless, PEGylated micelles often activate complement proteins, leading to accelerated blood clearance and negatively impacting drug efficacy and safety. Here, we have crafted amphiphilic block copolymers that merge hydrophilic sulfoxide-containing polymers (psulfoxides) with the hydrophobic drug 7-ethyl-10-hydroxylcamptothecin (SN38) into drug-conjugate micelles. Our findings show that the specific variant, PMSEA-PSN38 micelles, remarkably reduce protein fouling, prolong blood circulation, and improve intratumoral accumulation, culminating in significantly increased anti-cancer efficacy compared with PEG-PSN38 counterpart. Additionally, PMSEA-PSN38 micelles effectively inhibit complement activation, mitigate leukocyte uptake, and attenuate hyperactivation of inflammatory cells, diminishing their ability to stimulate tumor metastasis and cause inflammation. As a result, PMSEA-PSN38 micelles show exceptional promise in the realm of anti-metastasis and significantly abate SN38-induced intestinal toxicity. This study underscores the promising role of psulfoxides as viable PEG substitutes in the design of polymeric micelles for efficacious anti-cancer drug delivery.

Neutrophil Extracellular Traps-Inhibiting and Fouling-Resistant Polysulfoxides Potently Prevent Postoperative Adhesion, Tumor Recurrence, and Metastasis.

Peritoneal metastasis (PM) is considered one of the most dreaded forms of cancer metastases for both patients and physicians. Aggressive cytoreductive surgery (CRS) is the primary treatment for peritoneal metastasis. Unfortunately, this intensive treatment frequently causes clinical complications, such as postoperative recurrence, metastasis, and adhesion formation. Emerging evidence suggests that neutrophil extracellular traps (NETs) released by inflammatory neutrophils contribute to these complications. Effective NET-targeting strategies thus show considerable potential in counteracting these complications but remain challenging. Here, one type of sulfoxide-containing homopolymer, PMeSEA, with potent fouling-resistant and NET-inhibiting capabilities, is synthesized and screened. Hydrating sulfoxide groups endow PMeSEA with superior nonfouling ability, significantly inhibiting protein/cell adhesion. Besides, the polysulfoxides can be selectively oxidized by ClO- which is required to stabilize the NETs rather than H2O2, and ClO- scavenging effectively inhibits NETs formation without disturbing redox homeostasis in tumor cells and quiescent neutrophils. As a result, PMeSEA potently prevents postoperative adhesions, significantly suppresses peritoneal metastasis, and shows synergetic antitumor activity with chemotherapeutic 5-Fluorouracil. Moreover, coupling CRS with PMeSEA potently inhibits CRS-induced tumor metastatic relapse and postoperative adhesions. Notably, PMeSEA exhibits low in vivo acute and subacute toxicities, implying significant potential for clinical postoperative adjuvant treatment.

A new three-dimensional (3D) multilayer organic material: synthesis, swelling, exfoliation, and application.

A novel fully rigid, rod-shaped oligo(p-benzamide) (OPBA-6) molecule was designed and synthesized, which can be recrystallized into a three-dimensional (3D) multilayer material via an antiparallel molecular packing model. Intermolecular hydrogen bonding and π-π interaction are brought to ensure a strong intralayer interaction, while decoration of layer surface with sulfonic groups promotes water to enter interlayer space and facilitates the swelling and exfoliation of sample. With a simple dispersion in water, the obtained multilayer material can be easily swollen by water without destruction of in-plane morphology and subsequently delaminated into 2D nanosheets with thickness of about 5.38 nm. This achievement may be the first attempt to exfoliate layered organic materials and thus provide a new strategy to prepare 2D organic nanosheets without using any substrates or templates as required by conventional and widely used self-assembly routes. Based on exfoliated nanosheets, poly(vinyl alcohol) nanocomposites were prepared using a simple water solution processing method. A 64% increase in tensile stress and a 63% improvement in Young's modulus were achieved by addition of 7 wt % OPBA-6 loading.

Bioprinting EphrinB2-Modified Dental Pulp Stem Cells with Enhanced Osteogenic Capacity for Alveolar Bone Engineering.

Bioprinting, a technology that allows depositing living cells and biomaterials together into a complex tissue architecture with desired pattern, becomes a revolutionary technology for fabrication of engineered constructs. Previously, we have demonstrated that EphrinB2-modified dental pulp stem cells (DPSCs) are expected to be promising seed cells with enhanced osteogenic differentiation capability for alveolar bone regeneration. In this study, we aimed to bioprint EphrinB2-overexpressing DPSCs with low-concentrated Gelatin methacrylate (GelMA) hydrogels into three-dimensional (3D) constructs. The printability of GelMA (5% w/v) and the structural fidelity of bioprinted constructs were examined. Then, viability, proliferation, morphology, and osteogenic differentiation of DPSCs in bioprinted constructs were measured. Finally, the effect of EphrinB2 overexpression on osteogenic differentiation of DPSCs in bioprinted constructs was evaluated. Our results demonstrated that GelMA (5% w/v) in a physical gel form was successfully bioprinted into constructs with various shapes and patterns using optimized printing parameters. Embedded DPSCs showed round-like morphology, and had a high viability (91.93% ± 8.38%) and obvious proliferation (∼1.9-fold increase) 1 day after printing. They also showed excellent osteogenic potential in bioprinted constructs. In bioprinted 3D constructs, EphrinB2-overexpressing DPSCs expressed upregulated osteogenic markers, including ALP, BMP2, RUNX2, and SP7, and generated more mineralized nodules, as compared with Vector-DPSCs. Taken together, this study indicated that fabrication of bioprinted EphrinB2-DPSCs-laden constructs with enhanced osteogenic potential was possible, and 3D bioprinting strategy combined with EphrinB2 gene modification was a promising way to create bioengineered constructs for alveolar bone regeneration.

Transcriptome analysis of Termitomyces albuminosus reveals the biodegradation of lignocellulose.

OBJECTIVE: To study whether Termitomyces albuminosus can degrade lignocelluloses and to understand the symbiotic relationship between termite mushroom and fungus-growing termite. METHODS: cDNA library of T. albuminosus was sequenced by the Roche 454 GS FLX Titanium platform, and the diverse enzymes relevant to degradation of cellulose and lignin of symbiotic fungus T. albuminosus were analyzed. RESULTS: Eighth sequencing run resulted in a total of 82386 reads (express sequence tags, EST). After removing the vector and primer sequences, the remained 54410 reads were assembled into 3301 contigs and 3193 singletons. Comparing sequence similarity with known proteins, these sequences, representing approximately 2681 unique genes, were successfully annotated using BLAST searches (E-value < or = 1e(-10)) against the Nr, SwissProt and CDD databases. The T. albuminosus transcripts included 33 enzymes putatively involved in cellulose and hemicelluloses biodegradation. 5 enzymes could hydrolyze cellulose and others had catalytic activities for degradation of hemicelluloses, starch and glycogen and chitin. Moreover, four genes encoding laccases and a single aryl-alcohol oxidase which could degrade lignin were also identified. These results revealed symbiosis fungus T. albuminosus had many laccases and possibly decomposed phenolic compounds from plant litter. CONCLUSIONS: Data presented in this study indicated that T. albuminosus had the ability to degrade lignin, which made cellulose more easily degraded by the cellulase produced by fungus-growing termite.

In silico formulation prediction of drug/cyclodextrin/polymer ternary complexes by machine learning and molecular modeling techniques.

Ternary cyclodextrin (CD) complexes (drug/CD/polymer) can effectively improve the solubility of water-insoluble drugs with large size than binary CD formulations. However, ternary formulations are screened by a trial-and-error approach, which is laborious and material-wasting. Current research aims to develop a prediction model for ternary CD formulations by combined machine learning and molecular modeling. 596 ternary formulations data were collected to build a prediction model by machine learning. The random forest model achieved good performance with R2 = 0.887 in ST prediction and R2 = 0.815 in ST/SB prediction. Two ternary formulations (Hydrocortisone/ß-CD/HPMC and dovitinib/γ-CD/CMC) were used to validate the prediction model. Molecular modeling results showed that HPMC not only warped around hydrocortisone but also prevented CD molecules from self-aggregation to increase solubility. In conclusion, a prediction model for the ternary CD formulations was successfully developed, which will significantly accelerate the formulation screening process to benefit the formulation development of water-insoluble drugs.

Fabrication of Thick and Anisotropic Cardiac Tissue on Nanofibrous Substrate for Repairing Infarcted Myocardium.

In this chapter, we introduce the method for fabricating thick and anisotropic cardiac tissue for heart regeneration. Aligned and biodegradable nanofiber can be prepared by electrospinning Food and Drug Administration-approved poly (lactic-co-glycolic acid) on a rotating drum. After the nanofibers are transferred on to a polydimethylsiloxane frame, the cardiomyocytes could be plated on the nanofiber to form thick and anisotropic cardiac tissue rapidly. Cardiac tissue-like construct could be easily created by one-step method, and transplanted onto the hearts of myocardium infarction models and lead to their functional recovery.

Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium.

High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

Effects of canonical NF-κB signaling pathway on the proliferation and odonto/osteogenic differentiation of human stem cells from apical papilla.

BACKGROUND INFORMATION: NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. RESULTS: Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. CONCLUSION: Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

INTRODUCTION: Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. METHODS: SCAP was isolated and treated with 10⁻7 M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. RESULTS: MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. CONCLUSIONS: The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻7 M 17beta-estradiol via the activation of MAPK signaling pathway.

Adult stem cell-based apexogenesis.

Generally, the dental pulp needs to be removed when it is infected, and root canal therapy (RCT) is usually required in which infected dental pulp is replaced with inorganic materials (paste and gutta percha). This treatment approach ultimately brings about a dead tooth. However, pulp vitality is extremely important to the tooth itself, since it provides nutrition and acts as a biosensor to detect the potential pathogenic stimuli. Despite the reported clinical success rate, RCT-treated teeth are destined to be devitalized, brittle and susceptible to postoperative fracture. Recently, the advances and achievements in the field of stem cell biology and regenerative medicine have inspired novel biological approaches to apexogenesis in young patients suffering from pulpitis or periapical periodontitis. This review mainly focuses on the benchtop and clinical regeneration of root apex mediated by adult stem cells. Moreover, current strategies for infected pulp therapy are also discussed here.

Adjuvant effects of L. acidophilus LW1 on immune responses to the foot-and-mouth disease virus DNA vaccine in mice.

The adjuvant effects of Lactobacillus acidophilus on DNA vaccination are not fully understood. It has been hypothesized that swine-derived Lactobacillus acidophilus SW1 (LASW1) could function as an immune adjuvant to enhance antigen-specific immune responses after foot-and-mouth disease (FMD) DNA vaccination in mice. To evaluate the effect of oral LASW1 on the immune response to a DNA vaccine (pRC/CMV-vp1) harboring FMD VP1 gene, anti-FMDV antibody and its isotypes, T-cell proliferation, and cytokine detection were investigated. The results showed that LASW1 was able to enhance FMDV-specific antibody levels and FMDV-neutralizing antibodies. After a booster vaccine, the anti-FMDV antibody titers and FMDV-neutralizing antibodies levels induced by pRC/CMV-vp1 were higher in mice treated with LSAW1 than in the group immunized with pRC/CMV-vp1 alone (the control). Using T-cell proliferation, the stimulation index of the LASW1 group was significantly higher in response to ConA and 146S antigen (P<0.05) than in the control group. Importantly, higher concentrations of IFN-γ and IFN-γ-producing cells were also observed in splenocytes isolated from the experimental LASW1 mice, indicating that INF-γ secretion is important to the immune response to LASW1. The results indicate that LASW1 is a promising immune adjuvant in DNA vaccination against FMD when administrated orally.

Patterning of two-level topographic cues for observation of competitive guidance of cell alignment.

Cells display contact guidance when cultured on topographical cues. By combining standard photolithography, nanoimprint lithography, and soft lithography, we produced sophisticated patterns on two levels, including crossing microgrooves with different depth/spacing and microgrooves with superimposed submicrometer features. The results show that for narrowly spaced microgrooves, the contact guidance is more significant to the change of groove depth than to other geometry parameters. For crossing microgrooves, the shallow grooves take over the influence on cell alignment when the deeper grooves are well separated. Finally, the superimposed submicrometer features on the groove ridges decrease the efficiency of the contact guidance of microgrooves, due to increased adhesion of cells on patterned surfaces.

Encoding localized strain history through wrinkle based structural colors.

Surface wrinkles are created on a metallic film supported on a shape memory polymer substrate. The wrinkle wavelength approaches that of visible lights, resulting in diffraction colors. The spatial and geometric distribution of the surface wrinkles can be controlled in an arbitrary fashion, allowing the capture of a three dimensional arbitrary image on a macroscopically flat surface.