Stabilizer‐Free Poly(lactide‐co‐glycolide) Nanoparticles Conjugated with Quantum Dots as a Potential Carrier Applied in Human Mesenchymal Stem Cells

We report that human mesenchymal stem cells (hMSCs) were successfully labeled with poly(lactide‐co‐glycolide) nanoparticles (PLGA NPs) surface‐conjugated quantum dots (QDs) (PLGA‐QD NPs) via endocytosis pathway. These NPs were not toxicity even treated with PLGA‐QD NPs at high concentrations for at least four weeks. Besides, PLGA‐QD NPs‐labeled hMSCs did not change their proliferation and differentiation capability toward the cell fates of adipocytes, osteocytes, and chrondrocytes. It's known that PLGA has been widely employed to act as delivery carrier which encapsulates drugs and releases them under a controlled way. Currently, we have also demonstrated that FITC‐loaded PLGA‐QD NPs degraded in hMSCs to achieve intracellular release of FITC. The aim of this research is to investigate viability, proliferation and differentiation capability and the potential for gene delivery of MSCs labeled with PLGA‐QD NPs. In addition to PLGA‐QD NPs, QDs alone were used to serve as a control set for comparison.     

Polypyrrole/Alginate Hybrid Hydrogels: Electrically Conductive and Soft Biomaterials for Human Mesenchymal Stem Cell Culture and Potential Neural Tissue Engineering Applications

Electrically conductive biomaterials that can efficiently deliver electrical signals to cells or improve electrical communication among cells have received considerable attention for potential tissue engineering applications. Conductive hydrogels are desirable particularly for neural applications, as they can provide electrical signals and soft microenvironments that can mimic native nerve tissues. In this study, conductive and soft polypyrrole/alginate (PPy/Alg) hydrogels are developed by chemically polymerizing PPy within ionically cross‐linked alginate hydrogel networks. The synthesized hydrogels exhibit a Young's modulus of 20–200 kPa. Electrical conductance of the PPy/Alg hydrogels could be enhanced by more than one order of magnitude compared to that of pristine alginate hydrogels. In vitro studies with human bone marrow‐derived mesenchymal stem cells (hMSCs) reveal that cell adhesion and growth are promoted on the PPy/Alg hydrogels. Additionally, the PPy/Alg hydrogels support and greatly enhance the expression of neural differentiation markers (i.e., Tuj1 and MAP2) of hMSCs compared to tissue culture plate controls. Subcutaneous implantation of the hydrogels for eight weeks induces mild inflammatory reactions. These soft and conductive hydrogels will serve as a useful platform to study the effects of electrical and mechanical signals on stem cells and/or neural cells and to develop multifunctional neural tissue engineering scaffolds.

     

中国小型猪骨髓间充质干细胞的蛋白质组研究

报道了骨髓间充质干细胞(MSCs)的蛋白质组表达研究。从体外培养的MSCs提取细胞蛋白,经二维电泳分离后用银染方法可检出蛋白点约1600个,选取48个蛋白点进行胶内酶解及质谱分析,经数据库检索成功鉴定了37个蛋白,并对蛋白功能进行初步分析。本实验数据为进一步分析MSCs增殖、分化或凋亡的分子机理提供相关信息。     

Restraint of the Differentiation of Mesenchymal Stem Cells by a Nonfouling Zwitterionic Hydrogel

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem‐cell phenotype and multipotency, independent of differentiation‐promoting media, cytoskeletal‐manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem‐cell differentiation and highlights the importance of materials chemistry in maintaining stem‐cell multipotency and controlling differentiation.     

Scalable Production of Monodisperse Bioactive Spider Silk Nanowires

Elongated protein-based micro- and nanostructures are of great interest for a wide range of biomedical applications, where they can serve as a backbone for surface functionalization and as vehicles for drug delivery. Current production methods for protein constructs lack precise control of either shape and dimensions or render structures fixed to substrates. This work demonstrates production of recombinant spider silk nanowires suspended in solution, starting with liquid bridge induced assembly (LBIA) on a substrate, followed by release using ultrasonication, and concentration by centrifugation. The significance of this method lies in that it provides i) reproducability (standard deviation of length <13% and of diameter <38%), ii) scalability of fabrication, iii) compatibility with autoclavation with retained shape and function, iv) retention of bioactivity, and v) easy functionalization both pre- and post-formation. This work demonstrates how altering the function and nanotopography of a surface by nanowire coating supports the attachment and growth of human mesenchymal stem cells (hMSCs). Cell compatibility is further studied through integration of nanowires during aggregate formation of hMSCs and the breast cancer cell line MCF7. The herein-presented industrial-compatible process enables silk nanowires for use as functionalizing agents in a variety of cell culture applications and medical research.     

Adhesive peptides conjugated PAMAM dendrimer as a coating polymeric material enhancing cell responses

This present work aims to functionalize poly(amidoamine) (PAMAM) dendrimers with various reported adhesive peptides, including Arg-Gly-Asp (RGD), Tyr-Ile-Gly-Ser-Arg (YIGSR), and Ile-Lys-Val-Ala-Val (IKVAV) for enhancing cell responses. The RGD, YIGSR, or IKVAV functionalized PAMAM coated substrate could promote cell adhesion of bone marrow mesenchymal stem cells (BMSCs) within 1 h after incubation. The neurite differentiation and proliferation of pheochromocytoma (PC12) cells were also significantly enhanced after culturing on the peptide functionalized PAMAM dendrimers for two and four days. This peptide functionalized PAMAM dendrimers are considered as the potential candidates for various tissue engineering applications.     

基于可控定向PVDF电纺纤维构建细胞相容性的三维微环境

三维电纺纤维在生物医学领域, 如生物传感、 药物控制释放与组织工程等方面具有良好的应用前景. 然而, 现有的电纺技术在制备结构、 孔隙率与形貌均可调节的三维定向电纺纤维方面还存在一定不足. 因此亟需开发一种新型的电纺丝工艺以制备三维定向电纺纤维. 本文通过改进传统的电纺丝工艺, 开发了一种简单高效制备三维定向聚偏氟乙烯(PVDF)的电纺丝制备技术. 所制备的三维定向纤维的形貌、 直径及纤维密度均可控. 体外细胞实验结果表明, 该类三维定向纤维具有良好的生物相容性, 能够促进细胞活性, 诱导细胞沿着纤维的方向生长. 此外, 研究结果还表明, 将该三维定向纤维作为细胞培养支架时, 细胞的增殖高于利用传统的二维纤维膜. 该制备技术将极大地拓宽三维定向纤维在三维细胞培养、 组织工程及疾病诊断等生物医学领域的应用.     

Regulating the physicochemical and biological properties in vitro of octacalcium phosphate by substitution with strontium in a large doping range

Octacalcium phosphate (OCP) is a key precursor of biological apatite in bone and teeth with excellent osteoconductive and biodegradable properties for bone regeneration. In this work, strontium was employed for improving the poor bioactivity and osteogenesis of OCP by ionic substitution using a homogeneous precipitation method. Sr was successfully incorporated in the structure within a fairly large doping range, even up to 23.25 mol.%, leading to lattice expansion and preferred growth. Sr-OCP samples with better cellular affinity had significantly enhanced the viability, proliferation and osteogenic differentiation of bone mesenchymal stem cells, compared with control. Dynamically released biological ions were involved in the biological apatite deposition for biomineralization/cellular calcification, and remarkably promoted the osteogenic ability. Results indicated the effective enhancements of Sr substitution on the biocompatibility, bioactivity and osteogenesis of OCP biomaterials, providing a potential strategy for the accounts of ionic substitution in biomaterials and the following bone repair applications.     

Date Fruits-Assisted Synthesis and Biocompatibility Assessment of Nickel Oxide Nanoparticles Anchored onto Graphene Sheets for Biomedical Applications

Nanographene- and graphene-based nanohybrids have garnered attention in the biomedical community owing to their biocompatibility, excellent aqueous processability, ease of cellular uptake, facile surface functionalization, and thermal and electrical conductivities. NiO nanoparticle-graphene nanohybrid (G-NiO) was synthesized by first depositing Ni(OH)₂ onto the surface of graphene oxide (GO) sheets. The Ni(OH)₂-GO hybrids were then reduced to G-NiO using date palm syrup at 85 °C. The prepared G-NiO nanohybrids were characterized by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy (EDX). The NiO nanoparticles, with a diameter of approximately 20–30 nm, were uniformly dispersed over the surface of the graphene sheets. The G-NiO hybrids exhibit biocompatibility in human mesenchymal stem cells (hMSCs) up to 100 μg/mL. The nanohybrids do not cause any significant changes in cellular and nuclear morphologies in hMSCs. The as-synthesized nanohybrids show excellent biocompatibility and could be a promising material for biomedical applications.     

Myofibroblasts are important contributors to human hepatocellular carcinoma: Evidence for tumor promotion by proteome profiling

Liver cancer typically occurs with a background of chronic fibrosis, characterized by the accumulation of myofibroblast‐like cells. We performed 2D‐PAGE‐based comparative analyses with the aim to identify proteins expressed in human hepatocellular carcinoma (HCC) tissue but not in neighboring healthy liver tissue, and to make out which cell types are responsible for the expression of proteins most characteristic for HCC. LC‐MS/MS analysis of the most striking spots identified proteins that were mainly related to myofibroblast‐like cells. To gain more insights into the role of these cells in their contribution to HCC, we isolated myofibroblast‐like cells as well as hepatocytes, both derived from HCC tissues, and subjected them to proteome profiling based on shotgun experiments. Comparative analysis, also referring to proteome profiles of other cell types previously investigated by us, pointed again to a marked contribution of myofibroblast‐like cells to HCC. Intriguingly, secretome analysis of these cells identified several growth factors that may act as tumor promoters and several proteins that have been described as potential biomarkers for HCC including dickkopf‐1, connective tissue growth factor, and CXCL1. Other biomarker candidates presently identified in the secretome of myofibroblasts, including lipocalin‐1 and pappalysin‐1, may be selected for future clinical validation. The identification of myofibroblast‐like cells as important source of tumor‐promoters may open new avenues to therapeutic intervention by targeting these stroma cells in addition to the cancer cells.     

Fast and simple online sample preparation coupled with capillary LC-MS/MS for determination of prostaglandins in cell culture supernatants

An online 2-D strong cation exchange (SCX)-RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG) A(1), PGD(2), PGE(1), PGE(2), PGF(2a), 6-keto-(6k)PGF(1a), and 15-Delta(12,14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included dilution and filtration, and the analysis time including all sample preparation steps was less than 50 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 0.4-2.2 ng/mL cell culture supernatant, recoveries higher than 80% and within- and between-day precisions of less than 30% RSDs were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated T lymphocytes, and PG production was analyzed using the developed method. PGE(2 )was found in cultures from both untreated and stimulated hMSCs, while PGE(1) was present above the detection limit only in stimulated cells.     

生物材料表面性能调控骨髓间充质干细胞分化

结合细胞和生物可降解支架的组织工程和再生医学技术为组织、器官的修复和再生提供了一种新途径。骨髓间充质干细胞(BMSCs)具有多向分化潜能,因其取材简单、来源广泛、增殖能力强,无伦理争议,免疫排斥反应小而备受关注。BMSCs在特定区域定向分化成为靶细胞是干细胞治疗的一个重要前提,尤其受到生物材料表面正负电荷、亲疏水和不同的拓扑结构的影响。材料表面涂层蛋白或接枝多肽能够促进BMSCs的分化能力,而生物材料不同的机械性能、几何形状也会影响BMSCs的分化方向。本文综述了近期生物材料调控BMSCs分化的研究结果,为基于BMSCs的组织工程和再生医学材料的设计提供借鉴和指导。     

Enhanced critical-sized bone defect repair efficiency by combining deproteinized antler cancellous bone and autologous BMSCs

Previously we have demonstrated that calcinated antler cancellous bone(CACB)has great potential for bone defect repair,due to its highly similar composition and architecture to natural extracellular bone matrix.This study is aiming at seeking for an optimal strategy of combined application of CACB and bone marrow mesenchymal stem cells(BMSCs)in bone defect repair.In vitro study demonstrated that CACB promoted the adhesion,spreading and viability of BMSCs.Increased extracellular matrix production and expression of osteogenic markers in BMSCs were observed when seeded on CACB scaffolds.The cells ceased to proliferation in the dual effect of CACB and osteogenic induction at the early stage of incubation. Hence synergistic effect of CACB combined with autologous undifferentiated BMSCs in rabbit mandible critical-sized defect repair was further evaluated.Histological analysis results showed that loading the CACB with autologous BMSCs resulted in enhanced new bone formation and angiogenesis when compared with implanted CACB alone.These findings indicate that the combination of CACB and autologous BMSCs should become potential routes to improve bone repair efficiency     

Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.     

Determination of thromboxanes,leukotrienes and lipoxins using high-temperature capillary liquid chromatography–tandem mass spectrometry and on-line sample preparation

An on-line strong cation-exchange (SCX)–reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B₂, TXB₃, leukotriene (LT) B₄, LTD₄ and lipoxin (LX) A₄ in cell culture supernatants was developed and validated. In the present method, a high temperature (70 °C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Limits of detection in the range of 1–4 ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB₂ was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.     

Photocrosslinkable diazoresin/pectin films – Synthesis and application as cell culture supports

Multilayer polymeric films composed of diphenylamine-4-diazoresin (DR), a synthetic polycation, and pectin (Pec), a polyanionic natural polysaccharide, were obtained using a layer-by-layer method (LbL). The films was photocrosslinked by the irradiation with 350 nm light which resulted in their stabilization against different solvents. The nanostructure of the films was studied using AFM microscopy. Irradiation of the films resulted also in the change of the zeta potential of both DR and Pec outermost layer to more negative values and in the increase of the film hydrophobicity as found from the contact angle measurements. The DR/Pec films have been shown to support cell growth and osteogenic potential of cell culture supports in tissue engineering, as found from the experiments with human bone marrow stromal cells (hMSCs).     

Vibrational spectroscopy differentiates between multipotent and pluripotent stem cells

Over the last few years, there has been an increased interest in the study of stem cells in biomedicine for therapeutic use and as a source for healing diseased or injured organs/tissues. More recently, vibrational spectroscopy has been applied to study stem cell differentiation. In this study, we have used both synchrotron based FTIR and Raman microspectroscopies to assess possible differences between human pluripotent (embryonic) and multipotent (adult mesenchymal) stem cells, and how O(2) concentration in cell culture could affect the spectral signatures of these cells. Our work shows that infrared spectroscopy of embryonic (pluripotent) and adult mesenchymal (multipotent) stem cells have different spectral signatures based on the amount of lipids in their cytoplasm (confirmed with cytological staining). Furthermore, O(2) concentration in cell culture causes changes in both the FTIR and Raman spectra of embryonic stem cells. These results show that embryonic stem cells might be more sensitive to O(2) concentration when compared to mesenchymal stem cells. While vibrational spectroscopy could therefore be of potential use in identifying different populations of stem cells further work is required to better understand these differences.