Hyaluronic Acid Enhances the Mechanical Properties of Tissue-Engineered Cartilage Constructs

There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0–2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.     

Modeling the Insulin-Like Growth Factor System in Articular Cartilage

IGF signaling is involved in cell proliferation, differentiation and apoptosis in a wide range of tissues, both normal and diseased, and so IGF-IR has been the focus of intense interest as a promising drug target. In this computational study on cartilage, we focus on two questions: (i) what are the key factors influencing IGF-IR complex formation, and (ii) how might cells regulate IGF-IR complex formation? We develop a reaction-diffusion computational model of the IGF system involving twenty three parameters. A series of parametric and sensitivity studies are used to identify the key factors influencing IGF signaling. From the model we predict the free IGF and IGF-IR complex concentrations throughout the tissue. We estimate the degradation half-lives of free IGF-I and IGFBPs in normal cartilage to be 20 and 100 mins respectively, and conclude that regulation of the IGF half-life, either directly or indirectly via extracellular matrix IGF-BP protease concentrations, are two critical factors governing the IGF-IR complex formation in the cartilage. Further we find that cellular regulation of IGF-II production, the IGF-IIR concentration and its clearance rate, all significantly influence IGF signaling. It is likely that negative feedback processes via regulation of these factors tune IGF signaling within a tissue, which may help explain the recent failures of single target drug therapies aimed at modifying IGF signaling.     

Evaluation of Nanofiber-based Engineered Cartilage and its Integration with Native Cartilage

This article has no abstract.     

Keratinocyte Growth Factor Improves Epithelial Structure and Function in a Mouse Model of Intestinal Ischemia/Reperfusion

Background

Intestinal ischemia/reperfusion (I/R) induces the desquamation of the intestinal epithelium, increases the intestinal permeability, and in patients often causes fatal conditions including sepsis and multiple organ failure. Keratinocyte growth factor (KGF) increases intestinal growth, although little is known about KGF activity on intestinal function after intestinal I/R. We hypothesized that KGF administration would improve the intestinal function in a mouse model of intestinal I/R.

Methods

Adult C57BL/6J mice were randomized to three groups: Sham, I/R group and I/R+KGF group. Mice were killed on day 5, and the small bowel was harvested for histology, wet weight, RNA and protein content analysis. Epithelial cell (EC) proliferation was detected by immunohistochemistry for PCNA, and apoptosis was determined by TUNEL staining. The expressions of Claudin-1 and ZO-1 were detected by immunohistochemistry. Epithelial barrier function was assessed with transepithelial resistance (TER).

Results

KGF significantly increased the intestinal wet weight, contents of intestinal protein and RNA, villus height, crypt depth and crypt cell proliferation, while KGF resulted in the decrease of epithelial apoptosis. KGF also stimulated the recovery of mucosal structures and attenuated the disrupted distribution of TJ proteins. Moreover, KGF attenuated the intestinal I/R-induced decrease in TER and maintained the intestinal barrier function.

Conclusion

KGF administration improves the epithelial structure and barrier function in a mouse model of intestinal I/R. This suggests that KGF may have clinical applicability.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Stimulation of Mitogenic Pathways through Kinase-Impaired Mutants of the Epidermal Growth Factor Receptor

Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the γ-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [³H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [³H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [³H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.     

Receptor-Mediated Stimulation and Inhibition of Nerve Growth Factor Secretion by Vascular Smooth Muscle

Nerve growth factor (NGF) is a potent neurotrophin signaling protein, the best-known member of a family of similar neurotrophins. Specific neuronal populations depend upon the neurotrophins for normal function and disturbances in NGF and neurotrophin supply have been implicated in neurodegenerative disease, diabetes, and hypertension. This report details experiments in which the hourly pattern of NGF secretion by cultured vascular smooth muscle cells is examined. Vascular smooth muscle cells are major innervation targets of the neuronal population first discovered to be NGF-dependent: the sympathetic principal neurons. The results show that arginine vasopressin (AVP), angiotensin II (AngII), and α-adrenergic receptor activation, all contractile stimuli, elevate NGF secretion. However, AVP dependably does so alone while AngII requires coactivation of adenosine receptors. Adenosine alone inhibits secretion and the α-adrenergic increase in NGF output can be antagonized by activation of β-adrenergic receptors. A change to fresh culture medium is also a potent stimulus to increased NGF output.     

Magnitude of Ubiquitination Determines the Fate of Epidermal Growth Factor Receptor Upon Ligand Stimulation

Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modeling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor-α and epiregulin. Whereas only little differences in the order of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.     

Raman Spectral Dynamics of Single Cells in the Early Stages of Growth Factor Stimulation

Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not.     

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.     

Engineered Biocompatible and Stable Dipeptide Hydrogel with Tunable Mechanical and Cell Growth Properties to Embolden Neuroglial Cell Growth

International Journal of Peptide Research and Therapeutics - Physical properties of a tissue engineering scaffold play a crucial role in maneuvering the cellular fate. Scaffold properties have been...     

神经生长因子结构与功能研究进展

神经生长因子(NGF)是神经营养因子家族的典型代表, 它控制着脊椎动物周围和中枢神经系统中部分神经元的发育和存活.NGF的三维结构是以“胱氨酸结”和β折叠为基础,它以二聚体的形式结合细胞表面的受体从而发生生物学效应.参与这些反应的氨基酸残基已通过化学修饰和定点突变法加以确定,这有助于更进一步理解其结构与功能的关系.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

生长因子受体酪氨酸激酶的结构与功能

受体酪氨酸蛋白激酶是细胞信号转导进行的关键信号酶,在生长因子调控细胞生长、发育与功能的过程中起着重要的生理作用.本文主要介绍生长因子受体酪氨酸蛋白激酶的分类、结构与功能及其部分相关信号转导机制的研究进展.     

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (¹⁸⁸S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (¹⁰⁸T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on ⁸⁸S and ¹⁰⁰T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on ⁸⁸S and ¹⁰⁰T in response to EGF, which upregulates RhoA activity.     

自体ADSCs复合PRF修复家兔耳软骨缺损的实验研究

目的:将未诱导的自体脂肪干细胞(ADSCs)与富血小板纤维蛋白(PRF)复合,作为一种全新的软骨修复材料,探讨其对家兔耳软骨全层缺损修复的可行性.方法:取家兔10只,于每只家兔耳部做4处软骨全层缺损,随机分为A、B、C、D组,A组,作为空白对照;B组植入自体ADSCs;C组植入自体PRF;D组植入自体ADSCs与PRF的复合物.分别于术后1月、2月、3月取材,进行大体及HE染色观察,并使用IPP6.0软件对软骨生成量进行半定量分析.结果:HE染色显示,3月后,A组几乎无新生软骨生成,B、C、D三组软骨生成量依次增多,D组尤为明显.IPP6.0统计结果显示,移植物植入3月后,A组软骨缺损修复率为(1.68±0.17)％,B组为(15.4±0.91.)％,C组为(32.0±2.76)％,D组为(85.77±4.88)％.各组间有显著统计学差异,与HE染色结果相符.结论:未诱导的自体ADSC s复合自体PRF作为一种全新的软骨修复材料,可以有效的修复家兔耳软骨全层缺损,具有潜在的临床应用价值.     

Higher-Order Factor Invariance and Idiographic Mapping of Constructs to Observables

Theory, research, and application in developmental science share the conceptual and methodological challenges of integrating the study of the course of a person's intra-individual changes and the formulation of subgroup (differential) or nomothetic generalizations. One approach to these challenges has involved building tailored construct representations that filter out irrelevant idiosyncratic information hampering the articulation of nomothetic relations by means of individual level factor analysis (P–technique). Exploring this approach has provided initially promising results (Nesselroade, Gerstorf, Hardy, & Ram, 2007 Nesselroade , J. R. , Gerstorf , D. , Hardy , S. A. , & Ram , N. ( 2007 ). Idiographic filters for psychological constructs . Measurement: Interdisciplinary Research and Perspectives , 5 , 217 – 235 . doi: DOI: 10.1080/15366360701741807 [Taylor & Francis Online] , [Google Scholar]). We present a specification of an alternative to traditional factor invariance (higher-order invariance) that further formalizes these ideas and examine the results of some simulation experiments designed to evaluate the appropriateness of the implementation. The results also have implications for use in basic and applied work across the life span of between-persons variation and subgroup comparisons to establish relations among variables.     

胰岛素样生长因子1的结构与功能研究进展

胰岛素样生长因子1(IGF1)是一种多功能细胞调控因子.它的结构特征为其促生长和物质代谢功能提供依据.集中介绍了IGF1的三维结构、IGF1与相关受体和结合蛋白的结合区以及二硫键在蛋白质折叠中的重要作用.