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#28704392   2017/07/13 Save this To Up

Redifferentiation of aged human articular chondrocytes by combining bone morphogenetic protein-2 and melanoma inhibitory activity protein in 3D-culture.

Melanoma inhibitory activity (MIA) affects the differentiation to hyaline cartilage and can inhibit the osteogenic potential of bone morphogenetic protein (BMP)-2 in mesenchymal stem cells (MSC). The aim of this study was to investigate if MIA also inhibits the osteogenic potential of BMP-2 in human articular chondrocytes during redifferentiation, which may lead to a higher grade of differentiation without calcification. HAC of four female patients (mean age: 73.75 ±6.98) were seeded into 3D culture for 28 days; after adding the recombinant proteins, four groups were formed (Control, BMP-2, MIA, BMP-2+MIA). Samples were analysed for gene expression, glycosaminoglycan (GAG) content and histology on day 0, 14 and 28. Collagen type 2 (COL2A1) was significantly increased in the BMP-2 containing groups on day 28; BMP-2 (100-fold, p = 0.001), BMP-2+MIA (65-fold, p = 0.009) and similar to the level of native cartilage. Higher aggrecan (Agg) levels were present in the BMP-2 (3-fold, p = 0.007) and BMP-2+MIA (4-fold, p = 0.002) group after 14 days and in the BMP-2 (9-fold, p = 0.001) group after 28 days. Collagen type 10 (COL10A1) was increased in the BMP-2 containing groups (6-fold, p = 0.006) but these levels were significantly below native cartilage. Alkaline phosphatase (ALP), collagen type 1 (COL1A1) and the glycosaminoglycan (GAG) content did not reveal any relevant differences between groups. BMP-2 is a potent inducer for differentiation of HAC. A significant enhancement of this effect in combination with MIA could not be observed. Furthermore no significant reduction of osteogenic markers during re-differentiation of chondrocytes was present combining BMP-2 and MIA.

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#27836789   2016/11/12 Save this To Up

Evaluation of cytotoxicity of a purified venom protein from Naja kaouthia (NKCT1) using gold nanoparticles for targeted delivery to cancer cell.

In our earlier report, gold nanoparticle (GNP) and snake venom protein toxin NKCT1 were conjugated and primary characteristics were done. In this communication, further characteristics of GNP-NKCT1 were done with TGA, BET, Zeta potential, ICP-MS, FTIR, XPS, and in vitro release kinetics for its physicochemical, molecular nature and bonding. TGA and ICP-MS showed that the number of conjugation was 40 ± 5 to 90 ± 8 NKCT1 per gold nanoparticles. FTIR and XPS corresponding to (CO), (NH), (SS) reformulated the conjugation of GNP with NKCT1. The efficacy of GNP-NKCT1 on cancer cells were analyzed by MTT assay which demonstrated superior cytotoxic effects as compared to native NKCT1. IC50 dose of GNP-NKCT1 was less than 4 μg/ml in cancer cell lines, whereas in case of NKCT1 it was average 8 μg/ml. Twice dose of IC50 of GNP-NKCT1 even showed less toxicity compared to unconjugated NKCT1, towards normal epithelial or fibroblast cell and also in peripheral blood mononuclear lymphocytes. Flow cytometry analysis revealed that percentage of apoptotic C6 cells was much higher in GNP-NKCT1 treatment (54.58%) than that of NKCT1 treatment (26.79%). Flow cytometric analysis of cell cycle using GNP-NKCT1 on C6 cancer cells revealed that it arrested the cell cycle at Go/G1 phases. In diethylnitrosamine (DEN) induced in vivo hepatocarcinoma mice, the activities of hepatic enzymes- aspartate transaminase (AST) and alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and activities of antioxidant enzymes- superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) were restored by GNP-NKCT1. This study indicated the capability of gold nanoparticles in enhancing the cancer cell uptake of NKCT1 and also suggested that GNP-NKCT1 might be a good source of anti-carcinoma or anti-sarcoma targeted agent.

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#27353434   2016/06/29 Save this To Up

Extracellular Matrix of Current Biological Scaffolds Promotes the Differentiation Potential of Mesenchymal Stem Cells.

The purpose of this study was to quantitatively assess the ability of bone marrow-derived mesenchymal stem cells (bMSC) to differentiate toward bone, fat, cartilage, and tendon lineages when grown on commercially available scaffolds compared with control and native tendon tissue.

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#27340938   2016/07/07 Save this To Up

Heterodimeric BMP-2/7 for nucleus pulposus regeneration-In vitro and ex vivo studies.

Intervertebral disc (IVD) degeneration is the leading trigger of low back pain, which causes disability and leads to enormous healthcare toll worldwide. Biological treatment with growth factors has evolved as potential therapy for IVD regeneration. Bone morphogenetic protein 2 (BMP-2) and BMP-7 have shown promise in this regard. In the current study, we evaluated the effect of BMP-2/7 heterodimer for disc regeneration both in vitro and in organ culture. Nucleus pulposus (NP) cells isolated from bovine caudal disc were cultured in a fibrin-hyaluronan (FBG-HA) hydrogel for up to 14 days. BMP-2/7 heterodimer covalently incorporated within the hydrogel up-regulated the aggrecan and type II collagen gene expression, and glycosaminoglycan synthesis of NP cells. The activity of the BMP-2/7 heterodimer was dose dependent. The higher dose of BMP-2/7 was further assessed in an IVD whole organ system. After 14 days of culture with cyclic dynamic load, the BMP-2/7 heterodimer delivered into the nucleotomized region showed potential to stimulate the gene expression and synthesis of proteoglycan in the remaining NP tissue after partial nucleotomy. The gene expression level of type I collagen and alkaline phosphatase in the native disc tissue were not affected by BMP-2/7 treatment, indicating no adverse fibroblastic or osteogenic effect on the disc tissue. Intradiscal delivery of BMP-2/7 heterodimer may be a promising therapeutic approach for NP regeneration. The current IVD whole organ partial nucleotomy model may be utilized for screening of other biomaterials or drugs to treat early degenerative disc disorders. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:51-60, 2017.

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#27195765   2016/05/20 Save this To Up

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

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#27188244   2016/08/01 Save this To Up

Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells.

The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment.

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#27107902   2016/05/31 Save this To Up

The synergistic effect of nano-hydroxyapatite and dexamethasone in the fibrous delivery system of gelatin and poly(l-lactide) on the osteogenesis of mesenchymal stem cells.

Recently, electrospun nanofibrous scaffolds are vastly taken into consideration in the bone tissue engineering due to mimicking the natural structure of native tissue. In our study, surface features of nanofibers were modified through simultaneous electrospining of the synthetic and natural polymers using poly l-lactide (PLLA) and gelatin to fabricate the hybrid scaffold (PLLA/gelatin). Then, hydroxyapatite nanoparticles (nHA) were loaded in electrospun PLLA nanofibers (PLLA,nHA/gelatin) and also dexamethasone (DEX) was incorporated in these fibers (PLLA,nHA,DEX/gelatin) in the second experiment. Fabricated nanofibrous composite scaffolds were characterized via SEM, FTIR spectroscopy, contact angle, tensile strength measurements, DEX release profile and MTT assay. After seeding adipose derived mesenchymal stem cells, osteoinductivity and osteoconductivity of fabricated scaffolds were analyzed using common osteogenic markers such as alkaline phosphatase activity, calcium depositions and gene expression. These results confirmed that all properties of nanofibers were improved by modifications. Moreover, osteogenic differentiation of stem cells increased in PLLA,nHA/gelatin group in comparison with PLLA/gelatin. The sustained release of DEX was obtained from PLLA,nHA,DEX/gelatin which subsequently led to more osteogenic differentiation. Taken together, PLLA,nHA,DEX/gelatin showed significant potential to support the stem cell proliferation and ostogenic differentiation, and can be a good candidates for tissue engineering and regenerative medicine applications.

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#26953259   2016/04/18 Save this To Up

Regulatory Implications of Non-Trivial Splicing: Isoform 3 of Rab1A Shows Enhanced Basal Activity and Is Not Controlled by Accessory Proteins.

Alternative splicing often affects structured and highly conserved regions of proteins, generating so called non-trivial splicing variants of unknown structure and cellular function. The human small G-protein Rab1A is involved in the regulation of the vesicle transfer from the ER to Golgi. A conserved non-trivial splice variant lacks nearly 40% of the sequence of the native Rab1A, including most of the regulatory interaction sites. We show that this variant of Rab1A represents a stable and folded protein, which is still able to bind nucleotides and co-localizes with membranes. Nevertheless, it should be mentioned that compared to other wild-typeRabGTPases, the measured nucleotide binding affinities are dramatically reduced in the variant studied. Furthermore, the Rab1A variant forms hetero-dimers with wild-type Rab1A and its presence in the cell enhances the efficiency of alkaline phosphatase secretion. However, this variant shows no specificity for GXP nucleotides, a constantly enhanced GTP hydrolysis activity and is no longer controlled by GEF or GAP proteins, indicating a new regulatory mechanism for the Rab1A cycle via alternative non-trivial splicing.

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#26910665   2016/02/25 Save this To Up

Optimizing Biomaterials for Tissue Engineering Human Bone Using Mesenchymal Stem Cells.

Adequate biomaterials for tissue engineering bone and replacement of bone in clinical settings are still being developed. Previously, the combination of mesenchymal stem cells in hydrogels and calcium-based biomaterials in both in vitro and in vivo experiments has shown promising results. However, results may be optimized by careful selection of the material combination.

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#26898843   2016/02/22 Save this To Up

Morphogenetically active scaffold for osteochondral repair (polyphosphate/alginate/N,O-carboxymethyl chitosan).

Here we describe a novel bioinspired hydrogel material that can be hardened with calcium ions to yield a scaffold material with viscoelastic properties matching those of cartilage. This material consists of a negatively charged biopolymer triplet, composed of morphogenetically active natural inorganic polyphosphate (polyP), along with the likewise biocompatible natural polymers N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The porosity of the hardened scaffold material obtained after calcium exposure can be adjusted by varying the pre-processing conditions. Various compression tests were applied to determine the local (nanoindentation) and bulk mechanical properties (tensile/compression test system for force measurements) of the N,O-CMC-polyP-alginate material. Determinations of the Young's modulus revealed that the stiffness of this comparably water rich (and mouldable) material increases during successive compression cycles to values measured for native cartilage. The material not only comprises viscoelastic properties suitable for a cartilage substitute material, but also displays morphogenetic activity. It upregulates the expression of genes encoding for collagen type II and aggrecan, the major proteoglycan within the articular cartilage, in human chondrocytes, and the expression of alkaline phosphatase in human bone-like SaOS-2 cells, as revealed in RT qPCR experiments. Further, we demonstrate that the new polyP-based material can be applied for manufacturing 3D solid models of cartilage bone such as of the tibial epiphyseal plate and the superior articular cartilage surface. Since the material is resorbable and enhances the activity of cells involved in regeneration of cartilage tissue, this material has the potential to be used for artificial articular cartilage implants.

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