Gopal Pande

Mg–Zr–Sr alloys as biodegradable implant materials

Novel Mg-Zr-Sr alloys have recently been developed for use as biodegradable implant materials. The Mg-Zr-Sr alloys were prepared by diluting Mg-Zr and Mg-Sr master alloys with pure Mg. The impact of Zr and Sr on the mechanical and biological properties has been thoroughly examined. The microstructures and mechanical properties of the alloys were characterized using optical microscopy, X-ray diffraction and compressive tests. The corrosion resistance was evaluated by electrochemical analysis and hydrogen evolution measurement. The in vitro biocompatibility was assessed using osteoblast-like SaOS2 cells and MTS and haemolysis tests. In vivo bone formation and biodegradability were studied in a rabbit model. The results indicated that both Zr and Sr are excellent candidates for Mg alloying elements in manufacturing biodegradable Mg alloy implants. Zr addition refined the grain size, improved the ductility, smoothed the grain boundaries and enhanced the corrosion resistance of Mg alloys. Sr addition led to an increase in compressive strength, better in vitro biocompatibility, and significantly higher bone formation in vivo. This study demonstrated that Mg-xZr-ySr alloys with x and y ≤5 wt.% would make excellent biodegradable implant materials for load-bearing applications.

The influence of surface energy of titanium-zirconium alloy on osteoblast cell functions in vitro

The success of an implant used for bone regeneration and repair is determined by the events that take place at the cell-material interface. An understanding of these interactions in vitro gives insights into the formulation of ideal conditions for their effective functioning in vivo. Thus, it is not only important to understand the physico-chemical properties of the materials but, also necessary to assess the cellular responses to them to determine their long-term stability and efficacy as implants. In the present study, we have compared the physico-chemical and biological properties of titanium (Ti) and two Ti-based alloys, namely: Ti- Zirconium (TiZr) and Ti-Niobium (TiNb). The morphology, chemical analysis, surface roughness, and contact angle measurements of the alloys were assessed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), profilometer, and contact angle goniometer, respectively whereas the biological properties of the materials were evaluated by measuring the adhesion, proliferation, and differentiation of MC3T3-E1 osteoblast cells on the surfaces of these alloys. Our results indicate that the biological properties of osteoblasts were better on TiZr surface than on TiNb surface. Furthermore, the surface energy and substrate composition influenced the superior biological activity of the TiZr alloy.

The role of membrane lipids in regulation of integrin functions.

Recent evidence suggests that the biochemical and physical organization of lipid molecules in the plasma membrane can affect integrin-mediated cellular functions. The nature and mechanism of integrin-lipid interactions are unknown, but it is clear that they play specific roles in modulating the properties of integrins and integrin-associated proteins. A better knowledge of integrin functions, especially in the lipid milieu of plasma membranes, is necessary for the understanding of the phenomena that are regulated by integrins.

p53 Target Gene SMAR1 Is Dysregulated in Breast Cancer: Its Role in Cancer Cell Migration and Invasion

Tumor suppressor SMAR1 interacts and stabilizes p53 through phosphorylation at its serine-15 residue. We show that SMAR1 transcription is regulated by p53 through its response element present in the SMAR1 promoter. Upon Doxorubicin induced DNA damage, acetylated p53 is recruited on SMAR1 promoter that allows activation of its transcription. Once SMAR1 is induced, cell cycle arrest is observed that is correlated to increased phospho-ser-15-p53 and decreased p53 acetylation. Further we demonstrate that SMAR1 expression is drastically reduced during advancement of human breast cancer. This was correlated with defective p53 expression in breast cancer where acetylated p53 is sequestered into the heterochromatin region and become inaccessible to activate SMAR1 promoter. In a recent report we have shown that SMAR1 represses Cyclin D1 transcription through recruitment of HDAC1 dependent repressor complex at the MAR site of Cyclin D1 promoter. Here we show that downmodulation of SMAR1 in high grade breast carcinoma is correlated with upregulated Cyclin D1 expression. We also established that SMAR1 inhibits tumor cell migration and metastases through inhibition of TGFβ signaling and its downstream target genes including cutl1 and various focal adhesion molecules. Thus, we report that SMAR1 plays a central role in coordinating p53 and TGFβ pathways in human breast cancer.

Modulation of α5β1 integrin functions by the phospholipid and cholesterol contents of cell membranes

Several modifications of the α5β1 integrin, which alter its intracellular and extracellular interaction with fibronectin and other proteins, have been reported. However, the significance of the lateral mobility of integrin molecules in the plasma membrane, as a regulator of their distribution and function, is poorly understood. We examined this problem by increasing the cholesterol content of plasma membranes, and consequently modifying the fluidity of membrane phospholipids, in rat fibroblasts. Under these conditions, the clustering of α5β1 integrin molecules in focal adhesions, their adhesion to the cell‐binding domain of fibronectin, and their association with the cytoskeletal protein talin were significantly enhanced as compared to control cells. However, the activation of MAP‐kinase pathways by the association of fibronectin with α5β1 integrin, and its association with integrin‐linked kinase (ilk), were suppressed. The treated cells also showed distinct changes in shape, and their actin stress fiber network was more dense and thick as compared to control cells. The changes in fluidity of phospholipids occurred differentially and fluidity of phosphatidyl‐ethanolamine increased, while that of phosphatidyl‐choline was reduced. Our results suggest that proteins in focal adhesions could be partitioned in specific lipid domains, which regulate specific aspects of α5β1 integrin functions. J. Cell. Biochem.77:517–528, 2000.

Flow cytometric study of changes in the intracellular free calcium during the cell cycle

We measured the intracellular levels of free cytoplasmic calcium in different phases of the cell cycle in viable rat fibroblasts, using two parameter flow cytometric analysis with Hoechst 33342 as the DNA specific dye and Fluo-3 as the calcium sensitive dye. We studied changes in calcium levels during the G1 phase of cell cycle by arresting cells with chemical agents such as staurosporine and hydroxyurea or by density dependent arrest of cell growth. We show that levels of calcium are lowest at the beginning of G1 phase but rise steadily with its progression and culminate at the G1/S border. Our results suggest that complex changes occur in calcium levels during the process of mitotic division. During progression of S and G2 phases, calcium levels decline and increase respectively. Our results offer a new methodology to estimate intracellular calcium levels in specific phases of the cell cycle. Based upon these results we propose a general scheme representing the changes in the intracellular calcium concentration during the progression of the cell cycle.

Alterations in Cell-Extracellular Matrix Interactions during Progression of Cancers

Cancer progression is a multistep process during which normal cells exhibit molecular changes that culminate into the highly malignant and metastatic phenotype, observed in cancerous tissues. The initiation of cell transformation is generally associated with genetic alterations in normal cells that lead to the loss of intercellular- and/or extracellular-matrix- (ECM-) mediated cell adhesion. Transformed cells undergo rapid multiplication and generate more modifications in adhesion and motility-related molecules which allow them to escape from the original site and acquire invasive characteristics. Integrins, which are multifunctional adhesion receptors, and are present, on normal as well as transformed cells, assist the cells undergoing tumor progression in creating the appropriate environment for their survival, growth, and invasion. In this paper, we have briefly discussed the role of ECM proteins and integrins during cancer progression and described some unique conditions where adhesion-related changes could induce genetic mutations in anchorage-independent tumor model systems.

Lipopeptide with a RGDK Tetrapeptide Sequence Can Selectively Target Genes to Proangiogenic α5β1 Integrin Receptor and Mouse Tumor Vasculature

Integrins, the major class of alphabeta heterodimeric transmembrane glycoprotein receptors, play crucial roles in mediating tumor angiogenesis. Genetic ablation experiments combined with use of antibodies/peptide ligands for blocking either alpha(5) or beta(1) integrins have convincingly demonstrated alpha(5)beta(1) integrin to be unquestionably proangiogenic among the 24 known integrin receptors. Herein, we report on a novel RGDK-lipopeptide 1 that targets selectively alpha(5)beta(1) integrin and is capable of targeting genes to mouse tumor vasculatures.

Variation of mitochondrial size during the cell cycle: A multiparameter flow cytometric and microscopic study

Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line.

Lineage‐specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell‐free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell‐free matrices from a murine bone marrow stromal cell line (MS‐5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellular matrices were used as biological scaffolds for the lineage‐specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony‐forming assays. We could get up to 80‐fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering‐based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow. Copyright