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Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

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Chracterization of nanoparticles: High resolution TEM of (A) chitosan nanoparticles (B) HGF incorporated nanoparticles. Particles are flat and round shape (C). Cumulative release pattern of HGF from CNP. Sustainable release of growth factor could be seen from the release curve. Values are presented as mean ± S.D for triplicate experiment.
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Figure 1: Chracterization of nanoparticles: High resolution TEM of (A) chitosan nanoparticles (B) HGF incorporated nanoparticles. Particles are flat and round shape (C). Cumulative release pattern of HGF from CNP. Sustainable release of growth factor could be seen from the release curve. Values are presented as mean ± S.D for triplicate experiment.

Mentions: Morphological characteristics of CNP and the release pattern of HGF from CNP were studied. The loading efficiency of nanoparticles and entrapment efficiencies of HGF were 5 ng HGF/mg of nanoparticles and 85% respectively. This was achieved with chitosan to TPP ratio at 2:1. At lower ratio, more protein could be incorporated since more positive charge from chitosan will be available. Care was taken not to form crystal-like particles during the preparation. Plain and HGF loaded nanoparticles observed by HRTEM (Figure 1 A and 1B) appeared spherical in shape with a particle size range of 50-100 nm. The in vitro release profile of HGF from the nanoparticles in PBS at the predetermined time was expressed as percentage of HGF released with respect to the total amount of HGF encapsulated (Figure 1C). HGF release observed after 24 and 35 days was 82 and 85% respectively, indicating the monophasic release pattern of nanoparticles over a period of at least 25 days. This indicates that physical adsorption of the growth factor on the nanoparticles is unlikely, which is an essential characteristic feature for the nanoparticles required for sustained release.


Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Chracterization of nanoparticles: High resolution TEM of (A) chitosan nanoparticles (B) HGF incorporated nanoparticles. Particles are flat and round shape (C). Cumulative release pattern of HGF from CNP. Sustainable release of growth factor could be seen from the release curve. Values are presented as mean ± S.D for triplicate experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108285&req=5

Figure 1: Chracterization of nanoparticles: High resolution TEM of (A) chitosan nanoparticles (B) HGF incorporated nanoparticles. Particles are flat and round shape (C). Cumulative release pattern of HGF from CNP. Sustainable release of growth factor could be seen from the release curve. Values are presented as mean ± S.D for triplicate experiment.
Mentions: Morphological characteristics of CNP and the release pattern of HGF from CNP were studied. The loading efficiency of nanoparticles and entrapment efficiencies of HGF were 5 ng HGF/mg of nanoparticles and 85% respectively. This was achieved with chitosan to TPP ratio at 2:1. At lower ratio, more protein could be incorporated since more positive charge from chitosan will be available. Care was taken not to form crystal-like particles during the preparation. Plain and HGF loaded nanoparticles observed by HRTEM (Figure 1 A and 1B) appeared spherical in shape with a particle size range of 50-100 nm. The in vitro release profile of HGF from the nanoparticles in PBS at the predetermined time was expressed as percentage of HGF released with respect to the total amount of HGF encapsulated (Figure 1C). HGF release observed after 24 and 35 days was 82 and 85% respectively, indicating the monophasic release pattern of nanoparticles over a period of at least 25 days. This indicates that physical adsorption of the growth factor on the nanoparticles is unlikely, which is an essential characteristic feature for the nanoparticles required for sustained release.

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

Show MeSH
Related in: MedlinePlus