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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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Histological appearance of the liver: (A) Expression of collagen was viewed by sirius red staining: Collagen bridge was uniform in control and HSC treated groups but not in MSC and MSC+HGF-CNP treated groups (original magnification × 100). (B) Quantitative analysis of fibrosis is shown in histographic representation. The degree of fibrosis was expressed as the percentage of the total area measured. The fibrosis areas were significantly low in MSC and MSC+HGF-CNP treated groups (P < 0.05). However there were no statistical differences in liver fibrosis between control and HSC treated groups. (C) Hydroxyproline content was estimated by Neuman and Logan method. Hydroxyproline content did not vary significantly between control and HSC treated groups and but was significantly low in MSC and MSC+HGF+CNP groups (P < 0.05). (D) Formalin fixed liver tissues were sectioned into 5 μm size and stained with H&E dyes. Infiltration of inflammatory and necrotic cells was ubiquitous in control and HSC treated groups however, MSC treated groups did not show inflammatory cells (original magnification ×100) Control (1); HSC (2); MSC (3); HSC+HGF-CNP (4); MSC+HGF-CNP (5).
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Figure 4: Histological appearance of the liver: (A) Expression of collagen was viewed by sirius red staining: Collagen bridge was uniform in control and HSC treated groups but not in MSC and MSC+HGF-CNP treated groups (original magnification × 100). (B) Quantitative analysis of fibrosis is shown in histographic representation. The degree of fibrosis was expressed as the percentage of the total area measured. The fibrosis areas were significantly low in MSC and MSC+HGF-CNP treated groups (P < 0.05). However there were no statistical differences in liver fibrosis between control and HSC treated groups. (C) Hydroxyproline content was estimated by Neuman and Logan method. Hydroxyproline content did not vary significantly between control and HSC treated groups and but was significantly low in MSC and MSC+HGF+CNP groups (P < 0.05). (D) Formalin fixed liver tissues were sectioned into 5 μm size and stained with H&E dyes. Infiltration of inflammatory and necrotic cells was ubiquitous in control and HSC treated groups however, MSC treated groups did not show inflammatory cells (original magnification ×100) Control (1); HSC (2); MSC (3); HSC+HGF-CNP (4); MSC+HGF-CNP (5).

Mentions: Fibrotic conditions were assessed after fourth week of transplantation using sirius red staining and hyproxyproline content of the cirrhotic tissue. The representative images for fibrosis are shown in Figure 4A. Distribution of fibrosis was extensively reduced in MSC/+HGF-CNP group followed by MSC alone compared to control, HSC or HSC+HGF-CNP treated groups. Quantification of fibrosis by image analysis (Figure 4B) clearly indicated that the level of fibrosis has been significantly reduced in MSC, 4.56 ± 0.29 (P < 0.05) and MSC+HGF-CNP, 2.15 ± 0.14 (P < 0.01) treated groups but not in HSC (6.89 ± 0.24) or HSC+HGF-CNP (5.89 ± 0.18) groups compared to control. The hyproxyproline content (Figure 4C) in HSC or HSC+HGF-CNP treated groups did not show significant difference from the control group; however in the case of MSC or MSC+HGF-CNP treated groups, the hyproxyproline content was significantly low compared to the control (P < 0.01). These results provided direct evidence for an antifibrotic effect of MSC and MSC + HGF-CNP combination. Figure 4D shows the histology of liver tissue of control and treated groups. The microscopical view of the H&E stained specimens revealed the appearance of disrupted tissue architecture with large fibrous septa and infiltration of inflammatory and necrotic cells in the liver sections of control, HSC or HSC+HGF-CNP treated groups but not in MSC and MSC+HGF-CNP treated groups.


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)

Histological appearance of the liver: (A) Expression of collagen was viewed by sirius red staining: Collagen bridge was uniform in control and HSC treated groups but not in MSC and MSC+HGF-CNP treated groups (original magnification × 100). (B) Quantitative analysis of fibrosis is shown in histographic representation. The degree of fibrosis was expressed as the percentage of the total area measured. The fibrosis areas were significantly low in MSC and MSC+HGF-CNP treated groups (P < 0.05). However there were no statistical differences in liver fibrosis between control and HSC treated groups. (C) Hydroxyproline content was estimated by Neuman and Logan method. Hydroxyproline content did not vary significantly between control and HSC treated groups and but was significantly low in MSC and MSC+HGF+CNP groups (P < 0.05). (D) Formalin fixed liver tissues were sectioned into 5 μm size and stained with H&E dyes. Infiltration of inflammatory and necrotic cells was ubiquitous in control and HSC treated groups however, MSC treated groups did not show inflammatory cells (original magnification ×100) Control (1); HSC (2); MSC (3); HSC+HGF-CNP (4); MSC+HGF-CNP (5).
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Figure 4: Histological appearance of the liver: (A) Expression of collagen was viewed by sirius red staining: Collagen bridge was uniform in control and HSC treated groups but not in MSC and MSC+HGF-CNP treated groups (original magnification × 100). (B) Quantitative analysis of fibrosis is shown in histographic representation. The degree of fibrosis was expressed as the percentage of the total area measured. The fibrosis areas were significantly low in MSC and MSC+HGF-CNP treated groups (P < 0.05). However there were no statistical differences in liver fibrosis between control and HSC treated groups. (C) Hydroxyproline content was estimated by Neuman and Logan method. Hydroxyproline content did not vary significantly between control and HSC treated groups and but was significantly low in MSC and MSC+HGF+CNP groups (P < 0.05). (D) Formalin fixed liver tissues were sectioned into 5 μm size and stained with H&E dyes. Infiltration of inflammatory and necrotic cells was ubiquitous in control and HSC treated groups however, MSC treated groups did not show inflammatory cells (original magnification ×100) Control (1); HSC (2); MSC (3); HSC+HGF-CNP (4); MSC+HGF-CNP (5).
Mentions: Fibrotic conditions were assessed after fourth week of transplantation using sirius red staining and hyproxyproline content of the cirrhotic tissue. The representative images for fibrosis are shown in Figure 4A. Distribution of fibrosis was extensively reduced in MSC/+HGF-CNP group followed by MSC alone compared to control, HSC or HSC+HGF-CNP treated groups. Quantification of fibrosis by image analysis (Figure 4B) clearly indicated that the level of fibrosis has been significantly reduced in MSC, 4.56 ± 0.29 (P < 0.05) and MSC+HGF-CNP, 2.15 ± 0.14 (P < 0.01) treated groups but not in HSC (6.89 ± 0.24) or HSC+HGF-CNP (5.89 ± 0.18) groups compared to control. The hyproxyproline content (Figure 4C) in HSC or HSC+HGF-CNP treated groups did not show significant difference from the control group; however in the case of MSC or MSC+HGF-CNP treated groups, the hyproxyproline content was significantly low compared to the control (P < 0.01). These results provided direct evidence for an antifibrotic effect of MSC and MSC + HGF-CNP combination. Figure 4D shows the histology of liver tissue of control and treated groups. The microscopical view of the H&E stained specimens revealed the appearance of disrupted tissue architecture with large fibrous septa and infiltration of inflammatory and necrotic cells in the liver sections of control, HSC or HSC+HGF-CNP treated groups but not in MSC and MSC+HGF-CNP treated groups.

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