Journal of Tissue Engineering and Regenerative Medicine. 2015 July; 9(7): 745-858.
Cellular behavior of hepatocyte-like cells from nude mouse bone marrow-derived mesenchymal stem cells on galactosylated poly(D,L-lactic-co-glycolic acid)
Hyun Roh1,2, Dae Hyeok Yang1, Heung Jae Chun1,2* and Gilson Khang3
1Institute of Cell and Tissue Engineering, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea
2Department of Biomedical Sciences, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea
3Department of BIN Fusion Technology, Department of Polymer-Nanoscience and Technology and Polymer BIN Research Centre, Chonbuk National University, Deokjin, Jeonju, Republic of Korea
Prof. Heung Jae Chun, Ph. D.
Institute of Cell and Tissue Engineering, Department of Biomedical Sciences, College of Medicine, Catholic University of Korea, Seoul 137-701, Republic of Korea.
Phone: +82-2-2258-7033, E-mail: email@example.com
Previously, the galactosylation of poly(D,L-lactic-co-glycolic acid) (PLGA) surface was accomplished by grafting allylamine (AA), using inductively coupled plasma-assisted chemical vapour deposition (ICP-CVD) and conjugating lactobionic acid (LA) with AA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) activation for hepatic tissue-engineering purposes. As a continuation study, the cellular behaviour of hepatocyte-like cells (HLCs) on the surface of the galactosylated PLGA was investigated. Nude mouse bone marrow-derived mesenchymal stem cells (MSCs) were cultured under hepatogenic conditions and the differentiated cells were characterized by reverse-transcription polymerase chain reaction (RT–PCR), immunofluorescence and periodic acid–Schiff (PAS) staining. Galactosylated PLGA enhanced the proliferation rate of HLCs compared to the control; HLCs on the surface of the sample became aggregated and formed spheroids after 3 days of culture. A large number of cells on the surface of the sample exhibited increased liver-specific functional activities, such as albumin and urea secretions. In addition, multicellular spheroids in the sample strongly expressed phospholyated focal adhesion kinase (pFAK) (cell–matrix interactions), E-cadherin (cell–cell interactions) and connexin 32 (Cox32; gap junction).
KEYWORDS: adhesion molecule; galactosylated poly(d,l-lactic-co-glycolic acid); hepatocyte-like cell; mesenchymal stem cell; spheroid
Although the liver is known as a regenerative organ, it is difficult to bring back the liver to its normal condition once damaged . Therefore, understanding and manufacturing a bioartificial liver is often needed in the field of tissue engineering and regenerative medicine. In the present study, we investigated the cellular behavior of hepatocyte-like cells (HLCs), generated from nude mouse bone marrow-derived MSCs, on the surface of galactosylated poly(D,L-lactic-co-glycolic acid) (PLGA) . Galactose is known to induce the formation of hepatocyte aggregates, due to the interaction between asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes and galactose ligands [2-4]. The PLGA-g-AA/LA film showed the highest proliferation rate of the cells compared to the PLGA-g-AA and PLGA films (Figure 1). SEM images revealed the morphological changes of cultured on the surface of the films throughout the proliferation period. (Figure 1). Cellular interactions, specifically cell-matrix and cell-cell interactions, were analysed by expression of phosphorylated focal adhesion kinase (p-FAK) and E-cadherin, and connexin 32 (Cox32) during spheroid formation of HLCs. The amount of albumin and urea secretions of HLCs was examined as factors for liver-specific structural and functional maintenance. (Figure 2) Consequently, we found that galactosylated PLGA enhanced the proliferation rate of HLCs compared to pristine PLGA, and HLCs on the surface of the sample became aggregated and formed spheroids, due to the increase in pFAK (cell-matrix), E-cadherin (cell-cell) and Cox 32 expression (Figure 3) . Moreover, the spheroids of HLCs on the galactosylated surface increased and maintained liver-specific functions as the biomimetics of hepatic tissues
Figure 1. (A) In vitro proliferation of HLCs and (B) SEM images of HLCs cultured on PLGA, PLGA-g-AA and PLGA-g-AA/LA films for 1, 3, 5 and 7 days. The cell proliferation was evaluated by CCK-8.
Figure 2. Secretions of (A) albumin and (B) urea secretions in HLCs cultured on PLGA, PLGA-g-AA and PLGA-g-AA/LA films for 1, 3, 5 and 7 days.
Figure 3. Immunofluorescence images of (A) pFAK and (B) E-cadherin in HLCs cultured on PLGA, PLGA-g-AA and PLGA-g-AA/LA films
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 Roh H, Yang DH, Chun HJ et al. 2015; Cellular behavior of hepatocyte-like cells from nude mouse bone marrow-derived mesenchymal stem cells on galactosylated poly(D,L-lactic-co-glycolic acid). J Tissue Eng Regen Med 9: 745-858.
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