In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold.

Polymer scaffolds which can support cells to grow as well as deliver growth factors to the cells simultaneously have great potential for the successful regeneration of failed tissues. As popularly used vehicles to deliver anti-cancer drugs and growth factors, microspheres also show many advantages as substrates to guide the growth of cells. Therefore, we aimed to examine the feasibility of using microspheres as ideal scaffolds for liver tissue engineering. To determine the capabilities of previously used microsphere scaffold to deliver growth factors simultaneously, this work investigated a long-term (about three months) release of bovine serum albumin (BSA) from microsphere scaffolds fabricated by using two different polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), poly(lactide-co-glycolide) acid (PLGA, 5050) and a blend of PLGA and PHBV. BSA served as a model for hepatocyte growth factor (HGF) since both proteins have similar molecular weights and hydrophilicity. Furthermore, HGF was encapsulated into the PLGA/PHBV composite microsphere with a core-shell structure, and sustained delivery of HGF with maintained bioactivity was achieved for at least 40 days. The moderate degradation rate (about 55% loss of the initial mass) and well-preserved structure after three months of incubation indicated that the PLGA/PHBV composite microspheres would therefore be more suitable than the pure PHBV or PLGA microspheres as a scaffold for engineering liver tissue.

Delivery of basic fibroblast growth factor from gelatin microsphere scaffold for the growth of human umbilical vein endothelial cells.

One of the major obstacles for engineering large tissue or organs such as the liver in vitro is the insufficient supply of nutrients and oxygen to the cells growing inside the scaffold, which reduces cell viability significantly. Therefore, vascularization of the scaffolding system is necessary for successful engineering of such tissues. In this study, we investigated the use of gelatin microsphere as scaffold to culture human umbilical vein endothelial cells, which is considered to be the basis and premise for the formation of blood vessels. The gelatin microspheres were crosslinked with different concentrations of glutaraldehyde to study the effects of crosslinking extent on the growth of endothelial cells. The swelling ratios of the gelatin microspheres decreased from 5.9 +/- 0.8 to 3.9 +/- 0.6 with the increase of the crosslinking extent. Basic fibroblast growth factors (bFGFs), which can improve endothelial cell proliferation as well as stimulate the formation of capillary vessels, were incorporated into the gelatin microspheres through ionic complexation. Sustained delivery of the growth factors was achieved for at least 2 weeks. The proliferation of the cells cultured on the bFGF-encapsulated microspheres was improved by about two times as compared to control and about 1.3 times as compared to blank microspheres, which indicated that the bioactivity of bFGF was well maintained, and the delivery of the growth factors directly to the cells significantly improved the success of this tissue engineering system.

Characterization of porous poly(D,L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells.

This study presents the application of the porous poly(D,L-lactic-co-glycolic acid) (PLGA) sponges fabricated from an organic solvent free supercritical gas foaming technique. Two formulations of PLGA sponges with different co-polymer compositions (85:15 and 50:50) were fabricated as novel scaffolds to guide human hepatoma cell line, Hep3B cell growth in vitro. The PLGA sponges showed desirable biodegradability and exhibited uniform pore size distribution with moderate interconnectivity. It was observed in this study that cells cultured on PLGA sponges showed lower proliferation rate as compared to the control during 14 days of culture as measured by using total DNA and methylthiazol tetrazolium (MTT) assays. However, the cells cultured on the sponges tended to aggregate to form cell islets which were able to express better hepatic functions. The enzyme-linked immunosorbent assay (ELISA) results showed that the cell-sponge constructs secreted 1.5-3.0 times more albumin than the control when normalized to cellular content. In a similar fashion, its detoxification ability was also predominantly higher than that of the control as indicated by the ethoxyresorufin-O-deethylase (EROD) results. By comparing the cells growing on the two formulations of PLGA sponges, it was found that the PLGA 85:15 sponge exhibited better conductive and desirable environment for hep3B cells as justified by better cell infiltration, higher proliferation and hepatic function than the PLGA 50:50 sponge.

An integrin-specific collagen-mimetic peptide approach for optimizing Hep3B liver cell adhesion, proliferation, and cellular functions.

This study focused on mimicking collagen structurally and biologically using various peptide sequences toward realizing an artificial collagen-like biomaterial. Collagen-mimetic peptides (CMPs) incorporating integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) sequence from residues 502 to 507 of collagen alpha(1)(I) were used as a bioadhesive matrix and grafted onto poly(3-hydroxybutyrate-co3-hydroxyvalerate) microspheres to optimize cell adhesion, proliferation, and functions. Cell recognition of these biomolecules appeared to be conformation dependent, with the CMP1 of higher triple helix stability being preferred. Absence of the GFOGER hexapeptide in the CMP1' and CMP2' caused an adverse effect on the level of cell adhesion (<10%). The GFOGER-containing triple-helical CMPs effectively inhibited cell adhesion to collagen in a competition assay. The cell-adhesion activity of the CMP1 was approximately 50% of that of collagen. The cell spreading on the CMP1 was comparable with that observed on collagen. The presence of the CMP1 promoted cell attachment and spreading on the microspheres and extensive cell proliferation and bridging. Slower cell proliferation was observed on the blank microspheres. Live-dead assay showed that most cells are viable after 10-day culture. The presence of CMP1 on the microspheres maintained the albumin secretion and P-450 activity levels of the liver cells for up to 14 days. Our results established the potential of CMP1 to create a collagen-like microenvironment for optimizing cellular responses for liver tissue engineering.

Proteins combination on PHBV microsphere scaffold to regulate Hep3B cells activity and functionality: a model of liver tissue engineering system.

The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.

Growing tissue-like constructs with Hep3B/HepG2 liver cells on PHBV microspheres of different sizes.

In this study, an oil-in-water emulsion solvent evaporation technique was used to fabricate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), microspheres as scaffold, to guide liver cell growth. Human hepatoma cell lines, HepG2 and Hep3B, were cultured in vitro on both the microspheres and polymer films. SEM and optical microscope images showed that multilayer cells were formed among the microspheres to bridge them together and developed into cell-construct aggregates after 1 week of culture. MTT results showed that the cell proliferation on the microspheres was more than two times higher than that on the films after 12 days of culture. The cells seeded on microspheres secreted albumin 2-4 times more than that on the positive control after 1 week of culture, which indicated that this hepatic function was greatly improved by the aggregation of cells on microspheres. Although HepG2 failed to express P-450 activity, this hepatic function was preserved when Hep3B cultured on microspheres. All the results indicated that PHBV microspheres are appropriate scaffolds for liver tissue engineering.