Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R proportional to t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R proportional to t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.     

The influence of GFP-actin expression on the adhesion dynamics of HepG2 cells on a model extracellular matrix

Integrins belong to a family of important cell surface receptors which mediate the adhesion of most anchorage-dependent cells to nature extracellular matrix (ECM) and biomaterials. It is known that the binding of integrin with ECM proteins triggers mechanochemical responses of cytoskeleton. To date, the intricate interplay between integrin-ECM interaction and cytoskeleton dynamics leading to the regulation of cell morphogenesis on biomaterials remains largely unknown. In this study, green fluorescence protein (GFP)-actins were expressed in HepG2 cells for the temporal visualization of cytoskeletal structure of adherent cells on naturally derived materials. By combining confocal reflectance contrast microscopy and fluorescence microscopy, the adhesion contact dynamics, cytoskeleton remodeling and two-dimensional spreading of intact and GFP-actin expressing HepG2 cells on collagen and fibronectin-coated substrates are simultaneously probed during the initial cell seeding. First of all, our results show that the evolution of adhesion contact of HepG2 cells upon integrin-collagen or integrin-fibronectin interaction is impaired by GFP-actin expression. Also, the initial rate of cell deformation is reduced by 70% and 43% on fibronectin and collagen, respectively, upon GFP-actin expression. Interestingly, the steady-state adhesion energy of HepG2 cells remains unchanged and increases on fibronectin- and collagen-coated substrate, respectively, upon GFP-actin expression. Our highly integrated biophysical approach demonstrates that GFP-actins diffusively concentrate in the cytoplasmic cortex during initial cell seeding while adhesion contact evolves and cell spreads. Kinetics analysis on the adhesion contact formation demonstrates the intricate interplay between cytoskeleton property and ECM proteins in cell adhesion.     

High density of immobilized galactose ligand enhances hepatocyte attachment and function

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.     

Control of adhesion and detachment of parenchymal liver cells using lactose-carrying polystyrene as substratum.

Adhesion of hepatocytes on culture dishes whose surface was coated with a lactose-carrying styrene homopolymer (PVLA) was investigated. Hepatocytes maintained their round shape on PVLA substratum, which is in contrast to the usual spread shape characteristic of those cultured on collagen and fibronectin substrata. Calcium ion was indispensable for hepatocyte adhesion in PVLA substratum, and hence the hepatocytes on PVLA were easily detached when the culture was treated with ethylenediamine tetraacetic acid (EDTA). The recovered hepatocytes readheres to PVLA. The adhesion of hepatocytes to PVLA was not inhibited by cytochalasin B but by colchicine. Hepatocytes recognize the galactose moieties on the surface of asialoglycoproteins and removes these proteins from the blood stream by receptor mediated endocytosis. The mechanism of adhesion of hepatocytes on PVLA substratum which contains a high density of galactose residues was distinct from the attachment on collagen and fibronectin substrata, and showed great similarity to the receptor and ligand interactions which occurs in the clearance of asialoglycoproteins by hepatocytes.     

Mixed-ligand modification of polyamidoamine dendrimers to develop an effective scaffold for maintenance of hepatocyte spheroids

Compared with a monolayer culture, hepatocyte spheroids are known to maintain liver function for long periods. We found that hepatocytes formed spheroids when cultured on polyamidoamine dendrimers modified with fructose. Because galactose is a ligand for the asialoglycoprotein receptor on the hepatocyte cytoplasmic membrane, it was chosen as another ligand for modification in order to maintain adhesion of spheroids for long periods. Simultaneous modification of dendrimers with fructose and galactose had a marked effect on the time length of spheroid adhesion. Suppression of apoptosis and necrosis was observed in hepatocyte spheroids cultured on a dendrimer modified with fructose and galactose (F/G dendrimer). Moreover, the hepatocyte spheroids cultured on the F/G dendrimer had higher activities of liver-specific functions, such as urea synthesis and albumin gene expression, than did those cultured on single-ligand-modified dendrimers. The expression of heat shock protein (HSP) genes was examined to evaluate the stress response of cells to scaffolds. The hepatocytes cultured on the F/G dendrimer had very low expression levels of both HSP60 and HSP70 mRNAs. Thus immobilization of mixed-ligand-modified dendrimers could generate a suitable surface for hepatocyte spheroid formation. These dendrimers could be a powerful tool for generating custom-made scaffolds for cells other than hepatocytes by selecting the ligands suitable for each cell type.     

Hepatocyte behavior on synthetic glycopolymer matrix: inhibitory effect of receptor-ligand binding on hepatocyte spreading

The interaction of carbohydrate-based polymers with asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes has been used to design hepatocyte adhesion matrices. Therefore, we have characterized the interaction of ASGPR on the surface of hepatocytes with glycopolymer-coated surfaces. Since ASGPRs bound to glycopolymer surfaces escape from internalization and degradation, they were quantified by western blot analysis. The amount of hepatocyte ASGPRs that initially adhered to the glycopolymer surface was proportional to the concentration of the coated glycopolymer. We found that the initial adhesion of hepatocytes to the glycopolymer surface was enhanced by interactions with ASGPR, whereas interactions with ASGPR inhibited the post-adhesion process, a cell adhesion phenomenon that occurs following the initial adhesion. Furthermore, hepatocytes are much more spread on glycopolymer surfaces with lower coating density. Taken together, we suggest that the post-adhesion process triggered hepatocyte spreading on glycopolymer surfaces, and ASGPR-carbohydrate interactions act negatively on the post-adhesion mechanism as well as on hepatocyte spreading on glycopolymer surfaces depending on the density of coated glycopolymers.     

Control of adhesion and detachment of parenchymal liver cells using lactose-carrying polystyrene as substratum

Adhesion of hepatocytes on culture dishes whose surface was coated with a lactose-carrying styrene homopolymer (PVLA) was investigated. Hepatocytes maintained their round shape on PVLA substratum, which is in contrast to the usual spread shape characteristic of those cultured on collagen and fibronectin substrata. Calcium ion was indispensable for hepatocyte adhesion in PVLA substratum, and hence the hepatocytes on PVLA were easily detached when the culture was treated with ethylenediamine tetraacetic acid (EDTA). The recovered hepatocytes readheres to PVLA. The adhesion of hepatocytes to PVLA was not inhibited by cytochalasin B but by colchicine. Hepatocytes recognize the galactose moieties on the surface of asialoglycoproteins and removes these proteins from the blood stream by receptor mediated endocytosis. The mechanism of adhesion of hepatocytes on PVLA substratum which contains a high density of galactose residues was distinct from the attachment on collagen and fibronectin substrata, and showed great similarity to the receptor and ligand interactions which occurs in the clearance of asialoglycoproteins by hepatocytes.     

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.     

3D hepatocyte monolayer on hybrid RGD/galactose substratum

Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell-cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.     

Computer aided time-lapse video analysis of hepatocyte morphology during adhesion to cellulose membranes

An investigation was performed to demonstrate that time-lapse cinematography and computer aided video analysis of cell morphology is suitable to study and compare the characteristics of hepatocytes during the adhesion process to membranes. We chose to compare ordinary cellulose Cuprophan membranes and membranes coated with collagen or fibronectin. Striking differences between uncoated cellulose and fibronectin or collagen coating were seen in the cell count per square millimeter and adhesion behaviour. On the investigated uncoated Cuprophan the hepatocytes were found to attach but not to spread whilst on collagen coated Cuprophan most of the cells spread spherically, and on fibronectin coated membranes most of the cells flattened spherically or polygonally. Time-lapse video microscopy seems to be a valuable technique for assessing the morphologic behaviour of cells in a detailed and quantitative manner in order to improve the hepatocyte culture technique in bioreactors for hybrid systems.     

Adhesion contact dynamics of 3T3 fibroblasts on poly (lactide-co-glycolide acid) surface modified by photochemical immobilization of biomacromolecules

A simple and effective method of biomacromolecule immobilization on biomaterial surface for direct tuning of biophysical parameters such as the initial cell deformation rate, degree of cell spreading and adhesion kinetics is important for tissue engineering. The photochemical immobilization of azide-chitosan (Az-CS) on poly (lactide-co-glycolide) acid (PLGA) is applied here. Chitosan immobilization on PLGA through the photoactive azide group further facilitates subsequent grafting of other biocompatible biomacromolecules like gelatin (Gel) through the active amine groups on CS. This study quantitatively compares the 3T3 fibroblast adhesion dynamics on three PLGA surfaces (Gel-CS-PLGA, CS-PLGA and unmodified PLGA surfaces) using Confocal-Reflectance Interference Contrast Microscopy (C-RICM) together with phase contrast imaging. CS-PLGA and Gel-CS-PLGA surfaces developed were confirmed by X-ray photoelectron spectroscopy, atomic force microscopy and water contact angle and cell adhesion contact dynamics measurements. The cell adhesion was strongest on the Gel-CS-PLGA surface and lowest on unmodified PLGA. The steady state adhesion energy attained by the cells on gelatin modified PLGA surface is determined as 4.0 x 10(-8) J/m(2), which is about 400 times higher than that on PLGA surface (1.1 x 10(-10) J/m(2)). Significantly increased cell adhesion with Gel-CS-PLGA is postulated to result in increased cell spreading. Our integrated biophysical method can quantify the transient contact dynamics and is sufficiently accurate to discriminate even between Gel and CS modified surfaces.     

Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density

The regulation of cell motility by ligand density on substrates with variable microtopography is not well understood. In this report, we studied the adhesion and motility behavior of HepG2 cells on microtextured poly(glycolic-co-lactic)acid (PGLA) copolymer substrates, whose surface bioactivity was differentially modified through the adsorption of 0-5.5 ng/cm(2) collagen. Microtextured PGLA substrates were fabricated as thin films with a uniform surface distribution of micropores of median size of 3.1 +/- 1.5 microm and three-dimensional root mean squared roughness of 0.253 microm. Even in the absence of collagen, cells on microtextured substrates responded to substrate topography by exhibiting a 200% increase in adhesion strength compared with untextured controls and ventral localization of the intracellular adhesion protein vinculin. Further enhancement in adhesion strength (420% over untextured, untreated substrates) was demonstrated with bioactivated, microtextured surfaces, indicating that cell adhesion responses to topography and surface ligand density were cooperative. Our motility studies of cells on untextured substrates adsorbed with different levels of collagen demonstrated that a classical biphasic relationship between the cell population averaged migration rate, mu, and the collagen ligand density was preserved. However, comparison of cell motility responses between untextured and microtextured substrates indicates that the motility versus ligand density curve shifted, such that equivalent levels of cell motility were achieved at lower ligand density on microtextured surfaces. Furthermore, the maximum mu values achieved on the microtextured substrates exceeded those on untextured substrates by twofold. Taken together, we show that the magnitude of subcellular scale microtexture of a polymer substrate can sensitize the cell motility responsiveness to substrate ligand concentration; we suggest that the underlying mechanisms involve alteration in the degree of cell-substrate adhesivity as well as changes in the nature of ligand-induced cell activation processes.     

Xyloglucan as a synthetic extracellular matrix for hepatocyte attachment

The possibility of employing naturally derived xyloglucan (XG) having galactose moieties in the side chain for the development of synthetic extracellular matrix in tissue engineering was studied. Hepatocyte adhesion to the XG-coated polystyrene (PS) dish was 73.9% after 30 min incubation, whereas that to the PS dish as a negative control was 59.1%. The hepatocyte adhesion to the XG-coated surface was dependent on the presence of Ca2+ ions, whereas that to the XG-coated surface could not be induced by Mg2+ ions alone, indicating specific interaction between galactose moieties of XG and asialoglycoprotein receptors of hepatocytes. From the results of fluorescence, confocal laser micrographs and flow cytometry, it was suggested that XG was internalized by hepatocytes through a receptor-mediated mechanism. The DNA synthesis of hepatocytes attached to the XG-coated surface was decreased with an increase of the coating concentration of XG and in the presence of epidermal growth factor (EGF). The spreading shapes of the hepatocytes attached to the surface in the presence of EGF at low concentration of XG (1 microg/ml) were enhanced. The hepatocytes attached to the surface at a high concentration of XG (200 microg/ml) showed round shapes with spheroids after 16 h in the presence of EGF.     

The effect of adhesive ligands on bacterial and fibroblast adhesions to surfaces

The modification of medical device surface with adhesive ligands has been recently shown to be an effective means for making a bioselective surface which can inhibit bacterial adhesion while promoting host cell adhesion on device materials. Currently, the lack of quantitative correlation between the adhesion strength of bacteria, nature of adhesive ligand and adhesion kinetics of mammalian cells hinders the development of such device surface. In this study, the biophysical responses of bacteria and mammalian cells towards adhesive ligand on model device surfaces formed by the chemisorption of dopamine (a moderate antibiotic) on glass are elucidated. The effects of RGD, collagen and dopamine modification on the adhesion strength of two clinically significant bacteria including Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were investigated by the determination of minimum lateral forces for bacterial detachment and the density of adhering bacteria. The result indicates that RGD has no apparent effect on E. coli and S. aureus adhesion, while collagen reduces E. coli but enhances S. aureus. In order to assess the degree of host cell integration, the adhesion kinetics of 3T3 fibroblasts on the four surfaces was examined by confocal reflectance interference contrast microscopy (C-RICM). In contrast to the difference found in bacterial adhesion, the result indicates that both collagen and RGD significantly enhance the initial rate of deformation and adhesion energy for fibroblasts compared to those on glass and dopamine-glass. Overall, it is demonstrated that the choice of adhesive ligand is critical for designing a device surface which simultaneously minimizes bacterial adhesion and enhances host cell integrations.     

Effect of ligand orientation on hepatocyte attachment onto the poly(N p-vinylbenzyl-o-β-D-galactopyranosyl-D-gluconamide) as a model ligand of asialoglycoprotein

The orientation effect of galactose ligand on hepatocyte attachment was investigated. Poly(N-p-vinylbenzyl-o-β-D-galactopyranosyl-D-gluconamide)(PVLA, a β-galactose-carrying styrene homopolymer, was used as a model ligand for the asialoglycoprotein receptors on hepatocytes. PVLA was transferred onto the poly(γ-benzyl L-glutamate) (PBLG) or PBLG/poly(ethylene glycol) (PEG)PBLG Langmuir-Blodgett (LB) films as the monolayer level. The dichroic fluorescence values of the confocal microscope indicated that the PVLA transferred onto the LB films was located with a preferential orientation of its molecular axes with regard to the direction of the α-helix of polypeptide. Hepatocyte recognized well-oriented galactose moieties of the surface of PVLA through asialoglycoprotein receptors.     

Dual requirements of extracellular matrix protein and chitosan for inducing adhesion contact evolution of esophageal epithelia

It has been recently shown that chitosan (CHI)/collagen prostheses induced epithelization at the esophagus site of animal model. However, little is known on the biophysical mechanisms of cell adhesion on CHI-based material pertaining to esophagus tissue engineering. In this study, the adhesion contact dynamics of porcine esophageal epithelial cells seeded on CHI surface is probed using confocal-reflectance interference contrast microscopy in conjunction with phase-contrast microscopy. First of all, cells fail to form any adhesion contact on either CHI or elastin (ES)-coated surface. On CHI coated with fibronectin (CHI-FN) or elastin (CHI-ES), strong adhesion contact of cells evolved over time until they reached a steady-state level. The initial cell deformation rates of cells on CHI-FN and CHI-ES are 0.0138 and 0.0151 min(-1), respectively. Interestingly, cells on fibronectin (FN) coated substrate transiently form strong adhesion contact and eventually undergo deadhesion. Moreover, the steady-state adhesion energy of epithelial cells on CHI-FN is 1.73 and 148 times larger than that on CHI-ES and FN, respectively. The actin of cells on CHI-FN transforms from microfilament meshes at cell periphery to stress fibers throughout the cytoplasm during cell seeding. At the same time, vinculin staining demonstrated the evolution of focal adhesion complexes in cells on CHI-FN after 130 min of seeding. Interestingly, CHI-ES induces the formation of focal adhesion complexes in a lesser extent in cell but fails to lead to stress fiber formation. Overall, our study reveals that long-term adhesion contact evolution of esophageal epithelia is only triggered by both extracellular matrix protein and chitosan.     

Nanofabricated collagen-inspired synthetic elastomers for primary rat hepatocyte culture

Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture.     

Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold

Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760 nm were prepared by electrospinning of poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66 nmol/cm(2) was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50-300 microm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20-100 microm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.     

Effect of carbohydrates attached to polystyrene on hepatocyte morphology on sugar-derivatized polystyrene matrices

Sugar-carrying polymers have been utilized as artificial matrices for cell adhesion in tissue engineering. We have developed sugar-derivatized polystyrenes (PV-sugars) as artificial matrices, which control hepatocyte adhesion and hepatic function. Hepatocytes adhere to PV-sugar matrices in a receptor-mediated manner. In this study, we designed a new galactose-derivatized PV-sugar, poly-(6-O-p-vinylbenzyl-alpha-D-galactose) (PV6Gal) and evaluated the role of carbohydrate attached to polystyrene (PS) backbone in the morphological difference of hepatocyte cultured on PV-sugar matrices. Hepatocytes spread on monosaccharide-derivatized PV-sugars but not on disaccharide-derivatized PV-sugars. The actin filament remained aggregated in the central area of the cell body on disaccharide-derivatized PV-sugars. Hepatocyte cell bodies fully were spread on collagen, and the actin filament was almost completely reorganized. Hepatocyte spreading on monosaccharide-derivatized PV-sugars, however, was caused by protrusive cell-matrix contact like lamellipodia and the actin filament was not completely reorganized. This indicated that hepatocyte spreading on PV-sugar matrices was restricted compared with ECM-mediated cell spreading. In addition, typical spheroid formation of hepatocytes was promoted on disaccharide-derivatized PV-sugars compared with monosaccharide-derivatized PV-sugars. Although hepatocytes adhered with different affinities to PV-sugar matrices, hepatocyte morphology was not affected by the adhesion affinity. We suggest that the type of carbohydrate attached to the PS backbone governs the morphology of hepatocyte cultured on PV-sugar matrices.