Biomedical subjects

Prabhas V Moghe

Publications and source records attributed to Prabhas V Moghe.

16 recordsLinked to original sources

PubMed2006

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning.

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.

Adsorption↗

PubMed2006

Albumin-derived nanocarriers: substrates for enhanced cell adhesive ligand display and cell motility.

Cell-adhesive ligands organized on nanoscale synthetic biomaterials can potentially recapitulate the nanoscale organization of extracellular matrix and the consequent effects of cell dynamics. In this study, 100 nm albumin nanocarriers (ANC) were fabricated to serve as nanoscale organizational units for a well-defined ligand, the recombinant fragment from fibronectin comprised of the RGD-containing module 10 and the synergy-region-containing module 9. Conventional protein conjugation chemistry was employed to fabricate nanocarriers with increasing levels of displayed ligand. Presentation of ligand-functionalized ANCs adsorbed onto substrates was found to enhance keratinocyte attachment when compared to equivalent levels of adsorbed ligands, supported by ELISA data that the display of ligand on ANCs essentially increased the accessibility of the cell-binding domain and AFM data that the ligand was likely exposed due to ligand-ANC repulsion. The ligand presentation from ANCs converted the cellular morphology from a stationary phenotype to a motile phenotype, with the expression of filopodia-like microextensions, and a decrease in focal adhesions, indicating decreased cell adhesion strength. Consequently, cell motility was found to be significantly elevated on ligand-ANC substrates relative to substrates with equivalent levels of ligand. Overall, the ligand-functionalized albumin nanocarriers offer a unique model platform with two distinct properties: enhanced ligand exposure for enhancement of cell attachment to ligands at low concentrations; and enhanced cell detachment, motile phenotype, and migration kinetics.

Biocompatible Materials↗

PubMed2006

Tissue assembly guided via substrate biophysics: applications to hepatocellular engineering.

The biophysical nature of the cellular microenvironment, in combination with its biochemical properties, can critically modulate the outcome of three-dimensional (3-D) multicellular morphogenesis. This phenomenon is particularly relevant for the design of materials suitable for supporting hepatocellular cultures, where cellular morphology is known to be intimately linked to the functional output of the cells. This review summarizes recent work describing biophysical regulation of hepatocellular morphogenesis and function and focuses on the manner by which biochemical cues can concomitantly augment this responsiveness. In particular, two distinct design parameters of the substrate biophysics are examined--microtopography and mechanical compliance. Substrate microtopography, introduced in the form of increasing pore size on collagen sponges and poly(glycolic acid) (PGLA) foams, was demonstrated to restrict the evolution of cellular morphogenesis to two dimensions (subcellular and cellular void sizes) or induce 3-D cellular assembly (supercellular void size). These patterns of morphogenesis were additionally governed by the biochemical nature of the substrate and were highly correlated to resultant levels of cell function. Substrate mechanical compliance, introduced via increased chemical crosslinking of the basement membrane, Matrigel, and polyacrylamide gel substrates, also was shown to be able to induce active two-dimensional (2-D, rigid substrates) or 3-D (malleable substrates) cellular reorganization. The extent of morphogenesis and the ensuing levels of cell function were highly dependent on the biochemical nature of the cellular microenvironment, including the presence of increasing extracellular matrix (ECM) ligand and growth-factor concentrations. Collectively, these studies highlight not only the ability of substrate biophysics to control hepatocellular morphogenesis but also the ability of biochemical cues to further enhance these effects. In particular, results of these studies reveal novel means by which hepatocellular morphogenesis and assembly can be rationally manipulated leading to the strategic control of the expression of liver-specific functions for hepatic tissue-engineering applications.

Animals↗

PubMed2006

Nanoscale anionic macromolecules can inhibit cellular uptake of differentially oxidized LDL.

Nanoscale particles could be synthetically designed to potentially intervene in lipoprotein matrix retention and lipoprotein uptake in cells, processes central to atherosclerosis. We recently reported on lipoprotein interactions of nanoscale micelles self-assembled from amphiphilic scorpion-like macromolecules based on a lauryl chloride-mucic acid hydrophobic backbone and poly(ethylene glycol) shell. These micelles can be engineered to present varying levels of anionic chemistry, a key mechanism to induce differential retentivity of low-density lipoproteins (LDL) (Chnari, E.; Lari, H. B.; Tian, L.; Uhrich, K. E.; Moghe, P. V. Biomaterials 2005, 26, 3749). In this study, we examined the cellular interactions and the ability of carboxylate-terminated nanoparticles to modulate cellular uptake of differentially oxidized LDL. The nanoparticles were found to be highly biocompatible with cultured IC21 macrophages at all concentrations examined. When the nanoparticles as well as LDL were incubated with the cells over 24 h, a marked reduction in cellular uptake of LDL was observed in a nanoparticle concentration-dependent manner. Intermediate concentrations of nanoparticles (10(-6) M) elicited the most charge-specific reduction in uptake, as indicated by the difference in uptake due to anionic and uncharged nanoparticles. At these concentrations, anionic nanoparticles reduced LDL uptake for all degrees of oxidation (no oxidation, mild, high) of LDL, albeit with qualitative differences in the effects. The anionic nanoparticles were particularly effective at reducing the very high levels of uptake of the most oxidized level of LDL. Since complexation of LDL with anionic nanoparticles is reduced at higher degrees of LDL oxidation, our results suggest that anionic nanoparticles interfere in highly oxidized (hox) LDL uptake, likely by targeting cellular/receptor uptake mechanism, but control unoxidized LDL uptake by mechanisms related to direct LDL-nanoparticle complexation. Thus, anionically functionalized nanoparticles can modulate the otherwise unregulated internalization of differentially oxidized LDL.

Animals↗

PubMed2006

Engineered polymeric nanoparticles for receptor-targeted blockage of oxidized low density lipoprotein uptake and atherogenesis in macrophages.

Strategies to prevent the uptake of modified low density lipoproteins (LDLs) by immune cells, a major trigger of inflammation and atherogenesis, are challenged by complex interfacial factors governing LDL receptor-mediated uptake. We examine a new approach based on a family of "nanoblockers", which are designed to examine the role of size, charge presentation, and architecture on inhibition of highly oxidized LDL (hoxLDL) uptake in macrophages. The nanoblockers are macromolecules containing mucic acid, lauryl chloride, and poly(ethylene glycol) that self-assemble into 15-20 nm nanoparticles. We report that the micellar configuration of the macromolecules and the combined display of anionic (carboxylate) groups in the hydrophobic region of the nanoblockers caused the most effective inhibition in the uptake of hoxLDL by IC21 macrophages. The nanoblockers primarily targeted SR-A and CD36, the major scavenger receptors and modulated the "atherogenic" phenotype of cells in terms of the degree of cytokine secretion, accumulation of cholesterol, and "foam cell" formation. These studies highlight the promise of synthetically engineered nanoblockers against oxidized LDL uptake.

Arteriosclerosis↗

PubMed2005

E-cadherin synergistically induces hepatospecific phenotype and maturation of embryonic stem cells in conjunction with hepatotrophic factors.

Since effective cell sourcing is a major challenge for the therapeutic management of liver disease and liver failure, embryonic stem (ES) cells are being widely investigated as a promising source of hepatic-like cells with their proliferative and pluripotent capacities. Cell-cell interactions are crucial in embryonic development modulating adhesive and signaling functions; specifically, the cell-cell adhesion ligand, cadherin is instrumental in gastrulation and hepatic morphogenesis. Inspired by the role of cadherins in development, we investigated the role of expression of E-cadherin in cultured murine ES cells on the induction of hepatospecific phenotype and maturation. The cadherin-expressing embryonic stem (CE-ES) cells intrinsically formed pronounced cell aggregates and cuboidal morphology whereas cadherin-deficient cadherin-expressing embryonic stem (CD-ES) cells remained more spread out and corded in morphology. Through controlled stimulation with single or combined forms of hepatotrophic growth factors; hepatocyte growth factor (HGF), dexamethasone (DEX) and oncostatin M (OSM), we investigated the progressive maturation of CE-ES cells, in relation to the control, CD-ES cells. Upon growth factor treatment, the CE-ES cells adopted a more compacted morphology, which exhibited a significant hepatocyte-like cuboidal appearance in the presence of DEX-OSM-HGF. In contrast, the CD-ES cells exhibited a mixed morphology and appeared to be more elongated in the presence of DEX-OSM-HGF. Reverse-transcriptase polymerase chain reaction was used to delineate the most differentiating condition in terms of early (alpha-fetoprotein (AFP)), mid (albumin), and late-hepatic (glucose-6-phosphatase) markers in relation to growth factor presentation for both CE-ES and CD-ES cells. We report that following the most differentiating condition of DEX-OSM-HGF stimulation, CE-ES cells expressed increased levels of albumin and glucose-6-phosphatase, whereas the CD-ES cells showed low levels of AFP and marginal levels of albumin and glucose-6-phosphatase. These trends suggest that the membrane expression of E-cadherin in ES cells can elicit a marked response to growth factor stimulation and lead to the induction of later stages of hepatocytic maturation. Thus, cadherin-engineered ES cells could be used to harness the cross-talk between the hepatotrophic and cadherin-based signaling pathways for controlled acceleration of ES hepatodifferentiation.

Animals↗

PubMed2005

Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance.

In order to evaluate the sensitivity of hepatocellular cultures to variations in both substrate stiffness and bioactive ligand presentation, hepatocytes were cultured on differentially compliant polyacrylamide gel discs functionalized with varying amounts of the ECM ligand, fibronectin (FN). Subconfluent cell cultures were established in a multiwell plate format enabling the systematic evaluation of cellular response to both underlying substrate rigidity and substrate ligand concentration. Hepatocellular morphogenesis, regulated by a combination of both ligand density and substrate compliance, resulted in a broad spectrum of patterns of cellular reorganization and assembly ranging from highly two-dimensionally spread cells to highly compact, three-dimensional spheroids. Cell compaction was promoted by increasing levels of substrate mechanical compliance and generally inhibited by increasing concentrations of substrate-bound FN. We identified regimes of substrate compliance in which cells are highly responsive or relatively insensitive to the level of substrate-based ligands. For example, while FN presentation did not have a large impact on cell morphogenesis for cultures on highly compliant gels (G' = 1.9 kPa), hepatocytes on "firm" substrates of intermediate compliance (G' = 5.6 kPa) exhibited approximately a 2-fold increase in cell area between the highest and lowest FN concentrations used in this study. Further, we show that increasing substrate compliance at constant ligand concentration results in increased levels of liver-specific albumin secretion while increasing levels of FN at constant substrate rigidity yield reduced liver-specific functional activity. These substrate-elicited differences in cell function also coincided with analogous changes in the transcript levels of metabolic, growth-related, and liver-specific gene markers. Notably, these results also demonstrated that "firm" gel substrates elicit the most hepatocyte functional sensitivity to substrate-based FN presentation. Overall, our findings indicate that hepatocellular responsiveness to ligand concentration can be acutely regulated by gradation of substrate compliance, suggesting that concerted biochemical and biophysical design strategies may be critical toward the fabrication of hepatospecific biomaterials that effectively support desired levels of liver-specific function.

Animals↗

PubMed2005

Nanoscale anionic macromolecules for selective retention of low-density lipoproteins.

Synthetically designed anionic nanocarriers that mimic the charge properties of glycosaminoglycans can potentially sequester low-density lipoproteins (LDL) during the treatment of atherosclerosis. In this study, we explore the LDL retentivity of 15-20 nm anionic micelles formed from amphiphilic scorpion-like macromolecules (AScMs) as building blocks. The macromolecules comprise four aliphatic chains attached to mucic acid and a linear polyethylene glycol (PEG) segment to form micellar nanocarriers with a hydrophobic core and hydrophilic corona. Dynamic light scattering and transmission electron microscopy studies indicate that the carboxylate-terminated nanocarriers (20 nm) sequester LDL (22 nm), resulting in complexes with a diameter of 60-90 nm, but neutral ethoxy-terminated nanocarriers do not retain LDL. Further, carboxylate-terminated nanocarriers consistently bound to unoxidized LDL (Relative Electrophoretic Mobility, REM=1.0) and mildly oxidized LDL (REM=1.5), but not highly oxidized LDL (REM=3.6), whereas the neutral nanocarriers displayed no preference/affinity at all, indicating that the nanocarrier-LDL binding is charge-dependent. The binding affinity of unoxidized LDL for differentially charged nanocarriers, formed from varying ratios of carboxylate- and ethoxy-terminated macromolecules, was quantified. The 100% carboxylated nanocarriers elicited the highest binding affinity (K(d)=567 nm), whereas mixed micelles elicited significantly lower levels of binding affinity. Our results highlight the promise of synthetically designed nanomaterials in lipoprotein retention, a key step in managing the escalation of atherosclerosis.

Anions↗

PubMed2005

Glutathione preconditioning attenuates Ac-LDL-induced macrophage apoptosis via protein kinase C-dependent Ac-LDL trafficking.

Oxidized low-density lipoprotein (ox-LDL) incorporation into intimally resident vascular cells via scavenger receptors marks one of the early steps in atherosclerosis. Cellular apoptotic damage results from two major serial intracellular events: the binding and scavenger receptor-mediated uptake of oxidizable lipoproteins and the intracellular oxidative responses of accumulated lipoproteins. Most molecular approaches to prevent apoptotic damage have focused on singular events within the cascade of lipoprotein trafficking. To identify a multifocal strategy against LDL-induced apoptosis, we evaluated the role of cellular preconditioning by glutathione-ethyl ester (GSH-Et), a native redox regulator, in the prevention of the uptake and apoptotic effects of an oxidizable scavenger receptor-specific ligand, acetylated low-density lipoprotein (Ac-LDL). Our results indicate that GSH-Et-mediated protein kinase C (PKC) pathway modulation regulates Ac-LDL binding and incorporation into GSH-Et preconditioned cells and subsequently delays reactive oxygen intermediate generation and apoptotic conversion. The GSH-Et protective effects on apoptosis and Ac-LDL binding were reversed by calphostin C, a PKC inhibitor, and were accompanied by an increase in PKC phosphorylation. However, the rate of reactive oxygen intermediate accumulation was not increased following calphostin C treatment, suggesting that GSH-Et may play an important nonreactive oxygen-intermediate-based protective role in regulating apoptotic dynamics. Overall, we report on the novel role for GSH-Et preconditioning as a molecular strategy to limit lipoprotein entry into the cells, which presents a proactive modality to prevent cellular apoptosis in contrast with the prevalent antioxidant approaches that treat damage retroactively.

Animals↗

PubMed2004

Poly(ethylene glycol) enhances cell motility on protein-based poly(ethylene glycol)-polycarbonate substrates: a mechanism for cell-guided ligand remodeling.

The regulation of cell motility on ligand-adsorbed poly(ethylene glycol) (PEG)-based polymeric biomaterials is governed by variables that are not well characterized. In this report, we examined keratinocyte migratory responsiveness to PEG-variant tyrosine-derived polycarbonates adsorbed with equivalent levels of the cell adhesion ligand, fibronectin. The equivalently adsorbed ligand adopted differential distributions, confirmed via atomic force microscopy, and the total number of exposed cell-binding domains (CBD), quantified through immunosorbent fluorometry, varied as a function of PEG concentration. Specifically, the CBD exposure was maximized at 4 mol % PEG and diminished at 8 mol % PEG, suggesting, based on our previous work (Tziampazis et al., Biomaterials 2000;21:511-520), that activation of cell adhesion and motility could be potentially promoted through increased CBD exposure at intermediate levels of PEG. This was confirmed through cell migration studies wherein cell speed values increased from 11 to 22 microm/h as the PEG concentration was increased from 0 to 4 mol %. Unexpectedly, however, high cell motility rates were sustained at 8 mol % PEG despite diminished levels of initial CBD exposure beyond 4 mol % PEG, suggesting that factors other than the initial CBD exposure may additionally have a role in activating cell migration at higher levels of PEG. Through studies of direct ligand mobility, cell-ligand-polymer interactions via atomic force microscopy, and CBD variation and integrin receptor roles in ligand remodeling, we offer evidence that cell motility is enhanced by a new mechanism for the regimen of higher PEG concentration: upon cell attachment and spreading, the ligand exhibits greater "slippage" at the polymer interface, and undergoes cell-engendered remodeling, which further activates cell motility, likely through enhanced exposure of hitherto encrypted sites for cell binding and signaling.

Antibodies↗

PubMed2004

Exogenous cadherin microdisplay can interfere with endogenous signaling and reprogram gene expression in cultured hepatocytes.

We recently found that the basal micro substrate presentation of E-cadherin, a key cell-cell adhesion molecule in the liver, can modulate hepatocellular proliferative potential and differentiated function (Brieva and Moghe, in press). In the current study, we established a similar experimental model involving rat hepatocytes cultured on collagen and incorporated 5 microm polystyrene microbeads functionalized with Protein A-anchored E-cadherin/human lgG Fc chimeric fusion constructs. We investigated the cadherin governed dose-response of cell proliferative potential and quantified the underlying changes in intracellular gene signaling processes. Hepatocellular proliferative potential was found to be intensified with an increase in the microdisplay of acellular cadherins and this effect was offset by increased cell seeding density. Notably, we report that following overnight exposure to acellular cadherins, the expression of genes known to mediate the control of cell proliferation, cyclin D1 and c-myc, was upregulated, while the expression of differentiation-related genes, namely albumin and cytochrome p450 II B1, was reduced. The exposure of cell cultures to exogenous cadherins was found to markedly disrupt the localization of endogenous E-cadherin and beta-catenin to junctions at cell-cell contacts and cause a quantitative decrease in the endogenous cadherin protein levels. Based on all of our observations, we propose that the acellular presentation of E-cadherin chimeras competitively disrupts endogenous cadherin containing complexes at cell-cell junctions and increases intracellular cadherin turnover, thereby promoting beta-catenin mediated signaling, which ultimately engenders an increase in cell proliferative potential and a decrease in differentiated function.

Animals↗

PubMed2002

Alteration of leukocyte motility on plasma-conditioned prosthetic biomaterial, ePTFE, via a flow-responsive cell adhesion molecule, CD43.

The physiologic determinants of leukocyte migration on vascular prosthetic biomaterials remain poorly understood, despite their relevance to the control of periprosthetic infection. Using hemodynamic exposure of human polymorphonuclear leukocytes adherent to expanded polytetrafluoroethylene (ePTFE) in vitro, we investigated the role of fluid shear in regulating leukocyte migratory behavior on plasma-adsorbed, prosthetic vascular biomaterial. The presence of flow at a wall shear stress of 25 dyn/cm(2) increased the degree of leukocyte displacement along the flow direction without altering the degree of overall cell attachment. Moreover, plasma-ePTFE elicited a lower overall degree of displacement under flow in comparison with untreated ePTFE. We further probed the molecular level regulation of leukocyte migratory responses under flow through the immunocytochemical quantification of specific leukocyte adhesion molecules and determined that CD43, a cell adhesion molecule, was upregulated via flow exposure for leukocytes adherent to plasma-ePTFE, whereas basal levels of CD43 expression were not significantly altered on untreated ePTFE. When flow-exposed, adherent leukocytes were incubated in the presence of substrate immobilized anti-CD43 immunoglobulin, the degree of cell displacement along flow was found to be significantly enhanced on plasma-ePTFE. Quantification of the cell population redistribution under flow using a modified random motility model, indicated that the incorporation of anti-CD43 on plasma-ePTFE led to a significant increase (243 +/- 60%) in the cell dispersion coefficient, mu(D), whereas only a minimal increase (61 +/- 30%) was detected on non-adsorbed ePTFE. Overall, our results suggest that flow exposure can induce the migration of leukocytes adherent to prosthetic materials in a substrate-dependent manner. An important implication of our study is that, although biomaterials exposed to plasma intrinsically passivate leukocyte migration even under hemodynamic conditions, it may be possible to promote cell motility by targeting a specific, flow-responsive, adhesion molecule.

Antibodies↗

PubMed2002

Regulation of cell motility on polymer substrates via "dynamic," cell internalizable, ligand microinterfaces.

In vivo, motile epidermal tissues frequently encounter ligand microinterfaces that are dynamic, owing to rapid cell-mediated substrate phagocytosis. In this study, we have examined cell motility phenomena in response to the adhesion ligand, collagen, which was presented on cell-internalizable 400-nm colloidal gold microcarriers. Normal human keratinocytes were seeded onto collagen-adsorbed poly(lactide-co-glycolide) (PLGA) films that were predeposited with varying densities of the ligand-associated microcarriers (LAMs), such that the overall ligand density and the ligand loading per microcarrier were invariant. Cells seeded on LAMs exhibited rapid and distinct cytoskeletal redistribution resulting in numerous filopodial extensions, indicative of activation of cell motility processes. We report that the population-averaged cell migration rate of cells, mu, was increased from 2 microm2/min on collagen substrates lacking the microcarriers, to approximately 50 microm2/min on collagen presenting LAMs. An increase in LAM density to 1.2 LAMs per microm2 was found to maximize mu, as well as the directional persistence and speed of cell migration, whereas very high LAM densities saturated cell internalization and diminished migration rates. The cell-LAM interactions essential to enhanced migration were ligand-activated, as mu; values were reduced 400% on internalizable microcarriers lacking ligands; moreover, cooperative ligand elicitation was possible, for example, when 10 microg/mL soluble fibronectin was introduced as a costimulant of keratinocytes, yielding the highest reported mu values (80 microm2/min) on collagen LAM-PLGA substrates. Notably, cell migration rates were severely repressed when cell internalization processes were challenged through covalent conjugation of ligand carriers to the substrate, indicating that signals from cell-LAM binding alone were inadequate for elevated levels of cell migration. Further analysis indicated that the presence of LAMs did not alter the protease-resistant adhesivity between the cell and the underlying substrate, suggesting that the activation governing ligand interactions likely arose at the cell-LAM interface rather than the cell-substrate interface. This study highlights the novel use of secondary ligand-presenting microscale/nanoscale depots at polymer substrates to elicit, via dynamic cell internalization processes, significantly enhanced levels of cell migration over traditional interfaces with ligand-bearing substrates.

Cell Adhesion↗

PubMed2002

Cell migration on cell-internalizable ligand microdepots: a phenomenological model.

We have shown that collagen "ligand associated microdepots" (LAMs) at polymer substrates can significantly enhance levels of skin epidermal cell migration (Tjia and Moghe, Tissue Eng. 8:247-259, 2002). In this study, we have further examined the dynamics of cell-LAM interactions, primarily through a phenomenological model to examine the differential effects of LAM-cell binding and LAM internalization within the cells. Based on the experimental data of cell migration and LAM dynamics under selected conditions, the model was solved to yield rates of LAM binding and internalization at various LAM substrate densities. The clearance dynamics of LAMs computed at various times from the model matched well with the LAM clearance kinetics observed experimentally. The model was used to generate simulations of the rates of LAM binding and internalization over time, under conditions of differential exogenous activation. Our model analysis suggests that the rate of cell migration can be sensitively governed by rate of cellular sampling of LAMs, given by differential rates of LAM binding and internalization. Maximal cell migration was found to occur during LAM presentation regimes (LAM spatial density) that engendered concerted changes in the extent of cell activation, as measured via net tyrosine kinase activity, due to LAM sampling dynamics.

Biocompatible Materials↗

PubMedDate not supplied

Engineering the hepatocyte differentiation-proliferation balance by acellular cadherin micropresentation.

The successful development of bioartificial and cell-based liver support systems relies on the identification of molecular mechanisms controlling the balance between hepatocellular proliferation and differentiation. Although a definitive function-inductive role for the cell-cell adhesion molecule, E-cadherin, was established through lateral cadherin-cadherin engagement in hepatocyte cocultures (Brevia, T.A., and Moghe, P.V. Biotechnol. Bioeng. 76, 295, 2001), the roles of other modes of cadherin presentation are not well understood. Further, alternative cadherin display configurations promoting cell growth/proliferative pathways, a major requisite for sustainable engineered tissues, remain to be identified. In this report, we employed protein A-functionalized polymeric microsphere substrates that specifically bound self-dimerizing cadherin-IgG/Fc fusion chimeras via their Fc regions, thereby orienting them outward for active adhesion, and presented the E-cadherin chimeras basally to cultured rat hepatocytes to study the effects of cadherin display on cell proliferative potential and differentiated function. In contrast to the previously documented function-inductive roles of laterally expressed cadherin, basal acellular cadherin presentation resulted in an increase in hepatocyte DNA synthesis and cell divisions, accompanied by a decrease in the expression of a key marker of liver-specific function, albumin message levels. Next, we probed the relative effect of basal exogenous display of acellular cadherins on the inductive phase of differentiation within hepatocyte cocultures with cadherin-expressing L929 cells. When acellular cadherins were applied to hepatocyte cocultures involving chaperone cell-mediated cadherin presentation, the previously reported function-inductive effects of cadherins on hepatocyte function were reversed, resulting in lower levels of albumin and urea secretion indicating a dominance of acellular cadherin effects. Our results demonstrate that cadherins are important regulators of the balance between hepatocyte differentiation and proliferation, and furthermore that the direction of balance shift may be dependent on the method of cadherin presentation. Thus, the geometric display of cadherin could be a potential parameter to switch hepatocyte functional-proliferative balance and may aid in customizing scaffolds for regulating hepatic tissue dynamics.

Animals↗

PubMedDate not supplied

Cytomimetic engineering of hepatocyte morphogenesis and function by substrate-based presentation of acellular E-cadherin.

Although cadherin-mediated intercellular contacts can be integral to the maintenance of functionally competent hepatocytes in vitro, the ability to engineer hepatocellular differentiated function via acellular E-cadherin has yet to be thoroughly explored. To investigate the potential of substrate-presented, acellular E-cadherin to modulate hepatocellular self-assembly and functional fate, rat hepatocytes were cultured at sparse densities on surfaces designed to display recombinant E-cadherin/Fc chimeras. On these substrates, hepatocytes were observed to recognize microdisplayed E-cadherin/Fc and responded by modulating the spatial distribution of the intracellular cadherin-complexing protein beta-catenin. Substrate-presented E-cadherin/Fc was also found to markedly alter patterns of hepatocyte morphogenesis, as cellular spreading and two-dimensional reorganization were significantly inhibited under these conditions, leading to multicellular aggregates that were considerably more three-dimensional in nature. Increasing cadherin exposure was also associated with elevated levels of albumin and urea secretion, two markers of hepatocyte differentiation, over control cultures. This suggested that cell-substrate cadherin engagement established more functionally competent hepatocellular phenotypes, coinciding with the notion that E-cadherin is a differentiation-inducing ligand for these cells. The morphogenetic and function-promoting effects of substrate-bound E-cadherin/Fc were further enhanced under conditions in which protein A was utilized as an anchoring molecule to present cadherin molecules, suggesting that ligand mobility may play an important role in the effective establishment of cell-to-substrate cadherin interactions. Interestingly, the percent increase in function detected for conditions of high cadherin exposure versus control cultures was found to be substantially higher at extremely low cell densities. This observation indicated that hepatocytes respond to substrate-presented E-cadherin even in the absence of native intercellular interactions and associated juxtacrine signaling. The incorporation of acellular E-cadherin on biomaterial substrates may thus potentially present a means to prevent hepatocellular dedifferentiation by maintaining liver-specific function in otherwise severely functionally repressive culture conditions.

Animals↗