Bouhadir KH, Mooney DJ. In vitro and in vivo models for the reconstruction of intercellular signaling. Ann N Y Acad Sci. 1998;842 :188-94.Abstract
A critical need in both tissue-engineering applications and basic cell culture studies is the development of synthetic extracellular matrices (ECMs) and experimental systems that reconstitute three-dimensional cell-cell interactions and control tissue formation in vitro and in vivo. We have fabricated synthetic ECMs in the form of fiber-based fabrics, highly porous sponges, and hydrogels from biodegradable polymers (e.g., polyglycolic acid) and tested their ability to regulate tissue formation. Both cell seeding onto these synthetic ECMs and subsequent culture conditions can be varied to control initial cell-cell interactions and subsequent cell growth and tissue development. Three-dimensional tissues composed of cells of interest, matrix produced by these cells, and the synthetic ECM (until it degrades) can be created with these systems. For example, smooth muscle cells can be grown on polyglycolic acid fiber-based synthetic ECMs to produce tissues with cell densities in excess of 10(8) cells/mL. These tissues contain extensive elastin and collagen, and the smooth muscle cells within the tissue express the contractile phenotype (e.g., alpha-actin staining). Similar approaches can be used to grow a number of other tissues (e.g., dental pulp) that resemble the native tissue. These engineered tissues may provide novel experimental systems to study the role of three-dimensional intercellular signaling in tissue development and may also find clinical application as replacements to lost or damaged tissues.
Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol. 1998;16 (5) :224-30.Abstract
Tissue engineering may provide an alternative to organ and tissue transplantation, both of which suffer from a limitation of supply. Cell transplantation using biodegradable synthetic extracellular matrices offers the possibility of creating completely natural new tissues and so replacing lost or malfunctioning organs or tissues. Synthetic extracellular matrices fabricated from biocompatible, biodegradable polymers play an important role in the formation of functional new tissue from transplanted cells. They provide a temporary scaffolding to guide new tissue growth and organization, and may provide specific signals intended to retain tissue-specific gene expression.
Kim BS, Mooney DJ. Engineering smooth muscle tissue with a predefined structure. J Biomed Mater Res. 1998;41 (2) :322-32.Abstract
Nonwoven meshes of polyglycolic acid (PGA) fibers are attractive synthetic extracellular matrices (ECMs) for tissue engineering and have been used to engineer many types of tissues. However, these synthetic ECMs lack structural stability and often cannot maintain their original structure during tissue development. This makes it difficult to design an engineered tissue with a predefined configuration and dimensions. In this study, we investigated the ability of PGA fiber-based matrices bonded at their fiber crosspoints with a secondary polymer, poly-L-lactic acid (PLLA), to resist cellular contractile forces and maintain their predefined structure during the process of smooth muscle (SM) tissue development in vitro. Physically bonded PGA matrices exhibited a 10- to 35-fold increase in the compressive modulus over unbonded PGA matrices, depending on the mass of PLLA utilized to bond the PGA matrices. In addition, the bonded PGA matrices degraded much more slowly than the unbonded matrices. The PLLA bonding of PGA matrices had no effect on the ability of cells to adhere to the matrices. After 7 weeks in culture, the bonded matrices maintained 101 +/- 4% of their initial volume and an approximate original shape while the unbonded matrices contracted to 5 +/- 1% of their initial volume with an extreme change in their shape. At this time the bonded PGA matrices had a high cellularity, with smooth muscle cells (SMCs) and ECM proteins produced by these cells (e.g., elastin) filling the pores between PGA fibers. This study demonstrated that physically bonded PGA fiber-based matrices allow the maintenance of the configuration and dimensions of the original matrices and the development of a new tissue in a predefined three-dimensional structure. This approach may be useful for engineering a variety of tissues of various structures and shapes, and our study demonstrates the importance of matching both the initial mechanical properties and the degradation rate of a matrix to the specific tissue one is engineering.
Putnam AJ, Cunningham JJ, Dennis RG, Linderman JJ, Mooney DJ. Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells. J Cell Sci. 1998;111 ( Pt 22) :3379-87.Abstract
Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/-32 pg/cell (t=0) to 514+/-30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/-24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.
Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matrices formed with gas foaming. J Biomed Mater Res. 1998;42 (3) :396-402.Abstract
Engineering tissues utilizing biodegradable polymer matrices is a promising approach to the treatment of a number of diseases. However, processing techniques utilized to fabricate these matrices typically involve organic solvents and/or high temperatures. Here we describe a process for fabricating matrices without the use of organic solvents and/or elevated temperatures. Disks comprised of polymer [e.g., poly (D,L-lactic-co-glycolic acid)] and NaCl particles were compression molded at room temperature and subsequently allowed to equilibrate with high pressure CO2 gas (800 psi). Creation of a thermodynamic instability led to the nucleation and growth of gas pores in the polymer particles, resulting in the expansion of the polymer particles. The polymer particles fused to form a continuous matrix with entrapped salt particles. The NaCl particles subsequently were leached to yield macropores within the polymer matrix. The overall porosity and level of pore connectivity were regulated by the ratio of polymer/salt particles and the size of salt particles. Both the compressive modulus (159+/-130 kPa versus 289+/-25 kPa) and the tensile modulus (334+/-52 kPa versus 1100+/-236 kPa) of the matrices formed with this approach were significantly greater than those formed with a standard solvent casting/particulate leaching process. The utility of these matrices was demonstrated by engineering smooth muscle tissue in vitro with them. This novel process, a combination of high pressure gas foaming and particulate leaching techniques, allows one to fabricate matrices with a well controlled porosity and pore structure. This process avoids the potential negatives associated with the use of high temperatures and/or organic solvents in biomaterials processing.
Peters MC, Isenberg BC, Rowley JA, Mooney DJ. Release from alginate enhances the biological activity of vascular endothelial growth factor. J Biomater Sci Polym Ed. 1998;9 (12) :1267-78.Abstract
A primary factor which limits engineering tissues of substantial size is the lack of nutrients readily available to transplanted cells. One potential solution to this nutrient limitation is to encourage the rapid development of a vascular network within three-dimensional tissue engineering matrices. Vascular endothelial growth factor (VEGF) has been identified as a potent stimulator of angiogenesis in vivo. Though effective at stimulating endothelial cells to form blood vessels VEGF degrades rapidly. Spherical alginate beads (3.3+/-0.1 mm diameter) were examined as a means of delivering biologically functional VEGF at a controlled rate over extended times. The alginate beads demonstrated the ability to incorporate VEGF with an efficiency between 30 and 67%, depending on the processing conditions, and release it at a constant rate (5%/day) for up to 14 days in vitro. The released VEGF, when assayed for its ability to stimulate endothelial cells in culture, was found not only to be functional but more potent (three to five times) than the same mass of VEGF added directly to the culture medium. The release kinetics of freeze dried VEGF containing alginate beads were also examined and found to be comparable to non-freeze dried samples.
Bohl KS, Shon J, Rutherford B, Mooney DJ. Role of synthetic extracellular matrix in development of engineered dental pulp. J Biomater Sci Polym Ed. 1998;9 (7) :749-64.Abstract
In cases of damaged oral tissues, traditional therapies, such as a root canal, replace the injured tissue with a synthetic material. However, while the materials currently used can offer structural replacement of the lost tissue, they are incapable of completely replacing the function of the original tissue, and often fail over time. This report describes a tissue engineering approach to dental pulp tissue replacement utilizing cultured cells seeded upon synthetic extracellular matrices. Human pulp fibroblasts were obtained and multiplied in culture. These cells were then seeded onto three different synthetic matrices: scaffolds fabricated from polyglycolic acid (PGA) fibers, a type I collagen hydrogel, and alginate in an effort to examine which matrix is most suitable for dental pulp tissue formation. In addition, methods previously developed for seeding and culturing pulp cells on PGA were optimized. Culturing cells on PGA resulted in a very high cell density tissue with significant collagen deposition. No cell proliferation was observed on alginate, and the growth of cells in collagen gels after 45 days was only moderate. These studies indicate dental pulp-like tissues can be engineered, and this may provide the first step to engineering a complete tooth.
Kim BS, Putnam AJ, Kulik TJ, Mooney DJ. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng. 1998;57 (1) :46-54.Abstract
The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development.
Kaufmann PM, Heimrath S, Kim BS, Mooney DJ. Highly porous polymer matrices as a three-dimensional culture system for hepatocytes: initial results. Transplant Proc. 1997;29 (4) :2032-4.
Kaufmann PM, Heimrath S, Kim BS, Mooney DJ. Highly porous polymer matrices as a three-dimensional culture system for hepatocytes. Cell Transplant. 1997;6 (5) :463-8.Abstract
Hepatocyte-based therapies (e.g., hepatocyte transplantation and extracorporeal support devices) may provide alternative therapies to treat patients with liver disease, but suitable approaches to localize these cells to a given location while maintaining liver-specific gene expression must be developed. The suitability of highly porous three-dimensional sponges fabricated from poly (L-lactic acid) [PLLA] as an hepatocyte culture system was evaluated in this study. Sponges were fabricated utilizing a particulate leaching technique, and were approximately 95% porous, with an average pore diameter of 180 microns. Hepatocytes seeded into these sponges adhered and remained viable for 14 days. However, the secretion rate of albumin from these cells, an indication of liver-specific gene expression, was low (approximately 6 pg/cell/day at day 1), and decreased steadily over the 14 days of the experiment. Coating sponges with collagen, and more preferably, immobilizing cells within the PLLA sponges with a collagen gel, led to enhanced cell survival and albumin secretion at all time points. These data suggest that porous PLLA sponges may provide a novel system for long-term culture of hepatocytes, and proper design of the system may allow the liver-specific gene expression of hepatocytes transplanted in these matrices to be enhanced.
Mooney DJ, Sano K, Kaufmann PM, Majahod K, Schloo B, Vacanti JP, Langer R. Long-term engraftment of hepatocytes transplanted on biodegradable polymer sponges. J Biomed Mater Res. 1997;37 (3) :413-20.Abstract
Hepatocyte transplantation may provide an alternative to orthotopic liver transplantation to treat liver failure. However, suitable systems to transplant hepatocytes and promote long-term engraftment must be developed. In this study, highly porous, biodegradable sponges were fabricated from poly (L-lactic acid) (PLA), and poly (DL-lacticco-glycolic acid) (PLGA), and utilized to transplant hepatocytes into the mesentery of three groups of Lewis rats. The portal vein was shunted to the inferior vena cava in one group of rats (PCS). The second group of animals received a PCS and a 70% hepatectomy on the day of sponge-hepatocyte implantation (PCS + HEP), and the control group (CON) received no surgical stimulation. The sponges were vascularized by ingrowth of fibrovascular tissue over the first 7 days in vivo. Approximately 95-99% of the implanted hepatocytes (determined utilizing computer-assisted image analysis) died in all three experimental groups during this time. The number of engrafted hepatocytes in the CON group further decreased over the next 7 days to 1.3 +/- 1.1% of the original cell number. However, the number of engrafted hepatocytes in the PCS and PCS + HEP increased over this time to 6 +/- 1% and 5 +/- 2%, respectively. The number of engrafted hepatocytes in the PCS group continued to increase over the next 2.5 months to a value of 26 +/- 12% of the initial cell number, and a large number of engrafted hepatocytes was still present at 6 months. These results indicate that stable new tissues can be engineered by transplanting hepatocytes on biodegradable sponges into heterotopic locations if appropriate stimulation is provided.
Mooney DJ, Kaufmann PM, Sano K, Schwendeman SP, Majahod K, Schloo B, Vacanti JP, Langer R. Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes. Biotechnol Bioeng. 1996;50 (4) :422-9.Abstract
Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 +/- 12 mum) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal-caval shunt = PCS), and the second group of animals did not (non-PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two-fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non-PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF-containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells. (c) 1996 John Wiley & Sons, Inc.
Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R. Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents. Biomaterials. 1996;17 (14) :1417-22.Abstract
A novel method was developed to produce highly porous sponges for potential use in tissue engineering, without the use of organic solvents. Highly porous sponges of biodegradable polymers are frequently utilized in tissue engineering both to transplant cells or growth factors, and to serve as a template for tissue regeneration. The processes utilized to fabricate sponges typically use organic solvents, but organic residues remaining in the sponges may be harmful to adherent cells, protein growth factors or nearby tissues. This report describes a technique to fabricate macroporous sponges from synthetic biodegradable polymers using high pressure carbon dioxide processing at room temperature. Solid discs of poly (D,L-lactic-co-glycolic acid) were saturated with CO2 by exposure to high pressure CO2 gas (5.5 MPa) for 72 h at room temperature. The solubility of the gas in the polymer was then rapidly decreased by reducing the CO2 gas pressure to atmospheric levels. This created a thermodynamic instability for the CO2 dissolved in the polymer discs, and resulted in the nucleation and growth of gas cells within the polymer matrix. Polymer sponges with large pores (approximately 100 microns) and porosities of up to 93% could be fabricated with this technique. The porosity of the sponges could be controlled by the perform production technique, and mixing crystalline and amorphous polymers. Fibre-reinforced foams could also be produced by placing polymer fibres within the polymer matrix before CO2 gas processing.
Mooney DJ, Mazzoni CL, Breuer C, McNamara K, Hern D, Vacanti JP, Langer R. Stabilized polyglycolic acid fibre-based tubes for tissue engineering. Biomaterials. 1996;17 (2) :115-24.Abstract
Polyglycolic acid (PGA) fibre meshes are attractive candidates to transplant cells, but they are incapable of resisting significant compressional forces. To stabilize PGA meshes, atomized solutions of poly(L-lactic acid) (PLLA) and a 50/50 copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) dissolved in chloroform were sprayed over meshes formed into hollow tubes. The PLLA and PLGA coated the PGA fibres and physically bonded adjacent fibres. The pattern and extent of bonding was controlled by the concentration of polymer in the atomized solution and the total mass of polymer sprayed on the device. The compression resistance of devices increased with the extent of bonding, and PLLA bonded tubes resisted larger compressive forces than PLGA bonded tubes. Tubes bonded with PLLA degraded more slowly than devices bonded with PLGA. Implantation of PLLA bonded tubes into rats revealed that the devices maintained their structure during fibrovascular tissue ingrowth, resulting in the formation of a tubular structure with a central lumen. The potential of these devices to engineer specific tissues was exhibited by the finding that smooth muscle cells and endothelial cells seeded onto devices in vitro formed a tubular tissue with appropriate cell distribution.
Kilmartin DJ, Mooney DJ, Acheson RW, Payne SJ, Maher ER, Eustace P. von Hippel-Lindau disease and familial polyposis coli in the same family. Arch Ophthalmol. 1996;114 (10) :1294.
Mooney DJ, Powell C, Piana J, Rutherford B. Engineering dental pulp-like tissue in vitro. Biotechnol Prog. 1996;12 (6) :865-8.Abstract
Injury or infection of adult dental pulp often necessitates root canal therapy. This terminates dentin formation and subsequent tooth maturation. In addition, the synthetic materials currently utilized to replace lost tooth structure are not capable of completely replacing the function of the lost tissue, and often fail over time. This report describes a technique to engineer new pulp-like tissues utilizing cultured cells and synthetic extracellular matrices. Fibroblasts were obtained from human adult dental pulps and multiplied in culture. These cells were subsequently seeded onto synthetic matrices fabricated from fibers (approximately 15 microns in diameter) of polyglycolic acid (PGA). The pulp-derived fibroblasts adhered to the fibers, proliferated, and formed a new tissue over 60 days in culture with a cellularity similar to that of native pulp. These tissues may find application in the regeneration of oral tissues and may provide novel systems in which to study the biocompatibility of materials and chemicals used in dentistry.
Breuer CK, Shin'oka T, Tanel RE, Zund G, Mooney DJ, Ma PX, Miura T, Colan S, Langer R, Mayer JE, et al. Tissue engineering lamb heart valve leaflets. Biotechnol Bioeng. 1996;50 (5) :562-7.Abstract
Tissue engineered lamb heart valve leaflets (N - 3) were constructed by repeatedly seeding a concentrated suspension of autologous myofibroblasts onto a biodegradable synthetic polymeric scaffold composed of fibers made from polyglycolic acid and polylactic acid. Over a 2-week period the cells attached to the polymer fibers, multiplied, and formed a tissue core in the shape of the matrix. The tissue core was seeded with autologous large-vessel endothelial cells that formed a monolayer which coated the outer surface of the leaflet. The tissue engineered leaflets were surgically implanted in place of the right posterior pulmonary valve leaflet of the donor lamb while on cardiopulmonary bypass. Pulmonary valve function was evaluated by two-dimensional echocardiography with color Doppler which demonstrated valve function without evidence of stenosis and with only trivial regurgitation under normal physiologic conditions. Histologically, the tissue engineered heart valve leaflets resembled native valve leaflet tissue.
Putnam AJ, Mooney DJ. Tissue engineering using synthetic extracellular matrices. Nat Med. 1996;2 (7) :824-6.
Mooney DJ, Park S, Kaufmann PM, Sano K, McNamara K, Vacanti JP, Langer R. Biodegradable sponges for hepatocyte transplantation. J Biomed Mater Res. 1995;29 (8) :959-65.Abstract
Liver cell transplantation may provide a means to replace lost or deficient liver tissue, but devices capable of delivering hepatocytes to a desirable anatomic location and guiding the development of a new tissue from these cells and the host tissue are needed. We have investigated whether sponges fabricated from poly-L-lactic acid (PLA) infiltrated with polyvinyl alcohol (PVA) would meet these requirements. Highly porous sponges (porosity = 90-95%) were fabricated from PLA using a particulate leaching technique. To enable even and efficient cell seeding, the devices were infiltrated with the hydrophilic polymer polyvinyl alcohol (PVA). This reduced their contact angle with water from 79 to 23 degrees, but did not inhibit the ability of hepatocytes to adhere to the polymer. Porous sponges of PLA infiltrated with PVA readily absorbed aqueous solutions into 98% of their pore volume, and could be evenly seeded with high densities (5 x 10(7) cells/mL) of hepatocytes. Hepatocyte-seeded devices were implanted into the mesentery of laboratory rats, and 6 +/- 2 x 10(5) of the hepatocytes engrafted per sponge. Fibrovascular tissue invaded through the devices' pores, leading to a composite tissue consisting of hepatocytes, blood vessels and fibrous tissue, and the polymer sponge.
Mooney DJ, Langer R, Ingber DE. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix. J Cell Sci. 1995;108 ( Pt 6) :2311-20.Abstract
This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS)