Novel poly(L-lactide) PLLA/SWNTs nanocomposites for biomedical applications: material characterization and biocompatibility evaluation.

Poly(L-lactide) (PLLA)/single-walled carbon nanotubes (SWNTs) nanocomposite films were produced using the solvent casting method, and morphological, thermal and mechanical properties were investigated. Biocompatibility was evaluated by using human bone cells, performing adhesion and proliferation studies. The role of single-walled nanotube incorporation and functionalization on PLLA bio-polymers was investigated. Pristine (SWNTs) and carboxylated (SWNTs-COOH) carbon nanotubes were considered in order to control the interaction between PLLA and nanotubes. SWNTs and SWNTs-COOH showed a good dispersion in the polymer matrix and improved the PLLA crystallinity. Thermal, morphological and dynamic-mechanical analyses revealed that carboxylic groups on the tube sidewalls increased compatibility between PLLA and nanostructures. Mechanical properties demonstrated an enhancement related to introduction and functionalization of carbon nanotubes. Biological investigations showed osteoblasts cultured on PLLA/SWNTs-COOH nanocomposites has higher cell adhesion and proliferation than osteoblasts cultured on PLLA and PLLA/SWNTs nanocomposites. These studies suggest that combination of biodegradable polymers and SWNTs opens a new perspective in the self-assembly of nanomaterials and nanodevices for biomedical applications with tunable properties.

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

In vitro human osteoblast and extracellular matrix changes after transforming growth factor beta 1 treatment.

AIMS: Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia. METHODS: In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined. RESULTS: Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (p < or = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities. CONCLUSION: TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

Biomembranes enriched with TGFbeta1 favor bone matrix protein expression by human osteoblasts in vitro.

The use of growth factors in oral tissue regeneration is currently under investigation. When growth factors are combined with commercial materials, the in vitro mechanisms of action still remain unclear. The present study first evaluated the capacity of barrier membranes, used in oral surgery, to sequester TGFbeta(1). Resorbable HYAFF, paroguide, poly DL-lactide and nonresorbable PTFE membranes were immersed in MEM containing 0.2 ng (125)I-TGFbeta(1) for different periods of time. It was found that HYAFF membrane and paroguide sequestered the most TGFbeta(1), which was then released in its active form (as shown by the CCL64 cell line bioassay). Untreated membranes and membranes enriched with TGFbeta(1) were then used as substrate for human bone cells to evaluate the synthesis of the osteoblast phenotype, as indicated by specific parameters. Results showed that membranes enriched with TGFbeta(1) increased alkaline phosphatase activity, collagen, and osteocalcin production more than untreated membranes. HYAFF and paroguide membranes, which sequestered the most of TGFbeta(1), were the most suitable for stimulating bone matrix proteins.

Synthesis and secretion of transforming growth factor-beta1 by human desmoid fibroblast cell line and its modulation by toremifene.

The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1.

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

In vitro comparison of bioabsorbable and non-resorbable membranes in bone regeneration.

BACKGROUND: Barrier membranes are used to prevent down-growth of the oral mucosa along the root surface and to allow alveolar bone regeneration in guided tissue regeneration. Several studies have demonstrated bone regenerates in the presence of bioabsorbable and non-resorbable membranes, but no studies have compared multiple bioabsorbable barriers to one another and to non-resorbable barriers. This study evaluated the in vitro influence of bioabsorbable and non-resorbable membranes on specific parameters of human osteoblast activity. METHODS: Human osteoblasts were cultured on bioabsorbable membranes made of collagen, hyaluronic acid, and poly DL-lactide, and the most common non-resorbable membrane which is made of expanded polytetrafluoroethylene (ePTFE). The osteoblasts were cultured in vitro for 24 hours on barrier membranes in the presence of 3H-thymidine and 3H-proline to study cell proliferation and collagen synthesis. Transforming growth factor-beta1 (TGF-beta1) secretion was evaluated in conditioned media using an ELISA kit. RESULTS: The results showed that collagen and poly DL-lactide stimulated DNA synthesis more than ePTFE and hyaluronic acid. All bioabsorbable membranes significantly increased collagen synthesis and alkaline phosphatase activity. Collagen and hyaluronic acid increased secretion of TGF-beta1, a growth factor involved in bone remodeling. CONCLUSIONS: These data suggest bioabsorbable membranes, particularly collagen and hyaluronic acid, may promote bone regeneration through their activity on osteoblasts.

In vitro cytotoxic effects of orthodontic appliances.

The objective of this study was to evaluate the effects of an orthodontic appliance and of its components (brackets, bands, and arch wires) on some cell functions. Fibroblasts were cultured either in the presence of one unwashed orthodontic appliance, or one orthodontic appliance immersed in MEM for 28 days before use (washed appliance), or in the presence of MEM in which the appliances had been immersed. At the end of in vitro maintenance, morphological studies were carried out with SEM and TEM. Cell proliferation and GAG synthesis and secretion by radio-labeled precursors were assessed. The data indicated that unwashed appliances were more cytotoxic than washed ones. Moreover, the arch wire was the most biocompatible component of the orthodontic appliance, and the bracket was the least biocompatible. A comparative study into the effects on cell proliferation of the most common metal ions released by the appliances was also carried out. At the concentration released by one orthodontic appliance immersed for 28 days, the highest reduction in DNA synthesis was observed in the presence of Cu(++).

Biocompatibility of alloys used in orthodontics evaluated by cell culture tests.

The cytotoxicity of the most common alloys used in orthodontic appliances was determined by cell culture testing. Human gingival fibroblasts were cultured on 304 and 316 stainless steel, on brazing alloy composed of palladium (Pd), copper (Cu), and silver (Ag), and on plastic substrate (control). Studies were carried out with SEM and radiolabeled precursor incorporation. Cells were cultured in MEM without serum but with the addition of (3)H-thymidine to evaluate cell proliferation and (3)H-glucosamine to evaluate glycosaminoglycan (GAG) synthesis and secretion in the culture medium. Moreover, gingival fibroblasts were cultured in the presence of some metal ions generally released by orthodontic appliances to evaluate the cytotoxic effects of single ions. Morphologic observations with SEM and radiolabeled incorporation studies showed that 304 and 316 stainless steel were more biocompatible than the brazing alloy. Among the metal ions tested, Ag and Pd, constituents of the brazing alloy, showed the highest cytotoxicity.

Differential in vitro phenotype pattern, transforming growth factor-beta(1) activity and mRNA expression of transforming growth factor-beta(1) in Apert osteoblasts.

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.

Ion release from orthodontic appliances.

OBJECTIVE: The microbiological and enzymatic characteristics of the oral cavity would seem to provide a suitable environment for the corrosion of metals. We assayed the release of metal ions from one orthodontic appliance which included two 304 and 316 steel molar bands, ten 316 steel brackets, one nickel-titanium archwire and a brazing alloy to connect the elements of molar bands and brackets. METHODS: The orthodontic appliance was dipped in both inorganic (pH 3.5-6.5) and organic acid solutions (w/v 1% each of tartaric, citric and ascorbic acid at pH 2.2 or 1.5% each of lactic and acetic acid at pH 2.5). The release of nickel (Ni), chromium (Cr), copper (Cu), silver (Ag) and palladium (Pd) was determined using an atomic absorption spectrophotometer Varian AA 10. RESULTS: The release of Ni, Cr and Cu was markedly less at pH 6.5 than at pH 3.5 at all time points in acid solution. Daily release/single appliance after the first day decreased. Contrary to expectations, appliances immersed in organic acid solutions at pH 2.2 or 2.5 after 28 days generally released an amount of ions similar to that observed in inorganic acid solution at pH 3.5, with the exception of Cu. Release of silver and palladium, two metals present in the brazing alloy, proved to be very low (approximately 0.2 microgram after 28 days). CONCLUSIONS: The daily release of Ni, Cu and Cr by an orthodontic appliance in acid pH, particularly favourable to corrosion, was well below that ingested with a normal daily diet. It is therefore concluded that the quantities of metal ions released in our experimental conditions should not be cause for concern in utilising the appliance.

TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

Phenotype expression of human bone cells cultured on implant substrates.

Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [3H]-thymidine to evaluate cellular proliferation, and [3H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH4Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM.

Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration.

Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.

Exogenous heparin induces fibronectin overexpression parallel to angiogenesis in the extracellular matrix of the chick embryo chorioallantoic membrane.

Heparin (HE) was injected into the allantoic sac of chick embryo eggs on the 5th day of incubation. After 48 h, a morphometric analysis of angiogenic response and an immunohistochemical investigation of fibronectin (FN) and type IV collagen immunoreactivity in developing vasculature were performed in order to verify whether HE-related choriollantoic membrane (CAM) angiogenic activity was associated with overexpression of FN and/or type IV collagen changes in CAM extracellular matrix. Data to be presented show a close relationship between HE treatment, angiogenic processes, and overexpression of FN, but not of type IV collagen in CAM extracellular matrix. They agree with other studies proving a facilitating role of FN in angiogenic processes.

Metal substrates influence the release of glycosaminoglycan and transforming growth factor beta by human bone cells.

Bone cells derived from human jaw were isolated from explants and grown in vitro. Subcultures were cultured on plastic (control) and metal substrates for 24 and 48 hours in medium containing 3H-glucosamine and labeled glycosaminoglycan (GAG) accumulation was measured. In bone cells cultured on metal substrates there was an evident reduction in the synthesis and secretion of radiolabeled macromolecules compared to bone cells cultured on plastic. Moreover, the accumulation of single GAG classes was specific for each substrate tested. The results showed that titanium was the only metal substrate studied in which the percentage of individual GAG classes remained the same as control cultures. GAG reduction was due to a decreased synthesis and not to an increased degradation as shown by the decrement of exoglycosidase activity. The metals also reduced the activity of transforming growth factor beta (TGF beta), measured using interleukin-1 assay method, a factor involved in the various phases of bone remodeling; in this case, too, cells grown on titanium showed the highest TGF beta activity compared to the other metal substrates studied. The results indicate that the substrate to which the cells adhere do exhibit specific differences in GAG composition and TGF beta activity. The differences observed may be important during in vivo events such as guided tissue regeneration and bone deposition.

Glycosaminoglycan metabolism in otosclerotic bone cells.

Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.

Transforming growth factor beta 1-hyaluronic acid interaction.

Chick embryo skin fibroblasts release transforming growth factor beta 1 that is able to modulate glycosaminoglycan synthesis and secretion. When incubated with individual classes of glycosaminoglycans, the factor's modulatory activity was altered. To determine whether direct interactions between transforming growth factor beta 1 and glycosaminoglycans occur, we have assessed the activity of the growth factor after pre-incubation with single classes of glycosaminoglycans by assaying its inhibitory effect upon the proliferative response of thymocytes stimulated with interleukin-1. Untreated transforming growth factor beta 1 suppressed the proliferative response of thymocytes to interleukin-1, as did transforming growth factor beta 1 pre-incubated with sulphated glycosaminoglycans. By contrast, transforming growth factor beta 1 lost its inhibitory capacity when preincubated with high molecular weight hyaluronic acid. Digestion of transforming growth factor beta 1-hyaluronic acid complex with hyaluronidase released active transforming growth factor beta 1. Trypsin degraded transforming growth factor beta 1 alone, but did not degrade the transforming growth factor beta 1-hyaluronic acid complex. These results suggest that hyaluronic acid interacts with transforming growth factor beta 1, thus protecting the factor from tryptic degradation and may be a means of concentrating growth factor activity.

Exogenous heparin induces an increase in glycosaminoglycans of the chick embryo chorioallantoic membrane: its possible role in the regulation of angiogenic processes.

In this study heparin (HE) was injected into the allantoic sac of chick embryo eggs at 5, 9, 14 days of incubation. 48 h after injection glycosaminoglycan (GAG) concentration was measured in the chorioallantoic membrane (CAM) in order to verify if HE-related CAM angiogenic activity previously demonstrated [Ribatti et al: Acta Anat 1987; 130:257-263] might be correlated with changes in GAG concentration. The results showed that HE inoculation induced an increase of 3H-glucosamine incorporation into total GAGs in comparison to control CAMs. Furthermore, HE altered the balance between the GAG classes, and in particular it produced a significant increase in the accumulation of hyaluronic acid and heparan sulfate between 7 and 11 days of incubation in comparison to control CAMs.

Modulation of phenotypic expression of fibroblasts by alteration of the cytoskeleton.

Several studies indicate that the cytoskeleton may be involved in modulating the cellular response to environmental signals. We have studied the role of the cytoskeleton in regulating glycosaminoglycan (GAG) synthesis and secretion, hyaluronate (HA) endocytosis, the activities of hexoglycosidases, protein synthesis and secretion. Fibroblasts were treated with colchicine (1-8 microM) and nocodazole (1 or 4 microM) to alter microtubules or cytochalasin B (0.5-4 microM) to alter microfilaments. Colchicine inhibited GAG synthesis and secretion in a concentration-dependent manner. It reduced protein and sulphated GAG secretion, while HA secretion was not affected. Concentration-dependent disruption of microtubules from the periphery toward the cellular centre with nocodazole inhibited only the secretion of GAG. Centrosomal microtubles appeared to be required to promote GAG synthesis; intact microtubules promoted the transport of secretory products, intercompatmental transport of lysosomal enzymes and lysosome maturation, but not protein synthesis and HA secretion. Cytochalasin B treatment inhibited, in a concentration-dependent manner, the synthesis and secretion of GAGs and proteins, and the endocytosis of HA. Intact microfilament meshworks appeared to be required to promote synthesis and secretion of proteins and proteoglycans and to contribute to the transmembrane control of receptor-mediated endocytosis. Drug treatment of concanavalin A (Con A)-stimulated fibroblasts inhibited the stimulation of GAG synthesis. It is probable that this effect may result, in part, from drug-induced effects on Con A-mediated endocytosis.