Selective adhesion inhibition and hyaluronan envelope reduction of dermal tumor cells by cold plasma-activated medium.

The sensitivity to cold plasma is specific to tumor cells while leaving normal tissue cells unaffected. This is the desired challenge in cancer therapy. Therefore, the focus of this work was a comparative study concerning the plasma sensitivity of dermal tumor cells (A-431) versus non-tumorigenic dermal cells (HaCaT) regarding their adhesion capacity. We found a selective inhibiting effect of plasma-activated medium on the adhesion of tumor cells while hardly affecting normal cells. We attributed this to a lower basal gene expression for the adhesion-relevant components CD44, hyaluronan synthase 2 (HAS2), HAS3, and the hyaluronidases in A431. Noteworthy, after plasma exposure, we revealed a significantly higher expression and synthesis of the hyaluronan envelope, the HAS3 gene, and the transmembrane adhesion receptors in non-tumorigenic HaCaTs.

Synergistic effect of plasma-activated medium and novel indirubin derivatives on human skin cancer cells by activation of the AhR pathway.

Due to the increasing number of human skin cancers and the limited effectiveness of therapies, research into innovative therapeutic approaches is of enormous clinical interest. In recent years, the use of cold atmospheric pressure plasma has become increasingly important as anti-cancer therapy. The combination of plasma with small molecules offers the potential of an effective, tumour-specific, targeted therapy. The synthesised glycosylated and non glycosylated thia-analogous indirubin derivatives KD87 and KD88, respectively, were first to be investigated for their pharmaceutical efficacy in comparison with Indirubin-3'-monoxime (I3M) on human melanoma (A375) and squamous cell carcinoma (A431) cells. In combinatorial studies with plasma-activated medium (PAM) and KD87 we determined significantly decreased cell viability and cell adhesion. Cell cycle analyses revealed a marked G2/M arrest by PAM and a clear apoptotic effect by the glycosylated indirubin derivative KD87 in both cell lines and thus a synergistic anti-cancer effect. I3M had a pro-apoptotic effect only in A431 cells, so we hypothesize a different mode of action of the indirubin derivatives in the two skin cancer cells, possibly due to a different level of the aryl hydrocarbon receptor and an activation of this pathway by nuclear translocation of this receptor and subsequent activation of gene expression.

Efficacy of Plasma-Polymerized Allylamine Coating of Zirconia after Five Years.

Plasma-polymerized allylamine (PPAAm) coatings of titanium enhance the cell behavior of osteoblasts. The purpose of the present study was to evaluate a PPAAm nanolayer on zirconia after a storage period of 5 years. Zirconia specimens were directly coated with PPAAm (ZA0) or stored in aseptic packages at room temperature for 5 years (ZA5). Uncoated zirconia specimens (Zmt) and the micro-structured endosseous surface of a zirconia implant (Z14) served as controls. The elemental compositions of the PPAAm coatings were characterized and the viability, spreading and gene expression of human osteoblastic cells (MG-63) were assessed. The presence of amino groups in the PPAAm layer was significantly decreased after 5 years due to oxidation processes. Cell viability after 24 h was significantly higher on uncoated specimens (Zmt) than on all other surfaces. Cell spreading after 20 min was significantly higher for Zmt = ZA0 > ZA5 > Z14, while, after 24 h, spreading also varied significantly between Zmt > ZA0 > ZA5 > Z14. The expression of the mRNA differentiation markers collagen I and osteocalcin was upregulated on untreated surfaces Z14 and Zmt when compared to the PPAAm specimens. Due to the high biocompatibility of zirconia itself, a PPAAm coating may not additionally improve cell behavior.

Crystal structure of zirconia affects osteoblast behavior.

OBJECTIVES: Different approaches are currently undertaken to structure the endosseous part of zirconia implants. The purpose of the present study was to evaluate how surface roughness and monoclinic to tetragonal phase ratio of zirconia affect cell behavior of human osteoblasts. METHODS: Zirconia discs with five different surface structures were produced: machined; machined heat-treated; polished; polished heat-treated; sandblasted, etched and heat-treated (cer.face 14, vitaclinical). The specimen surfaces were then characterized in terms of monoclinic to tetragonal phase ratio, wettability, roughness and visualized using scanning electron microscopy. To determine the reaction of the human osteoblastic cells (MG-63) to the surface roughness and monoclinic to tetragonal phase ratio of zirconia, cell spreading, morphology, actin cytoskeleton, viability and gene expression of alkaline phosphatase (ALP), collagen type I (COL) and osteocalcin (OCN) were assessed. RESULTS: Heat-treatment of the specimens significantly improved the surface wettability. With increased surface roughness Ra of the specimens, cell spreading was reduced. Cell viability after 24h correlated linearly with the tetragonal phase ratio of the specimens. Gene expression after 24h and 3 d was comparable on all specimens irrespective their surface roughness or monoclinic to tetragonal phase ratio. SIGNIFICANCE: Smooth zirconia surfaces with a high tetragonal phase ratio revealed best surface conditions for MG-63 osteoblastic cells and may be considered to design the endosseous part of zirconia implants.

Copper as an alternative antimicrobial coating for implants - An in vitro study.

AIM: To investigate osteoconductive and antimicrobial properties of a titanium-copper-nitride (TiCuN) film and an additional BONIT® coating on titanium substrates. METHODS: For micro-structuring, the surface of titanium test samples was modified by titanium plasma spray (TPS). On the TPS-coated samples, the TiCuN layer was deposited by physical vapor deposition. The BONIT® layer was coated electrochemically. The concentration of copper ions released from TiCuN films was measured by atomic absorption spectrometry. MG-63 osteoblasts on TiCuN and BONIT® were analyzed for cell adhesion, viability and spreading. In parallel, Staphylococcus epidermidis (S. epidermidis) were cultivated on the samples and planktonic and biofilm-bound bacteria were quantified by counting of the colony-forming units. RESULTS: Field emission scanning electron microscopy (FESEM) revealed rough surfaces for TPS and TiCuN and a special crystalline surface structure on TiCuN + BONIT®. TiCuN released high amounts of copper quickly within 24 h. These release dynamics were accompanied by complete growth inhibition of bacteria and after 2 d, no planktonic or adherent S. epidermidis were found on these samples. On the other hand viability of MG-63 cells was impaired during direct cultivation on the samples within 24 h. However, high cell colonization could be found after a 24 h pre-incubation step in cell culture medium simulating the in vivo dynamics closer. On pre-incubated TiCuN, the osteoblasts span the ridges and demonstrate a flattened, well-spread phenotype. The additional BONIT®coating reduced the copper release of the TiCuN layer significantly and showed a positive effect on the initial cell adhesion. CONCLUSION: The TiCuNcoating inhibits the formation of bacterial biofilms on orthopedic implants by influencing the "race for the surface" to the advantage of osteoblasts.

Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

The generation of hybrid materials based on ß-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

Cellular Nutrition in Complex Three-Dimensional Scaffolds: A Comparison between Experiments and Computer Simulations.

Studies on bone cell ingrowth into synthetic, porous three-dimensional (3D) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro. Therefore, we developed an in vitro perfusion cell culture module, which allows the analysis of cells in the interior of scaffolds under different medium flow rates. For each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method. We found that the local cell viability correlates with the local oxygen concentration and the local shear stress. On the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow. On the other hand shear stress caused by high flow rates impedes cell vitality, especially at the surface of the scaffold. Our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate.

Microstructured zirconia surfaces modulate osteogenic marker genes in human primary osteoblasts.

In dentistry, zirconia has been used since the early 1990s for endodontic posts, more recently for implant abutments and frameworks for fixed dental prostheses. Zirconia is biocompatible and mechanically strong enough to serve as implant material for oral implants. Although several zirconia implant systems are available, currently the scientific and clinical data for zirconia implants are not sufficient to recommend them for routine clinical use. Here the influence of microstructured yttria-stabilized zirconia (YZ) on human primary osteoblast (HOB) behavior was determined. YZ surfaces were treated by sandblasting (YZ-S), acid etching (YZ-SE) and additionally heat treatment (YZ-SEH). Morphological changes of HOB were determined by scanning electron microscopy. Actin cytoskeleton was investigated by laser scanning microscopy and analyzed by novel actin quantification software. Differentiation of HOB was determined by real time RT-PCR. Improved mechanical interlocking of primary HOB into the porous microstructure of the acid etched and additionally heat treated YZ-surfaces correlates with drastically increased osteocalcin (OCN) gene expression. In particular, OCN was considerably elevated in primary HOB after 3 days on YZ-SE (13-fold) as well as YZ-SEH (12-fold) surfaces. Shorter actin filaments without any favored orientation on YZ-SE and YZ-SEH surfaces are associated with higher roughness (Ra) values. Topographically modified yttria-stabilized zirconia is a likely material for dental implants with cell stimulating properties achieving or actually exceeding those of titanium.

Persistent effectivity of gas plasma-treated, long time-stored liquid on epithelial cell adhesion capacity and membrane morphology.

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco's Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.

A novel modular device for 3-D bone cell culture and non-destructive cell analysis.

Synthetic materials have emerged as bone substitutes for filling bone defects of critical sizes. Because bone healing requires a mechanically resistant matrix (scaffold) attractive to osteogenic cells and must allow revascularization for nutrient and oxygen supply, scaffold-based strategies focus on the further development of chemical and physical qualities of the material. Cellular ingrowth towards the scaffold center is critical; therefore selective information from inner regions, in particular from the central part, is essential. In this paper we introduce a novel modular in vitro system for three-dimensional (3-D) in vitro bone cell cultures. This 3-D system is developed exclusively for in vitro research purposes, with special emphasis on the geometrical scaffold design (pore size, pore design). The system is composed of a stack of titanium slices which are mounted on a clamp and which enable the separate monitoring of cell growth patterns on every single slice of the slide stack. In this way we are able to gain selective information about the regulation of the cell physiology in the inner part of the 3-D construct which can be used for the development of an optimized scaffold design for orthopedic implants.

Osteoblast response to biomimetically altered titanium surfaces.

Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces.

Improved initial osteoblast functions on amino-functionalized titanium surfaces.

Adhesion and spreading of cells on biomaterials are integrin-mediated processes. But recent findings indicate a key role of the cell membrane associated matrix substance hyaluronan (HA) in interface interactions. Because HA is a negatively charged molecule we assume that a biomaterial surface with an opposed charge could boost the first contact of the cell to the surface. Polished cp titanium (R(a)=0.19 microm) was coated with an amino-group containing plasma polymer (Ti PPA). For this purpose, a microwave excited, pulsed, low-pressure plasma was used. Additionally, collagen was immobilized on Ti PPA with polyethylene glycol diacid (PEG-DA), catalyzed by carbodiimide (CDI). The physico-chemical surface analytical techniques like XPS, FT-IR, water contact angle and zeta-potential verified the retention of the allylamine precursor structure. Human osteoblasts were cultured in serum-free Dulbecco's modified Eagle medium (DMEM). Adhesion and cell cycle phases were calculated by flow cytometry. Spreading and actin cytoskeleton were visualized by confocal microscopy. Gene expression of osteogenic markers was detected by real-time RT-PCR. Ti PPA is significantly advantageous concerning initial adhesion and spreading during the first hours of the cell contact to the surface. The proliferation of osteoblasts is positively influenced. Gene expression of the differentiation marker bone sialoprotein was upregulated after 24h. Our results demonstrate that functionalization of titanium with positively charged amino-groups is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface.

The effect of positively charged plasma polymerization on initial osteoblastic focal adhesion on titanium surfaces.

The crucial factor of metal implant ingrowth in the bone is the rapid cellular acceptance. Therefore, the knowledge about additionally used adhesion mechanisms of osteoblasts, like their negatively charged hyaluronan coat, generates new surface functionalization strategies. Here, titanium was coated with a very thin, adherent, cross-linked, pinhole- and additive-free allylamine plasma polymer layer (PPAAm) resistant to hydrolysis and delamination and equipped with a high density of positively charged amino groups. This plasma polymer-functionalization of titanium is advantageous concerning osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development and, in consequence in differentiated cell functions, compared to a pure titanium surface-but similar such as the collagen I bonded surface via a polyethylenglycol-diacid (PEG DA)-spacer.

Development of monoclonal and polyclonal antibodies and an ELISA for the determination of glycodelin in human serum, amniotic fluid and cystic fluid of benign and malignant ovarian tumors.

OBJECTIVE: The role of glycodelin in human reproduction and gynecological malignancies has been investigated in a large number of studies in recent years. Three dominant functions of this glycoprotein were identified. Glycodelin is immunosuppressive, a morphological marker of differentiation and a contraceptive. Glycodelin is a glycoprotein with a molecular weight of 28 kDa and a carbohydrate content of 17.5%. Unusual LacdiNAc structures were identified on glycodelin A, isolated from amniotic fluid. Because no kit for glycodelin quantification is commercially available, we developed all reagents and a functional ELISA. MATERIALS AND METHODS: Glycodelin A was purified from amniotic fluid by chromatographic methods. The purity of the isolated protein was checked with SDS-PAGE. Polyclonal antibodies against glycodelin were generated in rabbits. Monoclonal antibodies against glycodelin were generated from immunized BALB/c mice. Positive hybridomas were cloned and cultured. Monoclonal antibodies were isolated by immunoadsorption chromatography from culture supernatants. The glycodelin ELISA was developed in two formats, namely coating with polyclonal antibodies and the use of monoclonal antibodies. RESULTS: The factors of variance for the ELISA were 7% (intraassay variance) and 15% (inter-assay variance). The glycodelin ELISA was used to determine the glycodelin A concentration in sera of fertile women during the proliferative and secretory phases of the endometrium. The glycodelin A concentration was insignificantly elevated in the secretory phase compared to the proliferative phase. Significantly higher levels of glycodelin A were found in women using oral contraceptives compared to women who were not (p<0.001). This is probably due to progesterone, which stimulates glycodelin production. We also found significantly increased glycodelin concentrations in the fluids of malignant ovarian cysts compared to benign ovarian tumors (p<0.001). Furthermore, we tested the monoclonal and polyclonal antibodies successfully in Western blot analysis and immunoadsorption chromatography. CONCLUSION: We consider the described ELISA for the quantification of glycodelin as a useful tool for the determination of glycodelin in amniotic fluid, serum and cystic fluids. Its most promising application is expected in the diagnosis of ovarian cancer. The antibodies generated are applicable to multiple techniques.

Measurement of glycodelin A in fluids of benign ovarian cysts, borderline tumours and malignant ovarian cancer.

BACKGROUND: Glycodelin A levels in fluids of benign ovarian cysts, borderline tumours and ovarian cancer, as well as serum-levels of glycodelin A, were analysed in patients with benign and malignant ovarian tumours. The aim of the study was to investigate if the level of glycodelin A in body fluids of patients with malignant ovarian tumours could be a marker for the disease. Additionally, immunohistochemical investigations of glycodelin A expression in tissue of ovarian cancer were performed. MATERIALS AND METHODS: A total of 158 samples of fluids from benign ovarian cysts, borderline tumours and ovarian cancer were collected during surgery in the Department of Obstetrics and Gynaecology at the University of Rostock, Germany. Additionally, 69 samples of serum from patients with benign ovarian cysts and ovarian cancer on the day before surgery were collected. An ELISA for determination of glycodelin A was used. Immunohistochemical detection of glycodelin A expression was performed on ovarian cancer tissue from 38 patients. RESULTS: Malignant cystic fluids showed higher glycodelin A values (mean: 1814.4 ng/ml) compared to fluids of benign ovarian cysts (mean: 784.4 ng/ml). The results of glycodelin A determination were compared using the Mann- Whitney U-test for comparison of the means. There was a statistically significant difference between benign ovarian cysts and malignant ovarian cancer for the fluid (p <0.001). In addition, serum samples of malignant ovarian tumours also showed significantly higher glycodelin A values compared to serum levels of benign tumours (p<0.001). Immunohistochemical staining on ovarian cancer tissue showed a glycodelin A expression in 25-30% of the carcinoma cells. CONCLUSION: High levels of glycodelin A were found in cystic fluids of ovarian cancer. In addition, we also found higher levels of glycodelin A in the serum of patients with ovarian cancer compared to the serum of patients with benign ovarian cysts. Furthermore, ovarian cancer tissues showed intense staining with a glycodelin A antibody. Further investigations are necessary to show if glycodelin A quantification could help to diagnose ovarian cancer.

Stimulation of endometrial glandular cells with genistein and daidzein and their effects on ERalpha- and ERbeta-mRNA and protein expresion.

UNLABELLED: Phytoestrogens seem to have estrogen-like effects in the human body as their structure is very similar to those estrogens produced in human glands. The aim of the present study was to analyse the effects of genistein and daidzein on estrogen receptor (ER)alpha- and ERbeta-mRNA and protein expression in the endometrium of premenopausal women. MATERIALS AND METHODS: Glandular endometrial cells were isolated from endometrial biopsies obtained from regularly menstruating women undergoing gynaecological abrasio or hysterectomy. Cells were stimulated with single doses of genistein or daidzein. ERalpha- and ERbeta-protein expression were determined by immunocytochemical analysis. In addition ERalpha- and ERbeta-mRNA expression were determined by quantitative real-time RT-PCR. Quantification was carried out by the deltadelta C(T)-method using glyceraldehyde phosphate dehydrogenase (GAPDH) as housekeeping gene. RESULTS: Endometrial glandular cells responded to stimulation with genistein and daidzein by alteration of both ERalpha- and ERbeta-mRNA expression. The effects were time- and dose-dependent. Detection of ERalpha- and ERbeta-protein expression by immunocytochemistry showed a dose-dependent regulation after stimulation of isolated endometrial cells with phytoestrogens. DISCUSSION: According to our results, we suggest that ER expression in endometrial glandular cells is regulated by genistein and daidzein on the mRNA and protein levels. We could detect a decrease in ERalpha- and an increase in ERbeta-mRNA expression after stimulation with tested phytoestrogens. Our results are in line with findings that phytoestrogens act as anti-estrogens in organs expressing more ERalpha and as estrogens in ERbeta-presenting organs.

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A.

AIM: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 microg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. CONCLUSIONS: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.

Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1.

Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGbeta gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)-polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galbeta1-4GlcNAc and Galbeta1-3GalNAc (TF) epitopes on BeWo cells may have regulatory effects on hCG and progesterone production.

Stimulation of hCG and inhibition of hPL in isolated human trophoblast cells in vitro by glycodelin A.

The immunosuppressive protein glycodelin A (formerly named PP14) is produced by human decidua and secreted in the maternal circulation. Glycodelin A concentrations in serum have been used as indicators of endometrial function. The purpose of this study was to investigate the effect of glycodelin A on human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release by freshly isolated cytotrophoblasts (in vitro). Cytotrophoblasts have been prepared from human term placenta by the three-step trypsin-DNase dispersion method of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the isolated mononuclear trophoblasts differentiated into syncytial counterparts within 12-72 h after plating. Trophoblasts were incubated with varying concentrations (60-300 microg/ml) of glycodelin A. Glycodelin A was isolated and purified by chromatographic methods from amnion fluid. Supernatants of the trophoblast cell cultures were assayed for hCG and hPL by immunological methods. The release of hCG is increased in glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. Glycodelin A inhibits the production of hPL in vitro. Differences in Glycodelin A stimulated cells and untreated controls are statistical significant. hCG and hPL are markers for the differentiation process of trophoblast cells to syncytial trophoblasts. The results imply that glycodelin A secreted by decidualised endometrium modulates endocrine function, as well as the differentiation of trophoblasts in culture.