Primary Ovarian Carcinomas and Abdominal Metastasis Contain 4,6-Disulfated Chondroitin Sulfate Rich Regions,Which Provide Adhesive Properties to Tumour Cells

High mortality in ovarian cancer patients is primarily caused through rapid metastasis of the tumour, but the underlying mechanisms are poorly understood. Glycosaminoglycans, are abundantly present in tumours and chondroitin sulfate-E (CSE), a highly 4,6-sulfated glycosaminoglycan, has been indicated to play a role in carcinogenesis. In this study we investigated the presence of CSE in ovarian cancer metastasis and studied its role in tumour cell adhesiveness and migration. CSE was studied immunohistochemically in primary ovarian carcinomas and abdominal metastases using the single chain antibody GD3G7. The role of CSE was studied in 2D (scratch assays) and 3D (collagen matrices, spheroids) systems using SKOV3 cells applying 1: overexpression of CSE by stable transfection with DNA encoding GalNAc4S-6 sulfotransferase, 2: enzymatic removal of CS, and 3: addition of CSE. In ovarian cancer tissue, CSE expression was predominantly seen in the stromal compartment of both primary ovarian carcinomas and metastases, with a comparable degree of intensity and extent. Overexpression of CSE disaccharide units by tumour cells increased their adhesive properties which was especially seen in tumour spheroid formation. Increased expression of CSE reduced cell migration. Addition of free CSE had similar effects. The data presented here indicate that CSE is associated with metastatic lesions and that it provides tumours with adhesive properties. CSE rich motifs are put forward as a potential target for ovarian cancer therapy.     

Sulfated glycosaminoglycan and collagen patterns in parietal yolk sac carcinoma (PYSC)

Electrophoretic analyses of collagenous material have shown that the parietal yolk sac carcinoma (PYSC) ascitic tumour synthesizes polypeptide chains that migrate as type IV procollagen. Having molecular weights of 185,000 and 160,000, these polypeptides are sensitive to collagenase. When the PYSC cells are injected subcutaneously, they form a solid tumour, and type I collagen predominates. The electrophoretic analyses of sulfated glycosaminoglycans and enzymatic degradation have shown a predominance of heparan sulfate in the ascitic tumour, and of chondroitin sulfate B in the solid tumour. Cells cultured from ascitic tumours have maintained the same collagen and sulfated glycosaminoglycan patterns as the original cells, whereas in the solid tumour culture only chondroitin sulfate AC has been detected.     

Fibroblast growth factors (FGF) in human myometrium and uterine leiomyomas in various stages of tumour growth

We previously described that the growth of human uterine leiomyomas was associated with a significant remodelling of the extracellular matrix of these tumours. Significant weight-related increase of collagen and heparan sulphate contents was detected. The latter was known as a component, which bound some peptide growth factors, mainly FGFs, therefore it was decided to evaluate the amounts of acidic FGF (aFGF) and basic FGF (bFGF) in human myometrium and in leiomyomas of various weight and FGF-binding to tissue components. It was found that myometrium and uterine leiomyomas contain picogram amount of aFGF and nanogram amounts of bFGF. No free aFGF was found. Slight amounts of free bFGF were detected both in myometrium and in the tumours. The aFGF and most of bFGF existed in a form of complex with a high molecular component(s). These complexes were very stable and they did not dissociate in denaturation conditions. In comparison to myometrium the tumours contained several times more FGFs and their amounts distinctly increased during the tumour growth. The expression of FGF-receptor I (FGF RI) in the tumours was more distinct in comparison to myometrium. The extracts from myometrium did not bind exogenous 125I-bFGF. In contrast to that the tumours of different weights contained at least two high molecular weight FGF-binding components. One of them (150 kDa) corresponded to FGF-receptor. The other one (190-200 kDa) might be a heparan sulphate-proteoglycan. It seems that aFGF and bFGF play an important role in transformation of normal myometrium into leiomyoma and further growth of this tumour. The action of FGFs on tumour cells enhances biosynthesis of collagen and sulphated glycosaminoglycans, especially heparan sulphate which binds FGFs in the vicinity of cells and facilitates their interaction with membrane receptors. The effect of these processes may be further stimulation of tumour growth and remodelling of tumour extracellular matrix.     

Localisation and expression of aquaporin subtypes in epithelial ovarian tumours

To characterise AQP subtype localisation and expression in epithelial ovarian tumours, immunohistochemistry was used to assess the localisation and expression of AQP1-9 in 30 benign tumour cases, 30 borderline tumour cases, 50 malignant tumour cases and 20 normal ovarian tissue cases. Multiple AQP subtypes were expressed in epithelial ovarian tumours, with each AQP subtype displaying a different pattern of localisation and expression. AQP1 was mainly expressed in the microvascular endothelium, and AQP 2-9 were mainly expressed in tumour cells. Most AQP subtypes co-localised in the basolateral membranes of the epithelia of benign tumours and plasma membranes of malignant tumour cells. The positive rates for AQP1, 5, 6, 7, 8, and 9 were over 50%, but those for AQP2, 3 and 4 were only 10-40%. The expression of AQP1, 5 and 9 in malignant and borderline tumours was significantly higher than that in benign tumours (P<0.05) and normal ovarian tissue (P<0.05). However, AQP6 expression in ovarian malignant and borderline tumours was significantly lower than that in benign tumours (P<0.01) or normal ovarian tissue (P<0.01). AQP1 expression was increased in cases with ascites volumes greater than 1000 mL (P<0.05), AQP5 expression was greater in cases with lymph node metastasis (P<0.05), and more AQP9 expression was observed in G3 cases versus G1 and G2 cases (P<0.01). These results suggest that changes in the distribution and expression of AQP subtypes may be involved in ovarian carcinogenesis. This study presents a novel avenue of research that could illuminate the mechanism of ovarian carcinogenesis and treatment.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

miR‐495 sensitizes MDR cancer cells to the combination of doxorubicin and taxol by inhibiting MDR1 expression

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR‐495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR‐495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol‐doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre‐treatment with miR‐495 before chemotherapy could improve the curative effect on MDR1‐based MDR cancer.     

Apoptosis and cell proliferation correlated with tumour grade in peritoneal fluids of patients with serous ovarian cancer

A. Kalogeraki, I. Karvela‐Kalogeraki, P. E. Petraki, I. Zois, D. Tamiolakis and E. N. Stathopoulos
Apoptosis and cell proliferation correlated with tumour grade in peritoneal fluids of patients with serous ovarian cancer Objective: Apoptosis and cell proliferation in peritoneal fluids of patients with ovarian serous adenocarcinoma have not been well described in cytology. To investigate the contribution of cell death to the growth of this tumour we analysed both apoptosis and cell proliferation in peritoneal fluids of patients with ovarian serous adenocarcinoma. Methods: We studied 40 tumours from 40 patients with ovarian serous adenocarcinoma. Twelve tumours were high grade, 13 were moderately differentiated and 15 were poorly differentiated. The detection of DNA fragments in situ using the terminal deoxyribonucleotidy transferase (TDT)‐mediated dUTP‐digoxigenin nick‐end labelling (TUNEL) assay was applied to investigate active cell death (apoptosis), and the MIB‐1 antigen was used to investigate cell proliferation. Results: The TUNEL indices were 0.29 ± 0.05, 0.79 ± 0.10 and 2.1 ± 0.90 in Grade I, Grade II and Grade III ovary carcinomas, respectively. The MIB‐1 antigen labelling indices were 6.5 ± 0.09, 12.9 ± 3 and 25.8 ± 6.2, respectively, in the same order of tumour differentiation. The differences in both TUNEL and MIB‐1 labelling indices were statistically significant between Grade I, Grade II and Grade III carcinomas and there was a positive correlation between the two indices (P < 0.001). Conclusions: Apoptosis and cell proliferation increased as the grade of tumour increased in ovarian serous adenocarcinoma, suggesting a rapid turnover of the tumour cells in tumours of higher grade, and may play an important role in the growth and the extension of such cancer cells in the peritoneal cavity.     

In vitro growth peculiarities of some human ovarian tumours.

The possibility of applying a short duration method of in vitro cultures for detecting malignancy of ovarian tumours with borderline lesions and with incipient malignant ones was investigated in 24 ovarian tumours. Three main types of histocultures were described in relation with the in vitro behaviour of ovarian tumours and their cytomorphology (atypic growth, mixed growth and normal one). The category of mixed type growth cultures is discussed, corresponding to ovarian tumours with histopathological borderline lesions, with incipient malignancy, as well as two cilio-epithelial cystadenomae rendered malignant with peritoneal seedings.     

Detection of trophoblastic beta 1-glycoprotein in tumor tissue and blood in ovarian neoplasms

Using immunochemical analysis methods (the reaction of precipitation in agar, immunoenzymatic method, immunofluorescence), trophoblastic beta 1-glycoprotein (TBG) concentration in tumour tissue and in the blood serum of patients with ovarian cancer was studied. By rabbits immunization with glycoprotein fraction of ovarian adenocarcinoma, dissolved in 0.6 M sulfosalicylic acid, the authors obtained antibodies to TBG. Immunoenzymatic method showed, that TBG level is raised during ovarian cancer (more than 3 micrograms/l): in 18% of tumour extracts, in 12.5% of blood sera samples and in 41.6% of cases in ascites fluid. Utilizing indirect immunofluorescence method morphological structures of trophoblastic type were identified in paraffin sections of ovarian adenocarcinoma. The authors suppose, that such structures may be responsible for TBG biosynthesis in ovarian tumours.     

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.     

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

BackgroundCurrent tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.MethodsPorcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.ResultsHuman chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.ConclusionsSynoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.     

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Tumour ploidy, morphometry, histological grading and clinical features in ovarian carcinoma: mutual relations

The relationship between tumour ploidy and qualitative and quantitative histopathology was assessed in a series of 95 ovarian carcinomas. 67% of the tumours were non-diploid (DNA aneuploid). 56% of the early stage (I-II) tumours were non-diploid and 81% of the tumours in advanced (III-IV) stages were aneuploid. Histological grading failed to show a clear relationship between increasing malignancy grade and ploidy. There was a close association between DNA ploidy and nuclear perimeter, area and shortest and longest nuclear diameter: the nuclei of non-diploid tumours were generally larger. Also the number of mitotic figures per square millimeter of epithelium in the microscope image (volume-corrected mitotic index, M/V-index) differed significantly between near-diploid and non-diploid tumours. Discriminant analysis showed that 74% of the learning-set tumours (67% of the test set tumours) could be correctly classified in low-ploidy and high-ploidy categories with morphometric features (nuclear perimeter, M/V-index and volume percentage of epithelium). Characteristic features of non-diploid ovarian tumours--rapid proliferation and large nuclear size--could be assessed with morphometric methods which allowed a relatively large aneuploid tumour group to be distinguished.     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

Stromal cells and extracellular matrix components in spontaneous canine transmissible venereal tumour at different stages of growth

Stromal cells and extracellular matrix (ECM) components are important for tumour cell behaviour. Little is known about the role of stromal cells and ECM components in the progression and regression of spontaneous canine transmissible venereal tumour (CTVT). In this study, the stromal cell type was determined by immunohistochemical labelling with antibodies to desmin, vimentin and alpha-smooth muscle actin (alpha-SMA) during the progressive and regressive stages of spontaneous CTVT. The distribution of ECM components tenascin-C, chondroitin sulphate and versican were determined immunohistochemically, and hyaluronan distribution was determined using a biotinylated protein complex with specific affinity for hyaluronan. Stromal cells of tumours in both the progressive and regressive stage were positive for vimentin and negative for desmin. The number of stromal cells expressing alpha-SMA was significantly higher (P=0.001) in regressing tumours, than progressing tumours. These results suggest that the modulation of stromal cells that occurs during the regression of CTVT is similar to that occurring during wound healing. Tenascin-C was weakly expressed in the stroma of tumours in the progressive stage and in regions of the regressing tumours with tumour infiltrating lymphocytes (TILs), but intensely expressed in the stroma of tumours in late regressive stage. In addition, tenascin-C was also expressed in the cytoplasm of some tumour cells in the late regressive stage. A strong stromal tenascin-C intensity was significantly associated with regressing tumours (P=0.001). Strong stromal hyaluronan intensity and a high proportion of hyaluronan-positive tumour cells were significantly associated with progressing tumours (P=0.001). This suggests that hyaluronan is involved in the growth of the tumour. There was no significant difference in the expression of chondroitin sulphate and versican in progressing and regressing tumours.     

Fine needle aspiration cytology in ovarian lesions: an institutional experience of 584 cases

N. Gupta, A. Rajwanshi, L. K. Dhaliwal, N. Khandelwal, P. Dey, R. Srinivasan and R. Nijhawan
Fine needle aspiration cytology in ovarian lesions: an institutional experience of 584 cases Objective: To assess the diagnostic value of fine needle aspiration cytology (FNAC) in ovarian lesions. Methods: This was a retrospective study of ultrasound‐guided (US) FNAC of 584 ovarian lesions from January 1998 to July 2010. The lesions were categorized into non‐neoplastic lesions, neoplastic lesions and inadequate aspirates. The results were compared with the corresponding histopathology whenever available. Results: Of the 584 lesions, 180 (30.8%) were reported as non‐neoplastic (48 non‐specific inflammation, 11 tuberculosis, 63 functional cysts and 58 endometriotic cysts), 249 (42.6%) as neoplastic (81 benign lesions/tumours and 168 malignant) and 155 (26.5%) as inadequate. Based on the subsequent histopathology, which was available in 121 (20.7%), the cases were divided into those that were concordant and discordant. Concordant cases comprised 92/121 (76%), including 28 non‐neoplastic lesions (seven non‐specific inflammation, nine functional cysts and 12 endometriotic cysts), 42 surface epithelial tumours (13 benign and 29 malignant), 10 germ cell tumours (five mature cystic teratomas and five mixed germ cell tumours), seven sex‐cord stromal tumours (three granulosa cell tumours, one sclerosing stromal tumour, one strümal leutoma, one Sertoli Leydig cell tumour and one malignant Sertoli cell tumour) and five miscellaneous lesions (one plasma cell tumour, two leiomyosarcomas and two cases of necrosis). Discordant cases comprised 29/121 (24%) (21were inconclusive or inadequate on cytology), including four endometriotic cysts, 14 surface epithelial tumours (one cystadenofibroma, one borderline mucinous tumour and 12 carcinomas), five germ cell tumours (two immature teratomas and three mature cystic teratomas), two thecomas, one fibroma, one sclerosing stromal tumour, one fibrosarcoma and one myxoma. FNAC sensitivity for a diagnosis of malignancy was 85.7%, specificity 98.0%, positive predictive value 97.7%, negative predictive value 87.7% and accuracy 92.0%, if 21 inconclusive/inadequate FNACs were excluded; with the latter taken as false negatives, sensitivity was 73.7% and accuracy 76.0%. Conclusion: FNAC has a high specificity for diagnosis of ovarian/adnexal lesions but greater experience is required for the accurate subtyping of neoplasms and sensitivity is limited by inconclusive/inadequate results.     

The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.