Preliminary results on a proposed histopathological assessment of predictive factors for basal cell carcinoma recurrence after primary free margin excision.

Background: Basal cell carcinoma (BCC) incidence is steadily increasing but therapeutic solutions remain limited and present a public health challenge. Aims: To identify predictive factors of BCC recurrence after primary free margin excision, with automated methods, by evaluating cell proliferation, the Hedgehog pathway activation and primary cilia. Materials and Methods: This case-control study included 32 patients (16 with recurrence occurring at least 6 months after complete resection, and 16 without recurrence) who underwent surgery for BCC. Formalin-fixed paraffin-embedded cutaneous resections were processed for immunohistochemistry or immunostaining with the following primary antibodies: mouse anti-MCM6, rabbit anti-ARL13B and rabbit anti-GLI1. Results: BCC recurrence after free margin excision was significantly linked to a higher proliferative index (p < 0.001) and a lower cilia count (p = 0.041) in the primary lesion. No significant differences were observed regarding cilia length (p = 0.39) or GLI1-positive nuclei. Discussion: The complex interplay between essential signaling pathways, cell proliferation and cilia requires further experimental investigations in the context of BCC recurrence. Conclusion: A higher proliferative index evaluated with MCM6 antibody could be a useful prognosis marker of BCC risk of recurrence. The lower cilia count in the primary lesion unveiled novel perspectives to understand BCC recurrence molecular mechanisms.

[Vulvar invasive squamous cell carcinoma and hidradenoma papilliferum. Case report]. / Carcinome épidermoïde et hidradénome papillifère vulvaire. A propos d'un cas.

We report a case of a vulvar invasive squamous cell carcinoma associated to a benign tumor with apocrine differenciation, the hidradenoma papilliferum, infiltrated by the carcinoma. Diagnosis was established by clinical and histopathological examination, first on biopsy and then on local vulvar excision. Classical association between hidradenoma papilliferum and Paget's disease is described, but to the best of our knowledge, there have been no previous reports of such case in published literature. A common physiopathological etiology cannot be completely excluded.

Metalloproteinase-2, -7 and -9 and tissue inhibitor of metalloproteinase-1 and -2 expression in normal, hyperplastic and neoplastic endometrium: a clinical-pathological correlation study.

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors are key-players in extracellular matrix and basement membrane degradation, and are involved in both physiological and malignant processes. The aim of this study was to examine MMP-2, -7 and -9 and TIMP-1 and -2 expression in normal, hyperplastic and malignant endometrium, and their relation to clinical and histological prognostic factors. MATERIALS AND METHODS: We performed qualitative and semi-quantitative immunohistochemical analysis of 20 samples of normal endometrium (10 in the proliferative phase, 10 in the secretory phase), 39 samples of hyperplastic endometrium (17 without atypia and 22 with atypia) and 38 samples of endometrioid carcinoma, by using specific monoclonal antibodies. RESULTS: In normal endometrium, epithelial expression of MMP-2 (P = 0.0007), MMP-7 (P = 0.0002) and TIMP-2 (P = 0.0004) was increased during the proliferative phase of the menstrual cycle. MMP-2 expression correlated negatively with TIMP-2 expression (P = 0.001, rho = 0.702). Endometrial stromal cells in the secretory phase showed strong MMP-2 expression (P = 0.004) and weak MMP-7 (P = 0.001) and TIMP-1 expression (P = 0.01). In hyperplastic endometrium, the presence of atypia was associated with lower TIMP-2 expression (P = 0.005) and was also associated with a trend towards higher MMP-2 expression. Endometrial stromal cell expression of MMP-2, -7 and -9 and TIMP-1 and -2 did not differ between hyperplastic endometrium with and without atypia. A gradient of MMP-2 and -9 expression was observed from hyperplastic endometrium to endometrial carcinomas. In endometrial carcinomas, MMP-2 expression increased (P = 0.0004) and TIMP-2 expression decreased (P = 0.0005) with the histological grade. TIMP-2 expression correlated with myometrial invasion (P = 0.005), lymphovascular space involvement (P = 0.008) and lymph node involvement (P = 0.007). CONCLUSION: These results support the involvement of MMPs and TIMPs in endometrial carcinogenesis. Strong MMP-2 and weak TIMP-2 expression were the most potent markers of endometrial malignancies with a high risk of local and distant spread.

Usefulness of HPV testing in the follow-up of untreated cervical low grade lesions.

The aim of the present work was to evaluate the usefulness of high-risk human papillomavirus (HR-HPV) testing for the follow-up of women with untreated low grade cervical squamous cell lesions (LSIL). For that, 412 women with a cytological diagnosis of LSIL at entry were monitored by cytology, HR-HPV testing with the Hybrid Capture II assay (HC-II) and colposcopy. Our primary endpoint was clinical progression defined by the presence of a high grade cervical intraepithelial neoplasia (CIN2 and CIN3) at the biopsy. At baseline, histological control revealed 10 CIN2 and 11 CIN3 only in the cohort of women HR-HPV+. In the follow-up, 4 CIN2 and 8 CIN3 were detected, always in the women initially HR-HPV+. Thus, the recurrence of a HR-HPV+ infection clearly selects a population at high-risk for CIN2-3. The semi-quantitative appreciation of the viral load with HC-II could not be used as a good prognostic factor for the follow-up of women with LSIL. HR-HPV testing reduces the number of cytology and colposcopy examinations in the follow-up of women aged >35 years when HPV testing is initially negative. Thus HR-HPV testing should be reserved for the follow-up of this population of women initially HR-HPV+ and proposed 6 to 12 months after the cytological diagnosis of LSIL.

[Gestational trophoblastic diseases: a retrospective study from 1997 to 2003]. / Maladies trophoblastiques gestationnelles: étude rétrospective de 1997 a 2003.

BACKGROUND: Trophoblastic diseases correspond to a very heterogeneous group of rare pathology in young women which fertility should be preserved. PATIENT AND METHODS: We conducted a retrospective study from 1997 to 2003, including all patients with molar pregnancy or trophoblatic tumor in our department of Obstetrics and Gynecology. RESULTS: Fifteen patients were identified with 9 molar pregnancies, 5 trophoblastic tumors and 1 placental tumor of implantation site. The outcome was favorable for 14, and one patient died from her metastatic disease. For 4 patients we asked our university colleague for the optimal approach. DISCUSSION: Management of molar pregnancies is well established. Persistent gestational disease is more rare and problematic with potential metastatic dissemination. Multidisciplinary care is often needed. CONCLUSION: Persistent gestational disease should be managed in a highly specialized centre, as developed in the Lyon University Hospital.

Comparison of liquid based cytology and histology for the evaluation of HER-2 status using immunostaining and CISH in breast carcinoma.

BACKGROUND: HER-2 amplification is an important prognostic biomarker and treatment determinant in breast carcinoma. AIMS: To correlate immunocytochemical (ICC) expression of HER-2 and gene amplification determined by chromogenic in situ hybridisation (CISH) using liquid based cytology (LBC) with immunohistochemistry (IHC) and CISH using histological samples of the same breast carcinomas. METHODS: Frozen sections and cytobrushings of 103 breast carcinomas were analysed. Four techniques were performed on each tumour: two on LBC samples (ICC, and CISH, both graded as positive, indeterminate, or negative) and two on histological samples (IHC and CISH). Two cell lines (MCF-7, negative; BT 474, positive) were used as controls for cytological analysis. A complementary fluorescence in situ hybridisation technique was carried out in histological samples with low amplification (4-10 dots/nucleus). RESULTS: Interobserver agreement for the four techniques calculated by the kappa coefficient indicated a substantial agreement. Nine cases failed in cytology because of poor cellularity. Among 94 cases, 19 were amplified; 73, 12, and 9 tumours were scored 0 or 1+, 2+, and 3+, respectively by IHC and 75, 13, and 6, respectively, by ICC. CISH found no amplification in 72 tumours. Correlations between the IHC and CISH results in the histological and cytological samples were always significant. CONCLUSIONS: Her-2 status could be determined in LBC samples and correlated well with reference histological methods using in situ hybridisation. ICC was less reliable because of the presence of the cytoplasmic membrane. However, these results should be confirmed by a large multicentre study.

DNA image cytometry on biopsies can help the detection of localized Gleason 3+3 prostate cancers.

PURPOSE: In spite of classifications based on digital rectal examination, prostate specific antigen (PSA), transrectal echography and histological analysis, 20% to 40% of operated prostate adenocarcinomas are not yet organ confined. New diagnostic features to predict extracapsular invasion before treatment are needed to avoid surgical extraction within positive margins. MATERIALS AND METHODS: A retrospective study was performed for 74 prostate adenocarcinomas with Gleason 3+3. A total of 54 organ confined tumors (T1T2) at digital rectal examination were compared with 20 nonorgan confined tumors (T3T4). Image cytometric DNA analysis was performed on prostate initial biopsies. DNA ploidy results were compared in both groups for values of PSA greater than 15, less than 15 and less than 10 ng/ml. RESULTS: For a PSA rate less than 15 ng/ml, 83.8% of T1T2 were diploid vs 33% T3T4 (p = 0.042). For a PSA rate less than 10 ng/ml, 96% of T1T2 were diploid vs 33% T3T4 (p = 0.025). CONCLUSIONS: DNA ploidy appears to be an interesting feature at diagnosis. When in doubt about the localized character of a tumor, DNA ploidy it makes it possible to predict a nonorgan confined tumor. Gleason 3+3 prostate cancers that are organ confined at digital rectal examination are more often diploid than T3T4 tumors. The prognostic interest in DNA ploidy is more reserved because of its correlation with PSA level.

Negative human papillomavirus testing in normal smears selects a population at low risk for developing high-grade cervical lesions.

High-risk human papillomaviruses (HR-HPV) are the necessary cause of cervical carcinomas and there is an increasing interest in using HR-HPV DNA detection in adjunction to cytological examination for primary cervical screening. To determine whether women with a normal smear negative for HR-HPV DNA detection with the Hybrid Capture II assay might represent a low-risk population for developing a high-grade squamous intraepithelial lesion (HSIL), 4401 women have been followed in a period of 12-72 months (median=34 months). During this follow-up, four HSIL and one microinvasive carcinoma have been detected in this cohort (three in the cohort of 3526 women >29 years). The global negative predictive value (NPV) of double-negative tests is thus of 99.9% (ninety-five percent confidence interval (95% CI): 99.8-100%), whereas cytology alone gives an NPV of 99.2% (95% CI: 98.9-99.5%). If we obtain a second negative HR-HPV test 1-2 years after the initial test, the NPV is 100%. The NPV is also of 100% in the cohort of women >49 years. We conclude that all these women could be safely screened at longer intervals between 3 and 5 years. This policy will offset the increased costs induced by an additional HR-HPV testing in primary screening.

Influence of physicochemical reactions of bioactive glass on the behavior and activity of human osteoblasts in vitro.

Bioactive glasses are characterized by a bond to bone with a hydroxyl carbonate apatite layer. They enhance bone tissue formation and for this purpose are used in orthopedic surgery and in dental implantology. In the current work, we studied the biological response of human osteoblasts with a bioactive glass. This bioactive glass is based on 50% Si0(2), 20% Na(2)O, 16% CaO, 6% P(2)O(5), 5% K(2)0, 2% Al(2)O(3) and 1% MgO and designated A9. Cracks and irregularities were observed on the material surface when it was immersed in the culture medium. In addition, energy dispersive X-ray analyses highlighted a selective release of the elements at the surface of the bioactive glass, such as Na(+) and K(+) ions, released from the first day, contrary to the Si, Al, Ca, P, and Mg elements, which were released more slowly. Cell proliferation kinetics, total protein synthesis, and DNA content of the osteoblasts in contact with bioactive glass were similar to control cells. The morphological studies by light and scanning electron microscopy revealed an increasing cellular density in culture with bioactive glass without contact inhibition. The immunohistochemical studies highlighted the expression of types I, III, and V collagens by osteoblasts cultured in the presence of bioactive glass. The pH measurement of the culture medium in the presence of bioactive glass demonstrated a slight alkalinization. We thus conclude that human osteoblasts preserve their properties in the presence of bioactive glass (A9).

Efficiency of amifostine as a protection against doxorubicin toxicity in rats during a 12-day treatment.

BACKGROUND: Our purpose was to determine the effects of amifostine, a cytoprotective agent, on doxorubicin tolerance and cardiotoxicity in rats. MATERIALS AND METHODS: Male Wistar rats were treated every other day with an intraperitoneal injection of amifostine or saline 30 minutes before intraperitoneal injection of doxorubicin or saline. Weight change was recorded, and contractile function was evaluated after 11 injections by means of the isolated heart. RESULTS: Weight evolution and cardiac function were significantly improved by 7 and 20 mg/kg amifostine (p < 0.001) but not by 50 mg/kg. The final weight were: controls 349 +/- 16 g; doxorubicin alone 258 +/- 54 g; with amifostine: 7 mg/kg 314 + 28 g; 20 mg/kg 312 +/- 32 g; 50 mg/kg 250 +/- 34 g. Left ventricular developed pressure were: controls 137 +/- 15 mmHg; doxorubicin alone 119 +/- 20 mmHg; with amifostine: 7 mg/kg 140 +/- 20 mmHg; 20 mg/kg 137 +/- 25 mmHg; 50 mg/kg 124 +/- 20 mmHg. CONCLUSION: Seven and 20 mg/kg amifostine protected rats from the toxicity of doxorubicin at the cumulative dose of 18 mg/kg during a 12-day treatment, with regard to weight loss and heart contraction.

[Malignant blue nevus: clinicopathologic study with AgNOR measurement. Seven cases]. / Naevus bleu malin: étude anatomo-clinique avec quantification des organisateurs nucléolaires argyrophiles (AgNOR): à propos de 7 cas.

BACKGROUND: Malignant blue nevus is a very rare tumor. Argyrophilic nucleolar organizer regions (AgNORs) have been reported to be both a diagnostic and prognostic clue in various tumors, especially if standardized using an image analysis systems. PATIENTS AND METHODS: Seven cases of malignant blue nevus were retrospectively recorded between 1974 and 1999, and their clinical and pathological features described. Using an image analysis system, AgNOR measurements were studied in all cases. These results were compared with those obtained in 10 cases of commun blue nevus, 10 cases of cellular blue nevus and 10 cases of malignant melanoma. RESULTS: The most frequent location of malignant blue nevus was the scalp. Clinically, the tumor generally consisted of a blue nodule, 2.5 cm in diameter. There was no single histopathological criterion for the diagnosis of malignant blue nevus. AgNOR measurement was significantly higher in malignant blue nevus in comparison with commun blue nevus (p<0.0004) or cellular blue nevus (p<0.012), whereas there was no difference between malignant blue naevus and malignant melanoma (p > 0.50). DISCUSSION: Our results confirm the severe prognosis of malignant blue nevus and highlight the necessity of removing all blue tumours located on the scalp. AgNOR measurement using an image analysis system appears to be a useful tool for the diagnosis of malignant blue nevus, but further studies remain necessary.

[Survival and infectious processes in pacients with AIDS: analysis based on initial serum vitamin A levels]. / Sobrevida e processos infecciosos em pacientes com AIDS: análise de acordo com os níveis séricos iniciais de vitamina A.

Patients with Aids (n = 39) were followed up for a maximum period of 36 weeks, after which the types and topographies of infectious complications presented and patient survival were analyzed and correlated with the vitamin A levels presented by the patients at the beginning of clinical follow-up. Twenty-one (53,8%) patients presented serum retinol levels below 1.6 micromol/L, 12 (57%) of whom had values lower than 1.05 micromol/L. There was no correlation between low serum vitamin A levels and the types or topographies of the infectious complications that occurred during the follow-up period. Although mean survival at the end of the 36 months follow-up period was similar for the two groups, patients with retinol deficiency presented a lower probability of survival during the first 24 months of follow-up compared to patients without hypovitaminosis A (8.44 x 1.42 months; p = 0.003).

Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women.

High-risk human papillomaviruses (HR-HPV) are the necessary cause of cervical carcinomas. To determine whether HPR-HPV DNA detection in primary routine screening could represent a sensitive and reliable technique for the detection of high-grade squamous intraepithelial lesions (HGSIL), laboratory analysis using 2 cytologic techniques (conventional and liquid-based), HPV testing with Hybrid Capture II assay (HC-II), followed by colposcopic examination of women with abnormal cervical finding and/or persistent HR-HPV infection, was conducted in 7932 women who had routine cervical examination. The sensitivity of HPV testing for detecting a histologically proven HGSIL was 100%, higher than that of conventional (68.1%) and liquid-based (87.8%) cytology. The low specificities of 85.6% and 87.3% of HPV testing slightly increased to 88.4% and 90.1% if HPV testing was reserved for woman >30 years old. The quantitative approach provided by the HC-II assay for the assessment of the viral load was not reliable for predicting HGSIL in normal smears. HR-HPV testing could be proposed in primary screening in association with cytology. With conventional cytology it significantly improves the detection of HGSIL. With the use of the same cervical scrape for HPV testing and liquid-based cytology, HR-HPV testing would allow to select positive samples treated in a second time for cytology which gives a good specificity.

DNA image cytometry and human papillomavirus (HPV) detection help to select smears at high risk of high-grade cervical lesions.

Three samples were submitted from women undergoing routine screening (n=910): two smears (one for routine cytology and one for DNA image cytometry) and a scrape for human papillomavirus (HPV) testing. DNA histograms were classified as suspect in cases of aneuploidy, polyploidy, and/or diploidy with a high proliferation rate. Follow-up was available in 239 cases. The primary end-point was the presence of a high-grade squamous intraepithelial lesion (HGSIL) at biopsy. Seventy women (7.7%) had a high-risk (HR) HPV infection and a suspect DNA profile. In 77 women with cytological abnormalities, 28 HGSILs were detected: four with a prior diagnosis of ASCUS (all HR-HPV infected including three with a suspect DNA profile), three with smears evocative of LGSIL (all with HR-HPV infection and a suspect DNA profile), and 21 with smears evocative of HGSIL (all with HR-HPV infection and 20 with a suspect DNA profile). During the follow-up period, out of 239 women with a cytologically normal smear at first entry, five developed a HGSIL; all were HR-HPV-positive and four had a suspect DNA profile at the first smear. HR-HPV detection alone gives a sensitivity of 100% for the detection of HGSIL, with a specificity of 84.3%, whereas DNA measurement associated with HPV testing significantly enhances the specificity to 95.4%. Thus, the combination of HPV testing and DNA measurement provides a highly sensitive and specific evaluation of the risk of HGSIL on cervical smears.

Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa.

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous DeltaF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0.001). The number of T lymphocytes was higher at the CF distal level (P = 0.035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0.033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.

Human papillomavirus detection by the hybrid capture II assay: a reliable test to select women with normal cervical smears at risk for developing cervical lesions.

The reliability of the Hybrid Capture II (HC-II; Digene, Silver Spring, MD, U.S.A.) assay was tested in detecting 18 human Papillomavirus (HPV) types for the screening of cervical lesions. Cytology, HPV testing, colposcopy, and biopsy were used to monitor 204 women with normal smears at the first entry. The median follow-up was 15 months (range, 4-27 months). The primary endpoint was clinical progression defined as the presence of a cervical intraepithelial lesion at the biopsy. In the patient population of 204 HPV-infected women, 81 (39.7%) had a persistent HPV infection at two or three examinations with a final histologic diagnosis of 14 high-grade and 13 low-grade squamous intraepithelial lesions (SIL) within 4 to 22 months. Women with regressive HPV infection did not develop any lesion during the same period. The evaluation of the viral load of high-risk HPV by the HC-II did not represent a sensitive approach to predict the persistence or the apparition of high-grade lesions. Thus, persistent high-risk HPV infection detected with HC-II represents a reliable tool to select populations at risk for the development of high-grade cervical lesions.

Image cytometry detection of breast cancer cells with > 5C DNA content and minor DNA stemlines.

OBJECTIVE: To determine if the presence of cells having a DNA content > 5c and occurring at very low frequency is related to breast cancer outcome. STUDY DESIGN: Feulgen-stained imprints of fresh tumors used for routine standard DNA image cytometry were reanalyzed, with the aim of detecting hyperploid (> 5c) cells or minor stemlines. Specially adapted software was used. RESULTS: The new DNA analysis showed discordance of 47.3% with standard DNA cytometry. Minor stemline or rarely occurring 5c exceeding cells were found. These were not detected by the first DNA analysis. The presence of both DNA hyperploid cells occurring as rare events and a DNA hyperploid stemline was related to outcome. CONCLUSION: The detection of DNA hyperploid cells, even in very small numbers, appears essential to outcome, particularly in diploid or single DNA aneuploid breast cancers.

Human papillomaviruses and DNA ploidy in anal condylomata acuminata.

Previous studies have emphasized the usefulness of DNA ploidy measurement and Human Papillomavirus (HPV) detection as prognostic markers in low grade cervical lesions. We addressed the eventual relationship between HPV type, DNA profile, and p53 tumor suppressor protein expression in anal condylomata acuminata to eventually determine parameters which may be considered as predictive risk factors for the development of cancer. DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of 45 anal condylomata acuminata without atypia containing HPV detected by in situ hybridization and Polymerase Chain Reaction (PCR). p53 expression was detected by immunohistochemistry. DNA aneuploidy was found in 53.3% of these lesions, 48.9% containing non oncogenic HPV types 6 and/or 11 and 4.4% harbouring HPV types 11 and 18. The DNA diploid lesions were all associated with non oncogenic HPV types 6 and/or 11 and one case also contained HPV type 33. There was no significant correlation between the detection of DNA aneuploidy and the presence of immuno-detected p53. DNA aneuploidy was not related to the presence of oncogenic HPV in anal condylomata acuminata. The DNA aneuploid profile frequently observed, especially in lesions associated with non oncogenic HPV types, is not yet well explained and cannot be considered as a prognostic factor. In contrast, a more intensive clinical follow-up should be proposed in patients with oncogenic HPV associated to DNA aneuploidy.

Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.