The Mechanisms Of The Calcification In Craniopharyngioma

Craniopharyngiomas (CP) are epithelial tumors arising along the path of the craniopharyngeal duct. The incidence of CP is about 1.3 per million, with one-third occurring in children under the age of 15.Despite their benign histological appearance, their often infiltrative tendency into critical parasellar structures and the propensity to recur result in significant morbidity and mortality, posing considerable medical and social problem.Two clinicopathological patterns are distinguished:the adamantinomatous craniopharyngioma (AC) and the papillary (PC). The AC is the most common and may occur at all ages;it is famous for the "motor oil" cyst fluid, fibrous tissue, and calcification, and usually has circumscribed lesions that are distinctive microscopically with peripheral palisades, stellate reticulum, wet keratin, and cholesterol clefts. The PC,in contrast to the AC,is composed of mature squamous epithelium forming pseudopapillae and of an anastomosing fibrovascular stroma without the presence of peripheral palisading of cells or stellate reticulin, as well as calcification.CP is one of the most commonly calcified tumors of the central nervous system. The calcification patterns vary from solid lumps to popcorn-like foci or less commonly to an eggshell pattern lining the cyst wall.Matrix vesicles, matrix giant bodies, or mitochondria are generally recognized as the initial mineralization foci both in physiological and pathological calcified tissue.Calcification has been reported in CP that initially started at the membrane-bounded vesicles (mitochondria or matrix vesicles) in the keratinized cells. Subsequently, hydroxyapatite crystals accumulate in these vesicles, and then tonofibrils adjacent to the calcified vesicles are mineralized. However, the molecular mechanisms that result in calcification of CP are unclear.So far, there hasn't been any main hypotheses explain the calcification of craniopharyngioma. The purpose of the study is to undertake molecular pathological and molecular biological studies on the dose-dependent effect of Osteopontin (OPN) on the calcification of craniopharyngioma as well as its relevant mechanisms.OPN is a 44-kDa, negatively charged, acidic protein that is almost always sialated and/or phosphorylated and that is found in large amounts in a variety of tissues and secreted into body fluids, including the stroma. The protein contains several conserved motifs, including the integrin binding sequence GRGDS.OPN can interact with a number of receptors, including RGD-mediated interactions with the integrinsαvβ3,αvβ5,α9β1 andα4β1.Also reported are non-RGD-mediated interactions of OPN with CD44, a family of surface receptors which regulatecell adhesion, movement, and activation. Other non-RGD cell binding interactions that are dependent only on the beta subunit or sialic acid moieties have also been demonstrated. Macrophages, T lymphocytes, NK cells, endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, and osteoclasts all express OPN in response to activation by various cytokines or inflammatory mediators.OPN has many diverse and incompletely understood functions, including the regulation of cell migration and proliferation, tissue repair and angiogenesis, and cellular chemotaxis and activation in inflammatory conditions. Materials and methods1,Subject:Patients with craniopharyngioma aged 3～66, male (n=31)and female (n=23),were studied from June 2004 to March in 2006.Cases acceptance standard:①Patients with craniopharyngioma who accepted neurosurgery in the period of study;②The craniopharyngioma were confirm by the postsurgery-pathology..2,Trial management:The study was consisting by two parts.a. To study the dose-dependent effect of OPN on the calcification of craniopharyngioma by immunohistochemistry, Western Blot, Rt-PCR; b. For the bionomics of the craniopharyngioma with light microscope, immunohistochemistry, Elisa, electron microscope, and immuno-electron microscope were performed.3,The results of the immunohistochemical staining were evaluated separately and in a blinded fashion by Huang and Liu. The proportion of positively stained cells in all epithelial cells of each section was determined and recorded as follows:Five representative microscopic areas at x400 magnificationwere randomly selected for examination. Expression of OPN, CD44v6, P65 were semiquantitatively assessed as percentage of positive cells with respect to the total number of epithelial cells.The samples were assigned to one of the four following categories:0 (none of the tumor epithelial cells were positively stained in the cytoplasmor on the cellmembrane);+ (<10% of positive cells were stained);++(10-50% of positive cells were stained); +++(>50% of positive cells were stained).4, Statistics treatment:The statistical analysis was performed using the SPSS software program (version 13.0; SPSS Inc.,Chicago, IL).Data appear as the mean(1 SD) for quantitative variables and proportions for categorical variables. The Kruskal-Wallis test was used to assess the statistical significance of the frequency distribution of all categories of OPN, CD44v6 or P65 expression by degree of clacification. Differences were significant with a P-value of<0.05.The nonparametric Spearman's correlation coefficient method was used to assess the statistical significance of the correlation between OPN expression vs.degree of clacification, CD44v6 positivity, or P65 positivity. Correlations were significant when the P-value was<0.05.ResultsSamples were from the 54 patients whose craniopharyngiomas had been surgically removed in our hospital from May 2004 to March 2006, followed by postoperative pathological diagnosis to find 41 cases of adamantinomatous craniopharyngiomas and 13 cases of papillary craniopharyngiomas.The 54 patients (31 male and 23 female) in the ages from 3 to 66 years were analyzed.The average age was 26.5 years.OPN expressed obviously in the admantinomatous craniopharyngioma cell, but absent or little expressed in the papillary types (Mann-Whitney U test, Z=-4.813,P <0.001).OPN stain grade was significantly in the admantinomatous craniopharyngioma and related to the grade of calcification, the more calcification apparent the OPN stain was (Spearman correlation, rs=0.533,P<0.001).OPN was aggregated in the epidermis like cell in the admantinomatous type.The western blot and RNA test results conformed the OPN protein test resultsPositive sections of OPN revealed a strong immunostaining in the cytoplasm. Fifty-four tumor specimens were analyzed:6(11.1%)were had no immunostaining, 18 (33.3%)presented cytoplasmic expression in<10% of tumor cells with moderate intensity,16 (29.6%) specimens showed 10-50% immunoreactive for OPN,and 14 (25.9%) presented cytoplasmic expression in more than 50% of tumor cells with strong intensity. All cases of AC showed cytoplasmic and membranous accumulation, while six cases of PC were absent positive staining. Sheets of epithelial cells showed weak to moderate cytoplasmic and membranous staining. Peripherally palisading cells showed somewhat stronger expression in both the cytoplasm and membrane. Clusters of cells showing strong membrane accumulation were seen among the whorl-like arrays of the epithelial cells. Among the 41 cases of AC,30 showed distinctive features in the stroma surrounding the epithelial cell nests. These were whorling and streaming fascicles of spindle cells around the epithelial tumor cell nests. The stroma of these specimens was more cellular than those of the other tumors, and the streaming arrangement of stromal cells was distinctive in these cases. In sections stained immunohistochemically for OPN, these spindle cells exhibited moderate to strong cytoplasmic and membranous staining observed in epithelial tumor cell nests. In these neoplasms, degenerative keratinized cell nests tended to be absent.Diffuse and strong membranous CD44v6 staining was found on the cells. Interestingly, in these samples, OPN expression coexisted with diffuse CD44v6 staining. In AC, the CD44v6 immunoreactivity was observed in both ameloblast-like and stellate reticulum-like cells. However,the central keratinized cells in tumor nests of acanthomatous ameloblastoma were usually negative for CD44v6.In PC, the CD44v6 was immunoreactive in squamous epithelium but not anastomosing fibrovascular stroma. The frequency distribution of the OPN expression level was significantly correlated with the degree of calcification. Moreover, samples were also analyzed for the expression of CD44v6, the receptor that is involved in OPN binding. The association between OPN and CD44v6 immunoreactivity was significant when analyzed by the Spearman's rank correlation test (Spearman correlation, rs=0.680, P <0.001).The neoplastic epithelial cells showed several common findings as shown below. The cells revealed intermediate filaments in the cytoplasm that increased in relation to maturation grade and desmosomes, which permitted us to distinguish epithelial cells from fibroblasts. Islands of keratinized cells, which contained numerous tonofibrils in the cytoplasm, were frequently seen in the epithelial cell zone. Some of these keratinized cell nests were observed to be in direct contact with fibrous connective tissue. Mesenchymal cells surrounded by collagen fibrils and amorphous ground matrices were occasionally observed alongside the keratinized cells.Ultrastructural analysis confirmed morphological and immunohistochemical findings. Immunoreactivity with gold-labeled anti-OPN was seen in the AC in both the cytoplasm and membrane of the epithelium. OPN in AC epithelial cells was localized to the Golgi complex, membrane-bound cytoplasmic granules, and the glycocalyx. It indicated that the OPN protein is probably synthesized and secreted by stellate reticulum-like cells and ameloblast-like cells.NF-κB stain grade was significantly over-expressing in the adamantinomatous craniopharyngioma; and related to the grade of OPN by the Spearman's correlation analysis. The tumor hydatid fluid, cerebrospinal fluid and serum hs-CRP level of adamantinomatous type were (4.280±0.900) mg/mL, (0.035±0.006)mg/mL, (1.720±0.540) mg/mL respectively. Inflammation plays an important role in the craniopharyngioma,and made its surgery difficaltly. The study of NF-κB signal transduction pathway maybe a way to identify the mechanism of inflammation and explode new target for the treatment of craniopharyngioma.The ossifications were found in the tumor with eggshell pattern lining the cyst wall and popcorn-like foci calcification, but absent neither in the papillary craniopharyngioma nor those adamantinomatous craniopharyngioma that absent calcification or solid lumps calcification. Calcification was the basement for the formation of ossification. ConlusionsIn conclusion, our results show that OPN secreted by epithelial cells probably plays a core role in the formation of calcification in CP.However, there may be discrepancies in the tumor as compared to the histological specimen being analyzed, as the CPs are notorious for having focal calcium deposits in some areas and none in other areas. Further studies using an OPN-siRNA or an OPN-knockout mouse model may be required to reach a definitive conclusion. This is based on the notion that mechanistic insight is a first step toward a rational design of therapeutic intervention.