Multinucleated giant cells within the in vivo implantation bed of a collagen-based biomaterial determine its degradation pattern.

OBJECTIVES: The aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days. MATERIALS AND METHODS: Ex vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern. RESULTS: The membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane. CONCLUSION: The present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs. CLINICAL RELEVANCE: Understanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.

Modification of collagen-based sponges can induce an upshift of the early inflammatory response and a chronic inflammatory reaction led by M1 macrophages: an in vivo study.

BACKGROUND: The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group. MATERIAL AND METHODS: First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group. RESULTS: At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days. CONCLUSION: Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial's degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo. CLINICAL RELEVANCE: A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

Platelet-rich fibrin secretome induces three dimensional angiogenic activation in vitro.

Different tissue engineering techniques are used to support rapid vascularisation. A novel technique is the use of platelet-rich fibrin (PRF), an autologous source of growth factors. This study was the first to investigate the influence of PRF matrices, isolated following different centrifugation protocols, on human dermal vascular endothelial cells (ECs) in mono-culture and co-culture with human primary fibroblasts (HFs) as an in vitro model for tissue regeneration. Focus was placed on vascular structure formation and growth factor release. HFs and ECs were cultivated with PRF prepared using a high (710 ×g) or low (44 ×g) relative centrifugation force (RCF) over 14 d. Immunofluorescence staining and immunohistochemistry were used to evaluate the microvascular formation. Cell culture supernatants were collected for evaluation of growth factor release. The results showed a PRF-mediated effect on the induction of angiogenesis in ECs. Microvessel-like structure formation was promoted when ECs were combined with low-RCF PRF as compared to high-RCF PRF or control group. The percentage of vascular lumen area was significantly higher in low-RCF PRF, especially at day 7, which coincided with statistically significantly higher growth factor [vascular endothelial factor (VEGF), transforming growth factor ß1 (TGF-ß1) and platelet derived growth factor (PDGF)] concentration measured in low-RCF PRF as compared to high-RCF PRF or control group. In conclusion, reducing the RCF according to the low-speed centrifugation concept (LSCC) resulted in increased growth factor release and angiogenic structure formation with EC mono-culture, suggesting that PRF may be a highly beneficial therapeutic tool for tissue engineering applications.

Endoplasmic reticulum-resident chaperones modulate the inflammatory and angiogenic responses of endothelial cells.

BACKGROUND: Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. OBJECTIVES: To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. METHODS: Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on their inflammatory response and angiogenic potential. RESULTS: In all chronic wounds investigated, the levels of these ER-resident chaperones were elevated in endothelial cells and leucocytes. The proangiogenic role of BiP has been shown in tumour growth studies before and was confirmed in this study. Proangiogenic activity of the cochaperone Mdg1 has been postulated before but could not be confirmed in this study. The chemokine tumour necrosis factor (TNF)-α was shown to trigger the presentation of proinflammatory adhesion molecules and the release of proinflammatory cytokines. Here we show that TNF-α does not affect endogenous chaperone levels, but that the ER-resident chaperones BiP and Mdg1 modulate the cellular TNF-α-induced proinflammatory response. CONCLUSIONS: According to the presented data we assume that in chronic wounds upregulated levels of ER-resident chaperones might contribute to persistent inflammation in chronic wounds. Therapies to downregulate chaperone levels might provide a tool that switches the imbalanced chronic wound microenvironment from inflammation to healing.

Expression of CD34 and maspin in ameloblastoma from a West African subpopulation.

Ameloblastoma is a locally invasive odontogenic tumor with a high recurrence rate. Its local invasiveness is aided by angiogenesis, which can be correctly estimated by CD34. On the other hand, maspin decreases the local invasive and metastatic capability of cancer cells and functions as an angiogenesis inhibitor. We aim to assess the association between maspin expression and microvessel density in ameloblastoma. Twenty-five formalin-fixed paraffin-embedded (FFPE) blocks of ameloblastoma cases were prepared for antibody processing to CD34 and maspin. Positive immunohistochemical staining was marked by brown cytoplasmic/membrane coloration for CD34 and by nuclear/cytoplasmic coloration for maspin. At the ×40 magnification, we counted blood vessels in two areas of dimension; 300 × 400 µm (area A) and 150 × 200 µm (area B) adjacent to the tumor region to assess relative dispersion of the vessels bordering the tumor. The overall approximate microvessel density (MVD) for area A = 11 (minimum 2, maximum 21) and that for area B = 5 (minimum 1, maximum 10). The MVD in the area A of plexiform ameloblastoma was similar to that of the unicystic, while the hemangiomatous variant had the highest MVD for area A. Maspin positivity was present only in the cytoplasm of ameloblast, stellate reticulum, and the fibrous connective tissue in varying proportions. There was no evidence of the anti-angiogenesis effect of maspin in ameloblastoma from this study. The significance of cytoplasmic localization of maspin in the ameloblasts and stellate reticulum cells needs further investigation.

Early endothelial progenitor cells as a source of myeloid cells to improve the pre-vascularisation of bone constructs.

According to present knowledge, blood derived endothelial progenitor cells (EPC) might act as proangiogenic myeloid cells, which play a fundamental role in the regulation of angiogenesis and blood vessel reorganisation. In this context, we have evaluated the contribution of endogenous myeloid cells in co-cultures of blood derived outgrowth endothelial cells (OEC) and osteogenic cells. In addition, we investigated the role of EPC as a potential source of myeloid cells in the formation of vascular structures in an in vitro model consisting of mesenchymal stem cells (MSC) and OEC. For this purpose, we added EPCs to co-cultures of MSC and OECs. Vascular structures and the co-localisation of myeloid cells were analysed by confocal laser microscopy (CLSM) for endothelial and myeloid markers and quantitative image analysis. The molecular effects of myeloid cells were evaluated by quantitative real time PCR, ELISA and protein arrays from cell culture supernatants and lysates. Endogenous myeloid cells were significantly co-localised with angiogenic structures in co-cultures of OEC and osteogenic cells. The active addition of EPC to co-cultures of OEC and MSC resulted in a statistically approved increase in the formation of prevascular structures at early stages of the co-culture process. In addition, we observed an increase of endothelial markers, indicating beneficial effects of EPC or myeloid cells on endothelial cell growth. Furthermore, real time PCR indicated high expression levels of CD68, CD11b and CD163 in co-cultures of EPC and MSC indicating that EPC act at least partly as macrophage like-cells.

Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts.

The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

["Hic gaudet mors succurere vitae", autopsy findings of the Mainz Institute of Pathology 1971-2010. An analysis on the occasion of the 100 year jubilee]. / "Hic gaudet mors succurere vitae", Sektionsbefunde der Mainzer Pathologie 1971-2010. Eine Analyse aus Anlass des 100-jährigen Jubiläums.

In the past numerous analyses have studied several aspects of autopsies in particular with regard to the decline of frequency; however, long-term studies spanning more than one decade have rarely been published, especially in recent years. On the occasion of the 100 year jubilee the archive data of the Institute of Pathology of the University of Mainz were analyzed for autopsies performed between 1971 and 2010. In this cohort, we focused on patients over 14 years old (n = 14,724) who died in the University hospital. We compared the number of autopsies with the total number of deceased patients and, in addition, studied several epidemiological aspects with special relevance for the cause of death (COD). In 1973 the peak autopsy frequency was reached with a value of 73.4 % followed by a decrease to 49.1 % in 1980. In the following decade a relatively steady state was achieved (frequency 53.3 % in 1985, and 43.2 % in 1990), followed by a remarkable decline after the 1990s (1997: 26.4 %, 1998: 15.9 % and 2010: 5.6 %). The mean overall age increased during the observation period (59.1 years in 1971 and 67.5 years in 2008). Among the COD groups cardiovascular diseases were predominantly recorded (between 35 % in the 1970s and 39 % in 1995-2010), followed by infectious diseases (between 20 and 25 %). Malignancies represented the third most common COD group with an increase in frequency from approximately 10.5 % in the 1970s to 17 % observed in the last decade. Among the single specific CODs, pulmonary embolism was most often encountered in the 1970s (about 11.5 %), while in the following decades myocardial infarction predominated (up to 15.8 % between 1995 and 2010). In the overall period, lung cancer was the single most common malignancy of the CODs (between 2.5 and 3.9 %). These data confirmed previous studies showing that in Germany the autopsy frequency began to decline remarkably in the 1990s. Besides general aspects, the specific local causes for these phenomena are discussed.

[Rectal goblet cell carcinoid. Primary tumor or metastasis?]. / Becherzellkarzinoid des Rektums. Primarius oder Metastase?

Goblet cell carcinoids are biphasic neoplasms of the gastrointestinal tract composed of a glandular and neuroendocrine differentiation. Typically, goblet cell carcinoids are localized in the vermiform appendix. We report the case of a 60-year-old female patient with the diagnosis of a 1.2-cm rectal goblet cell carcinoid tumor discovered during prophylactic proctocolonoscopy. Because of the known aggressive behavior of this entity, a rectosigmoidectomy was performed. The preoperative staging revealed neither local nor systemic spread. After 8 months, the patient is in good health. As a primary tumor of the extraappendiceal gastrointestinal tract, goblet cell carcinoids are a rarity. It is generally recommended to exclude metastasis of a primary appendiceal neoplasm. However, since the patient underwent an appendectomy in 1974, primary origin in the rectum is favored.

[Tubulosquamous vaginal polyp. Case report with respect to histogenesis]. / Tubulosquamöser Vaginalpolyp. Fallbericht unter Berücksichtigung der Histogenese.

Tubulosquamous vaginal polyp is a rare form of lesion. It is thought to derive from displaced paraurethral Skene's gland which is the female equivalent of the prostate gland in men. We report on a case of tubulosquamous vaginal polyp in a 77-year-old female patient. Histological examination showed prominent epithelial nests in a spindle cell-rich stroma. The nests were predominantly squamous in type with small tubules at the periphery of the island. Using immunohistochemistry the tubules were found to be positive for CK7, prostate-specific acidic phosphatase (PAP) and androgen receptor (AR) but negative for CK 5/6 and prostate-specific antigen (PSA). The solid parts were positive for CK5/6 and most interestingly, also expressed AR but not CK7, PAP or PSA. The stroma also showed strong staining for AR but not for the other markers applied. To our knowledge this is the first report on AR expression in this benign polyp. Our findings confirm the view that tubulosquamous polyp of the vagina is derived from paraurethral Skene's gland.

Comparative study assessing effects of sonic hedgehog and VEGF in a human co-culture model for bone vascularisation strategies.

The morphogen sonic hedgehog (Shh) seems to mediate adult repair processes in bone regeneration and vascularisation. In this study we investigated the effects of Shh on co-cultures consisting of human primary osteoblasts and outgrowth endothelial cells in terms of angiogenic activation and vessel maturation in comparison to the treatment with the commonly used proangiogenic factor, VEGF. Both, stimulation with VEGF or Shh, leads to an increase in the formation of microvessel-like structures compared to untreated controls. In contrast to VEGF, proangiogenic effects by Shh could already be observed after 24 h of treatment. Nevertheless, after 14 days the angiogenic activity of OEC was comparable in VEGF- or Shh-treated co-cultures. Furthermore, Shh and VEGF resulted in different growth factor expression or release profiles. Compared to VEGF, Shh stimulates also the expression and secretion of angiopoietins which was detected as early as 24 h of treatment. Moreover, smooth muscle cell-related markers, such as alpha-smooth muscle actin, desmin and myocardin, as well as basement membrane components were clearly upregulated in response to Shh treatment compared to VEGF- or untreated controls. In terms of growth factors relevant for vessel stabilisation and maturation increased levels of PDGF-BB, angiopoietin-1 and TGF-beta were observed in cell culture supernatants when treated with Shh. This was in accordance with higher levels of smooth muscle actin in Shh-treated samples indicating the potential of Shh to improve the angiogenic activity and vessel stabilisation of human tissue engineered constructs. Experiments using cyclopamine, a Shh pathway inhibitor, blocked the effects of Shh.

Tissue reaction to sealing materials: different view at biocompatibility.

The biodegradability of root canal sealers in areas other than the root canal system is crucial to the overall success rate of endodontic treatment. The aim of the present study was to investigate, the cell and tissue reaction to GuttaFlow and AHPlus, both in vitro and in vivo. For the in vitro experiments the materials were incubated with Human Periodontal Ligament Fibroblasts and cell proliferation and cytotoxicity analyses were performed. Additional fluorescence-microscope stainings were carried out in order to visualize cell growth and morphology. For assessment of the tissue reaction to the materials a subcutaneous implantation model in Wistar rats was employed and the inflammatory response to the materials was visualized by means of general and specific histology after 6 weeks. Human gingival fibroblasts proliferation seemed to be dependent upon dental material and cultivation time. After an incubation period of 96 hrs AHPlus proved to be significantly (p<0.002) more cytotoxic than GuttaFlow, as only a small number of fibroblasts survived on AHPlus. In vivo, GuttaFlow was surrounded by a fibrous capsule and no degradation took place, while AHPlus induced a well-vascularized granulation tissue in which the material was phagocyted by macrophages. The results of this study demonstrate that a potential cytotoxic effect of a sealing material may beneficial in order to have antibacterial properties and induce self degradation when accidentally extruded over the apical foramen.

Expression of E-selectin and vascular cell adhesion molecule-1 in so-called 'negative' appendices: first results to support the pathological diagnosis in borderline cases.

BACKGROUND: Since the rate of histologically 'negative' appendices still ranges between 15 and 20%, appendicitis in 'borderline' cases remains a challenging disease. As previously described, cell adhesion molecule expression correlates with different stages of appendicitis. Therefore, it was of interest to determine whether the 'negative' appendix correlated with the absence of E-selectin or vascular cell adhesion molecule-1 (VCAM-1). METHODS: Nineteen grossly normal appendices from a series of 120 appendectomy specimens from patients with suspected appendicitis were analysed in frozen sections for the expression of E-selectin and VCAM-1. As control, 5 normal appendices were stained. RESULTS: This study showed a coexpression of E-selectin and VCAM-1 in endothelial cells in early and recurrent appendicitis. In patients with symptoms for less than 6 h, only E-selectin was detected. Cases with fibrosis and luminal obliteration were only positive for VCAM-1. In cases of early appendicitis with symptoms of less than 6 h duration, a discordance between histological and immunohistochemical results was found. CONCLUSIONS: This report indicates that E-selectin and VCAM-1 expression could be useful parameters in the diagnosis of appendicitis in borderline cases.

In situ synthesis of calcium phosphate-polycaprolactone nanocomposites with high ceramic volume fractions.

Biodegradable calcium phosphate-PCL nanocomposite powders with unusually high ceramic volume fractions (80-95%) and uniform PCL distribution were synthesized by a non-aqueous chemical reaction in the presence of the dissolved polymer. No visible polymer separation occurred during processing. Depending on the reagents combination, either dicalcium phosphate (DCP) or Ca-deficient HA (CDHA) was obtained. CDHA-PCL composite powders were high pressure consolidated at room temperature yielding dense materials with high compressive strengths. Such densification route provides the possibility of incorporating drug and proteins without damaging their biological activity. The CDHA-PCL composites were tested in osteoblastic and endothelial cell line cultures and were found to support the attachment and proliferation of both cell types.

Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling.

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.

[Littoral cell angioma of the spleen. Association with colon and hepatocellular carcinoma]. / Littoralzellangiom der Milz. Assoziation mit Kolon- und hepatozellulärem Karzinom.

Littoral cell angioma is a rare vascular tumor of the spleen. An increased association with concomitant malignancies has been described. We report the case of a 67-year-old man suffering from colon carcinoma and presenting with a tumor of the spleen which could be seen using radiography. Splenectomy was performed and histology revealed a 2.1 cm vascular tumor with a characteristic immunophenotype (CD31/68+). In addition a hepatocellular carcinoma was found. Due to the relationship with malignant tumors littoral cell angioma should be considered in every case of a splenic tumoral space-occupying mass, particularly in carcinoma patients.

Prediction model of lymph node metastasis in superficial esophageal adenocarcinoma and squamous cell cancer including D2-40 immunostaining.

BACKGROUND: It was the aim of our study to establish a model for prediction of lymph node metastases in superficial esophageal cancer. METHODS: We analyzed the clinical and histopathological data of 50 consecutive patients with pT1-esophageal cancer who underwent oncological resection. Submucosal carcinomas (pT1b) were classified according to sm levels 1-3. D2-40 immunostaining was investigated using the ABC technique. In a first step, we performed univariate analysis (One-way ANOVA: Sigma restricted parameterization; test of SS whole vs. SS predicted) to test the predictive value of the following categorical parameters for lymph node status (positive/negative): sex, histologic tumor type, localization, surgical technique (transhiatal/transthoracic), grading, pT1-subclassification (pT1a, pT1b sm 1-3), pL-, pV-status, and D2-40 labeling. Simple regression was applied for the following continuous predictors: age and tumor size. All significant variables of univariate analysis were included in the multivariate analysis. For this purpose, we used the General Liner Models's analysis (forward stepwise). In a third step, the Kruskal-Wallis test with post hoc comparisons was intended to define the cut-off value of parameters tested. RESULTS: Only the following variables gained statistical significance in univariate analysis: sex, histological tumor type, grading, pT1-subclassification, lymphatic infiltration, microvascular infiltration, D2-40 immunostaining, and tumor size (P < 0.05). Variables reaching significance in multivariate analysis were tumor size (P = 0.017) and pV-status (P = 0.037). In the Kruskal-Wallis test with post hoc comparisons, the cut-off value of tumor size was 2 cm (model P = 0.002) and between the categories (P < 0.05). CONCLUSIONS: Lymph node positivity and lymphatic vessel infiltration did not linearly increase with sm tumor infiltration depth. The risk category of lymph node involvement in superficial esophageal cancer exists according to our prediction model on the basis of tumor size of >2 cm and microvascular infiltration. The hitherto common sm levels 1-3 classification of submucosal cancers appears to display a lesser impact than previously assumed with regard to prediction of potential lymph node metastases and consequently the indication for endoscopic or surgical therapy.

Endothelial VEGFR-3 expression in colorectal carcinomas is associated with hematogenous metastasis.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a major inducer of lymphangiogenic signalling and seems to be involved also in angiogenesis. Since both processes are closely linked with tumor metastasis this study investigated the expression of VEGFR-3 in tumor-associated vessels in colorectal carcinomas and evaluated its relevance for lymphogenous and hematogenous metastasis. In a comparative study between microvascular endothelial cells isolated from the tumor (HCTEC) and the corresponding non-neoplastic tissue (HCMEC) from five patients with colorectal cancer VEGFR-3 expression was measured using a specific ELISA. The expression pattern was individually different, with cases showing reduced, elevated and unchanged protein levels. Under hypoxic culture conditions (3% O2 for 24 h), which are more realistic for the tumor situation, the levels remained unchanged. In contrast, hypoxia exposure of macrovascular human umbilical vein endothelial cells (HUVEC) led to a consistent downregulation of VEGFR-3 protein. These data indicate a 'hypoxia-resistant' behaviour of VEGFR-3 in colonic microvasculature. Using immunohistochemistry the endothelial expression pattern of VEGFR-3 in 74 non-metastatic, lymphogenously-metastatic and hematogenously-metastatic colorectal carcinoma specimens was assessed. Positive VEGFR-3 expression was highly significantly associated with those cases showing distant metastasis (p=0.0003). In contrast, significant differences in the expression of VEGFR-3 between non-metastatic tumors and carcinomas with lymph node metastasis were not found. The majority of the detectable intratumoral VEGFR-3-positive vessels were of blood vascular origin (CD31 positive, D2-40 negative). Whereas intratumoral lymphatic vessels were collapsed, VEGFR-3 positive peritumoral lymphatic vessels had mostly open lumina. These morphological observations provide evidence for a predominant significance of VEGFR-3-positive, possibly angiogenesis-mediated, tumor-associated blood vessels in hematogenous metastasis of colorectal cancer. In addition, due to their patency VEGFR-3-positive peritumoral, but not intratumoral lymphatics could be the vascular substrate functionally mediating lymphogenous metastasis.

Do we truly see what we think we see? The role of cognitive bias in pathological interpretation.

In the histomorphological grading of prostate carcinoma, pathologists have regularly assigned comparable scores for the architectural Gleason and the now-obsolete nuclear World Health Organization (WHO) grading systems. Although both systems demonstrate good correspondence between grade and survival, they are based on fundamentally different biological criteria. We tested the hypothesis that this apparent concurrence between the two grading systems originates from an interpretation bias in the minds of diagnostic pathologists, rather than reflecting a biological reality. Three pathologists graded 178 prostatectomy specimens, assigning Gleason and WHO scores on glass slides and on digital images of nuclei isolated out of their architectural context. The results were analysed with respect to interdependencies among the grading systems, to tumour recurrence (PSA relapse > 0.1 ng/ml at 48 months) and robust nuclear morphometry, as assessed by computer-assisted image analysis. WHO and Gleason grades were strongly correlated (r = 0.82) and demonstrated identical prognostic power. However, WHO grades correlated poorly with nuclear morphology (r = 0.19). Grading of nuclei isolated out of their architectural context significantly improved accuracy for nuclear morphology (r = 0.55), but the prognostic power was virtually lost. In conclusion, the architectural organization of a tumour, which the pathologist cannot avoid noticing during initial slide viewing at low magnification, unwittingly influences the subsequent nuclear grade assignment. In our study, the prognostic power of the WHO grading system was dependent on visual assessment of tumour growth pattern. We demonstrate for the first time the influence a cognitive bias can have in the generation of an error in diagnostic pathology and highlight a considerable problem in histopathological tumour grading.

Er:YAG laser osteotomy: preliminary clinical and histological results of a new technique for contact-free bone surgery.

BACKGROUND: The purpose of this retrospective study was to evaluate the surgical performance, clinical usability and outcome of a new variable square pulsed (VSP) Er:YAG laser for bone cutting in oral and maxillofacial surgery. MATERIALS AND METHODS: In 40 patients an Er:YAG laser with pulse energy of 1,000 mJ, pulse duration of 300 mus and a frequency of 12 Hz was used for different intraoral osteotomies. The spot size was 0.9 mm, and the handpiece was kept at a distance of 10 mm from the bone surface. Additionally, histological analyses of the fresh osteotomy rims of lasered bone were performed. RESULTS: Er:YAG laser osteotomy revealed a remarkable cutting efficiency without any visible, negative, thermal side effects. There was no damage of adjacent soft tissue structures. However, depth control was limited to visual inspection. Histologically, a 5- to 10-microm-wide zone of a characteristic laser fingerprint appeared on the cut edges. However, there was no sign of thermal tissue damage to the underlying bone structures. CONCLUSIONS: VSP Er:YAG laser osteotomy is clinically practicable without any signs of charred tissue and wound healing disturbances. However, the lack of depth control and the necessity for careful handling are still technical limitations to be overcome.