Osteogenic Potential of Human Dental Pulp Stem Cells (hDPSCs) Growing on Poly L-Lactide-Co-Caprolactone and Hyaluronic Acid (HYAFF-11TM) Scaffolds.

Bone tissue engineering using different scaffolds is a new therapeutic approach in regenerative medicine. This study explored the osteogenic potential of human dental pulp stem cells (hDPSCs) grown on a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold and a non-woven hyaluronic acid (HYAFF-11™) mesh. The adhesion, immunophenotype, and osteogenic differentiation of hDPSCs seeded on PLCL and HYAFF-11™ scaffolds were analyzed. The results showed that PLCL and HYAFF-11™ scaffolds significantly supported hDPSCs adhesion; however, hDPSCs' adhesion rate was significantly higher on PLCL than on HYAFF-11™. SEM analysis confirmed good adhesion of hDPSCs on both scaffolds before and after osteogenesis. Alizarin red S staining showed mineral deposits on both scaffolds after hDPSCs osteogenesis. The mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Coll-I), osterix (Osx), osteocalcin (Ocn), osteopontin (Opn), bone sialoprotein (Bsp), and dentin sialophosphoprotein (Dspp) gene expression and their proteins were higher in hDPSCs after osteogenic differentiation on both scaffolds compared to undifferentiated hDPSCs on PLCL and HYAFF-11™. These results showed that PLCL scaffolds provide a better environment that supports hDPSCs attachment and osteogenic differentiation than HYAFF-11™. The high mRNA of early osteogenic gene expression and mineral deposits observed after hDPSCs osteogenesis on a PLCL mat indicated its better impact on hDPSCs' osteogenic potential than that of HYAFF-11™, and hDPSC/PLCL constructs might be considered in the future as an innovative approach to bone defect repair.

Prognostic Value of WNT1, NOTCH1, PDGFRß, and CXCR4 in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: The biological behaviour of oral squamous cell carcinoma (OSCC) might be an effect of aberrant activation of several signalling pathways, like WNT and NOTCH pathways. The purpose of this study was to investigate the prognostic significance of WNT1 and NOTCH1 in patients with OSCC in relation to pro-angiogenic factors (PDGFRß, CXCR4). MATERIALS AND METHODS: Immunohistochemistry was performed in 60 OSCC tissue samples to compare WNT1, NOTCH1, PDGFRß, and CXCR4 expression and correlate it with clinicopathological parameters. RESULTS: Immunoreactivity of WNT1, NOTCH1, PDGFRß, and CXCR4 was found in 51.7%, 25.0%, 63.3%, and 70.0% of the patients, respectively. WNT1 expression was positively correlated with NOTCH1 (r=0.269, p=0.037) and CXCR4 (r=0.268, p=0.038). Positive correlation was observed also between NOTCH1 and CXCR4 (r=0.493, p<0.001). WNT1 and PDGFRß expression was significantly associated with shorter overall survival (p=0.042, p=0.033, respectively). Multivariate Cox regression model revealed that WNT1 and CXCR4 were independent prognostic factors (p=0.009, p=0.021, respectively). CONCLUSION: WNT1 and NOTCH1 expression is associated with CXCR4 expression in OSCC, suggesting that WNT and NOTCH pathways are important in oral tumour angiogenesis. Moreover, WNT1 and CXCR4 could serve as independent prognostic factors for patients with OSCC.

Comparison of biomarker expression in oral lichen planus and oral lichenoid lesions.

BACKGROUND: Oral lichen planus (OLP) and oral lichenoid lesions (OLL) comprise a group of oral mucosal disorders that have similar clinical and histological features. OBJECTIVES: To compare the levels of investigated biomarkers in biopsied OLP and OLL, and to determine the pattern of biomarkers, which could be useful for the biological characterization of these 2 disorders. MATERIAL AND METHODS: A total of 56 biopsy specimens in 2 groups were analyzed in this study. One group consisted of 25 idiopathic OLP lesions, and the other included 31 OLL from patients treated with antihypertensive and cardiac medications. The expression of protein p53, topoisomerase I (topo I), heat shock protein 90 (HSP90), and E-cadherin was analyzed using immunohistochemistry. RESULTS: The p53 protein expression showed a trend to a positive correlation with topo I expression in the total sample (p = 0.067, R = 0.25). The p53 protein and HSP90 expression was higher in the OLL group compared to the OLP group, but the difference was not statistically significant. No association was found between topo I and E-cadherin expression for either the OLP or OLL group. CONCLUSIONS: The findings of this study suggest that the slightly higher protein p53 and HSP90 expression in the OLL group might be caused by the medications used. The slight association between p53 and topo I expression indicates that the cooperation between these proteins might be essential for the growth of OLP/OLL in general. We conclude that the overexpression of p53 protein and high expression of topo I found in both types of lesions might induce their biologically aggressive behavior.

Characterization of Biological Properties of Dental Pulp Stem Cells Grown on an Electrospun Poly(l-lactide-co-caprolactone) Scaffold.

Poly(l-lactide-co-caprolactone) (PLCL) electrospun scaffolds with seeded stem cells have drawn great interest in tissue engineering. This study investigated the biological behavior of human dental pulp stem cells (hDPSCs) grown on a hydrolytically-modified PLCL nanofiber scaffold. The hDPSCs were seeded on PLCL, and their biological features such as viability, proliferation, adhesion, population doubling time, the immunophenotype of hDPSCs and osteogenic differentiation capacity were evaluated on scaffolds. The results showed that the PLCL scaffold significantly supported hDPSC viability/proliferation. The hDPSCs adhesion rate and spreading onto PLCL increased with time of culture. hDPSCs were able to migrate inside the PLCL electrospun scaffold after 7 days of seeding. No differences in morphology and immunophenotype of hDPSCs grown on PLCL and in flasks were observed. The mRNA levels of bone-related genes and their proteins were significantly higher in hDPSCs after osteogenic differentiation on PLCL compared with undifferentiated hDPSCs on PLCL. These results showed that the mechanical properties of a modified PLCL mat provide an appropriate environment that supports hDPSCs attachment, proliferation, migration and their osteogenic differentiation on the PLCL scaffold. The good PLCL biocompatibility with dental pulp stem cells indicates that this mat may be applied in designing a bioactive hDPSCs/PLCL construct for bone tissue engineering.

Comparison of p53, HSP90, E-cadherin and HPV in oral lichen planus and oral squamous cell carcinoma.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease. There are no markers that can be used to identify the risk of a malignant transformation of OLP to oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical staining was performed among 56 patients with OLP and 66 patients with OSCC for p53, HSP90 and E-cadherin expression and presence of HPV16/18. RESULTS: Significant differences in p53 and HSP90 expression between OLP and OSCC were found (p = 0.01 and p = 0.006, respectively). A positive correlation between HSP90 and p53 expression was seen in OLP (p = 0.017). Univariate analysis identified HSP90 expression and HPV16/18 presence as prognostic factors for overall survival time (OS) (p < 0.05). In multivariate analysis, only HSP90 expression was an independent prediction factor for shorter OS of OSCC patients (p = 0.016). CONCLUSIONS: The present study suggests that cooperation between p53 and HSP90 as well as between HPV16/18 and HSP90 exists in OLP and may affect the biological behaviour of OLP. The observed expression of HSP90 and p53 in OLP and their increase in OSCC suggests that these proteins participate in the malignant transformation of OLP. HSP90 may be a potential independent prognostic biomarker that can predict poor prognosis in OSCC.

SHH Expression Is Significantly Associated With Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a highly invasive malignancy with poor prognosis. Recent reports suggest that Sonic Hedgehog (SHH) plays a key role in tumor progression and worsens the response to therapy, possibly through an association with a cancer stem cell (CSC) phenotype. The objective of our study was to investigate the relationship between SHH expression and CSC markers in OSCC. MATERIALS AND METHODS: A total of 67 OSCC specimens were immunostained for SHH and CSC markers using specific antibodies and expression was correlated with clinicopathological parameters. RESULTS: SHH expression was significantly correlated with CD133 (p=0.026, r=0.272) and SRY-box transcription factor 2 (SOX2; p<0.001, r=0.793). SHH and SOX2 expression were associated with worse survival in OSCC (p=0.003 and p=0.003, respectively). In multivariate analysis SHH and CD44 were independent prognostic biomarkers in patients with OSCC (p=0.001 and p=0.008, respectively). CONCLUSION: Our study revealed that SHH overexpression is closely associated with CSC markers, contributing to tumor progression and worse outcomes of patients with OSCC.

Dental Pulp Stem Cell-Derived Secretome and Its Regenerative Potential.

The therapeutic potential of the dental pulp stem (DSC) cell-derived secretome, consisting of various biomolecules, is undergoing intense research. Despite promising in vitro and in vivo studies, most DSC secretome-based therapies have not been implemented in human medicine because the paracrine effect of the bioactive factors secreted by human dental pulp stem cells (hDPSCs) and human exfoliated deciduous teeth (SHEDs) is not completely understood. In this review, we outline the current data on the hDPSC- and SHED-derived secretome as a potential candidate in the regeneration of bone, cartilage, and nerve tissue. Published reports demonstrate that the dental MSC-derived secretome/conditional medium may be effective in treating neurodegenerative diseases, neural injuries, cartilage defects, and repairing bone by regulating neuroprotective, anti-inflammatory, antiapoptotic, and angiogenic processes through secretome paracrine mechanisms. Dental MSC-secretomes, similarly to the bone marrow MSC-secretome activate molecular and cellular mechanisms, which determine the effectiveness of cell-free therapy. Many reports emphasize that dental MSC-derived secretomes have potential application in tissue-regenerating therapy due to their multidirectional paracrine effect observed in the therapy of many different injured tissues.

Sonic Hedgehog is a novel prognostic biomarker in patients with oral squamous cell carcinoma.

Recent studies revealed that Sonic Hedgehog (SHH) signaling pathway plays an important role in initiation and tumor progression in various malignancies, however, its role in oral squamous cell carcinoma (OSCC) remains unclear. The objective of this study was to investigate the prognostic significance of SHH expression in patients with OSCC in relation to p53 protein expression and human papillomavirus (HPV) presence. SHH, p53, HPV expression was analyzed in surgical specimens from 70 patients with primary OSCC by immunohistochemistry (IHC) and correlated with clinicopathological parameters. The presence of SHH, p53, HPV was found in 51/70 (72.9%), 32/70 (45.7%), 11/70 (15.7%) cases, respectively. No correlation between SHH, p53 overexpression, and HPV presence was found. SHH expression was associated with tumor stage (p=0.026). P53 immunoreactivity correlated with tumor grade (p=0.040). By Kaplan-Meier analysis, SHH expression was significantly associated with shorter overall survival (p=0.005). Multivariate cox regression analysis showed that SHH was an independent prognostic factor for overall survival (HR=2.93; 95% CI=1.40-6.13; p=0.004). Our findings revealed that the SHH signaling pathway contributes to the poor survival of patients with OSCC and should be considered as a new prognostic biomarker in patients with OSCC. Inhibition of the SHH pathway may be used as a new potential target in cancer therapy.

Utility of direct 3D co-culture model for chondrogenic differentiation of mesenchymal stem cells on hyaluronan scaffold (Hyaff-11).

This study presents direct 2D and 3D co-culture model of mesenchymal stem cells (MSCs) line with chondrocytes isolated from patients with osteoarthritis (unaffected area). MSCs differentiation into chondrocytes after 14, 17 days was checked by estimation of collagen I, II, X, aggrecan expression using immunohistochemistry. Visualization, localization of cells on Hyaff-11 was performed using enzymatic technique and THUNDER Imaging Systems. Results showed, that MSCs/chondrocytes 3D co-culture induced suitable conditions for chondrocytes grow and MSCs differentiation than 2D monoculture. Despite that differentiated cells on Hyaff-11 expressed collagen X, they showed high collagen II (80%) and aggrecan (60%) expression with simultaneous decrease of collagen I expression (10%). The high concentration of differentiated cells on Hyaff-11, indicate that this structure has an impact on cells cooperation and communication. In conclusion, we suggest that high expression of collagen II and aggrecan in 3D co-culture model, indicate that cooperation between different subpopulations may have synergistic impact on MSCs chondrogenic potential. Revealed the high concentration and localization of cells growing in deeper layers of membrane in 3D co-culture, indicate that induced microenvironmental enhance cell migration within scaffold. Additionally, we suggest that co-culture model might be useful for construction a bioactive structure for cartilage tissue regeneration.

PDGFRα/HER2 and PDGFRα/p53 Co-expression in Oral Squamous Cell Carcinoma.

AIM: The purpose of this study was to explore the parallel expression of platelet-derived growth factor receptor α (PDGFRα) and human epidermal growth factor receptor 2 (HER2) or p53 in relation to clinicopathological parameters of oral squamous cell carcinoma (OSCC) to define their role in progressive growth of tumor. MATERIALS AND METHODS: Expression of PDGFRα, HER2 and p53 was evaluated in 71 OSCC samples by immunohistochemistry. HER2 status was verified by fluorescence in situ hybridization. RESULTS: PDGFRα and p53 expression were associated with tumor grade (p=0.043 and p=0.040, respectively). HER2 expression was more frequent in advanced (III/IV) cancer (p=0.006). A positive correlation of PDGFRα with HER2 (r=0.267; p=0.024) and with p53 (r=0.266; p=0.025) was noted. PDGFRα/HER2 and PDGFRα/p53 co-expression was found more often in G3 than in G1 and G2 tumors (p=0.008 and p=0.015, respectively). CONCLUSION: Our study revealed that PDGFRα/HER2 and PDGFRα/p53 co-expression exists in poorly differentiated OSCCs, suggesting that cooperation between these proteins might enhance aggressive behavior of tumor.

The Association Between HSP90/topoisomerase I Immunophenotype and the Clinical Features of Colorectal Cancers in Respect to KRAS Gene Status.

AIM: The aim of this study was to investigate heat shock protein 90 (HSP90) and topoisomerase I (Topo I) expression and the association between both proteins and clinicopathological parameters of colorectal cancer (CRC), in order to describe their role in tumor biology regarding to Kirsten Ras (KRAS) - positive/negative cases. MATERIALS AND METHODS: Expression of HSP90 and Topo I, and KRAS gene mutations were estimated in primary CRCs. RESULTS: HSP90/Topo I immunophenotype correlated with gender, Duke staging, tumor grade and lymph node metastasis (p<0.01). Positive correlation was found between KRAS mutation and HSP90 expression (p=0.02). HSP90, Topo I expression, and co-expression of HSP90/Topo I correlated with unfavorable parameters of CRCs in respect to KRAS gene status (p<0.001). CONCLUSION: Our results revealed that cooperation between HSP90 and Topo I expression exists in CRCs, independently of KRAS gene status, suggesting that co-expression of both proteins might be considered as a double target on individual tumor cells.

K-ras gene mutation as an early prognostic marker of colon cancer.

UNLABELLED: Due to increased colorectal cancer incidence there is a necessity of seeking new both prognostic and prediction factors that will allow to evolve new diagnostic tests. K-ras gene seems to be such a factor and its mutations are considered to be an early marker of progression of colorectal cancer. The aim of the study was to find a correlation between K-ras gene mutation in patients with diagnosed colorectal cancer and selected clinical parameters. MATERIAL AND METHODS: A total of 104 patients (41 women and 63 men) with diagnosed colorectal cancer were included in this study. The average age of male group was 68.3 and in female group - 65.9. Samples were taken from paraffine blocks with tissue from diagnosed patients and K-ras gene mutation were identified. Afterwards the statistical analysis was made seeking the correlation between K-ras gene mutation incidence and clinical TNM staging system, tumour localisation, histological type, sex, age. RESULTS: K-ras gene mutations were detected in 20.1% of all colorectal cancers. Significantly higher rate of K-ras gene mutations were diagnosed among patients classified at stage I (40%), stage IIC (50%) and stage IV (50%) according to the TNM classification. CONCLUSIONS: The results of our study are compatible with other studies and indicate the correlation between K-ras gene mutation and colorectal cancer incidence. Identification of K-ras gene mutation may complement other diagnostic methods at early stage of colorectal cancer.

[The role of cancer stem cells in progressive growth and resistance of ovarian cancer: true or fiction?]. / Udzial nowotworowych komórek macierzystych w progresywnym wzroscie i opornosci raka jajnika: prawda czy mit?

Growing evidence indicates that biological heterogeneity of ovarian cancer is associated with a small subpopulation of cancer cells existing within tumor tissue and defined as cancer stem cells (CSCs). This small group of ovarian cells possesses the capacity of self-renewal. Recent data revealed that progression, metastasis and relapse of ovarian cancers are related to the behavior of cancer stem cells. However, how ovarian CSCs maintain their migration properties is still unclear. The clinical relevance of CSCs has been supported by emerging evidence, showing that CSCs are resistant to conventional chemotherapy of ovarian cancer. Identification of biomarkers of ovarian cancer stem cells seems to be important for target therapy. Therapeutic strategies aimed at eliminating CSCs in ovarian cancers might extend disease survival and limit recurrence. This review will describe the current knowledge of ovarian CSCs biology and contribution of these cells to metastasis and chemoresistance of ovarian cancer as well as the possibility to use target therapy of ovarian CSCs.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.

Expression and Interactions Between Cell Adhesion Molecules CD44v6 and E-Cadherin in Human Gliomas.

BACKGROUND: Gliomas are a heterogenous group of tumors that show the same histological features but differ in their behavior. Gliomas are characterized by biological aggressiveness and extensive infiltrative growth into surrounding healthy brain tissue. OBJECTIVES: In this study we estimated CD44v6 and E-cadherin expression and correlation between CD44v6 and E-cadherin in relation to glioma malignancy. We also analyzed simultaneous expression of CD44v6 and E-cadherin in the same tumor sample in order to determine the biological tumor behavior. MATERIAL AND METHODS: Expression of CD44v6 and E-cadherin was evaluated on ninety-two formalin-fixed paraffin-embedded glioma tissue blocks using immunohistochemistry (IHC). RESULTS: CD44v6 expression was found in 71.6% of gliomas. There was a statistically significant difference between the frequency of positive cases for CD44v6 expression in low (grade I) vs. high (grade IV) as well as in grade I vs. grade II of glioma malignancy (p = 0.001). E-cadherin membrane staining was observed in 28.8% of gliomas. No significant differences were observed between E-cadherin expression and grade of gliomas (p > 0.05). However, re-expression of E-cadherin was found in grade II gliomas. In this group, E-cadherin expression was revealed in 43.3% of the cases. In order to define the relationship between CD44v6 expression and E-cadherin, we analyzed the simultaneous expression of CD44v6 and E-cadherin in the same glioma sample in the whole group and in respect to the degree of glioma malignancy. A positive correlation between studied biomarkers was observed in the analyzed gliomas (p = 0.004) but a simultaneous expression of CD44v6 and E-cadherin revealed no significant differences in respect to glioma malignancy. CONCLUSIONS: Our results showed that the level of E-cadherin might reflect different biological features of gliomas, whereas CD44v6 is associated with tumor cell malignancy. The simultaneous presence of CD44v6 and E-cadherin in a set of low-grade gliomas indicates that both these molecules might strengthen cell migration and may be a hallmark of glioma invasive growth.

Determination of basic fibroblast growth factor levels in serum of tumor-bearing BALB/c mice treated with photodynamic therapy.

In the present study we have checked whether photodynamic therapy (PDT) may influence concentration of basic Fibroblast Growth Factor (bFGF) in in vivo conditions. We have implanted malignant tumor, i.e. BFS1 fibrosarcoma into BALB/c mice and have them treated using well established photosensitizer, hematoporphyrin derivative and new compound, hydroxygallium (III) phthalocyanine tetrasulfonic acid tetrasodium salt, BON-6. The administration of those compounds was followed by light irradiation using a halogen lamp at proper wavelengths. Our results indicate that in vivo photodynamic therapy may cause a significant decrease in bFGF concentration and this phenomenon is accompanied by prolongation of survival of treated animals.

Determination of vascular-endothelial growth factor levels in serum from tumor-bearing BALB/c mice treated with photodynamic therapy.

MATERIAL/METHODS: We implanted a malignant tumour, BFS1 fibrosarcoma, into BALB/c mice and then treated them using a new photosensitizer, hydroxygallium (III) phthalocyanine tetrasulfonic acid tetrasodium salt, BON-6. The administration of this compound was followed by light irradiation using a halogen lamp at 680 nm. VEGF concentrations were measured in sera from the mice and compared to the time of tumor growth. RESULTS: BON-6 was found to be effective in PDT. This feature was accompanied by low levels of VEGF after BON-6+PDT, and also prolongation of the time of survival of treated animals. The mice which received BON-6+PDT survived 83.8 days (SD 10.23). The mean survival time in control groups did not exceed 35 days. Additionally, measurement of tumor size showed total regression in single cases after BON-6+PDT. CONCLUSIONS: PDT, by decreasing VEGF serum levels, may influence the capability of tumor tissue to form new vessels.