Isolation and characterization of cells derived from human epithelial rests of Malassez

The epithelial rests of Malassez (ERMs) might represent a valuable source of oral epithelial cells with stem cell properties. The purpose of this study was to isolate and characterize cells derived from human ERM, and compare them with cells derived from matched normal oral mucosa (NOM). Matched tissue specimens of the periodontal ligament of extracted tooth and NOM were collected. Cells were isolated in culture, then characterized by immunohistochemistry and flow cytometry for expression of pancytokeratin, ESA, PDGFRB, CD31 and CD44. 3D organotypic cultures were constructed by growing epithelial cells on top of fibroblast-populated collagen gels. Both ERM and NOM-isolated cells expressed the markers of epithelial lineage (ESA and pancytokeratin), and to some extent PDGFR, an indicator of a more mesenchymal phenotype, but not the endothelial cell marker CD31. Cells with epithelial morphology were isolated from periodontium of cervical, middle and apical parts of the root, but contained a significantly lower percentage of ESA and pancytokeratin-positive cells than when isolating cells from NOM (p < 0.001). ERM cells expressed a significantly higher percentage of the stem cell-related molecule CD44 (cervical 92.93 ± 0.25%, middle 93.8 ± 0.26%, apical 94.36 ± 0.41%) than cells isolated from NOM (27.8 ± 1.47%, p < 0.001). When grown in 3D organotypic cultures and in collagen gels, ERM cells formed a less differentiated epithelium than NOM cells, but expressing pancytokeratin and vimentin. In conclusion, epithelial cells could be isolated from human periodontium and grown in culture; their in vitro characterization indicates that they have a less differentiated phenotype compared with cells derived from normal oral epithelium.publishedVersio

MicroRNA-138 abates fibroblast motility with effect on invasion of adjacent cancer cells

Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.publishedVersio

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).publishedVersio

Pathological mechanisms in oral lichen planus : a atudy of apoptosis - regulatory proteins and risk markers for malignant transformation

Oral lichen planus (OLP) is a chronic mucocutaneous disease characterized by basal cell destruction and a subepithelial band-like mononuclear inflammatory cell infiltrate predominated by T cells. Molecular biological changes in the basal cell compartment and keratinocyte cell death have been a matter of particular interest in later OLP research. The majority of OLP lesions run a benign course. However, OLP have been associated with an increased risk of malignant transformation. The aims of this study were to investigate apoptotic cell death and putative regulatory proteins in keratinocytes (Papers I-III), as well as potential risk markers for malignant transformation in OLP (Paper III). Biopsies from clinically and histologically verified OLP were investigated (Papers I-II), as well as a biopsy material from OLP patients where a certain amount of them had developed epithelial dysplasia and oral squamous cell carcinoma (OSCC) (Paper III). Tissues were evaluated by histomorphometry, imunnohistochemistry (Papers I-III), TUNEL method (Paper I), mRNA in situ hybridization (Paper II) and image cytometry for measurement of DNA content (Paper III). Our data show that apoptosis is increased within the epithelium of OLP compared with normal oral mucosa (OM), and that both Fas receptor (FasR) and Fas-ligand (FasL) apoptosis regulatory proteins are expressed within the epithelium and subepithelial cell infiltrate of OLP (Paper I). In actively diseased OLP lesions, basal keratinocytes are CD40 negative and epithelial (E)-cadherin negative in focal areas (Papers II-III). Cyclooxygenase-2 (Cox-2) is up-regulated within the epithelium of OLP lesions, compared with normal OM (Paper III). According to DNA content measurements, all biopsies were classified as diploid including OLP, epithelial dysplasia and OSCC, except for one biopsy from an OLP lesion with epithelial dysplasia which was tetraploid (Paper III). Based on these findings we conclude that basal keratinocytes in OLP die by apoptosis and regulation of apoptosis appears to involve following mechanisms; (1) dysfunction in FasR/FasL system; (2) by down-regulating CD40 in diseased areas, basal keratinocytes may escape CD40-CD40L mediated apoptosis; (3) an up-regulation of Cox-2 , which may inhibit apoptosis; (4) loss of E-cadherin in basal keratinocytes may promote apoptosis and contribute to reduced basal cell structural integrity in OLP, allowing T cells to enter the epithelial compartment. Our biopsy material indicates that neither DNA content, nor expression of Cox-2 and E-cadherin are reliable as prognostic markers to select the OLP patients at risk for development of OSCC

Patient experience following iliac crest alveolar bone gradfting av implant replacement

Background The objective of this study was to assess patient-reported outcomes such as satisfaction and quality of life after advanced alveolar bone augmentation with anterior iliac crest grafting and implant treatment in orally compromised patients. Methods This cross-sectional retrospective cohort study included 59 patients (29 women and 30 men) with major functional problems, who underwent advanced alveolar augmentation with autologous iliac bone grafts during a 10-year period (2002–2012). The self-administered questionnaire included 36 validated questions related to (1) demographics, (2) perceived general and oral health, (3) donor site and hospitalization, (4) status of implants and/or prosthesis, and (5) oral health-related quality of life (OHRQoL). Results Questionnaires were completed by 44 patients: 24 women and 20 men (response rate, 74.6%). Most patients reported good tolerance of the operative iliac bone harvesting (85%) and implant (90%) procedures. Post-operative pain at the donor site was reported by 38%, lasting 18.1 ± 16.1 days. An average of 4.3 ± 3.5 days of hospitalization and 20.2 ± 18.5 days of sick leave was reported. The overall satisfaction with prosthetic reconstruction was 90.5%. OHRQoL was reported with a mean Oral Health Impact Profile-14 (OHIP-14) score of 8.4. Conclusion Favorable OHRQoL and satisfaction were reported after advanced reconstruction of alveolar ridges with iliac crest-derived grafting and implants in severely compromised patients. However, this treatment requires substantial resources including hospitalization and sick leave

Isolation and characterization of cells derived from human epithelial rests of Malassez

The epithelial rests of Malassez (ERMs) might represent a valuable source of oral epithelial cells with stem cell properties. The purpose of this study was to isolate and characterize cells derived from human ERM, and compare them with cells derived from matched normal oral mucosa (NOM). Matched tissue specimens of the periodontal ligament of extracted tooth and NOM were collected. Cells were isolated in culture, then characterized by immunohistochemistry and flow cytometry for expression of pancytokeratin, ESA, PDGFRB, CD31 and CD44. 3D organotypic cultures were constructed by growing epithelial cells on top of fibroblast-populated collagen gels. Both ERM and NOM-isolated cells expressed the markers of epithelial lineage (ESA and pancytokeratin), and to some extent PDGFR, an indicator of a more mesenchymal phenotype, but not the endothelial cell marker CD31. Cells with epithelial morphology were isolated from periodontium of cervical, middle and apical parts of the root, but contained a significantly lower percentage of ESA and pancytokeratin-positive cells than when isolating cells from NOM (p < 0.001). ERM cells expressed a significantly higher percentage of the stem cell-related molecule CD44 (cervical 92.93 ± 0.25%, middle 93.8 ± 0.26%, apical 94.36 ± 0.41%) than cells isolated from NOM (27.8 ± 1.47%, p < 0.001). When grown in 3D organotypic cultures and in collagen gels, ERM cells formed a less differentiated epithelium than NOM cells, but expressing pancytokeratin and vimentin. In conclusion, epithelial cells could be isolated from human periodontium and grown in culture; their in vitro characterization indicates that they have a less differentiated phenotype compared with cells derived from normal oral epithelium

Rapid adherence to collagen IV enriches for tumour initiating cells in oral cancer.

Background: Although several approaches for identification and isolation of carcinoma cells with tumour initiating properties have been established, enrichment of a population of pure and viable tumour-initiating cells (TICs) is still problematic. This study investigated possibilities to isolate a population of cancer cells with tumour initiating properties based on their adherence properties, rather than expression of defined markers or clonogenicity. Methods: Several human cell lines derived from oral dysplasia and oral squamous cell carcinoma (OSCC), as well as primary cells derived from patients with OSCC were allowed to adhere to collagen IV-coated dishes sequentially. Rapid adherent cells (RAC), middle adherent cells (MAC) and late adherent cells (LAC) were then harvested and further investigated for their morphology, stem cell-like properties and molecular profile while grown in vitro and tongue xenotransplantation in NOD-SCID mice at serial dilutions. Results: RAC showed significantly higher colony forming efficiency (p < 0.05), sphere forming ability, greater migration ability (p < 0.05), exhibited longer G2 phase and displayed higher expression of integrin b1 and other stem-cell related molecules as compared to MAC and LAC. RAC induced tongue tumours in NOD-SCID mice with the highest incidence. These tumours were also bigger and metastasised more frequently in loco-regional lymph nodes than MAC and LAC. Conclusions: These findings prove for the first time that OSCC cells with tumour initiating properties can be enriched based on their rapid adhesiveness to collagen IV. This separation procedure provides a potentially useful tool for isolating TICs in OSCC for further studies on understanding their characteristics and drug-resistant behaviour

Nano-TiO2 penetration of oral mucosa: in vitro analysis using 3D organotypic human buccal mucosa models

BACKGROUND: Oral cavity is a doorway for a variety of products containing titanium dioxide (TiO2) nanoparticles (NPs) (nano-TiO2) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined. OBJECTIVES: To evaluate the ability of nano-TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM). METHODS: RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano-TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh-resolution imaging (URI) microscopy (CytoVivaTM, Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues. RESULTS: Ultrahigh-resolution imaging microscopy demonstrated the presence of nano-TiO2 mostly in the epithelium of RNHBM at both 20 min and 24-h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness. CONCLUSION: Nano-TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze- and timedependent manner

MicroRNA-138 abates fibroblast motility with effect on invasion of adjacent cancer cells

Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s)