Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo.

To study cellular characteristics of human cementoblasts using a cellular model is important for understanding the mechanisms of homeostasis and regeneration of periodontal tissues. However, at present no immortalized human cementoblast cell line has been established due to limitation of the life span. In the present study, therefore, we attempted to establish human cementoblast-like cell lines by transfection with telomerase catalytic subunit hTERT gene. Two stable clones (HCEM-1 and -2) with high telomerase activity were obtained and they grew over 200 population doublings without significant growth retardation. The expression of mRNA for differentiation markers, type I collagen, alkaline phosphatase (ALP), runt-related transcription factor 2, osteocalcin, bone sialoprotein and cementum-derived protein was revealed in these clones by RT-PCR. Moreover, these cells showed high ALP activity and calcified nodule formation in vitro. Interestingly, HCEM-2 showed cementum like formation on the surface of hydroxyapatites granules by subcutaneous transplantation into immunodeficient mice with hydroxyapatite granules. Thus, we established human cementoblast-like cell lines. We suggest that HCEM cell lines can be useful cell models for investigating the characteristics of human cementoblasts.

Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene.

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39 degrees C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39 degrees C than 33 degrees C. High ALP activity and mineralized nodule formation were observed at 39 degrees C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.

The effect of experimental cartilage damage and impairment and restoration of synovial lubrication on friction in the temporomandibular joint.

AIMS: To evaluate how the frictional coefficient of the porcine temporomandibular joint (TMJ) is affected by an impairment of the synovial lubrication produced by an experimental abrasion of the articular cartilage and the application of hyaluronic acid (HA) with different molecular weights to the abraded cartilage surfaces. METHODS: Erosion of the articular cartilage was produced by scouring it with sandpaper. Impairment and restoration of synovial lubrication were modeled by washing the joint space with phosphate-buffered saline (PBS) and by the application of HA with different molecular weights. After measuring the frictional coefficients in the intact TMJs (n = 10), the effects of washing with PBS, sandpaper scouring, and the application of HA were subsequently examined. RESULTS: The mean frictional coefficient in the intact joint was 0.0154 (SD 0.0043). After PBS washing and sandpaper scouring, it increased significantly to 0.0235 (SD 0.0052) and 0.0520 (SD 0.0088), respectively. Subsequent application of HA resulted in a significant decrease (43% to 56%) of the frictional coefficient. Observations by scanning electron microscopy showed that after sandpaper scouring, the superficial cartilage layer was disrupted and inner layer was exposed, creating an irregular surface. CONCLUSION: Joint friction may increase by approximately 350% following an experimental scouring of the cartilage surface and impairment of synovial lubrication. Lubrication by means of HA decreased joint friction by approximately 50%.

Synthetic ameloblastin peptide stimulates differentiation of human periodontal ligament cells.

OBJECTIVE: This study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC). DESIGN: We used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, we examined quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey-Lowry enzymological method, mineral nodule formation by Dahl's method, and expression of mineralization-related genes by RT-PCR. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide. RESULTS: At all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by an anti-ameloblastin antibody, which caused ALP levels to revert to their approximate levels in the untreated condition. At concentrations greater than 1ng/ml, the peptide promoted mineralized nodule formation of PDLC. And the peptide induced higher expressions of ALP and bone sialoprotein (BSP) than the control. CONCLUSION: Our results show that the ameloblastin peptide upregulate ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide promotes the differentiation activity of PDLC.

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide.

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.

Upregulated CD44v9 expression inhibits the invasion of oral squamous cell carcinoma cells.

OBJECTIVES: CD44 is one of the cell surface molecules that play an important role in cancer metastasis. In oral squamous cell carcinoma (OSCC), the downregulation of CD44v9 has been shown to be associated with tumor metastasis. We found that treatment with an anti-CD44v9 antibody enhanced the invasive potential of OSCC cell lines. Based on previous studies and our results, reduced expression of CD44v9 may be correlated with an increased invasive potential, i.e. the overexpression of CD44v9 may inhibit the invasive activity of OSCC cells. METHODS: To study this correlation, we transfected the CD44v9 gene into HSC-4 cells with low CD44v9 expression and examined their invasive potential using the cell culture invasion assay and a three-dimensional culture invasion assay. RESULTS: Overexpression of CD44v9 resulted in a downregulation of the invasive potential of HSC-4 cells. Moreover, CD44v9-transfected cells did not invade reorganized stroma, while parent HSC-4 cells exhibited diffuse invasion into reorganized stroma by the three-dimensional culture invasion assay. CONCLUSIONS: Overall, these findings suggest that the inhibition of the invasive potential by upregulation of CD44v9 expression may be due to enhanced cell-cell adhesion. In our opinion, the upregulation of CD44v9 may be a target for future cancer treatment.