Initial attachment of osteoblasts to various guided bone regeneration membranes: an in vitro study

Guided bone regeneration (GBR) has proved to be a suitable and somehow predictable technique for promoting bone regeneration. Avariety of synthetic and naturally derived GBR barriers have been used in clinics to facilitate bone regeneration. These barriers may differ in composition and structure and these may affect the outcomes of GBR. Therefore, the present study was undertaken to evaluate the in vitro ability of osteoblasts (MC3T3-E1) to attach to various GBR membranes. Materials and methods:  Six GBR/GTR (guided tissue regeneration) membranes [BioMend ® (BM), Resolut ® (RL), Guidor ® (GD), EpiGuide ® (EG), Gore-Tex ® (GT) and Millipore filter ® (MP)] were tested. For controls, cells were directly plated on culture dishes (CD). Each test membrane was secured to the bottom of a culture dish with a double-sided adhesive tape. All samples were triplicate. At 1.5 and 24 h after plating of 2 ml (5 × 10 4 cells/ml) of MC3T3-E1 (passage 7) cells, the specimens were rinsed with phosphate-buffered saline to wash out any unattached cells and then fixed with a 10% buffered formalin solution for 1 d. After washing with distilled water, the cells were stained with hematoxylin. The number of attached cells was counted under a light microscope equipped with an ocular-micrometer in a unit area of 0.25 mm 2 (five areas on each membrane). In addition, cell morphology attached to the membranes was evaluated under scanning electron microscope. Results:  Data were presented as mean ± standard error and analyzed for statistical difference using a generalized Wilcoxon's test. Cell attachment at 1.5 h was as follows: MP (27.5 ± 2.1) > RL (17.0 ± 1.4) ≈ BM (14.5 ± 1.4) ≈ EG (11.4 ± 1.0) > GD (5.2 ± 0.8) ≈ GT (3.1 ± 0.6); and at 24 h was: MP (67.6 ± 3.6) > RL (35.8 ± 1.8) > BM (15.4 ± 0.9) ≈ EG (13.3 ± 1.3) > GD (5.9 ± 0.7) ≈ GT (5.6 ± 1.3). At 24 h, the scanning electron microscope finding revealed that cells attached on MP, RL, BM and EG were flatter in shape, like cells on CD, than cells on GD and GT, where cells were rather round. Conclusions:  Results from this study suggested that MP, BM, RL and EG enhanced the early osteoblast attachment. However, the true benefit of this observation in clinic remains to be determined.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65252/1/j.1600-0765.2002.01625.x.pd

Periostin promotes invasion and anchorage-independent growth in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. Typically HNSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. However, molecular mechanisms associated with invasion and metastasis of HNSCC remain poorly understood. Here we identified Periostin as an invasion promoting factor in HNSCC by comparing the gene expression profiles between parent HNSCC cells and a highly invasive clone. Indeed, Periostin overexpression promoted the invasion and anchorage independent growth both in vitro and in vivo in HNSCC cells. Moreover, Periostin overexpressing cells spontaneously metastasized to cervical lymph nodes and to the lung through their aggressive invasiveness in an orthotopic mouse model of HNSCC. Interestingly, Periostin was highly expressed in HNSCCs in comparison with normal tissues, and the level of Periostin expression was well correlated with the invasiveness of HNSCC cases. In summary, these findings suggest that Periostin plays an important role for invasion and anchorage independent growth in the metastatic process of HNSCC

Periostin overexpression and oral cancer

Oral squamous-cell carcinoma (OSCC) is one of the most common types of human cancer. Typically OSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. We previously identified Periostin as the gene demonstrating the highest fold change expression in the invasive clone by comparing the transcriptional profile of parent OSCC cell line and a highly invasive clone. Here, we demonstrated that Periostin overexpression enhanced invasiveness in oral cancer cell lines. To know the role of Periostin in invasion, angiogenesis and metastasis in OSCC cases, we first examined the expression of Periostin mRNA in 31 OSCC cases by RT–PCR and Periostin protein in 74 OSCC cases by immunohistochemistry. Then, we compared the Periostin expression with invasion pattern, metastasis and blood vessel density. Periostin mRNA and protein overexpression were frequently found in OSCC cases and Periostin expression was well correlated with the invasion pattern and metastasis. Moreover, blood vessel density of Periostin-positive cases was higher than those of Periostin-negative cases. Interestingly, recombinant Periostin enhanced capillary formation in vitro in a concentration-dependant manner. In summary, these findings suggest that Periostin may promote invasion and angiogenesis in OSCC, and that Periostin can be a strong marker for prediction of metastasis in oral cancer patients

Migration of osteoblastic cells on various guided bone regeneration membranes

To evaluate the biological effects of guided bone regeneration (GBR) barrier materials on osteoblastic cell migration, migration of mouse osteoprogenitor cells (MC3T3-E1) was examined, in vitro , on various membranes. Eight commercially available GBR membranes – bovine type I collagen (BioMend ® ; BM), porcine type I collagen (BioGide ® ; BG), bovine type I atelocollagen (Tissue Guide ® ; TG), polylactic acid (Epi-Guide ® ; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute ® ; RL, Resolut XT ® ; RL-XT), expanded polytetrafluoroethylene (e-PTFE; Gore Tex ® ; GT) and co-polymer of cellulose acetate and nitrocellulose (Millipore filter ® ; MP) – were tested. A 3×5 mm section of the membrane was fixed to the bottom of a culture dish with double-sided adhesive tape, and half of the membrane was closely covered by PARAFILM ® (American National Can TM ) to leave an unexposed area for cell migration. The border between exposed and unexposed areas was marked as a baseline of cell migration. Membranes were then plated with 3 ml of cell suspension at an initial density of 1×10 5 cells/ml in α-MEM culture medium with 10% fetal bovine serum and ascorbic acid. After a 5-hour incubation, non-attached cells were completely washed out with phosphate buffered saline and the PARAFILM ® cover was removed. After 3 days cultivation, specimens were fixed with 10% buffered formalin and stained briefly with hematoxylin. The area of cell migration on a membrane was analyzed using a LA 500 Image Analysis System and migration area per unit length of the baseline (mm 2 /mm) was compared among membranes. Results demonstrated that cell migration was greater in the order: RL>RL-XT, BM, TG, MP>EG, BG. Membranes except for BG, EG and GT showed the migration rate equal to or higher than a plastic culture cover slip (Celldesk ® ) ( P <0.01) on which cells generally grow favorably. Only a small number of the cells attached to GT, and the net cell migration for the membrane could not be determined. These results indicate that GBR barrier materials per se may influence the process of bone regeneration in vivo through the effects of their presence on cell migration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75078/1/j.1600-0501.2001.012004332.x.pd

Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1α, IL-1β, and TNF-α was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1β but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-α, IL-1α, and IL-1β reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42233/1/418-116-1-57_s004180100298.pd

Protein kinase A-dependent substance P expression by pituitary adenylate cyclase-activating polypeptide in rat sensory neuronal cell line ND7/23 cells.

The neurotrophic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on rat sensory neuronal cell line ND7/23 cells were investigated. PACAP caused a concentration-dependent increase in the number of neurite-bearing cells and the expression of the substance P precursor (PPT) mRNA in 24 h. The effects of PACAP were mimicked by vasoactive intestinal polypeptide with lower potency and dibutyryl-cyclic AMP, and inhibited by inhibitors of protein kinase A, ERK kinase or p38 kinase, KT5720, U0126, or SB203580, respectively. In a PPT promoter luciferase reporter assay, the increase of PPT mRNA was the result of an increase in PPT gene transcriptional activity by PACAP. The increasing effects of PACAP on PPT mRNA were similarly observed in primary cultured rat dorsal root ganglion cells. Thus, PACAP could induce differentiation-like phenomena in sensory neurons in a cAMP-, protein kinase A-, ERK kinase-, and p38 kinase-dependent manner. These results provide evidence of the neurotrophic action of PACAP, which may function to rescue damaged neurons or to switch the neuronal phenotype in injured or inflamed sensory neurons.The neurotrophic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on rat sensory neuronal cell line ND7/23 cells were investigated. PACAP caused a concentration-dependent increase in the number of neurite-bearing cells and the expression of the substance P precursor (PPT) mRNA in 24 h. The effects of PACAP were mimicked by vasoactive intestinal polypeptide with lower potency and dibutyryl-cyclic AMP, and inhibited by inhibitors of protein kinase A, ERK kinase or p38 kinase, KT5720, U0126, or SB203580, respectively. In a PPT promoter luciferase reporter assay, the increase of PPT mRNA was the result of an increase in PPT gene transcriptional activity by PACAP. The increasing effects of PACAP on PPT mRNA were similarly observed in primary cultured rat dorsal root ganglion cells. Thus, PACAP could induce differentiation-like phenomena in sensory neurons in a cAMP-, protein kinase A-, ERK kinase-, and p38 kinase-dependent manner. These results provide evidence of the neurotrophic action of PACAP, which may function to rescue damaged neurons or to switch the neuronal phenotype in injured or inflamed sensory neurons

Ameloblastin regulates osteogenic differentiation

Ameloblastin, the most abundant non-amelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin was abrogated by treatment with anti-CD63 antibody and overexpression of constitutive active Src, respectively. Moreover, amelobastin upregulated the formation of stress-fibre and focal adhesions and downregulated cell migration in association with RhoA regulation via Src activity. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1

Odontogenic stem cells

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig’s epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis

Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65287/1/j.1600-0765.2001.360508.x.pd