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Öğe Bone Morphogenetic Protein-7 Enhances Cementoblast Function In Vitro(Wiley, 2010) Hakkı, Sema S.; Foster, Brian L.; Nagatomo, Kanako J.; Bozkurt, S. Buket; Hakkı, Erdoğan E.; Somerman, Martha J.; Nohutcu, Rahime M.Background: Bone morphogenetic protein (BMP)-7 is a potent bone-inducing factor and was shown to promote periodontal regeneration in vivo and in vitro; however, to our knowledge, the specific effect of BMP-7 on cementoblasts has not been defined. We aimed to investigate the effects of BMP-7 on cementoblasts, which are cells responsible for tooth root-cementum formation. We hypothesized that BMP-7 would regulate mineralized tissue-associated genes in cementoblasts and influence the expression profile of genes associated with cementoblast extracellular matrix (ECM) and cell adhesion molecules (CAMs). Methods: A murine immortalized cementoblast cell line (OCCM.30) was cultured with and without 50 ng/ml BMP-7. After 72 hours, total RNA was isolated, and mRNA levels for bone/cementum markers, including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor-2 (Runx2), were investigated by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). In vitro mineral nodule formation was assayed on day 8 using von Kossa staining. A pathway-specific gene-expression array was used to determine BMP-7-responsive ECM and CAM genes in cementoblasts. Results: Mineralized tissue markers were strongly regulated by BMP-7, with an almost three-fold increase in BSP and OCN transcripts and significant increases in OPN and Runx2 mRNA expressions. BMP-7 treatment markedly stimulated cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. BMP-7 treatment altered the OCCM.30 expression profile for ECM and CAM functional gene groups. BMP-7 tended to increase the expression of collagens and matrix metalloproteinases (MMPs), mildly decreased tissue inhibitors of MMPs (TIMPs), and had mixed regulatory effects on integrins. Using Q-PCR, selected array results were confirmed, including a significant BMP-7-induced increase in MMP-3 and a decrease in TIMP-2 mRNA expression. Conclusion: These results support the promising applications of BMP-7 in therapies aimed at regenerating periodontal tissues lost as a consequence of disease.Öğe Bone Sialoprotein Gene Transfer to Periodontal Ligament Cells May Not Be Sufficient to Promote Mineralization In Vitro or In Vivo(Amer Acad Periodontology, 2006) Hakkı, Sema S.; Wang, Dian; Franceschi, Renny T.; Somerman, Martha J.Background: To improve regenerative therapies, it is important to understand the cells and factors modulating periodontal tissues. Our group has focused on bone sialoprotein (BSP), a mineralized tissue-selective protein considered to be involved in the initiation of cementogenesis and osteogenesis. In this study, we examined whether gene transfer of BSP into periodontal ligament (PDL) cells would result in an increased ability of PDL cells to promote mineralization in vitro and in vivo. Methods: PDL cells obtained from CD-1 mice were immortalized using simian virus (SV) 40 large T antigen (TAg) and designated SV-PDL cells. SV-PDL cells were infected in vitro with LacZ gene-expressing control adenovirus vector. A 1,000 plaque-forming unit (pfu) titer was selected (based on X-gal staining) and cells were infected with mouse BSP-expressing replication-deficient adenoviral vector to determine the mRNA expression and protein level of BSP. Total RNA was isolated from cells on days 2, 4, and 6. Media were obtained on days 3, 5, and 7 for protein determination. Northern blot analysis was performed for mRNA expression and Western blot analysis for protein expression. To test the effect of BSP gene transfer on the mineralization of PDL cells, in vitro (von Kossa) and in vivo (severe combined immunodeficiency [SCID] mice) experiments were performed. Results: Under normal conditions, PDL cells do not express BSP transcripts and do not promote significant mineralization. SV-PDL cells infected with a BSP viral vector expressed and secreted substantial levels of BSP as confirmed by Northern and Western blot analysis. BSP mRNA and protein levels were strong on day 2 and still apparent on day 6, although not as great. However, no mineral nodule formation was noted either in vitro or in vivo. Conclusions: Although BSP is an important and necessary protein for mineralization, it may not be sufficient for promoting mineralization without the addition or removal of other factors. Further studies will help to clarify the specific factors required for promoting mineralization, a required step for designing predictable periodontal regenerative therapies.Öğe Dexamethasone and basic-fibroblast growth factor regulate markers of mineralization in cementoblasts in vitro(AMER ACAD PERIODONTOLOGY, 2005) Hakkı, Sema S.; Nohutçu, Rahime M.; Hakkı, Erdoğan E.; Berry, Janice E.; Akkaya, Mahinur S.; Somerman, Martha J.Background: The aim of this study was to determine the effects of basic-fibroblast growth factor (b-FGF) and/or dexamethasone (Dex) on cementoblasts in vitro. Methods: Murine cementoblasts were treated as follows: 1) 5% FBS (fetal bovine serum) + ascorbic acid (AA, 50 mu g/ml, control); 2) 5% FBS+Dex (10(-7)M)+AA; 3) 5% FBS+b-FGF (50 ng/ml)+AA; or 4) 5% FBS+Dex (10(-7) M)+b-FGF (50 ng/ml)+AA and then evaluated by Northern analysis for changes in specific genes and by von Kossa stain for changes in mineral nodule formation. Results: Mitotic activity: b-FGF stimulated DNA synthesis significantly versus negative control. Gene expression: osteocalcin (OCN): Dex or b-FGF or the combination resulted in a decrease in expression versus control. Bone sialoprotein (BSP): Dex increased expression of BSP mRNA levels, b-FGF decreased transcript for BSP at 6 and 24 hours. Long-term (8 days) Dex, b-FGF, or Dex plus b-FGF caused a decrease in BSP expression versus control; osteopontin (OPN): both Dex and b-FGF increased transcripts for OPN seen by 6 hours, with a greater increase noted with b-FGF versus Dex. No apparent additive effect of Dex with b-FGF was noted; matrix gamma-carboxyglutamic acid protein (MGP): b-FGF induced transcripts for MGP and addition of Dex increased this effect, while Dex alone had no effect on expression. Biomineralization: Dex increased cementoblast-mediated biornineralization, while b-FGF blocked this activity, and addition of Dex to b-FGF did not alter FGF associated inhibition. Conclusion: Dex and FGF alone and in combination alter cementoblast behavior, but additional studies are required to determine whether these factors have beneficial effects at the clinical level.Öğe Periodontal ligament hücrelerinde platelet derived growth factor-BB mRNA ekspresyonunun Basic-fibroblast growth factor ve dexamethasone ile regülasyonu(2004) Hakkı, Sema S.; Nohutçu, Rahime M.; Somerman, Martha J.Amaç: Periodontal Ligamentin (PDL), periodontal rejenerasyon için gerekli olan sementoblast ve/veya osteoblastlara farklılaşabilen heterojen bir hücre populasyonuna sahip olması nedeniyle bu çalışmanın amacı, basic-Fibroblast Growth Factor (b-FGF) ve Dexamethasone (Dex) gibi biyolojik faktörlerin, PDL hücrelerinin mitotik aktivitesi, Platelet Derived Growth Factor-BB (PDGF-BB) mRNA ekspresyonu üzerine etkilerinin belirlenmesidir. Ayrica PDL hücrelerinde b-FGF'nin mitotik aktiviteyi artırıcı etkisinde, PDGF-BB'nin transkripsiyonel seviyede mRNA ekspresyonun regülasyonunun bir rolünün olup olmadığının da incelenmesi amaçlanmıştır. Gereç ve Yöntem: PDL hücreleri ortodontik nedenlerle çekilen premolar dişlerden elde edildi. Hücrelerin mitotik aktivitelerinin tayini 3H thymidine kullanılarak değerlendirildi. Mitotik aktivite deneyinde 0.1 ng/ml, 1 ng/ml, 10 ng/ml ve 100 ng/ml b-FGF konsantrasyonları kullanıldı. Serum içermeyen ortam negatif kontrol, %5 FBS (fetal bovine serum) içeren ortam ise pozitif kontrol olarak kullanıldı. Hücrelerin ataçmanını takiben, kültürler b-FGF (1 veya 10 ng/ml), Dex (10-7 M) ve bu faktörlerin kombinasyonu ile muamele edildi. Faktörlerle muameleyi takiben, PDL hücrelerinden 3. ve 7. günlerde RNA izolasyonu yapılarak Northern blot analizi ile PDGF-BB mRNA ekspresyon miktarları değerlendirildi. Bulgular: Proliferasyonu artırıcı etki açısından en uygun b-FGF konsantrasyonunun lng/ml ve 10 ng/ml olduğu belirlendi. Northern blot analizlerinden elde edilen sonuçlara göre, 10 ng/ml b-FGF'nin, diğer gruplarla kıyaslandığında hem 3. günde hem de 7. günde PDL hücrelerinin PDGF mRNA ekspresyonunu artırdığı saptandı. Sonuç: Elde edilen veriler b-FGF'nin periodontal doku rejenerasyonunda potansiyel bir büyüme faktörü olarak rol oynayabileceğini, aynı zamanda bFGF'nin PDL hücrelerinin mitotik aktivitesini artırıcı etkisinde, PDGF-BB mRNA ekspresyonundaki regülasyonunun da önemli olabileceğini düşündürmektedir.
