Collagen Membranes Adsorb the Transforming Growth Factor‐β Receptor I Kinase‐Dependent Activity of Enamel Matrix Derivative

Background: Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)‐β signaling in oral fibroblasts. Methods: Three commercially available collagen products were exposed to EMD or recombinant TGF‐β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF‐β target genes interleukin (IL)‐11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. Results: EMD or TGF‐β1 provoked a significant increase of IL‐11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF‐β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin‐coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL‐11 and PRG4. Conclusion: These in vitro findings suggest that collagen products adsorb a TGF‐β receptor I kinase‐dependent activity of EMD and make it available for potential target cells.     

Transforming growth factor‐β‐regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF‐β), as a novel biomarker for correct prediction and early detection of OSCC‐associated bone invasion. TGF‐β knockdown and treatment with a TGF‐β‐neutralizing antibody decreased the level of fractalkine in the culture media of HSC‐2 and YD10B OSCC cells. Treatment with a fractalkine‐neutralizing antibody reduced TGF‐β‐stimulated invasion by HSC‐2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild‐type or TGF‐β knockdown OSCC cells in mouse calvaria. TGF‐β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor‐bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF‐β and mediates TGF‐β‐induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC‐induced bone destruction.     

Immunoexpression of TNF-α and TGF-β in central and peripheral giant cell lesions of the jaws

J Oral Pathol Med (2012) 41 : 194–199 Background: Peripheral giant cell lesion (PGCL) is a reactive process associated with a local irritating factor that shows low recurrence after treatment, especially if the irritating factor is eliminated. On the other hand, central giant cell lesion (CGCL) presents a variable clinical behavior ranging from slow and asymptomatic growth without recurrence to rapid, painful and recurrent growth. Our aim was to compare the immunoexpression of tumor necrosis factor‐alpha (TNF‐α) and transforming growth factor‐beta (TGF‐β) in CGCL and PGCL. Methods: Twenty CGCL and 20 PGCL were selected for analysis of the immunoexpression of TNF‐α and TGF‐β in multinucleated giant cells (MGC) and mononucleated cells (MC). Results: The PGCL showed lightly higher expression of TNF‐α than CGCL. In comparison with PGCL, the CGCL showed higher expression of TGF‐β in MC and MGC (P < 0.05) and in total cells (P < 0.05). Significant positive correlation was found between expressions of TGF‐β and TNF‐α in CGCL (P < 0.05). Conclusions: Our results suggest that, in CGCL, coordinated interactions between TGF‐β and TNF‐α may be important for osteoclastogenesis and bone resorption. PGCL occasionally cause bone resorption but to a lower extent, a fact that might be explained by the lower expression of TGF‐β in these lesions.     

Influence of demineralized bone matrix's embryonic origin on bone formation: an experimental study in rats

Background: There are results suggesting that differences regarding bone‐inducing potential, in terms of amount and/or rate of bone formation, exist between demineralized bone matrices (DBMs) of different embryonic origins. Purpose: The aim of the present study was to examine whether the embryonic origin of DBM affects bone formation when used as an adjunct to guided tissue regeneration (GTR). Materials and Methods: Endomembranous (EM) and endochondral (ECH) DBMs were produced from calvarial and long bones of rats, respectively. Prior to the study the osteoinductive properties of the DBMs were confirmed in six rats following intramuscular implantation. Following surgical exposure of the mandibular ramus, a rigid hemispheric Teflon capsule loosely packed with a standardized quantity of DBM was placed with its open part facing the lateral surface of the ramus in both sides of the jaw in 30 rats. In one side of the jaw, chosen at random, the capsule was filled with EM‐DBM, whereas in the other side ECH‐DBM was used. Groups of 10 animals were sacrificed after healing periods of 1, 2, and 4 months, and undecalcified sections of the capsules were produced and subjected to histologic analysis and computer‐assisted planimetric measurements. Results: During the experiment increasing amounts of newly formed bone were observed inside the capsules in both sides of the animals' jaws. Limited bone formation was observed in the 1‐ and 2‐month specimens, but after 4 months of healing, the newly formed bone in the ECH‐DBM grafted sides occupied 59.1% (range 45.6–74.7%) of the area created by the capsule versus 46.9% (range 23.0–64.0%) in the EM‐DBM grafted sides (p =.01). Conclusion: It is concluded that the embryonic origin of DBM influences bone formation by GTR and that ECH‐DBM is superior to EM‐DBM.     

In Vitro Evaluation of Acellular Dermal Matrix as a Three‐Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three‐dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P <0.05); B16F10 exhibited an increase in cell number within 7 days (P <0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P <0.05) compared to inside was observed for all cell types. 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) values indicated higher cell viability in samples cultured with HGF compared to CGF (P = 0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non‐CM was observed at 48 and 72 hours (P <0.05). Conclusions: ADM is not suitable as a three‐dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability.     

Comparison of applying particulate demineralized bone matrix (DBM), putty DBM and open flap debridement in periodontal horizontal bone defects. A 12-month longitudinal, multi-center, triple-blind, split-mouth, randomized, controlled clinical study. Part 1 – clinical and radiographic evaluation

Summary Putty form graft materials may have additional favourable effects when compared with particulate ones in periodontal bone defects. The purpose of this study was to assess clinical and also radiographic changes following application of (i) putty form demineralized bone matrix (DBM), (ii) particulate form DBM and (iii) open flap debridement (control), using modified curtain suturing technique in the treatment of interproximal suprabony (horizontal) defects. Twenty‐five chronic periodontitis patients with 125 sites (radiologically ≥4 mm horizontal bone defect) were selected to participate in this triple‐blind, split mouth, randomized, controlled clinical trial. Putty and particulate form DBM grafts were placed at experimental sites. Clinical measurements included probing depth (PD), relative attachment level (RAL), gingival recession and bone probing depth (BPD) were made at baseline and repeated 12 months after the operations. Standardized digital radiographs were also taken to measure radiographic bone level (RBL) at baseline and 12 months later to be compared in a software. Probing depth reductions and RAL gains were significantly improved in all treatment groups (P < 0·001). No significant differences in soft tissue parameters were found among three groups (P > 0·05). Bone probing depth measurements indicated comparable significant bone gain in graft applied groups (P < 0·01) and a significant bone resorption in open flap debridement group (P < 0·01). Radiographic evaluation did not show any significant bone gain or resorption in all treatment groups (P > 0·05). The results of this study indicate that either putty or particulate DBM demonstrates similar enhancements in soft and hard tissue parameters. Applying putty or particulate form DBM results with slight bone formation when compared with open flap debridement in horizontal bone defects at 1‐year post‐operative examination according to BPD measurements.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

MicroRNA profiling reveals dysregulated microRNAs and their target gene regulatory networks in cemento‐ossifying fibroma

Background

Cemento‐ossifying fibroma (COF) is a benign fibro‐osseous neoplasm of uncertain pathogenesis, and its treatment results in morbidity. MicroRNAs (miRNA) are small non‐coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis.

Methods

Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human microRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA‐miRPath.

Results

Eleven miRNAs were downregulated (hsa‐miR‐95‐3p, hsa‐miR‐141‐3p, hsa‐miR‐205‐5p, hsa‐miR‐223‐3p, hsa‐miR‐31‐5p, hsa‐miR‐944, hsa‐miR‐200b‐3p, hsa‐miR‐135b‐5p, hsa‐miR‐31‐3p, hsa‐miR‐223‐5p and hsa‐miR‐200c‐3p), and five were upregulated (hsa‐miR‐181a‐5p, hsa‐miR‐181c‐5p, hsa‐miR‐149‐5p, hsa‐miR‐138‐5p and hsa‐miR‐199a‐3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant KEGG pathways revealed by enrichment analysis were proteoglycans in cancer, miRNAs in cancer, pathways in cancer, p53‐, PI3K‐Akt‐, FoxO‐ and TGF‐beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis.

Conclusion

miRNA dysregulation occurs in COF, and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation.     

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

J Oral Pathol Med (2010) 39 : 250–256 Background: Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL‐4, TGF‐β, tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Methods: Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme‐linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. Results: TNF‐α and IFN‐γ were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL‐4 was reactive in 24% of cysts, and TGF‐β was positive in all samples. Patients with tenderness showed significantly higher levels of IFN‐γ and IL‐4 (P < 0.05). Swelling was associated with high levels of TNF‐α, IFN‐γ, and IL‐4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Conclusions: Periapical granulomas display a regulatory environment characterized by high TGF‐β and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN‐γ, TNF‐α, and IL‐4.     

Folic acid rescue of ATRA‐induced cleft palate by restoring the TGF‐β signal and inhibiting apoptosis

J Oral Pathol Med (2010) 40 : 433–439 Background: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF‐β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all‐trans retinoic acid (ATRA)‐induced cleft palate in organ culture were tested. Methods: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. Results: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase‐9 in MEPM cells were detected in the ATRA group than in the control or the ATRA + FA group. Compared with the control or the ATRA + FA group, ATRA had little effect on TGF‐β3 in MEPM cells but significantly inhibited TGF‐β receptor II. Conclusions: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA‐induced cleft palate.     

Increased expression of Smad proteins,and in particular Smad3, in oral lichen planus compared to normal oral mucosa

J Oral Pathol Med (2010) 39 : 639–644 Backgound: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organisation (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. EMT occurs during embryogenesis and wound healing but also in some human diseases such as cancer and fibrosis. A factor known to induce EMT is transforming growth factor‐β (TGF‐β), which uses the Smad proteins as mediators for its signalling. TGF‐β is also often over‐expressed in squamous cell carcinoma of the head and neck (SCCHN). Methods: In the present study we mapped expression of Smad proteins in OLP lesions by immunohistochemistry, and compared to expression in normal and sensitive oral mucosa. The latter group of patients had developed SCCHN after shorter or longer periods of diffuse oral symptoms. The aim was to see if there were any signs of EMT related changes in the OLP lesions, as judged by changes in the TGF‐β pathway. Conclusion: Changes in the TGF‐β pathway related to EMT are seen in the very earliest stages of oral malignancy and become more severe as lesions progress.     

Comparison of applying particulate demineralized bone matrix (DBM), putty DBM and open flap debridement in periodontal horizontal bone defects. A 12-month longitudinal, multi-centre, triple-blind, split-mouth, randomized, controlled clinical study. Part 2 – evaluation of the interdental soft tissue

Summary To date, there have been many studies clinically evaluating periodontal regenerative procedures by the help of routinely used hard and soft tissue parameters; however, these parameters are not capable of assessing interdental soft tissue located above the regenerative periodontal surgery area. The purpose of this study was to assess interproximal soft tissue changes following application of (i) particulate form demineralized bone matrix (DBM), (ii) putty form DBM and (ii) open flap debridement (OFD, control), using modified curtain technique in the treatment of interproximal suprabony (horizontal) defects located in anterior maxillary region, as previously reported. Twenty‐five chronic periodontitis patients with 125 interproximal surgery sites (radiologically ≥4 mm horizontal bone defect) were also participate in this second stage of the triple‐blind, split mouth, randomized, controlled clinical trial. Surgery sites were assessed by (i) plaque index (PI), (ii) gingival index (GI), (iii) the presence of interdental soft tissue clefts or craters and (iv) the loss of interdental papilla height by using papilla presence index (PPI), during the healing period. At the baseline and 3, 6, 9 and 12 months after the operations, these measurements were repeated. In all groups, there is a significant increase in the prevalence of soft tissue cleft and crater formation (P < 0·01), with increase in PI and GI scores at interdental soft tissue defect areas (P < 0·001), 3 months after the operations. There was also an increase in PPI scores after the operations in all treatment groups (P < 0·01). Three procedures affected the interproximal soft tissues similarly. There was no significant difference among groups in terms of all parameters (P > 0·05). Particulate DBM, putty DBM and OFD demostrated similar interproximal soft tissue changes especially increasing interproximal PI and GI scores in 3 months follow‐up.     

Low‐temperature particulate calcium phosphates for bone regeneration

Background: Low‐temperature synthesized calcium phosphates are produced by mixing calcium phosphate powders in an aqueous solution resulting in a precipitated phase. These compounds can be formulated in several forms (e.g. injectable cements and implantable blocks), and are commonly used as bone substitutes and drug delivery systems for the treatment of bone defects. As bone substitutes, calcium phosphates in general offer the advantages of being biocompatible and osteoconductive. Aims: The present work employed a machine‐based process to derive a reproducible preparation method for low‐temperature calcium phosphate particulate (LTCP). The in vivo outcomes of LTCP were compared with those of three commercially available bone substitutes by histomorphometric measurements of bone formation and material degradation in a rat femur implantation model. Materials & Methods: Specifically, LTCP, anorganic bovine bone (AB), bioactive glass (BG), and demineralized bone matrix (DBM) were implanted in defects created in the distal aspect of rat femora. Reparative bone and particulate volumes of these biomaterials were evaluated post‐operatively using micro‐computed tomography and histological analyses at 3, 6, 12, and 16 weeks. Results & Discussion: Results showed that, despite invoking bone formation, AB, BG, and DBM were found un‐resorbed in situ at 16 weeks. Conversely, LTCP showed an early increase in bone formation as well as clear evidence of complete degradation and reparative bone remodelling, resulting in the total reconstitution of the marrow cavity and marrow tissue. Conclusion: LTCP promoted increased early bone formation, associated with an improved degradation rate, compared with the other three bone‐substitute biomaterials tested. To cite this article:
Araújo MVF, Mendes VC, Chattopadhyay P, Davies JE. Low‐temperature particulate calcium phosphates for bone regeneration.
Clin. Oral Impl. Res. 21 , 2010; 632–641.     

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor‐β supergene family

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor‐β (TGF‐β) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF‐β₃ (hTGF‐β₃) in growth factor‐reduced Matrigel^® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non‐human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF‐β₃ in Matrigel^® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF‐β₃ in Matrigel^® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF‐β supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine‐tuning of speciation‐related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.     

Transfection of truncated bone morphogenetic protein receptor‐II into oral squamous cell carcinoma cell line Tca8113 and inhibitory effect on proliferation and inductive effect on apoptosis

J Oral Pathol Med (2010) 40 : 490–496 Bone morphogenetic proteins (BMPs), one of the crucial regulators in embryonic development and bone formation, have been implicated in epithelium‐derived tumors. Previous results showed the involvement of overexpression of BMP 2, 4, 5 in the carcinogenesis of oral epithelia. The ability of BMP receptor‐II mutant to modify the malignant phenotype of oral squamous cell carcinoma cell line Tca8113 by blocking the BMP signal transduction pathway has been proposed. In this study, a negative truncated mutant of the BMP receptor‐II (tBMPR‐II) was transfected into Tca8113 cells. The effects were evaluated though RT‐PCR, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) assay, BrdU staining, cell cyclin assay, TdT‐mediated dUTP nick end labeling (TUNEL) staining, and cell cycle protein detection. Overexpression of tBMPR‐II gene transfection truncates the expression of BMPR‐II mRNA expression, but not BMP 2, 4, 5. tBMPR‐II resulted in a remarkable inhibition of cell proliferation and viability compared with control Tca8113. The inhibitory effects were partly attributed to the induction of apoptosis and cell cycle arrest in G₀/G₁ accompanied by downregulation of the intracellular cell cycle proteins of cyclin D1 and cyclin‐dependent kinases 4, as well as the upregulation of p27 and p57. Loss of BMP signals correlates tightly with suppression of cell proliferation, induction of apoptosis, and benign transformation of Tca8113 cells phenotype.     

Divergence of the systemic immune response following oral infection with distinct strains of Porphyromonas gingivalis

Periodontitis is a polymicrobial oral infection characterized by the destruction of tooth‐supporting structures that can be linked to systemic diseases such as cardiovascular disease, diabetes or rheumatoid arthritis. Porphyromonas gingivalis, a bacterium implicated in the etiology of periodontitis, has shown variation in inducing T‐cell responses among different strains. Therefore, in this study we investigated the strain‐specific immune response using a murine experimental model of periodontitis. Periodontitis was induced by P. gingivalis strains A7A1‐28, W83 and W50, and later confirmed by the presence of P. gingivalis in the oral microflora and by alveolar bone resorption. Splenocytes were evaluated for gene expression, cellular proteins and cytokine expression. Dendritic cells were stimulated in vitro for T helper cell–cytokine profiling. Results showed that P. gingivalis had the ability to alter the systemic immune response after bacterial exposure. Strains W50 and W83 were shown to induce alveolar bone loss, whereas the A7A1‐28 strain did not significantly promote bone resorption in mice. Splenocytes derived from mice infected with strains W50 and W83 induced expression of high levels of interleukin‐4 (IL‐4) but A7A1‐28 stimulated increased IL‐10. Stimulation of dendritic cells in vitro showed a similar pattern of cytokine expression of IL‐12p40, IL‐6 and transforming growth factor‐β among strains. A distinct systemic response in vivo was observed among different strains of P. gingivalis, with IL‐10 associated with the least amount of alveolar bone loss. Evaluation of pathogen‐driven systemic immune responses associated with periodontal disease pathogenesis may assist in defining how periodontitis may impact other diseases.     

Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts

Agis H, Watzek G, Gruber R. Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts. J Periodont Res 2012; 47: 165–173. © 2011 John Wiley & Sons A/S Background and Objective: Pharmacological inhibitors of prolyl hydroxylases (PHDs) can induce a proangiogenic response that favors wound healing and bone regeneration. However, the response of periodontal cells to PHD inhibitors is unknown. Material and Methods: To determine the effects of PHD inhibitors on periodontal cells, we exposed human fibroblasts from the gingiva and the periodontal ligament to dimethyloxallyl glycine, desferrioxamine, l ‐mimosine and CoCl₂. Viability, proliferation, and protein synthesis were assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), [³H]thymidine, and [³H]leucine incorporation, respectively. The levels of Ki67, hypoxia‐inducible factor 1α (HIF‐1α), p27, phosphorylated c‐Jun N‐terminal kinase (JNK) and phosphorylated p38 were determined by immunohistochemistry and western blotting. Vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative PCR. Protein levels of VEGF and interleukin (IL)‐6 were evaluated by immunoassays. Results: We found that PHD inhibitors, while leaving cell viability unchanged, reduced proliferation and protein synthesis. This was paralleled by decreased Ki67 levels and increased p27 levels, suggesting that PHD inhibitors provoke growth arrest. Independently from this response, PHD inhibitors stabilized HIF‐1α and increased the production of VEGF. This increase of VEGF was observed in the presence of proinflammatory IL‐1 and pharmacological inhibitors of JNK and p38 signaling. Moreover, PHD inhibitors did not modulate expression of IL‐6 and the phosphorylation of JNK and p38. Conclusion: These results suggest that PHD inhibitors enhance the production of VEGF in periodontal fibroblasts, even in the presence of proinflammatory IL‐1. The data further suggest that PHD inhibitors do not provoke a significant proinflammatory or anti‐inflammatory response in this in vitro setting.     

Differential effects of bone graft substitutes on regeneration of bone marrow

Background: This study used a rat tibial marrow ablation model to test the hypothesis that bone remodeling within the medullary canal varies with bone graft materials of different chemical compositions and structural properties, impacting marrow cavity restoration. Bone graft materials were selected based on their relative resorption or degradation in vivo and their osteogenic properties. Methods: Following ablation of the right tibial marrow in male Sabra‐strain rats, materials were implanted in the proximal marrow cavity: poly‐d,l ‐lactide‐co‐glycolide 75 : 25 (PLGA); coralline‐hydroxyapatite (HA), calcium‐sulfate (CaSO4), collagen–HA–tricalcium phosphate granules, anorganic bovine bone mineral, demineralized bone matrix (DBM), 45S5 Bioglass (BG), PLGA with BG 50 : 50, PLGA : BG 80 : 20, and PLGA and PLGA:BG 50 : 50 plus bone marrow (BM). Control tibias were ablated but received no implants. At 2 (endosteal bone healing), 4 (marrow cavity remodeling) and 8 weeks (marrow restoration), six to eight animals per group were euthanized and tibias processed for histomorphometry of proximal and distal medullary canals. Results: Control tibias showed primary bone in proximal and distal medullary canals at 2 weeks, with trabeculae surrounded by cellular marrow. At 4 and 8 weeks, control trabeculae were thinned and marrow had more fat cells. In the treated tibias, trabecular bone volume (TBV) varied with time and was material specific. Most implants supported comparable TBV at 2 weeks. Sites with CaSO4 or DBM exhibited decreased TBV with time whereas trabecular bone was retained in proximal tibias containing other materials, closely juxtaposed to the implants. TBV did not always correlate directly with implant volume, but changes in BM volume were correlated inversely with TBV. Addition of BM increased marrow restoration in sites containing PLGA; however, BM reduced restoration of marrow when added to PLGA : BG. Although the presence of implants in the proximal tibia resulted in retention of trabecular bone, there was a time‐dependent reduction in TBV in distal canals; the rate and extent of the distal TBV reduction were implant dependent. Conclusions: Thus, although many materials can support bone formation in the marrow cavity, bone quality, quantity, and physical relationship to the implant, and its rate of resorption differ in a material‐dependent manner, resulting in differences in the restoration of marrow. Clinical relevance: Bone graft materials should be selected not only for their ability to support new bone formation but also for their impact on the remodeling phase of bone healing.