Dexamethasone promotes regeneration of crushed inferior alveolar nerve by inhibiting NF-κB activation in adult rats

PurposeNuclear factor kappa B (NF-κB), which is closely related to inflammation, has become a topic of interest for research. The aim of this study is to investigate the effects of dexamethasone (Dex), an inhibitor of NF-κB, on inferior alveolar nerve injury in adult rats.Materials and methodsThe crushed inferior alveolar model is established in Wistar rats and they are randomly divided into three groups according to treatment: pyrrolidine dithiocarbamate (PDTC), dexamethasone (Dex), and saline (physiological saline). After treatment, the rats are respectively sacrificed at 3, 7, and 14 d, and inferior alveolar nerves are extracted for histochemical and western blot analysis.ResultCompared with the PDTC and saline groups, nerve fibers in the Dex group are regularly arranged with few vacuoles, which is similar to normal inferior alveolar nerves. Immunofluorescent results show significantly decreased NF-κB expression in the Dex group. Western bolt shows higher expression of GAP-43 and lower expression of NF-κB.ConclusionTaken together, all results show that dexamethasone significantly improved the regeneration of crushed inferior alveolar nerves by inhibiting NF-κB activation in adult rats.     

Association between facial measurements and polymorphisms in human epidermal growth factor and transforming growth factor β1

The aim of the study was to evaluate the association between genetic polymorphisms in human epidermal growth factor (EGF) (rs4444903) and transforming growth factor β1 – (TGF-β1) (rs1800470) with facial measurements in patients with dentofacial deformities. A total of 144 adult patients with dentofacial deformities were included. Facial linear and angular measurements were traced in lateral cephalometric radiographs used Dolphin 2D software. Cells from oral mucosa were collected for DNA to be extracted. The polymorphisms were genotyped using real-time polymerase chain reaction (PCR). Probabilites of less than 0.05 were accepted as significant. The rs4444903 heterozygous patients had a decrease in the mandibular length (p = 0.043) and the length of the mandibular base (p = 0.008), and homozygous A patients also had a reduction in the length of the mandibular base (p = 0.013) compared with homozygous G patients. Patients AG had an increase in measurement of the anterior facial height (p = 0.032) and in ANS-Me distance (p = 0.022) when compared with homozygous A. To the rs1800470, heterozygous patients had an increase in the length of the mandibular base (p = 0.043) when compared with homozygous A. Heterozygous AG patients had an increase in angular measurements in TGF-β1 polymorphism for the upper gonial angle, when compared with the homozygous AA (p = 0.032). Genetic polymorphisms in EGF and TGF-β1 are associated with facial measurements in a Brazilian population of patients with dentofacial deformities.     

Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein-1 and human transforming growth factor-β3 in Class II furcation defects of Papio ursinus

Teare JA, Petit J‐C, Ripamonti U. Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein‐1 and human transforming growth factor‐β ₃ in Class II furcation defects of Papio ursinus. J Periodont Res 2012; 47: 336–344. © 2011 John Wiley & Sons A/S Background and Objective: Binary applications of recombinant human osteogenic protein‐1 (hOP‐1) and transforming growth factor‐β3 (hTGF‐β3) synergize to induce pronounced bone formation. To induce periodontal tissue regeneration, binary applications of hOP‐1 and hTGF‐β₃ were implanted in Class II furcation defects of the Chacma baboon, Papio ursinus. Material and Methods: Defects were created bilaterally in the furcation of the first and second mandibular molars of three adult baboons. Single applications of 25 μg hOP‐1 and 75 μg hTGF‐β₃ in Matrigel^® matrix were compared with 20:1 binary applications, i.e. 25 μg hOP‐1 and 1.25 μg hTGF‐β₃. Morcellated fragments of autogenous rectus abdominis striated muscle were added to binary applications. Sixty days after implantation, the animals were killed and the operated tissues harvested en bloc. Undecalcified sections were studied by light microscopy, and regenerated tissue was assessed by measuring volume and height of newly formed alveolar bone and cementum. Results: The hOP‐1 and hTGF‐β₃ induced periodontal tissue regeneration and cementogenesis. Qualitative morphological analysis of binary applications showed clear evidence for considerable periodontal tissue regeneration. Quantitatively, the differences in the histomorphometric values did not reach statistical significance for the group size chosen for this primate study. The addition of morcellated muscle fragments did not enhance tissue regeneration. Binary applications showed rapid expansion of the newly formed bone against the root surfaces following fibrovascular tissue induction in the centre of the treated defects. Conclusion: Binary applications of hOP‐1and hTGF‐β₃ in Matrigel^® matrix in Class II furcation defects of P. ursinus induced substantial periodontal tissue regeneration, which was tempered, however, by the anatomy of the furcation defect model, which does not allow for the rapid growth and expansion of the synergistic induction of bone formation, particularly when additionally treated with responding myoblastic stem cells.     

Effect of β tricalcium phosphate particle size on recombinant human platelet-derived growth factor-BB-induced regeneration of periodontal tissue in dog

The aim of this study was to investigate the effect of β tricalcium phosphate (β-TCP) particle size on recombinant human platelet-derived growth factor-BB (rhPDGF-BB)-induced regeneration of periodontal tissue in dog. The control group (rhPDGF-BB alone) was characterized by incomplete, newly formed bone. The large-particle β-TCP (L-TCP(O))/rhPDGF-BB group showed a statistically significant increase in both new bone and cementum formation compared to the small-particle β-TCP (S-TCP(G))/rhPDGF-BB group. These findings suggest that L-TCP(O)-particle promotes rhPDGF-BB-induced formation of bone and cementum.     

β-Tricalcium phosphate promotes cell proliferation,osteogenesis and bone regeneration in intrabony defects in dogs

ObjectiveThis study investigates the effect of the new synthetic bone grafting material, high pure-phase β-tricalcium phosphate (Cerasorb^® M, granule size 500–1000 μm), on the osteogenesis process and proliferation marker in bone marrow stromal cells (BMSCs) and its regenerative effect in the periodontal intrabony defects in dogs.DesignThe effect of Cerasorb^® M (20 and 40 mg ml^?1 for 1 and 2 weeks) on the proliferation rate of BMSCs was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on the proliferating cell nuclear antigen (PCNA) by immunoblotting and on alkaline phosphatase level by colourimetric assay. The regenerative effect of Cerasorb^® M in the periodontal intrabony defects in dogs was investigated by histological and immunohistochemical analysis after 3 and 6 months of grafting.ResultsIncubation of BMSCs with Cerasorb^® M for 2 weeks led to significant increase in cell proliferation rate, which was associated with increased PCNA. Cerasorb^® M significantly increased the production of alkaline phosphatase as a marker for the osteogenic stromal lineage and for differentiation and bone formation in BMSCs after 2 weeks. In the histological features and immunohistochemical analysis of PCNA of the intrabony defects in dogs augmented with Cerasorb^® M, osteoid tissue with a plate-like structure and cellular mesenchymal proliferation besides osteoid islands joined by bridges were observed after 3 months. Six months after the implantation, the Cerasorb^® M granules were replaced by abundant new plate-like bone besides PCNA-enriched, small, oval-shaped mononuclear cells and multinucleated-giant cells that were attached to newly formed bones. No remains of the Cerasorb^® M granules could be seen after 3 and 6 months with the newly formed plate-like bones and no histological sign of inflammatory reaction or formation of foreign-body granulomas.ConclusionCerasorb^® M may induce cell proliferation via induction of PCNA that may induce early osteogenesis and bone formation. Cerasorb^® M regenerated the bone completely in intrabony defects and that this regeneration was highly associated with PCNA expression in different cell lineage.     

Detection of mutation-specific epidermal growth factor receptor (E746–A750del) and lack of detection of human papillomavirus in oral squamous cell carcinoma

The clinical impact of epidermal growth factor receptor (EGFR) (E746–A750del) mutation and human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) is unclear. EGFR (E746–A750del) expression was analyzed in OSCC specimens (n = 161) by immunohistochemistry. The expression results were correlated with clinical characteristics and impact on survival. Using INNO-LiPA Extra, high-risk HPV types were genotyped and analyzed in 211 OSCC specimens. Positive EGFR (E746–A750del) expression (n = 40/161, 25%) was not associated with any clinicopathological characteristics, prognostic factors, social habits (smoking, alcohol consumption), or tumour-specific survival. HPV16 DNA was detected in three out of 211 samples (HPV16-positive: n = 3/211, 1.4%). This study shows that mutation-specific EGFR (E746–A750del) expression and HPV do not appear to be relevant to the survival of patients with OSCC.     

Anaesthetic efficacy of bupivacaine 2-hydroxypropyl-β-cyclodextrin for dental anaesthesia after inferior alveolar nerve block in rats

Bupivacaine is a long-acting local anaesthetic that is widely used in medicine and dentistry. The duration and intensity of its sensory blockade in animal models is increased by its inclusion in complexes with cyclodextrins. The aim of the present study was to evaluate the anaesthetic efficacy of bupivacaine 2-hydroxypropyl-β-cyclodextrin (HPβCD) inclusion complex for dental anaesthesia after inferior alveolar nerve block in rats. Thirty rats were each given an injection close to the mandibular foramen of 0.2 ml of one of the following formulations: 0.5% bupivacaine alone; 0.5% bupivacaine with 1:200,000 epinephrine; and 0.5% bupivacaine-HPβCD inclusion complex (bupivacaine-HPβCD). The other sides were used as controls, with either 0.9% saline or anaesthetic-free HPβCD solution being injected. The onset, success, and duration of pulpal anaesthesia were assessed by electrical stimulation (“pulp tester”) on inferior molars. Results were analysed using ANOVA (Tukey), log rank, and chi square tests (α = 5%). There were no differences among the formulations in onset of anaesthesia (p = 0.59) or between the bupivacaine plus epinephrine and bupivacaine plus HPβCD in duration of anaesthesia, but bupivacaine plus epinephrine gave significantly higher values than bupivacaine alone (p = 0.007). Bupivacaine plus epinephrine was a better anaesthetic than bupivacaine alone (p = 0.02), while Bupi-HPβCD gave intermediate results, and therefore did not differ significantly from the other 2 groups (p = 0.18 with bupivacaine alone; and p = 0.44 with bupivacaine plus epinephrine). The bupivacaine-HPβCD complex showed similar anaesthetic properties to those of bupivacaine with epinephrine.     

Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in osteopetrotic (op/op) mice

ObjectiveExpression of osteoclasts in osteopetrotic (op/op) mice is substantially reduced by the absence of functional macrophage colony-stimulating factor (M-CSF). However, it has been reported that osteoclasts do gradually appear in the bones of op/op mice and spontaneously correct the osteopetrosis.DesignAge-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in op/op mice were examined.ResultsThe number of femoral osteoclasts, and the serum levels of VEGF, both gradually increased in op/op mice after birth and reached a peak in 120- and 60-day-old mice, respectively. However, the serum levels of RANKL showed an inverse relationship to osteoclast number.ConclusionsThese findings suggest that the appearance of osteoclasts may be influenced by the serum levels of VEGF and that the serum levels of RANKL may be influenced by the appearance of osteoclasts.     

Role of vascular endothelial growth factor in reconstructive surgery after surgical excision of malignant tumor

As a key mediator of normal physiological angiogenesis, vascular endothelial growth factor(VEGF) has been regarded as an emancipator to plastic surgeon, and yet a misfortune to oncology surgeon, due to its singular biological effect. Therefore in some clinical cases, especially for some malignant tumor patients having endured radical surgery and being craving for a reconstructive surgery, VEGF plays a role full of paradoxes. To make a clinical balance, we should find a point to inhibit tumor cell from utilizing VEGF and make a permission to normal tissues to employ it.     

Genetic polymorphism of tumor necrosis factor alpha (TNF-α) and tumor necrosis factor beta (TNF-β) genes and risk of oral pre-cancer and cancer in North Indian population

Objective

There are inconclusive data connecting single-nucleotide polymorphisms (SNPs) of TNF-α (rs361525) and TNF-β (rs909253) to potential malignant oral disorder (PMOD) such as lichen planus and oral fibrosis. Here, we have investigated the risk of oral squamous cell carcinoma as well as oral pre-cancerous lesions in North Indian population with the polymorphism of the TNFα/ β genes.

Material and methods

A total 500 patients with oral pre-cancer and OSCC and 500 healthy volunteers were genotypes for the TNF-α (-238) G/A (rs361525) and TNF-β (252) A/G (rs909253) gene polymorphism. Genotypes were identified by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). Genotype frequencies were evaluated by Chi-square test.

Results

Compared to the GG genotype, the GA genotype of TNF-α (G238A) polymorphism (rs361525) has been found to significantly increase the risk of oral disease (OR = 1.99) and especially the risk of lichen planus and OSCC (OR = 2.805 and 5.790, respectively). Similarly, the risk of oral disease was also more in the heterozygote (AG) than the common allele homozygote (AA) of TNF-β (A252G) polymorphism (rs909253) (OR = 1.483).

Conclusion

We conclude that the SNPs rs361525 and rs909253 were significantly associated with oral pre-cancer and OSCC.

     

Downregulated gene expression of TGF-βs in diabetic oral wound healing

BackgroundHealing of tooth extraction sockets in poorly controlled diabetic patients is often delayed and accompanied by severe infection. The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are still not fully understood.ObjectivesThe purpose of this study was to investigate molecular changes associated with delayed oral wound healing in diabetes.Materials and methodsSix to eight weeks old male type 2 diabetes and age matched control inbred mice were used and maxillary molar tooth extractions were performed. At 4 and 7 days after tooth extraction, the edentulous mucosa of the mice were harvested, and analyzed for histology and gene expression of key wound healing factors.ResultsIn the diabetic model, histological analysis showed that epithelial tissue migration for wound closure was delayed after tooth extraction compared to the control. Quantitative real-time PCR revealed that expression of the TGF-β1, TGF-β2, TGF-β3, TGFβRII and TGFβRIII genes was significantly downregulated in the diabetic model at 4 and 7 days after tooth extraction.ConclusionThese results suggest that delayed wound healing of oral mucosa in diabetes may be associated with decreased expression levels of these regulatory genes which play important roles in controlling epithelial wound closure.     

Expression of osteoprotegerin and receptor activator of nuclear factor ��B ligand in root resorption induced by heavy force in rats

Objective

The aim of this study was to investigate the expression patterns of osteoprotegerin (OPG) and the receptor activator of nuclear factor ??B ligand (RANKL) in root resorption during orthodontic tooth movement.

Material and methods

Forty 12-week-old male SD rats were used with the right maxillary side as the experimental group and the left maxillary side as the control group. After 1?N (100g) force was loaded on the right maxillary first molar, the rats were sacrificed on days 0, 1, 4, 8, and 12. Mesial root resorption of the first molar, the number of odontoclasts and osteoclasts, and OPG and RANKL mRNA expression were determined by hematoxylin-eosin and scanning electron microscopy, tartrate-resistant acid phosphate staining, and in situ hybridization, respectively.

Results

Serious root resorption was apparent on the pressure side of the mesial root of the right maxillary first molar on days 8 and 12. The number of odontoclasts in the cementum lacuna was elevated on days 8 and 12. OPG expression rose significantly on the tensile side, while RANKL expression increased on the pressure side. The mRNA level of RANKL was significantly elevated on days 4, 8, and 12. Moreover, the RANKL/OPG mRNA ratio was increased on the pressure side, but decreased on the tensile side.

Conclusion

Changes in the expression of RANKL mRNA and the RANKL/OPG mRNA ratio are accompanied by a parallel alteration in the number of odontoclasts and tooth resorption, suggesting crucial involvement of RANKL and OPG in tooth resorption.     

Plasma rich in growth factors promote gingival tissue regeneration by stimulating fibroblast proliferation and migration and by blocking transforming growth factor-β1-induced myodifferentiation

Background: Periodontitis involves inflammation and infection of the ligaments and bones that support the teeth. Gingival fibroblasts are the most abundant cells in periodontal tissue, and they play a role in maintaining the structural integrity of the tissue. Plasma rich in growth factors contain a pool of proteins and growth factors that promote wound healing and tissue regeneration. In the present study, we evaluate the potential of different formulations obtained with this approach to stimulate several biologic processes involved in wound healing, including fibroblast proliferation, migration, adhesion, and the autocrine release of some angiogenic factors and extracellular matrix components. Furthermore, the ability of this technology to prevent and inhibit transforming growth factor β1‐induced myodifferentiation was also determined. Methods: Cell proliferation was evaluated through a colorimetric assay, cell migration was performed on culture inserts, and cell adhesion was studied through a fluorescence‐based method. Enzyme‐linked immunosorbent assay was used to determine some of the biomolecules released by gingival fibroblasts. Smooth muscle actin expression was assessed through immunofluorescence microscopy. Results: Results showed that plasma rich in growth factors significantly increased gingival fibroblast proliferation, migration, and cell adhesion on type I collagen matrix. In addition, it stimulated the autocrine expression of vascular endothelial growth factor, hepatocyte growth factor, and hyaluronic acid. The myofibroblast phenotype, which is characterized by expressing α‐smooth muscle actin, was inhibited and reverted by treating with this technology. Conclusion: These findings suggest that plasma rich in growth factors is capable of promoting regeneration of gingival connective tissue by stimulating some of the main processes involved in wound regeneration.     

Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvβ6 and transforming growth factor beta signalling in a three-dimensional in vitro wound healing model

This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 × 10⁵ cm² each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2 mM Ca²⁺ EpiLife for 5 days, and then in an air–liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 μM zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin αvβ6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-β1) production into the supernatant, but down-regulated TGF-β receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin αvβ6 expression, cause the down-regulation of TGF-β/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.