Cholesterol depletion induces mesenchymal properties in oral squamous cell carcinoma cell line.

BACKGROUND: Cholesterol in cell membranes is crucial for cell signaling, adhesion, and migration. Membranes feature cholesterol-rich caveolae with caveolin proteins, playing roles in epithelial-mesenchymal transition and cancer progression. Despite elevated cholesterol levels in tumors, its precise function and the effects of its depletion in oral squamous cell carcinoma remain unclear. The aim of this study was to evaluate the influence of cholesterol depletion in oral squamous cell carcinoma cell line and epithelial-mesenchymal transition process. METHODS: Cholesterol depletion was induced on SCC-9 cells by methyl-ß-cyclodextrin and cell viability, proliferation, apoptosis, and colony formation capacities were evaluated. Gene and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-qPCR) and Western Blot, respectively, and cell sublocalization was assessed by immunofluorescence. RESULTS: Cholesterol depletion resulted in alteration of oral squamous cell carcinoma cell morphology at different concentrations of methyl-ß-cyclodextrin, as well as decreased cell proliferation and viability rates. Analysis of CAV1 transcript expression revealed increased gene expression in the treated SCC-9 during the 24 h period, at different concentrations of methyl-ß-cyclodextrin: 5 , 7.5, 10, and 15 mM, in relation to parental SCC-9. CAV1 protein expression was increased, with subsequent dose-dependent decrease. A statistically significant difference was observed in samples treated with 5 mM of methyl-ß-cyclodextrin (p = 0.02, Kruskal-Wallis test). The immunofluorescence assay showed lower cytoplasmic and membrane labeling intensity in the treated samples for CAV1. CONCLUSION: These findings indicate the modulation of cholesterol as a possible mechanism underlying the regulation of these molecules and activation of epithelial-mesenchymal transition in oral squamous cell carcinoma.

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

BACKGROUND: Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). METHODS: Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. RESULTS: Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. CONCLUSION: Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines.

INTRODUCTION: Cholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. MATERIALS AND METHODS: Cell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-ß-cyclodextrin (MßCD - 7.5, 10 or 15 mM) RESULTS: Decreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein ß-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. CONCLUSION: Cholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

A common SNP risk variant MT1-MMP causative for Dupuytren's disease has a specific defect in collagenolytic activity.

Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.

Extracellular Matrix Composition and Remodeling: Current Perspectives on Secondary Palate Formation, Cleft Lip/Palate, and Palatal Reconstruction.

Craniofacial development comprises a complex process in humans in which failures or disturbances frequently lead to congenital anomalies. Cleft lip with/without palate (CL/P) is a common congenital anomaly that occurs due to variations in craniofacial development genes, and may occur as part of a syndrome, or more commonly in isolated forms (non-syndromic). The etiology of CL/P is multifactorial with genes, environmental factors, and their potential interactions contributing to the condition. Rehabilitation of CL/P patients requires a multidisciplinary team to perform the multiple surgical, dental, and psychological interventions required throughout the patient's life. Despite progress, lip/palatal reconstruction is still a major treatment challenge. Genetic mutations and polymorphisms in several genes, including extracellular matrix (ECM) genes, soluble factors, and enzymes responsible for ECM remodeling (e.g., metalloproteinases), have been suggested to play a role in the etiology of CL/P; hence, these may be considered likely targets for the development of new preventive and/or therapeutic strategies. In this context, investigations are being conducted on new therapeutic approaches based on tissue bioengineering, associating stem cells with biomaterials, signaling molecules, and innovative technologies. In this review, we discuss the role of genes involved in ECM composition and remodeling during secondary palate formation and pathogenesis and genetic etiology of CL/P. We also discuss potential therapeutic approaches using bioactive molecules and principles of tissue bioengineering for state-of-the-art CL/P repair and palatal reconstruction.

E-cadherin regulators are differentially expressed in the epithelium and stroma of keratocystic odontogenic tumors.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is the process where cells lose their epithelial features and acquire properties of typical mesenchymal cells. The dissociation of tumor cells due to changes in cell-cell adhesion is one of the key principles of tumor invasion and EMT. Thus, the knowledge of the molecular features of EMT in keratocyst odontogenic tumor (KOT) can provide useful markers to aid in the diagnosis and prognosis and perhaps contribute to an alternative therapeutic approach as it shows an aggressive clinical behavior and high recurrence rates. This study aimed to evaluate the EMT in KOT by the immunoexpression of E-cadherin, N-cadherin, Snail, and Slug and comparing to radicular cysts and dental follicles. METHODS: Thirty-two KOTs, 15 radicular cysts, and 08 dental follicles were used for immunohistochemistry, evaluating the extent, intensity, labeling pattern, cellular compartment in the epithelium and stroma, and the presence of inflammation. RESULTS: E-cadherin was preserved in most cases of keratocystic odontogenic tumor. N-cadherin was increased in the tumor epithelium, a result that was positively correlated with the heterogeneous and nuclear immunoexpression of Slug in the epithelium; Slug also correlated with high Snail immunoexpression. N-cadherin was positively correlated with Slug in the stroma of keratocystic odontogenic tumors. CONCLUSIONS: The high immunoexpression of Snail and nuclear Slug in keratocystic odontogenic tumors suggests these proteins as transcription factors without necessarily participating in 'cadherin switching'. However, the knowledge of their induction of the epithelial-mesenchymal transition in odontogenic tumors is still limited.

Immunohistochemical expression of WNT5A and MMPs in odontogenic epithelial tumors and cysts.

OBJECTIVE: The aim of this study was compare the expression of WNT5A and MMP2, 7 and 20, in frequent benign odontogenic tumors and odontogenic cysts, since these lesions have a different biological behavior. MATERIALS AND METHODS: Eighty-one paraffin-embedded specimens of odontogenic tumors, including ameloblastoma and keratocystic odontogenic tumor, and thirty-two odontogenic cysts were used for immunohistochemical analysis. RESULTS: The expression of WNT5A in odontogenic tumors and inflammatory cyst was higher than in developmental odontogenic cyst. There was no statistical difference (p<0.05) in the expression of WNT5A when comparing the analyzed tumors. The expression of MMP7 was lower in RC with a statistical difference when compared with all tumors and cysts. Statistical differences also occurred when comparing glandular odontogenic cyst (GOC) to keratocyst odontogenic tumor (KOT) and calcifying cystic odontogenic tumor (CCOT). MMP20 expression was higher in ameloblastoma when compared to adenomatoid odontogenic tumor (AOT), DC and GOC. The expression of MMP20 was lower in CCOT when compared to all tumors and cysts. CONCLUSIONS: The expression of WNT5A in a group of odontogenic lesions suggests the participation of a non-canonical WNT signaling pathway in the progression and maintenance of these lesions. These molecules are possibly involved in the biological differences between odontogenic tumors and cysts. Considering previous studies, WNT5A may help promote the calcification seen in AOT, CCOT and CEOT by activating MMP7.

Involvement of the NF-кB/p50/Bcl-3 complex in response to antiangiogenic therapy in a mouse model of metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) represents approximately 2-3% of human malignancies. Nuclear transcription factor кB (NF-кB) is composed of a family of transcription factors that have been associated with the development and progression of RCC. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we evaluated the expression of NF-кB in metastatic tumor cells from animals treated with ES. Balb/c-bearing Renca-EGFP cells were treated with NIH/3T3-LendSN or NIH/3T3-LXSN cells as a control. At the end of the in vivo experiment, plasma Renca-EGFP-sorted cells and tissue lung samples were collected. A real-time PCR array for NF-κB target genes revealed that ES therapy led to down regulation of Bcl-3 (P<0.031), NF-кB1 (P<0.001) and c-Rel (P<0.004) in the ES-treated group. Using an electrophoretic mobility shift assay (EMSA), we observed a reduction in NF-kB binding activity in ES-treated Renca-EGP cells. Furthermore, a supershift assay showed a clear shift of the NF-кB DNA band in samples incubated with a p50 antibody. By immunohistochemistry analysis, ES treatment resulted in a significant reduction in expression of p50. (ES vs. control P<0.05). The immunoprecipitation experiments confirmed the presence of a p50/Bcl-3 complex in nuclear extracts from cells of metastatic lung tissues. Our findings indicate that p50 and Bcl-3 plays a regulatory role in gene transcription in RCC.

Bone tissue remodeling and development: focus on matrix metalloproteinase functions.

Bone-forming cells originate from distinct embryological layers, mesoderm (axial and appendicular bones) and ectoderm (precursor of neural crest cells, which mainly form facial bones). These cells will develop bones by two principal mechanisms: intramembranous and endochondral ossification. In both cases, condensation of multipotent mesenchymal cells occurs, at the site of the future bone, which differentiate into bone and cartilage-forming cells. During long bone development, an initial cartilaginous template is formed and replaced by bone in a coordinated and refined program involving chondrocyte proliferation and maturation, vascular invasion, recruitment of adult stem cells and intense remodeling of cartilage and bone matrix. Matrix metalloproteinases (MMPs) are the most important enzymes for cleaving structural components of the extracellular matrix (ECM), as well as other non-ECM molecules in the ECM space, pericellular perimeter and intracellularly. Thus, the bioactive molecules generated act on several biological events, such as development, tissue remodeling and homeostasis. Since the discovery of collagenase in bone cells, more than half of the MMP members have been detected in bone tissues under both physiological and pathological conditions. Pivotal functions of MMPs during development and bone regeneration have been revealed by knockout mouse models, such as chondrocyte proliferation and differentiation, osteoclast recruitment and function, bone modeling, coupling of bone resorption and formation (bone remodeling), osteoblast recruitment and survival, angiogenesis, osteocyte viability and function (biomechanical properties); as such alterations in MMP function may alter bone quality. In this review, we look at the principal properties of MMPs and their inhibitors (TIMPs and RECK), provide an up-date on their known functions in bone development and remodeling and discuss their potential application to Bone Bioengineering.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma.

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

ORAOV1 is amplified in oral squamous cell carcinoma.

BACKGROUND: Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. METHODS: Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. RESULTS: ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. CONCLUSION: The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification.

Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9.

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice.

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos--E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel's cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial-mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation.

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance.

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

Avaliação da biocompatibilidade de uma membrana de pericárdio bovino acelular e seu potencial como carreador de osteoblastos / Evaluation of the biocompatibility of an acellular bovine pericardium membrane and its potential as an osteoblast scaffold

A biocompatibilidade de uma membrana de pericárdio bovino foi avaliada em tecido subcutâneo de camundongos 3,7, 15, 30 e 60 dias após a implantação. Os componentes celulares da resposta inflamatória, a degradação da membranae as características do colágeno foram analisadas em cortes histológicos corados pela hematoxilina-eosina, tricrômico de Masson e Picro-Sírius, respectivamente. Para verificar seu potencial como carreador celular, osteoblastos humanos(hFOB1.19, ATCC) foram semeados sobre a membrana e mantidos em DMEM/F12 por 7 dias. Os resultados in vitro mostraram que os osteoblastos proliferaram em monocamada na superfície da membrana, mas sem penetrar em seu interior. A análise dos cortes histológicos demonstrou 3 dias após a implantação apenas a formação da rede de fibrina. Aos 7 dias, o material implantado estava circundado por células inflamatórias mononucleares, com pouca penetração celular no seu interior. Após 15 dias foi observado um intenso infiltrado inflamatório em contato e dentro do material,bem como sinais de degradação interna e externa. No período de 30 dias, o material, em processo bastante avançado de absorção, estava totalmente tomado por fibroblastos e macrófagos. Aos 60 dias pós-implantação, o material não foi maisdetectado em quaisquer dos animais e a tecido subcutâneo apresentava-se normal. Os cortes corados com Picro-Sírius e observados sob luz polarizada mostraram o remodelamento tecidual. Em conclusão, a membrana de pericárdio é bioabsorvívele biocompatível, porém, in vitro, não proporciona uma adequada matriz tridimensional para osteoblastos.

The biocompatibility of a pericardium membrane was evaluated in the subcutaneous tissue of mouse killed 3, 7, 15, 30 and 60 days post implantation. The cellular components of inflammatory infiltrate, the membrane degradation, and the collagen characteristic were analyzed in histological sections stained with hematoxilyn and eosin, Tricromic of Masson and Sirius Red, respectively. The potential features as a tissue engineering scaffold was tested in vitro using human osteoblasts (h.Fob 1.19, ATCC) seeded over the membrane and maintained for 7 days in DMEM/F12. We observed in vitro the monolayer proliferation of osteoblasts, but without penetrating in the membrane. The histological sections showed after 3 days of implantation only the presence of a fibrin net. At the 7-day period, mononuclear inflammatory cells were observed around the implant, but a few one were observed inside the membrane. After 15 days the inflammatory infiltrate was more intense than in the previous period and the cells were inside and in close contact to the material showing evident signs of internal and external degradation. The implant degradation was intense after 30 days and theresidual material was fulfilled of fibroblasts and macrophages. No signs of membrane were observed after 60 days in any animals and the subcutaneous tissue presented normal aspect. Sirius Red staining at polarized light had evidenced the tissue remodeling throughout the experimental periods. In conclusion, the pericardium membrane is bioabsorbable and biocompatible, but, in vitro, do not fulfill the requirements as a tridimensional scaffold to osteoblast.

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat.

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.

Receptor activator NFkappaB-ligand and osteoprotegerin protein expression in human periapical cysts and granulomas.

OBJECTIVE: The purpose of this study was to determine the expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG) associated with bone destruction in periapical cysts and granulomas. STUDY DESIGN: Forty human dental chronic periapical lesions were collected after periapical surgery. The lesions collected were fixed in 10% buffered formalin and histologically processed. At least 2 sections of each specimen were stained with hematoxylin and eosin for microscopic diagnosis. After that, 10 human periapical granulomas and 10 cysts were selected for immunohistochemical analysis for RANKL, OPG, and CD68+. RESULTS: Polymorphonuclear neutrophils, macrophages, endothelial cells, and lymphocytes were stained for RANKL and OPG in both lesions. Epithelial cells were also stained for RANKL and OPG in periapical cysts. Quantitative analysis was conducted and the results were expressed as a ratio of the number of immunostained cells over the total number of cells in the field (n = 100). The ratio of RANKL+/total cells was higher than OPG+/total cells in periapical granulomas (0.553 +/- 0.153 and 0.483 +/- 0.189, respectively; P < .0012; paired t test) and in cysts (0.519 +/- 0.09 and 0.339 +/- 0.117, respectively; P < .0001; paired t test). The ratios of OPG+/total cells (P < .0001; paired t test) and RANKL+/total cells (P < .0322; paired t test) were greater in granulomas than in cysts. However, the ratio RANKL+/OPG+ in granulomas (1.336 +/- 0.723) and cysts (1.404 +/- 0.385) was not significantly different. The ratio of CD68+/total cells was significantly higher in granulomas (0.381 +/- 0.040) than in cysts (0.307 +/- 0.068) (P < .0001; unpaired t test with Welch correction). CONCLUSION: Taking into account the limitations of the experimental approach employed, our findings indicate the presence of RANKL and OPG in cysts and granulomas, strongly suggesting the involvement of these gene products in the development of periapical lesions.

Expression of metalloproteinase 2 in the cell response to porous demineralized bovine bone matrix.

The purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 microm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin-biotin-peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 +/- 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 +/- 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 +/- 10.4) to day 28 (19.5 +/- 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.